COMPOSITIONS AND METHODS FOR RECOVERY OF STRANDED GAS AND OIL

Abstract

The present disclosure provides compositions and methods for using recombinant C.sub.1 metabolizing microorganisms capable of metabolizing sulfur containing compounds and other contaminants to biologically convert sour or acidic natural gas into high-value molecules, and to allow recovery of stranded oil.

Description

STATEMENT REGARDING SEQUENCE LISTING

[0001] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200206 412C1 SEQUENCE LISTING.txt. The text file is 255 KB, was created on Mar.20, 2019, and is being submitted electronically via EFS-Web.

BACKGROUND

[0002] Oil deposits are associated, in many cases, with natural gas, which can often be tainted with significant levels of sulfur (referred to as `sour gas`) and other contaminants (like CO.sub.2). Natural gas is highly flammable and potentially explosive, hence the gas must be dealt with to allow access to an associated oil deposit. Historically, gas was transferred to a pipeline for sale (requiring scrubbing to remove contaminants as well as additional costs to pressurize the gas for introduction into the pipeline), flared (e.g., as waste gas or smokeless), incinerated, and sometimes simply vented to the atmosphere. However, current environmental regulations prevent the flaring and venting of gas in many locations, especially due to the highly polluting effects of sulfur and other contaminants. Further, the current low price of natural gas means that introducing the gas into a pipeline is often unprofitable because more money will be required to make the gas suitable for the pipeline than can be recovered from its sale. An additional complication is that as new oil drilling and recovery technologies have come online, the majority of new oil deposits are located far from existing gas pipelines. Thus, even if a well operator was willing to spend money to remove the gas, the infrastructure does not exist to enable the operation.

[0003] For these and other reasons, there currently exist a number of known oil deposits which cannot be accessed due to the difficulties in mitigating the associated gas deposits. Given the high price of oil, there is a need in the art for alternative methods for converting gas (and associated contaminants) into safe and non-polluting forms in a cost-effective manner. The present disclosure meets such needs, and further provides other related advantages.

BRIEF SUMMARY

[0004] In brief, the present disclosure provides a recombinant C.sub.1 metabolizing microorganism having a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing an S substrate, wherein the recombinant microorganism is capable of assimilating and/or oxidizing the S substrate, a C.sub.1 substrate, or both.

[0005] In some aspects, the present disclosure provides a method for treating gas comprising culturing a first recombinant C.sub.1 metabolizing microorganism with a tainted gas feedstock comprised of a C.sub.1 substrate and an S substrate; wherein the recombinant microorganism includes a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate and the recombinant C.sub.1 metabolizing microorganism assimilates and/or oxidizes each substrate.

[0006] In some aspects, the present disclosure provides a system for treating gas comprising a source of gas comprising a C.sub.1 substrate and an S substrate; a bioreactor comprising a recombinant C.sub.1 metabolizing microorganism comprising a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate; and a connector disposed between the gas source and the bioreactor to allow flow of the gas into the bioreactor; wherein the recombinant C.sub.1 metabolizing microorganism assimilates and/or oxidizes each substrate.

[0007] In another aspect, the present disclosure provides a system for recovering stranded gas and/or oil, comprising a mechanism for recovering gas from an underground formation, wherein the gas comprises a C.sub.1 substrate and an S substrate, and the mechanism for recovering comprises a well; a mechanism for assimilating and/or oxidizing at least a portion of each substrate from the recovered gas, the mechanism for assimilating and/or oxidizing comprising a bioreactor, wherein the bioreactor comprises a recombinant C.sub.1 metabolizing microorganism comprising a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate; and a mechanism for recovering the bioremediated stranded oil from the underground formation, wherein the mechanism for recovering comprises a well.

[0008] In some embodiments, a polypeptide capable of metabolizing an S substrate is a hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, sulfur dioxygenase, sulfite oxidase, or any combination thereof. For example, the polypeptide capable of metabolizing the S substrate is (1) hydrogen sulfide:NADP.sup.+ oxidoreductase, sulfite oxidase, or both; (2) hydrogen sulfide:ferredoxin oxidoreductase, sulfite oxidase, or both; (3) sulfide:flavocytochrome-c oxidoreductase, sulfite oxidase, or both; (4) sulfide:quinone oxidoreductase, sulfite oxidase, or both; (5) a hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, or sulfide:quinone oxidoreductase, and wherein the endogenous sulfite oxidase activity is increased; or (6) a sulfur oxygenase.

[0009] In some embodiments, the polypeptide capable of metabolizing the S substrate is encoded by a nucleic acid wherein the nucleic acid comprises a sequence as set forth in any one of SEQ ID NOS.:21-54. In some embodiments, the polypeptide capable of metabolizing the S substrate comprises an amino acid sequence as set forth in any one of SEQ ID NOS.:55-88.

[0010] In some embodiments, the C.sub.1 substrate, the S substrate, or both are converted into a biological material, such as an animal feed, a fertilizer or an oil composition. In other embodiments, the S substrate is oxidized to a sulfate or a sulfide.

[0011] In some embodiments, a tainted gas feedstock is a light alkane gas, natural gas, unconventional natural gas, syngas, casinghead gas, wellhead condensate, or any combination thereof. In some embodiments, a tainted gas feedstock is an acid gas or a sour gas.

[0012] In any of the aforementioned embodiments, a recombinant C.sub.1 metabolizing microorganism may further comprise a second exogenous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof; and wherein the recombinant C.sub.1 metabolizing microorganism converts the C.sub.1 substrate into an oil composition. For example, the oil composition produced may be substantially located in the cell membrane of the C.sub.1 metabolizing microorganism.

[0013] In certain embodiments, the present disclosure further provides the step of obtaining the oil composition by extraction. In further embodiments, the extracted oil composition is further refined into a fuel, such as jet fuel, diesel fuel, paraffinic kerosene, gasoline, or any combination thereof.

[0014] In any of the aforementioned embodiments, the present disclosure further provides a second recombinant C.sub.1 metabolizing microorganism or cell lysate thereof, wherein the second recombinant C.sub.1 metabolizing microorganism comprises an exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing light alkanes; and wherein the second recombinant C.sub.1 metabolizing microorganism or cell lysate thereof oxidizes the C.sub.1 substrate into an alcohol composition. In some embodiments, the first recombinant C.sub.1 metabolizing microorganism further comprises a second exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing light alkanes such that the recombinant microorganism or cell lysate thereof oxidizes the C.sub.1 substrate into an alcohol composition. In any of these embodiments, a polypeptide capable of oxidizing light alkanes may be a monooxygenase selected from a MMO, AMO, BMO, PMO or P450.

[0015] In certain embodiments, the exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing light alkanes comprises a sequence as set forth in any one of SEQ ID NOS.:1-20. In some embodiments, the polypeptide capable of oxidizing light alkanes comprises a sequence as set forth in any one of SEQ ID NOS.:89-108.

[0016] In some embodiments, the recombinant C.sub.1 metabolizing microorganism further comprises a second exogenous nucleic acid molecule encoding a fatty acid converting enzyme; and wherein the recombinant C.sub.1 metabolizing microorganism converts the C.sub.1 substrate into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a hydroxy fatty acid, a dicarboxylic acid, or any combination thereof. In certain embodiments, the fatty acid converting enzyme is a fatty acyl-CoA reductase capable of forming a fatty alcohol. In some embodiments, the fatty acyl-CoA reductase capable of forming a fatty alcohol is FAR, CER4, or Maqu 2220. In certain embodiments, the fatty acid converting enzyme is a fatty acyl-CoA reductase capable of forming a fatty aldehyde. In certain embodiments, the fatty acyl-CoA reductase capable of forming a fatty aldehyde is acrl. In some embodiments, the fatty acid converting enzyme is a carboxylic acid reductase.

[0017] In some embodiments, the recombinant C.sub.1 metabolizing microorganism further comprises an exogenous nucleic acid molecule encoding a thioesterase. In certain embodiments, the thioesterase is a tesA lacking a signal peptide, UcFatB or BTE. In certain embodiments, endogenous thioesterase activity is reduced, minimal or abolished as compared to unaltered endogenous thioesterase activity.

[0018] In some embodiments, the recombinant C.sub.1 metabolizing microorganism further comprises an exogenous nucleic acid molecule encoding an acyl-CoA synthetase. In certain embodiments, the acyl-CoA synthetase is FadD, yng1, or FAA2. In certain embodiments, endogenous acyl-CoA synthetase activity is reduced, minimal or abolished as compared to unaltered endogenous acyl-CoA synthetase activity.

[0019] In some embodiments, the present disclosure further provides a recombinant nucleic acid molecule encoding a monooxygenase to produce w-hydroxy fatty acid. In certain embodiments, endogenous alcohol dehydrogenase activity is reduced, minimal or abolished as compared to unaltered endogenous alcohol dehydrogenase activity.

[0020] In some embodiments, endogenous alcohol dehydrogenase activity is increased or elevated as compared to unaltered endogenous alcohol dehydrogenase activity to produce dicarboxylic acid.

[0021] In some embodiments, the C.sub.1 metabolizing microorganism is selected from the group consisting of Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, and Pseudomonas. In other embodiments, the C.sub.1 metabolizing microorganism is selected from the group consisting of Candida, Yarrowia, Hansenula, Pichia, Torulopsis, and Rhodotorula.

[0022] In some embodiments, the C.sub.1 metabolizing microorganism is a bacterium. In certain aspects, the C.sub.1 metabolizing bacterium is a methanotroph or methylotroph. In certain aspects, the C.sub.1 metabolizing bacterium is a methanotroph. In certain embodiments, the methanotroph is a Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, or a combination thereof. In some aspects, the methanotroph is a Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402), Methylosinus trichosporium OB3b (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris, Methylocella palustris (ATCC 700799), Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystis bryophila, Methylocapsa aurea KYG, Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylomicrobium alcaliphilum, or a combination thereof. In some aspects, the methanotroph is Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomonas sp. 16a, Methylomicrobium alcaliphilum, or a high growth variant thereof.

[0023] In some embodiments, the C.sub.1 metabolizing bacterium is a methylotroph. In certain embodiments, the methylotroph is Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylobacterium nodulans, or a combination thereof.

[0024] In some embodiments, the C.sub.1 metabolizing bacterium is a natural gas, unconventional natural gas, or syngas metabolizing bacterium. In certain embodiments, the syngas metabolizing bacterium is Clostridium, Moorella, Pyrococcus, Eubacterium, Desulfobacterium, Carboxydothermus, Acetogenium, Acetobacterium, Acetoanaerobium, Butyribaceterium, Peptostreptococcus, or a combination thereof In certain aspects, the syngas metabolizing bacterium is Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium woodii, Clostridium neopropanologen, or a combination thereof. In some aspects, the C.sub.1 metabolizing microorganism is an obligate C.sub.1 metabolizing microorganism.

[0025] In certain embodiments, the culture further comprises a heterologous bacterium.

DETAILED DESCRIPTION

[0026] The instant disclosure provides compositions and methods for biologically converting gas, along with any unwanted impurities or contaminants, into useful compositions, such as high-value molecules (e.g., alcohols, fatty acid derivatives, oil composition), biological material (e.g., animal feed), or a combination thereof. For example, oil may be stranded because it is associated with gas, such as tainted gas (e.g., acidic, sour gas). Such oil associated gas can be fed to recombinant C.sub.1 metabolizing microorganisms comprising a nucleic acid molecule encoding a sulfur utilizing (e.g., sulfide converting) enzyme, to generate one or more different compounds and allow recovery of the previously stranded oil. This new approach allows for the use of methylotroph or methanotroph bacteria as a new host system to bioremediate stranded oil.

[0027] By way of background, natural gas from a well may contain a number of undesirable compounds that must be removed or reduced prior to distribution and sale, or the natural gas and any associated contaminants must be removed to access stranded oil. Hydrogen sulfide is one of the most common problems in the gas industry because it is a toxic gas that is very corrosive in the presence of water. Current regulations require that natural gas destined for the fuel market contain no more than 0.25 grains per 100 standard cubic feet or 4 parts per million (ppm) on a volume basis. The most common process to remove hydrogen sulfide involves a two-step treatment: (1) an amine process (also known as the Girdler process, usually using alkanolamines (e.g., monoethanolamine, diethanolamine) to remove hydrogen sulfide from natural gas; Maddox, Gas and Liquid Sweetening, 2.sup.nd Edition, Norman, OK: Campbell Petroleum Series, 1974) followed by (2) the Claus process (for elemental sulfur recovery) (Canjur and Manning, Thermodynamic Properties and Reduced Correlations for Gases, Gulf Publishing Co., 1967). But, the hydrophobic elemental sulfur produced by the Claus process requires the use of surfactants since sulfur tends to float and aggregate, which leads to foaming and plugging. Moreover, when the sulfur content is not at sufficient levels or when the CO.sub.2 content is greater than the H.sub.2S content, the Claus process is not economical. The instant disclosure provides compositions and methods for biologically consuming gas associated with oil (or removing other sources of gas), along with any contaminants contained in a gas, to simplify the process for mitigating a major barrier to stranded oil and to eliminate much of the processing equipment needed to scrub tainted gas.

[0028] In one aspect, the present disclosure provides compositions, methods, and systems for treating gas (e.g., deacidifying, desulfurizing), comprising use of a recombinant C.sub.1 metabolizing microorganism in the presence of an acid gas feedstock, wherein the recombinant C.sub.1 metabolizing microorganism comprises an exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing or assimilating sulfur. In certain embodiments, the recombinant C.sub.1 metabolizing microorganism may further comprise another exogenous nucleic acid molecule that encodes a biocatalytic enzyme capable of oxidizing light alkanes into various compounds of interest, as described herein.

[0029] In another aspect, this disclosure provides a system for treating gas comprising a source of tainted gas comprising an S substrate, a bioreactor comprising a recombinant C.sub.1 metabolizing microorganism (e.g., methanotroph) that includes a first exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing or assimilating sulfur, and optionally includes a second exogenous nucleic acid molecule that encodes a biocatalytic enzyme capable of oxidizing light alkanes, and a connector disposed between the gas source and bioreactor to allow flow of the tainted gas into the bioreactor; wherein the recombinant microorganism utilizes the sulfur and light alkanes to produce one or more high-value molecules (e.g., alcohols, fatty acid derivatives), biological material (e.g., animal feed, oil composition), or a combination thereof.

[0030] In still a further aspect, the present disclosure provides a system for bioremediation of stranded oil, comprising a well for recovering oil associated gas from an underground formation, wherein the gas comprises at least one acidic compound; a bioreactor comprising a recombinant C.sub.1 metabolizing microorganism having a first exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing or assimilating sulfur, wherein the C.sub.1 metabolizing microorganism uses the gas as a carbon and energy source and substantially converts the acidic compounds from the recovered oil associated gas into, for example, compounds of interest; whereby the bioremediation process substantially removes the oil associated gas and allows recovery of previously stranded oil from the underground formation.

[0031] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

[0032] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means.+-.20% of the indicated range, value, or structure, unless otherwise indicated. The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic and novel characteristics of the claimed invention. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have" and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0033] As used herein, the term "C.sub.1 substrate" or "C.sub.1 compound" refers to any carbon containing molecule or composition that lacks a carbon-carbon bond. C.sub.1 substrates include natural gas, unconventional natural gas, syngas, methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines (e.g., methylamine, dimethylamine, trimethylamine, etc.), methylated thiols, methyl halogens (e.g., bromomethane, chloromethane, iodomethane, dichloromethane, etc.), cyanide, or any combination thereof.

[0034] As used herein, "C.sub.1 metabolizing microorganism" or "C.sub.1 metabolizing microorganism" refers to any microorganism having the ability to use a C.sub.1 substrate as a source of energy or as its primary source of energy or as its sole source of energy and biomass, and may or may not use other carbon substrates (such as sugars and complex carbohydrates) for energy and biomass. For example, a C.sub.1 metabolizing microorganism may oxidize a C.sub.1 substrate, such as methane, natural gas, or methanol. C.sub.1 metabolizing microorganisms include bacteria (such as methanotrophs and methylotrophs) and yeast. In certain embodiments, a C.sub.1 metabolizing microorganism does not include a photosynthetic microorganism, such as algae. In certain embodiments, a C.sub.1 metabolizing microorganism will be an "obligate C.sub.1 metabolizing microorganism," meaning its primary source of energy are C.sub.1 substrates. In further embodiments, a C.sub.1 metabolizing microorganism (e.g., methanotroph) will be cultured in the presence of a C.sub.1 substrate feedstock (i.e., using the C.sub.1 substrate as a source of energy).

[0035] As used herein, the term "methanotroph," "methanotrophic bacterium" or "methanotrophic bacteria" refers to a methylotrophic bacteria capable of utilizing C.sub.1 substrates, such as methane or unconventional natural gas, as its primary or sole carbon and energy source. As used herein, "methanotrophic bacteria" include "obligate methanotrophic bacteria" that can only utilize C.sub.1 substrates for carbon and energy sources and "facultative methanotrophic bacteria" that are naturally able to use multi-carbon substrates, such as acetate, pyruvate, succinate, malate, or ethanol, in addition to C.sub.1 substrates as their carbon and energy source.

[0036] As used herein, the term "methylotroph" or "methylotrophic bacteria" refers to any bacteria capable of oxidizing organic compounds that do not contain carbon-carbon bonds. In certain embodiments, a methylotrophic bacterium may be a methanotroph. For example, "methanotrophic bacteria" refers to any methylotrophic bacteria that have the ability to oxidize methane as it primary source of carbon and energy. Exemplary methanotrophic bacteria include Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, or Methanomonas. In certain other embodiments, the methylotrophic bacterium is an "obligate methylotrophic bacterium," which refers to bacteria that are limited to the use of C.sub.1 substrates for the generation of energy.

[0037] As used herein, "gas" refers to one or mixture of light alkane gases (light alkane refers to saturated or unsaturated C.sub.1-C.sub.6 alkanes optionally substituted), such as methane, ethane, propane, butane, pentane; natural gas; unconventional natural gas; synthesis gas (syngas); casinghead gas; wellhead condensate; or other hydrocarbon gas taken from the earth or water, whether produced by conventional or unconventional methods, or whether produced from a gas well or a well also producing oil, distillate or condensate or both, or other products. "Casinghead gas" means gas or vapor indigenous to an oil stratum and produced from the stratum with oil. "Condensate" means liquid hydrocarbon that is or can be recovered from gas by a separator, or may be liquid hydrocarbon recovered from gas by refrigeration or absorption and separated by a fractionating process.

[0038] As used herein, "tainted gas" refers to gas having unwanted contaminant(s), such as CO.sub.2 or H.sub.2S or both as may be found in acid gas, H.sub.2S as found in sour gas, or the like. "Sour gas" means gas having at least one "S substrate," wherein an S substrate may be any sulfur-containing compound associated with or mixed in gas, such as hydrogen sulfide (H.sub.2S), thiosulfate, sulfite, carbon disulfide, elemental sulfur, other organosulfur compounds (e.g., mercaptans such as thiols (R--SH, where R is a hydrocarbon), thiol carboxylic acids (RCO--SH), dithio acids (RCS--SH), or the like. In a certain embodiments, more than 50% of sulfur-containing compounds of an "S substrate" will be comprised of H.sub.2S. In a certain embodiments, sour gas comprises more than 0.25 grains (gr) of H.sub.2S per 100 standard cubic feet (scf) or 6 parts per million (ppm) on a volume basis, or 10 gr of sulfur per 100 scf, or about 0.1% to about 25% sulfur content. A "standard cubic foot" is a measure of quantity (not volume) at 70.degree. F. and one atmosphere (atm =14.7 pounds per square inch (psi) absolute). "Sweet gas" means gas other than acid gas, sour gas, or casinghead gas (e.g., gas treated to remove unwanted contaminants). In certain embodiments, sweet gas will contain 0.25 gr or less (4 ppm or less) of H.sub.2S or other organosulfur compounds, or have a heating value of at least 920 to 1,000 Btu/scf.

[0039] As used herein, "methane" refers to the simplest (CO alkane compound with the chemical formula CH.sub.4, which is a colorless and odorless gas at room temperature and pressure. Sources of methane include natural sources (such as natural gas fields), "unconventional natural gas" sources (such as shale gas or coal bed methane, wherein methane content will vary from about 75% to about 97%, depending on the source), and biological sources where it is synthesized by, for example, methanogenic microorganisms (biogenic natural gas), and industrial or laboratory synthesis. Methane includes pure methane, substantially purified compositions, such as "pipeline quality natural gas" or "dry natural gas", which is 95-98% percent methane, and unpurified compositions, such as "wet natural gas", wherein other hydrocarbons have not yet been removed and methane comprises more than 60% of the composition.

[0040] As used herein, "natural gas" refers to naturally occurring gas mixtures that have formed in porous reservoirs and can be accessed by conventional processes (e.g., drilling, waterflooding) and are primarily made up of methane, but may also have other light alkane gases (e.g., ethane, propane, butane, pentane), carbon dioxide, nitrogen, hydrogen sulfide, or the like, or any combination thereof.

[0041] As used herein, "unconventional natural gas" refers to a naturally occurring gas mixtures created in formations with low permeability that must be accessed by unconventional methods, such as hydraulic fracturing, horizontal drilling or directional drilling. Exemplary unconventional natural gas deposits include tight gas sands formed in sandstone or carbonate, coal bed methane formed in coal deposits and adsorbed in coal particles, shale gas formed in fine-grained shale rock and adsorbed in clay particles or held within small pores or microfractures, methane hydrates that are a crystalline combination of natural gas and water formed at low temperature and high pressure in places such as under the oceans and permafrost. Unconventional natural gas tends to have a more variable composition, including having potentially higher levels of ethane, propane, butane, CO.sub.2, or any combination thereof, as compared to natural gas.

[0042] As used herein, "synthesis gas" or "syngas" refers to a mixture of carbon monoxide (CO) and hydrogen (H.sub.2), which may be produced, for example, by steam reforming of natural gas or liquid hydrocarbons, or by gasification of coal, biomass or waste. Syngas may also include methane, CO.sub.2, H.sub.2S, and other gases in smaller quantities relative to CO and H.sub.2.

[0043] As used herein, "nucleic acid molecule," also known as a polynucleotide, refers to a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), both of which may be single or double stranded. DNA includes cDNA, genomic DNA, synthetic DNA, semi-synthetic DNA, or the like.

[0044] As used herein, "biological material" refers to organic material having a biological origin, which may include whole cells, lysed cells, extracellular material, or the like. For example, the material harvested from a cultured microorganism (e.g., bacterial or yeast culture) is considered the biological material, which can include secreted products. Such a culture may be considered a renewable resource.

[0045] As used herein, "oil composition" refers to the lipid content of a biological material, including fatty acids, triglycerides, phospholipids, polyhyroxyakanoates, isoprenes, terpenes, or the like. An oil composition contained in biological material may be extracted from the rest of the biological material by methods known in the art, such as by hexane extraction. In addition, an "oil composition" may be found in any one or more areas of a culture, including the cell membrane, cell cytoplasm, inclusion bodies, secreted or excreted in the culture medium, or any combination thereof In certain embodiments, an oil composition functions as a fuel precursor since it can be refined into a fuel, such as jet fuel, diesel fuel, paraffinic kerosene, gasoline, or any combination thereof.

[0046] As used herein, the term "host" refers to a cell or microorganism (e.g., methanotroph) that may be genetically modified with an exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., sulfide oxidoreductase, monooxygenase, cysteine synthase). In certain embodiments, a host cell may optionally already possess other genetic modifications that confer desired properties related or unrelated to the exogenous polypeptide being expressed (e.g., sulfur oxidation as disclosed herein). For example, a host cell may possess genetic modifications conferring additional or enhanced sulfite oxidase activity, additional or enhanced light alkane oxidation activity, high growth, tolerance of contaminants or particular culture conditions (e.g., H.sub.2S tolerance, biocide resistance), ability to metabolize additional carbon substrates, or ability to synthesize desirable products or intermediates.

[0047] For example, an exogenous nucleic acid molecule may encode a polypeptide capable of oxidizing or assimilating sulfur. Exemplary polypeptides capable of oxidizing or assimilating sulfur include hydrogen sulfide:NADP.sup.+ oxidoreductase (also known as sulfite reductase (NADPH) or cysJ/cysl), hydrogen sulfide:ferredoxin oxidoreductase (also known as sulfite reductase (ferredoxin) or SIR), sulfide:flavocytochrome-c oxidoreductase (also known as sulfide-cytochrome-c reductase (flavocytochrome c) or flavocytochrome c sulfide dehydrogenase or FCC), sulfide:quinone oxidoreductase (also known as sulfide quinone reductase or SQR), sulfur dioxygenase, sulfur oxygenase (also known as sulfur oxygenase/reductase or SOR), or the like, which may be useful in converting, for example, hydrogen sulfide into elemental sulfur, sulfite, sulfate, or any combination thereof.

[0048] As used herein, the term "recombinant" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alternation or has been modified by introduction of an exogenous nucleic acid molecule, or refers to a cell that has been altered such that the expression of an endogenous nucleic acid molecule or gene can be controlled, where such alterations or modifications are introduced by genetic engineering. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of the cell's genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous or homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.

[0049] Recombinant methods for expression of exogenous or heterologous nucleic acids in microbial organisms are well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999). Exemplary exogenous proteins or enzymes to be expressed include those involved in sulfur oxidation (e.g., hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, sulfur dioxygenase, sulfur oxygenase, sulfite oxidase, or any combination thereof) or in sulfur assimilation (e.g., sulfate adenylyltransferase, sulfite reductase). Genetic modifications to nucleic acid molecules encoding enzymes, or functional fragments thereof, can confer a biochemical or metabolic capability to a recombinant cell that is altered from its naturally occurring state.

[0050] As used herein, "transformation" refers to the introduction of a nucleic acid molecule (e.g., exogenous or heterologous nucleic acid molecule) into a host cell. The transformed host cell may carry the exogenous or heterologous nucleic acid molecule extra-chromosomally or integrated in the chromosome. Integration into a host cell genome and self-replicating vectors generally result in genetically stable inheritance of the transformed nucleic acid molecule. Host cells containing the transformed nucleic acids are referred to as "recombinant" or "non-naturally occurring" or "genetically engineered" or "transformed" or "transgenic" cells (e.g., bacteria).

[0051] As used herein, the term "endogenous" or "native" refers to a gene, protein, compound or activity that is normally present in a host cell.

[0052] As used herein, "heterologous" or "exogenous" nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, a nucleic acid molecule or portion of a nucleic acid molecule native to a host cell that has been altered or mutated, or a nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous control sequence (e.g., promoter, enhancer) may be used to regulate expression of a native gene or nucleic acid molecule in a way that is different from the way a native gene or nucleic acid molecule is normally expressed in nature or culture. In certain embodiments, heterologous or exogenous nucleic acid molecules may not be endogenous to a host cell or host genome, but instead may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self replicating vector). In addition, "heterologous" can refer to an enzyme, protein or other activity that is different or altered from that found endogenous to a host cell, or is not native to a host cell but instead is encoded by a nucleic acid molecule introduced into the host cell. The term "homologous" or "homolog" refers to a molecule or activity found in or derived from a host cell, species or strain. For example, a heterologous or exogenous nucleic acid molecule may be homologous to a native host cell gene, but may have an altered expression level or have a different sequence or both.

[0053] In certain embodiments, more than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof, and still be considered as more than one heterologous or exogenous nucleic acid. For example, as disclosed herein, a C.sub.1 metabolizing microorganism can be modified to express two or more heterologous or exogenous nucleic acid molecules encoding desired sulfur and light alkane oxidizing components (e.g., hydrogen sulfide:NADP.sup.+ oxidoreductase or hydrogen sulfide:ferredoxin oxidoreductase, optionally a sulfite oxidase, optionally a monooxygenase, optionally a fatty acid converting enzyme). When two or more exogenous nucleic acid molecules are introduced into a host C.sub.1 metabolizing microorganism, it is understood that the two more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, and each of these embodiments is still to be considered two or more exogenous nucleic acid molecules. Thus, the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.

[0054] The term "chimeric" refers to any nucleic acid molecule or protein that is not endogenous and comprises sequences joined or linked together that are not normally found joined or linked together in nature. For example, a chimeric nucleic acid molecule may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source but arranged in a manner different than that found in nature.

[0055] The "percent identity" between two or more nucleic acid sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions.times.100), taking into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. The comparison of sequences and determination of percent identity between two or more sequences can be accomplished using a mathematical algorithm, such as BLAST and Gapped BLAST programs at their default parameters (e.g., Altschul et al., J. Mol. Biol. 215:403, 1990; see also BLASTN at www.ncbi.nlm.nih.gov/BLAST).

[0056] A "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published Mar. 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:N.Y. (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, Mass. (1990), p. 8).

[0057] "Inhibit" or "inhibited," as used herein, refers to an alteration, reduction, down regulation or abrogation, directly or indirectly, in the expression of a target gene or in the activity of a target molecule (e.g., alcohol dehydrogenase) relative to a control, endogenous or reference molecule, wherein the alteration, reduction, down regulation or abrogation is statistically, biologically, industrially, or clinically significant.

[0058] As used herein, the term "derivative" refers to a modification of a compound by chemical or biological means, with or without an enzyme, which modified compound is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. A derivative may have different chemical, biological or physical properties from the parent compound, such as being more hydrophilic or having altered reactivity as compared to the parent compound. Derivatization (i.e., modification) may involve substitution of one or more moieties within the molecule (e.g., a change in functional group). For example, a hydrogen may be substituted with a halogen, such as fluorine or chlorine, or a hydroxyl group (--OH) may be replaced with a carboxylic acid moiety (--COOH). Other exemplary derivatizations include glycosylation, alkylation, acylation, acetylation, ubiqutination, esterification, and amidation.

[0059] The term "derivative" also refers to all solvates, for example, hydrates or adducts (e.g., adducts with alcohols), active metabolites, and salts of a parent compound. The type of salt depends on the nature of the moieties within the compound. For example, acidic groups, such as carboxylic acid groups, can form alkali metal salts or alkaline earth metal salts (e.g., sodium salts, potassium salts, magnesium salts, calcium salts, and also salts with physiologically tolerable quaternary ammonium ions and acid addition salts with ammonia and physiologically tolerable organic amines such as, for example, triethylamine, ethanolamine or tris-(2-hydroxyethyl)amine). Basic groups can form acid addition salts with, for example, inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or with organic carboxylic acids or sulfonic acids such as acetic acid, citric acid, lactic acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, methanesulfonic acid or p-toluenesulfonic acid. Compounds that simultaneously contain a basic group and an acidic group, for example, a carboxyl group in addition to basic nitrogen atoms, can be present as zwitterions. Salts can be obtained by customary methods known to those skilled in the art, for example, by combining a compound with an inorganic or organic acid or base in a solvent or diluent, or from other salts by cation exchange or anion exchange.

C.sub.1 Metabolizing Microorganisms--Host Cells

[0060] The C.sub.1 metabolizing microorganisms of the instant disclosure may be a natural strain, strain adapted (e.g., performing fermentation to select for strains with improved sulfite oxidase activity, improved growth rates, or increased total biomass yield compared to the parent strain), or recombinantly modified to treat gas (e.g., desulfurize), convert alkanes or alkenes to their corresponding alcohol or epoxide, to have increased sulfite oxidase activity, to have increased growth rates, or any combination thereof. In certain preferred embodiments, the C.sub.1 metabolizing microorganisms are not photosynthetic microorganisms, such as algae or plants.

[0061] In certain embodiments, the present disclosure provides C.sub.1 metabolizing microorganisms that are prokaryotes or bacteria, such as Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, or Pseudomonas.

[0062] In further embodiments, the C.sub.1 metabolizing bacteria are a methanotroph or a methylotroph. Exemplary methanotrophs include Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylocella, or a combination thereof. Exemplary methylotrophs include Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylobacterium nodulans, or a combination thereof.

[0063] In certain embodiments, methanotrophic bacteria are genetically engineered with the capability to sweeten gas or convert C.sub.1 substrate feedstock into, for example, alcohols or biomass. Methanotrophic bacteria have the ability to oxidize methane as a carbon and energy source. Methanotrophic bacteria are classified into three groups based on their carbon assimilation pathways and internal membrane structure: type I (gamma proteobacteria), type II (alpha proteobacteria, and type X (gamma proteobacteria). Type I methanotrophs use the ribulose monophosphate (RuMP) pathway for carbon assimilation whereas type II methanotrophs use the serine pathway. Type X methanotrophs use the RuMP pathway but also express low levels of enzymes of the serine pathway. Methanotrophic bacteria include obligate methanotrophs, which can only utilize C.sub.1 substrates for carbon and energy sources, and facultative methanotrophs, which naturally have the ability to utilize some multi-carbon substrates as a sole carbon and energy source.

[0064] Exemplary facultative methanotrophs include some species of Methylocella, Methylocystis, and Methylocapsa (e.g., Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystis bryophila, and Methylocapsa aurea KYG), Methylobacterium organophilum (ATCC 27,886), Methylibium petrolelphilum, or high growth variants thereof. Exemplary obligate methanotrophic bacteria include Methylococcus capsulatus Bath, Methylomonas 16a (ATCC PTA 2402), Methylosinusi trichosporium OB3b (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylomonas flagellata sp AJ-3670 (FERM P-2400), Methylacidiphilum infernorum, Methylacidiphilum fumariolicum, Methylomicrobium alcahphilum, or high growth variants thereof.

[0065] In still further embodiments, the present disclosure provides C.sub.1 metabolizing microorganisms that are syngas metabolizing bacteria, such as Clostridium, Moorella, Pyrococcus, Eubacterium, Desulfobacterium, Carboxydothermus, Acetogenium, Acetobacterium, Acetoanaerobium, Butyribaceterium, Peptostreptococcus, or a combination thereof. Exemplary methylotrophs include Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium woodii, Clostridium neopropanologen, or a combination thereof.

[0066] In certain other embodiments, C.sub.1 metabolizing microorganisms are eukaryotes such as yeast, including Candida, Yarrowia, Hansenula, Pichia, Torulopsis, or Rhodotorula.

[0067] In certain other embodiments, the C.sub.1 metabolizing microorganism is an obligate C.sub.1 metabolizing microorganism, such as an obligate methanotroph or methylotroph.

[0068] In certain embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph comprising a heterologous polynucleotide encoding a hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: quinone oxidoreductase, sulfide:flavocytochrome-c oxidoreductase, or sulfur dioxygenase. In certain embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph comprising a heterologous polynucleotide encoding a sulfur oxygenase (e.g., SOR).

[0069] In certain embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph comprising a first heterologous polynucleotide encoding a hydrogen sulfide:NADP.sup.+ oxidoreductase (e.g., cysJ/cysl), and optionally a second heterologous nucleic acid molecule that encodes a biocatalytic enzyme capable of oxidizing light alkanes, such as an alkane monooxygenase (e.g., AMO, BMO, PMO, MMO), alkene monooxygenase or alkane hydroxylase, or a cell lysate thereof; or optionally a second heterologous nucleic acid molecule that encodes a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof or optionally a second heterologous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, capable of converting a gas (e.g., natural gas) into an oil composition; or any combination thereof.

[0070] In certain embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph comprising a first heterologous polynucleotide encoding a hydrogen sulfide:ferredoxin oxidoreductase (SIR), and optionally a second heterologous nucleic acid molecule that encodes a biocatalytic enzyme capable of oxidizing light alkanes, such as an alkane monooxygenase (e.g., AMO, BMO, PMO, MMO), alkene monooxygenase or alkane hydroxylase, or a cell lysate thereof; or optionally a second heterologous nucleic acid molecule that encodes a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof; or optionally a second heterologous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, capable of converting a gas (e.g., natural gas) into an oil composition; or any combination thereof.

[0071] In certain embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph comprising a first heterologous polynucleotide encoding a sulfide:quinone oxidoreductase (SQR), and optionally a second heterologous nucleic acid molecule that encodes a biocatalytic enzyme capable of oxidizing light alkanes, such as an alkane monooxygenase (e.g., AMO, BMO, PMO, MMO), alkene monooxygenase or alkane hydroxylase, or a cell lysate thereof; or optionally a second heterologous nucleic acid molecule that encodes a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof; or optionally a second heterologous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, capable of converting a gas (e.g., natural gas) into an oil composition; or any combination thereof.

[0072] In certain embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph comprising a first heterologous polynucleotide encoding a sulfide:flavocytochrome-c oxidoreductase (FCC), and optionally a second heterologous nucleic acid molecule that encodes a biocatalytic enzyme capable of oxidizing light alkanes, such as an alkane monooxygenase (e.g., AMO, BMO, PMO, MMO), alkene monooxygenase or alkane hydroxylase, or a cell lysate thereof; or optionally a second heterologous nucleic acid molecule that encodes a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof; or optionally a second heterologous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, capable of converting a gas (e.g., natural gas) into an oil composition; or any combination thereof.

[0073] In certain embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph comprising a first heterologous polynucleotide encoding a sulfur dioxygenase, and optionally a second heterologous nucleic acid molecule that encodes a biocatalytic enzyme capable of oxidizing light alkanes, such as an alkane monooxygenase (e.g., AMO, BMO, PMO, MMO), alkene monooxygenase or alkane hydroxylase, or a cell lysate thereof; or optionally a second heterologous nucleic acid molecule that encodes a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof; or optionally a second heterologous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, capable of converting a gas (e.g., natural gas) into an oil composition; or any combination thereof.

[0074] In certain embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph comprising a first heterologous polynucleotide encoding a sulfur oxygenase, and optionally a second heterologous nucleic acid molecule that encodes a biocatalytic enzyme capable of oxidizing light alkanes, such as an alkane monooxygenase (e.g., AMO, BMO, PMO, MMO), alkene monooxygenase or alkane hydroxylase, or a cell lysate thereof; or optionally a second heterologous nucleic acid molecule that encodes a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof; or optionally a second heterologous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, capable of converting a gas (e.g., natural gas) into an oil composition; or any combination thereof.

[0075] In any of the aforementioned embodiments, a non-natural C.sub.1 metabolizing microorganism is a recombinant methanotroph that encodes a biocatalytic enzyme capable of oxidizing light alkanes, such as an alkane monooxygenase (e.g., AMO, BMO, PMO, MMO), alkene monooxygenase or alkane hydroxylase, or a cell lysate thereof, further comprises a genetic modification to attenuate, inhibit, substantially reduce or functionally delete an alcohol dehydrogenase activity when the compound or composition to be produced comprises alcohol.

[0076] In any of the aforementioned embodiments, the C.sub.1 metabolizing microorganism may be a C.sub.1 metabolizing non-photosynthetic microorganism.

[0077] Codon Optimization

[0078] Expression of recombinant proteins may be difficult outside their original host. For example, variation in codon usage bias has been observed across different species of bacteria (Sharp et al., Nucl. Acids. Res. 33:1141, 2005). Over-expression of recombinant proteins even within their native host may also be difficult. In certain embodiments, nucleic acid molecules (e.g., nucleic acids encoding sulfur or alkane oxidizing enzymes) to be introduced into a host as described herein may be subjected to codon optimization prior to introduction into the host to ensure protein expression is enhanced. Codon optimization refers to alteration of codons in genes or coding regions of nucleic acids before transformation to reflect the typical codon usage of the host without altering the polypeptide encoded by the DNA molecule. Codon optimization methods for optimum gene expression in heterologous hosts have been previously described (see, e.g., Welch et al., PLoS One 4:e7002, 2009; Gustafsson et al., Trends Biotechnol. 22:346, 2004; Wu et al., Nucl. Acids Res. 35:D76, 2007; Villalobos et al., BMC Bioinformatics 7:285, 2006; U.S. Patent Publication Nos. 2011/0111413 and 2008/0292918; disclosure of which are incorporated herein by reference, in their entirety).

[0079] In some embodiments, exogenous nucleic acid molecules of this disclosure are codon optimized for C.sub.1 metabolizing microorganisms, such as bacteria. In certain embodiments, exogenous nucleic acid molecules of this disclosure are codon optimized for methanotrophs and methylotrophs as described herein. In particular embodiments, exogenous nucleic acid molecules of this disclosure are codon optimized for Methylococcus capsulatus Bath.

[0080] Exemplary codon optimized nucleic acid molecules for expression in a C.sub.1 metabolizing microorganism are provided in SEQ ID NOS.:1-54.

[0081] Similarly, exogenous nucleic acid molecules of this disclosure encoding polypeptide variants may be designed using the phylogenetic-based methods described in the references noted above (U.S. Pat. No. 8,005,620; Gustafsson; Welch etal.; Villalobos etal.; Minshull et al.). Each variant polypeptide generated by these methods will retain at least 50% activity (preferably 100% or more activity) and have a polypeptide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to a reference or parental wild-type polypeptide sequence. In certain embodiments, variant polypeptides will include at least one amino acid substitution (e.g., 1, 2, 3, 5, 6, 7, 8, 9 or 10 or more or up to 20, 25, or 30 substitutions) at a pre-determined position relative to a reference or parental wild-type enzyme, provided that a variant retains an activity of interest (e.g., sulfur oxidation or assimilation, light alkane oxidation, fatty acid production, fatty acid conversion, formaldehyde assimilation).

Transformation Methods

[0082] Any of the recombinant C.sub.1 metabolizing microorganisms or methanotrophic bacteria described herein may be transformed to comprise at least one exogenous nucleic acid to provide the host with a new or enhanced activity (e.g., enzymatic activity) or may be genetically modified to remove or substantially reduce an endogenous gene function using any of a variety of methods known in the art.

[0083] Transformation refers to the transfer of a nucleic acid molecule (e.g., exogenous nucleic acid) into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid molecules are referred to as "non-naturally occurring" or "recombinant" or "transformed" or "transgenic" cells. Expression systems and expression vectors useful for the expression of heterologous nucleic acids in C.sub.1 metabolizing microorganisms or methanotrophic bacteria are known.

[0084] Electroporation of C.sub.1 metabolizing bacteria has been previously described in, for example, Toyama et al., FEMS Microbiol. Lett. 166:1, 1998; Kim and Wood, Appl. Microbiol. Biotechnol. 48:105, 1997; Yoshida et al., Biotechnol. Lett. 23:787, 2001, and U.S. Patent Appl. Pub. No. 2008/0026005.

[0085] Bacterial conjugation, which refers to a particular type of transformation involving direct contact of donor and recipient cells, is more frequently used for the transfer of nucleic acids into C.sub.1 metabolizing bacteria. Bacterial conjugation involves mixing "donor" and "recipient" cells together in close contact with each other. Conjugation occurs by formation of cytoplasmic connections between donor and recipient bacteria, with unidirectional transfer of newly synthesized donor nucleic acid molecules into the recipient cells. A recipient in a conjugation reaction is any cell that can accept nucleic acids through horizontal transfer from a donor bacterium. A donor in a conjugation reaction is a bacterium that contains a conjugative plasmid, conjugative transposon, or mobilized plasmid. The physical transfer of the donor plasmid can occur through a self-transmissible plasmid or with the assistance of a "helper" plasmid. Conjugations involving C.sub.1 metabolizing bacteria have been previously described in Stolyar et al., Mikrobiologiya 64:686, 1995; Motoyama et al., Appl. Micro. Biotech. 42:67, 1994; Lloyd et al., Arch. Microbiol. 171:364, 1999; PCT Publication No. WO 02/18617; and Ali et al., Microbiol. 152:2931, 2006.

[0086] Expression of heterologous nucleic acids in C.sub.1 metabolizing bacteria is known in the art (see, e.g., U.S. Pat. No. 6,818,424, U.S. Patent Appl. Pub. No. 2003/0003528). Mu transposon based transformation of methylotrophic bacteria has been described (Akhverdyan et al., Appl. Microbiol. Biotechnol. 91:857, 2011). A mini-Tn7 transposon system for single and multicopy expression of heterologous genes without insertional inactivation of host genes in Methylobacterium has been described (U.S. Patent Appl. Pub. No. 2008/0026005).

[0087] Various methods for inactivating, knocking-out, or deleting endogenous gene function in C.sub.1 metabolizing bacteria may be used. Allelic exchange using suicide vectors to construct deletion/insertional mutants in slow growing C1 metabolizing bacteria have also been described in, for example, Toyama and Lidstrom, Microbiol. 144:183, 1998; Stolyar et al., Microbiol. 145:1235, 1999; Ali et al., Microbiol. 152:2931, 2006; Van Dien et al., Microbiol. 149:601, 2003.

[0088] Suitable homologous or heterologous promoters for high expression of exogenous nucleic acids may be utilized. For example, U.S. Pat. No. 7,098,005 describes the use of promoters that are highly expressed in the presence of methane or methanol for heterologous gene expression in Cl metabolizing bacteria. Additional promoters that may be used include deoxy-xylulose phosphate synthase methanol dehydrogenase operon promoter (Springer et al., FEMS Microbiol. Lett. 160:119, 1998); the promoter for PHA synthesis (Foellner et al., Appl. Microbiol. Biotechnol. 40:284, 1993); or promoters identified from native plasmid in methylotrophs (EP 296484). Non-native promoters include the lac operon Plac promoter (Toyama et al., Microbiol. 143:595, 1997) or a hybrid promoter such as Ptrc (Brosius et al., Gene 27:161, 1984). In certain embodiments, promoters or codon optimization are used for high constitutive expression of exogenous nucleic acids encoding sulfur utilization pathway enzymes in host methanotrophic bacteria. Regulated expression of an exogenous nucleic acid in a host methanotrophic bacterium may also be utilized. In certain embodiments, regulated expression of exogenous nucleic acids encoding sulfur utilization enzymes may be desirable to optimize growth rate of the non-naturally occurring methanotrophic bacteria. Controlled expression of nucleic acid molecules encoding sulfur utilization enzymes for response to the presence of an S substrate may improve bacterial growth in a variety of carbon source conditions. For example, an inducible/regulatable system of recombinant protein expression in methylotrophic and methanotrophic bacteria as described in, for example, U.S. Patent Appl. No. US 2010/0221813 may be used.

C.sub.1 Metabolizing Microorganisms--Recombinant

[0089] The C.sub.1 metabolizing microorganisms of this disclosure can be recombinantly modified to include nucleic acid molecules that express or over-express polypeptides of interest, which results in recombinant microorganisms useful for converting (e.g., assimilating, oxidizing) various components of gas or tainted gas (e.g., acid or sour natural gas) into other useful compounds.

[0090] For example, a C.sub.1 metabolizing microorganism (such as a methanotroph or methylotroph) can be recombinantly transformed to produce a polypeptide capable of metabolizing an S substrate (e.g., sulfide oxidoreductase), recombinantly transformed to produce an alcohol composition (e.g., alkyl monooxygenase or hydroxylase), recombinantly transformed to produce fatty acid derivatives from light alkanes (e.g., fatty acyl-CoA reductase, carboxylic acid reductase, and optionally a thioesterase, acyl-CoA synthetase, monooxygenase), recombinantly transformed to produce an oil composition (e.g., a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof), recombinantly transformed or genetically modified to optionally increase production of native sulfite oxidase, or any combination thereof. Exemplary amino acid sequences suitable for production by a C.sub.1-metabolizing microorganism are provided in SEQ ID NOS.:55-108.

[0091] In certain embodiments, any of the recombinant polypeptides produced by C.sub.1 metabolizing microorganisms as described herein may be stable in the presence of a chemical or environmental stress. The modifications to C.sub.1 metabolizing microorganisms described herein can be through genomic alterations, addition of extrachromosomal recombinant expression systems, or a combination thereof.

[0092] By way of background, several biological pathways exist for the oxidation or assimilation of sulfur. Different steps in the pathway are catalyzed by various enzymes and, therefore, each of these may be over-expressed to increase the amount of enzyme and thus drive the oxidation or assimilation of sulfur. Nucleic acid molecules encoding enzymes required for the pathway may also be recombinantly added to a C.sub.1 metabolizing microorganism lacking such enzymes. Finally, steps that would compete with the pathway leading to oxidation or assimilation of sulfur can be attenuated or blocked in order to maximize the removal of sulfur from, for example, sour gas. Elemental sulfur can be produced by partial oxidation of sulfide (Equation 1), which can then be removed by sedimentation. Complete oxidation of sulfide results in the production of sulfate (Equation 2), which is innocuous and water soluble.

2HS.sup.-+O.sub.2.fwdarw.2S.sup.0+2OH.sup.- (1)

2HS.sup.-+4O.sub.2.fwdarw.2SO.sub.4.sup.-+2H.sup.+ (2)

[0093] Inorganic reduced sulfur compounds serve as electron donors in many phototrophic and chemotrophic bacteria (Friedrich, Adv. Microb. Physiol. 39:235, 1998). Hydrogen sulfide, the most reduced form of inorganic sulfur, occurs in hydrothermal vents and in sediments, where it is generated by sulfate reducing bacteria (Jannasch, Autotrophic Bacteria, Schlegel and Bowien (eds.) Springer Verlag, Berlin Heidelberg, pages 147-167, 1989; Trudinger, Early Organic Evolution, Schidlowski (ed.) Springer Verlag, Berlin Heidelberg, pages 367-377, 1992). Although hydrogen sulfide is toxic for most organisms, mainly because of the inhibition of aerobic respiration (Gosselin et al., Hydrogen sulfide In: Clinical toxicology of commercial products, 5th ed. Baltimore, Md., Williams and Wilkins, pages 111-198-111-202, 1984), it serves as an electron donor for the energy generating systems of photo- and chemolithotrophic bacteria (Kelly et al., Antonie Van Leeuwenhoek 71:95, 1997; Stetter, FEMS Microbiol. Rev. 18:149, 1996).

[0094] In bacteria, the transport of electrons from sulfide to NAD.sup.+ is mediated by membrane bound electron transport. The electrons from sulfide enter the chain either at the level of quinone via a sulfide:quinone oxidoreductase (SQR; EC 1.8.5.4), or at the level of c type cytochromes via a sulfide:cytochrome c oxidoreductase (flavocytochrome c, FCC; EC 1.8.2.3). In contrast, to assimilate sulfur into biosynthetic pathways (e.g., cysteine synthesis), hydrogen sulfide is required and therefore is produced by reduction of sulfate. Sulfate is first reduced to sulfite (see, e.g., Kopriva et al., J. Biol. Chem. 277:21786, 2002), which in turn can be further reduced to sulfide by sulfite reductases (Lillig, Arch. Biochem. Biophys. 392:303, 2001). Two types of sulfite reductase enzymes are known--hydrogen sulfide:NADP.sup.+ oxidoreductase (also known as sulfite reductase (NADPH); EC 1.8.1.2) and hydrogen sulfide:ferredoxin oxidoreductase (also known as sulfite reductase (ferredoxin) or SIR, EC 1.8.7.1). But, the reduction of sulfite to sulfide is a reversible reaction and, therefore, can instead be an oxidation reaction of sulfide to sulfite, which in turn can be further oxidized to sulfate.

[0095] Any sulfide that is produced or is present for use by a C.sub.1 metabolizing microorganism can enter the cysteine biosynthesis pathway, wherein cysteine synthase incorporates H.sub.2S into O-acetyl-serine to produce cysteine. Alternatively, sulfide can enter the homocysteine biosynthesis pathway wherein O-acetylhomoserine sulfhydrylase incorporates H.sub.2S into O-acetylhomoserine to produce homocysteine, which can be further converted into methionine by methionine synthase (cobalamin dependent or independent) or homocysteine methyltransferase.

[0096] An alternate pathway for inorganic sulfur (including elemental sulfur) oxidation is through a pathway found in several sulfur-oxidizing organisms, including the facultative chemolithotroph Starkeya novella, thermoacidophilic Sulfobacillus sibiricus, and acidophilic Thiobacilli, such as Thiobacillus thioparus or Thiobacillus denitrificans. Another group is Acidithiobacilii, which are bacteria capable of catalyzing the oxidation of inorganic sulfur compounds under acidic conditions and ambient temperatures. Several members of Acidithiobacillus (e.g., Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans) and Acidiphilium (e.g., Acidiphilium acidophilum) can grow chemolithoautotrophically with sulfide, elemental sulfur, thiosulfate or polythionates (Hirai shi et al., Int. J. Syst. Bacteriol. 48:1389, 1998; Kelly and Wood, Int. J. Syst. Evol. Microbiol. 50:511, 2000). Another exemplary group having this pathway are the Acidianus spp. (e.g., Acidianus ambivalens, Acidianus brierleyi), which are obligately chemolithotrophic, facultatively aerobic archaea isolated from acidothermal springs. This alternate pathway comprises the use of sulfur dioxygenase or glutathione-dependent sulfur dioxygenase (EC 1.13.11.18) to oxidize sulfide via S-sulfanylglutathione (GSSH), a product of the non-enzymatic reaction of glutathione disulfide (GSSG) with H.sub.2S.

[0097] Another pathway for inorganic sulfur oxidation involves sulfur oxygenase reductase (SOR, EC 1.13.11.55), which is found in thermophilic microorganisms. SOR simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. Exemplary organisms that produce this enzyme include Acidianus ambivalens, Acidianus tengchongensis, Aquifex aeolicus, Acidithiobacillus caldus, Halothiobacillus neopolitanus, Sulfolobus metallicus, Sulfolobus tokodaii, Sulfobacillus acidophillus, and Sulfobacillus thermosulfidooxidans

[0098] In certain embodiments, C.sub.1 metabolizing microorganisms and C.sub.1 metabolizing non-photosynthetic microorganisms of this disclosure may be engineered to express or overproduce hydrogen sulfide:NADP.sup.+ oxidoreductase (also known as sulfite reductase (NADPH) or cysJ/cysI; EC 1.8.1.2), including both subunits (.alpha. and .beta.). One advantage of using this enzyme is that the sulfide to sulfate reaction will generate reducing equivalents that can provide energy for the cells to grow faster and for carbon fixation.

[0099] For example, to express or overproduce sulfide:NADP.sup.+ oxidoreductase, one or more genes from Bacillus subtilis (cysJ and cysI), Escherichia coli (cysJ and cysI), or Saccharomyces cerevisiae (met10 and mets) can be introduced into and expressed or overexpressed in a C.sub.1 metabolizing microorganism or a C.sub.1 metabolizing non-photosynthetic microorganism (e.g., non-natural methanotroph bacteria), thereby producing or overproducing exogenous sulfite reductase (NADPH) alpha and beta subunit polypeptides or functional fragments thereof. Other sources of sulfite reductase (NADPH) alpha and beta subunit polypeptides or functional fragments thereof can be from Rhodobacter capsulatus, Shewanella putrefaciens, or Acidithiobacillus ferrooxidans. In certain embodiments, sulfite reductase (NADPH) alpha and beta subunit polypeptides for use in the compositions and methods disclosed herein are from Rhodobacter capsulatus SB 1003 (Genbank Accession Nos. YP_003579141.1 [.alpha.] and YP_003577746.1 MA Escherichia coli K-12 substrain MG1655 (Genbank Accession No. AAA69273.1 [a] and AAA69274.1 Shewanella putrefaciens CN-32 (Genbank Accession No. ABP76777.1 [.alpha.] and ABP76776.1 WA Bacillus subtilis MB73/2 (Genbank Accession No. EME08247.1 [.alpha.] and EME08683.1 WA or Acidithiobacillus ferrooxidans ATCC 23270 (Genbank Accession No. YP_002427483.1[.alpha.] and YP_002427484.1 [.beta.]).

[0100] In certain embodiments, sulfite reductase (NADPH) alpha and beta subunit polypeptides or functional fragments thereof are derived or obtained from Rhodobacter capsulatus SB 1003 or Escherichia coli K-12 substrain MG1655 and have an amino acid sequence that is at least at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in Genbank Accession Nos. YP_003579141.1 [.alpha.] and YP_003577746.1 [.beta.] or AAA69273.1 [.alpha.] and AAA69274.1 [.beta.], respectively, or a functional fragments thereof. In another embodiment, the recombinant encoded sulfite reductase (NADPH) alpha and beta subunit polypeptides have amino acid sequences that are identical to the sequences set forth in Genbank Accession Nos. YP_003579141.1 [.alpha.] and YP_003577746.1 [.beta.] or AAA69273.1 [.alpha.] and AAA69274.1 [.beta.] or comprises a consensus sequence of known sulfite reductase (NADPH) .alpha.-subunits and a consensus sequence of known sulfite reductase (NADPH) .beta.-subunits.

[0101] In certain embodiments, the sulfite reductase (NADPH) alpha and beta subunit polypeptides or functional fragments thereof are encoded by a nucleic acid sequence that has been codon optimized. The codon optimized sulfite reductase (NADPH) alpha and beta subunit polypeptides or functional fragments thereof may be encoded by nucleic acids comprising any one of SEQ ID NOS.:21-35. In certain embodiment, the recombinant encoded sulfite reductase (NADPH) alpha and beta subunit polypeptides have amino acid sequences that are identical to the sequences set forth in any one of SEQ ID NOS.:55-69. In some embodiments, the recombinant encoded sulfite reductase (NADPH) alpha and beta subunit polypeptides have amino acid sequences that are at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in any one of SEQ ID NOS.:55-69.

[0102] In certain embodiments, C.sub.1 metabolizing microorganisms and C.sub.1 metabolizing non-photosynthetic microorganisms as described herein may be engineered to express or overproduce hydrogen sulfide:ferredoxin oxidoreductase (SIR, EC 1.8.7.1). One advantage of using this enzyme is that the sulfide to sulfate reaction will generate reducing equivalents that can provide energy for the cells to grow faster and for carbon fixation.

[0103] For example, a nucleic acid molecule from Cyanidioschyzon merolae encoding a SIR enzyme can be introduced into and expressed or overexpressed in a C.sub.1 metabolizing microorganism or a C.sub.1 metabolizing non-photosynthetic microorganism (e.g., non-natural methanotroph bacteria), thereby producing or overproducing exogenous SIR polypeptides or functional fragments thereof. Other sources of exogenous SIR polypeptides or functional fragments thereof can be from Cyanidioschyzon merolae, Aphonathece halophytica, Oscillatoria nigro-viridis, Pseudomonas putida, or Anabaena cyhndrica. In certain embodiments, an SIR enzyme for use in the compositions and methods disclosed herein is from Cyanidioschyzon merolae 10D (Genbank Accession No. BAM79554.1), Oscillatoria nigro-viridis PCC 7112 (Genbank Accession No. YP_007113209.1), Pseudomonas putida GB-1 (Genbank Accession No. YP_001669502.1), Anabaena cylindrica PCC 7220 (Genbank Accession No. YP 007159823.1), or Aphonathece halophytica 7418 (Genbank Accession No. YP_007168206.1).

[0104] In certain embodiments, a SIR polypeptide or functional fragment thereof is derived or obtained from Cyanidioschyzon merolae 10D or Oscillatoria nigro-viridis PCC 7112 and has an amino acid sequence that is at least at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in Genbank Accession No. BAM79554.1 or YP 007113209.1, respectively, or a functional fragment thereof. In another embodiment, the recombinant encoded SIR polypeptide has an amino acid sequence that is identical to the sequence set forth in Genbank Accession No. BAM79554.1 or YP_007113209.1 or comprises a consensus sequence of known SIR polypeptide sequences.

[0105] In certain embodiments, C.sub.1 metabolizing microorganisms and C.sub.1 metabolizing non-photosynthetic microorganisms as described herein may be engineered to express or overproduce sulfide:quinone oxidoreductase (SQR; EC 1.8.5.4). The SQR polypeptide appears to transfer electrons from sulfide directly into the quinone pool. One advantage of using this enzyme is that the sulfide can be oxidized to elemental sulfate and will likely precipitate, which may be in the form of intracellular globules or granules as observed in native sulfur metabolizing microorganisms. Also, the native cofactor (quinone) can be used to provide the reducing power to the cells.

[0106] Exemplary SQR polypeptides (encoded by sqr gene) or functional fragments thereof can be found in Rhodobacter capsulatus, Shewanella putrefaciens, Paracoccus denitrificans, Acidithiobacillus ferrooxidans, Thiobacillus ferrooxidans, Aquifex aeolicus, Oscillatoria limnetica, Chlorobaculum tepidum, Chlorobium limicola, Anabaena ATCC 7120, and Aphonathece halophytica. SQR polypeptide sequences are publicly available and exemplary sequences are provided in FIG. 3 of Griesbeck et al., Recent Res. Dev. Microbiol. 4:179, 2000, which figure and sequences therein are hereby incorporated by reference in their entirety. In certain embodiments, an SQR enzyme for use in the compositions and methods disclosed herein is from Rhodobacter capsulatus SB 1003 (Genbank Accession No. YP_003576957.1), Oscillatoria limnetica `Solar Lake` (Genbank Accession No. AAF72962.1), Acidithiobacillus ferrooxidans ATCC 23270 (Genbank Accession No. YP_002424774.1), Aquifex aeolicus VF5 (Genbank Accession No. AAC07903.1), Aphonathece halophytica 7418 (Genbank Accession No. YP_007167227.1).

[0107] In certain embodiments, an SQR polypeptide or functional fragment thereof is derived or obtained from Rhodobacter capsulatus SB 1003 or Oscillatoria limnetica `Solar Lake` and has an amino acid sequence that is at least at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in Genbank Accession No. YP_003576957.1 or AAF72962.1, respectively, or a functional fragment thereof. In another embodiment, the recombinant encoded SQR enzyme has an amino acid sequence that is identical to the sequence set forth in Genbank Accession No. YP_003576957.1 or AAF72962.1, or comprises a consensus sequence of known SQR polypeptide sequences.

[0108] In certain embodiments, the SQR polypeptide or functional fragments thereof is encoded by a nucleic acid sequence that has been codon optimized. The codon optimized SQR polypeptide or functional fragments thereof may be encoded by nucleic acids comprising any one of SEQ ID NOS.:36-40. In certain embodiment, the recombinant encoded SQR polypeptide has an amino acid sequence that is identical to the sequence set forth any one of SEQ ID NOS.:70-74. In some embodiments, the recombinant encoded SQR polypeptide has an amino acid sequences that is at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in any one of SEQ ID NOS.:70-74.

[0109] In certain embodiments, C.sub.1 metabolizing microorganisms and C.sub.1 metabolizing non-photosynthetic microorganisms as described herein may be engineered to express or overproduce sulfide:cytochrome c oxidoreductase (flavocytochrome c sulfide dehydrogenase, FCC; EC 1.8.2.3). The sulfide dehydrogenases are generally either soluble periplasmic heterodimeric enzymes having a flavoprotein subunit and a heme subunit, or are monomeric membrane-bound enzymes having a single heme c.sub.554 subunit. The FCC polypeptide appears to shuttle electrons from sulfide to a cytochrome c. One advantage of using this enzyme is that the sulfide can be oxidized to elemental sulfate and will likely precipitate, which may be in the form of intracellular globules or granules as observed in native sulfur metabolizing microorganisms. Also, the native cofactors (cytochromes) can be used to provide the reducing power to the cells.

[0110] Exemplary polypeptides (encoded by fcc gene) can be found in Allochromatium vinosum, Thiobacillus spp. W5, Chlorobium limicola, Ectothiorhodospira shaposhnikovii, Chlorobaculum tepidum, Thiobacillus denitrificans, or Thiocystis violascens. Representative FCC polypeptide sequences are publicly available, and an exemplary polypeptide sequence from Allochromatium vinosum is provided in FIG. 3 of Griesbeck et al., Recent Res. Dev. Microbiol. 4:179, 2000, which figure and sequence therein are hereby incorporated by reference in their entirety. In certain embodiments, FCC flavoprotein and heme subunit polypeptides for use in the compositions and methods disclosed herein are from Allochromatium vinosum DSM 180 (Genbank Accession No. AAB86576.1 [flavo] and AAA23316.1 [heme]), Chlorobium limicola (Genbank Accession No. ACD89119.1 [flavo] and AAL68891.1 [heme]), Chlorobaculum tepidum TLS (Genbank Accession No. AAM72249.1 [flavo] and Q8KAS5.1 [heme]), Thiobacillus denitrificans ATCC 25259 (Genbank Accession No. YP_315793.1 [flavo] and YP_315792.1 [heme]), or Thiocystis violascens DSM 198 (Genbank Accession No. YP_006416402.1 [flavo] and YP_006416403.1 [heme]).

[0111] In certain embodiments, FCC flavoprotein and heme subunit polypeptides or functional fragment thereof are derived or obtained from Allochromatium vinosum DSM 180 or Thiocystis violascens DSM 198 and have an amino acid sequence that is at least at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in Genbank Accession Nos. AAB86576.1 [flavo] and AAA23316.1 [heme] or YP_006416402.1 [flavo] and YP_006416403.1 [heme], respectively, or a functional fragment thereof. In another embodiment, the recombinant encoded FCC flavoprotein and heme subunit polypeptides have amino acid sequences that are identical to the sequence set forth in Genbank Accession Nos. AAB86576.1 [flavo] and AAA23316.1 [heme] or YP_006416402.1 [flavo] and YP_006416403.1 [heme], or comprise a consensus sequence of known FCC flavo subunits and a consensus sequence of known FCC heme subunits, respectively.

[0112] In certain embodiments, the FCC flavoprotein and heme subunit polypeptides or functional fragments thereof are encoded by a nucleic acid sequence that has been codon optimized. The codon optimized FCC flavoprotein and heme subunit polypeptides or functional fragments thereof may be encoded by nucleic acids comprising any one of SEQ ID NOS.:41-48. In certain embodiment, the recombinant encoded FCC flavoprotein and heme subunit polypeptides have amino acid sequences that are identical to the sequences set forth in any one of SEQ ID NOS.:75-84. In some embodiments, the recombinant encoded FCC flavoprotein and heme subunit polypeptides have amino acid sequences that are at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in any one of SEQ ID NOS.:75-84.

[0113] In certain embodiments, C.sub.1 metabolizing microorganisms and C.sub.1 metabolizing non-photosynthetic microorganisms as described herein may be engineered to express or overproduce sulfur oxygenase (sulfur oxygenase/reductase, SOR). Exemplary SOR polypeptides for use in the microorganisms, compositions and methods disclosed herein include those from Acidianus tegchongensis (Genbank Accession No. AAK58572.1), Sulfolobus metallicus (Genbank Accession No. ABN04222.1), Acidithiobacillus caldus ATCC 51756 (Genbank Accession No. AIA55075.1), Sulfobacillus thermosulfidooxidans (Genbank Accession No. WP_028963476.1), or any combination thereof.

[0114] In certain embodiments, SOR polypeptides or functional fragment thereof are derived or obtained from Acidianus tegchongensis or Sulfolobus metallicus and have an amino acid sequence that is at least at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in Genbank Accession Nos. AAK58572.1 or ABN04222.1, respectively, or a functional fragment thereof. In another embodiment, the recombinant encoded SOR polypeptides have amino acid sequences that are identical to the sequence set forth in Genbank Accession Nos. AAK58572.1 or ABN04222.1 or comprise a consensus sequence of known SOR polypeptides, respectively.

[0115] In certain embodiments, the SOR polypeptides or functional fragments thereof are encoded by a nucleic acid sequence that has been codon optimized. The codon optimized SOR polypeptides or functional fragments thereof may be encoded by nucleic acids comprising any one of SEQ ID NOS.:51-54. In certain embodiment, the recombinant encoded SOR polypeptides have amino acid sequences that are identical to the sequences set forth in any one of SEQ ID NOS.:85-88. In some embodiments, the SOR polypeptides have amino acid sequences that are at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence set forth in any one of SEQ ID NOS.:85-88.

[0116] In some embodiments, the recombinant C.sub.1 metabolizing microorganism is expressing a polypeptide capable of metabolizing an S substrate that is a hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide:quinone oxidoreductase, sulfur dioxygenase, or any combination thereof, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity.

[0117] In certain embodiments, the recombinant C.sub.1 metabolizing microorganism is expressing a polypeptide capable of metabolizing an S substrate that is a sulfide:flavocytochrome-c oxidoreductase, sulfide:quinone oxidoreductase, and sulfur dioxygenase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity.

[0118] In some embodiments, the recombinant C.sub.1 metabolizing microorganism is expressing a polypeptide capable of metabolizing an S substrate that is a sulfur oxygenase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity.

[0119] In some embodiments, the recombinant microorganism is expressing a polypeptide capable of metabolizing an S substrate that is hydrogen-sulfide:NADP.sup.+ oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity.

[0120] In some embodiments, the recombinant microorganism is expressing a polypeptide capable of metabolizing an S substrate that is hydrogen sulfide:ferredoxin oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity.

[0121] In some embodiments, the recombinant microorganism is expressing a polypeptide capable of metabolizing an S substrate that is sulfide:flavocytochrome-c oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity.

[0122] In some embodiments, the recombinant microorganism is expressing a polypeptide capable of metabolizing an S substrate that is sulfide:quinone oxidoreductase, and optionally expresses an exogenous sulfite oxidase, has increased endogenous sulfite oxidase activity, or is expressing a sulfide:flavocytochrome-c oxidoreductase encoded by an exogenous nucleic acid molecule.

[0123] In some embodiments, the recombinant microorganism is expressing a polypeptide capable of metabolizing an S substrate that is sulfur dioxygenase, and optionally expresses an exogenous sulfite oxidase, has increased endogenous sulfite oxidase activity, is expressing a sulfide:flavocytochrome-c oxidoreductase encoded by an exogenous nucleic acid molecule, or is expressing a sulfide:quinone oxidoreductase encoded by an exogenous nucleic acid molecule.

[0124] In any of the aforementioned embodiments, a polypeptide capable of metabolizing an S substrate is stable in the presence of a chemical or environmental stress.

[0125] In further aspects, several additional different modifications can be made to a recombinant C.sub.1 metabolizing microorganism or a recombinant C.sub.1 metabolizing non-photosynthetic microorganism as described herein, either in combination with the sulfur oxidizing or assimilation activity or individually, to utilize the C.sub.1 substrate feedstock to obtain , for example, light alkane conversion to alcohol; light alkane conversion to a C.sub.8-C.sub.24 fatty aldehyde, fatty alcohol, fatty ester wax, hydroxy fatty acid, dicarboxylic acid, or any combination thereof light alkane conversion to biological material (including oil composition); or any combination thereof.

[0126] For example, a recombinant C.sub.1 metabolizing microorganism of the present disclosure may further comprise an exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing light alkanes, such as a biocatalytic enzyme with monooxygenase or hydroxylase activity, to convert C.sub.1 substrates contained in gas into high-value molecules (e.g., alcohol, fatty acid derivatives, fuel precursor). In certain embodiments, the polypeptide capable of oxidizing light alkanes is stable in the presence of a chemical or environmental stress. In further embodiments, a recombinant C.sub.1 metabolizing microorganism of the present disclosure may be engineered to have at least one inactivated alcohol dehydrogenase or at least one alcohol dehydrogenase with reduced activity to facilitate specific oxidation of light hydrocarbons, including mixed gas substrates, into an alcohol composition or a mixed alcohol composition.

[0127] Monooxygenases, expressed by methanotrophic bacteria, utilize an enzyme-associated metal center to split the O--O bond of dioxygen (O.sub.2), wherein one oxygen atom is reduced to form H.sub.2O, while the other oxygen atom attacks a C--H bond of a light alkane (e.g., methane) and is incorporated into the light alkane to form the corresponding alcohol (e.g., methanol will be produced from methane). A reducing agent, such as formate, duroquinol, and hydrogen gas (H.sub.2) (see, e.g., Shiemke et al., Arch. Biochem. Biophys. 321:421, 1995; U.S. Patent Publication No. 2003/0203456), can help complete this oxidation reaction and regenerate the monooxygenase.

[0128] Exemplary monooxygenases include methane monooxygenases (MMOs, which may be soluble, sMMO, or membrane-bound, pMMO), ammonia monooxygenases (AMO), butane monooxygenases (BMOs, which may be soluble, sBMO, or membrane-bound, pBMO, and optionally associate with a hydroxylase, reductase or chaperonin-like protein), propane monooxygenase (PMO, which may be soluble, sPMO or associated with P450, referred to as PMO:P450), alkene monooxygenases, alkane hydroxylases, or P450 (also known as cytochrome P450 or CYP). Moreover, monooxygenases can utilize a broad range of substrates beyond methane, including ethane, propane, butane or pentane, into their corresponding alcohols (see, e.g., Jiang et al., Biochem. Engineering J. 49:277, 2010; Colby etal., Biochem. J. 165:395, 1977; Hyman et al., Applied Environ. Microbiol. 54:3187, 1988; Chen et al., Protein Eng. Design Selection 25:171, 2012; Chen, 2011, Directed evolution of cytochrome P450 for small alkane hydroxylation. Dissertation (Ph.D.), California Institute of Technology). Additionally, monooxygenases can oxidize propene into propene oxide, but-1-ene into 1,2-epoxybutane, 1,3-butadiene into 1,2-epoxybut-3-ene, cis-but-2-ene into cis-2,3-epoxybutane and crotonaldehyde, and trans-but-2-ene into trans-2,3-epoxybutane, crotonyl alcohol and crotonaldehyde. sMMO can oxidize ethane, propane, butane, hexane, octane, and 2-methylpropane into their associated alcohols, as well as oxide ethene into epoxyethane, propene into epoxypropane, but-1-ene into 1,2-epoxybutane, cis-but-2-ene into cis-2,3-epoxybutane and cis-2-buten-l-ol, and trans-but-2-ene into trans-2,3-epoxybutane and trans-2-buten-1-ol. Also, alkene monooxygenases can catalyze aromatic monohydroxylation of benzene, toluene, and phenol (see, e.g., Zhou et al. pplied Environ. Microbiol. 65:1589-95, 1999).

[0129] Numerous monooxygenases and P450 genes have been sequenced and characterized (see, e.g., Stainthorpe et al., Arch. Microbiol. 152:154, 1989; Stainthorpe et al., Gene 91:27, 1990; Coufal et al., Eur. J. Biochem. 267:2174, 2000; Cardy et al., Mol. Microbiol. 5:335, 1991; Cardy et al., Arch. Microbiol. 156:477, 1991; Semrau et al., J. Bacteriol. 177:3071, 1995; Stolvar et al., Microbiol. 145:1235, 1999; Gilbert et al., Appl. Environ. Microbiol. 66:966, 2000; Bodrossy et al., Applied Environ. Microbiol. 61:3549, 1995; Bodrossy et al., FEMS Microbiol. Lett. 170:335, 1999; Lin et al., Appl. Environ. Microbiol. 71:6458, 2005; Hou et al., Biol. Direct. 3:26, 2008; McTavish et al., J. Bacteriol. 175:2436, 1993; Norton et al., Arch. Microbiol. 177:139, 2002; Nelson et al., Pharmacogenetics 6:1, 1996; Funhoff et al., J. Bacteriol. 188:5220, 2006; Kubota et al., Biosci. Biotechnol. Biochem. 69:2421, 2005). Exemplary pMMO amino acid sequences and AMO amino acid sequences are provided in International Patent Publication No. WO 2014/062703, which sequences are incorporated herein in their entirety.

[0130] In certain embodiments, MMO, BMO, PMO, AMO, alkene monooxygenase, alkane hydroxylase, or P450 polypeptides or functional fragments thereof are encoded by a nucleic acid sequence that has been codon optimized. A codon optimized MMO, BMO, PMO, AMO, alkene monooxygenase, alkane hydroxylase, or P450 polypeptide, or functional fragment thereof, may be encoded by nucleic acids as set forth in any one of SEQ ID NOS.:1-20. In certain embodiments, a MMO, BMO, PMO, AMO, alkene monooxygenase, alkane hydroxylase, or P450 polypeptide as used herein has an amino acid sequence that is identical to the corresponding sequence set forth in any one of SEQ ID NOS.:89-108. In some embodiments, a MMO, BMO, PMO, AMO, alkene monooxygenase, alkane hydroxylase, or P450 polypeptide has an amino acid sequence that is at least 75%, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the corresponding sequence set forth in any one of SEQ ID NOS.:89-108.

[0131] As provided herein, methods for oxidizing hydrocarbons, including converting alkanes into their corresponding alcohols or alkenes into their corresponding epoxides, comprises providing a genetically engineered C.sub.1 microorganism or cell lysate thereof in the presence of air or oxygen and a reducing agent. In certain embodiments, a gas substrate comprising a light alkane (e.g., methane, ethane, propane, butane) is converted into a corresponding alcohol, a light alkene (e.g., ethylene, propylene, butylene, butadiene) is converted into a corresponding epoxide, or a mixed gas composition comprising light alkanes, alkenes, or both are converted into their corresponding alcohol(s), their corresponding epoxide(s), or mixture of alcohols and epoxides, respectively.

[0132] In certain embodiments, there are provided recombinant C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms, or a cell lysate thereof, comprising an exogenous nucleic acid molecule that encodes a polypeptide capable of oxidizing light alkanes, such as a methane monooxygenase (MMO), an ammonia monooxygenases (AMO), or P450 enzyme.

[0133] In further embodiments, any one of the aforementioned C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms, or a cell lysate thereof, comprising an exogenous hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: quinone oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfur dioxygenase, or any combination thereof, may further comprise a second exogenous nucleic acid molecule that encodes a polypeptide capable of oxidizing light alkanes, such as a methane monooxygenase (MMO), a butane monooxygenase (BMO), a propane monooxygenase (PMO), an ammonia monooxygenase (AMO), an alkane hydroxylase, or a P450 enzyme.

[0134] In further embodiments, any one of the aforementioned C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms, or a cell lysate thereof, comprising an exogenous sulfur oxygenase, may further comprise a second exogenous nucleic acid molecule that encodes a polypeptide capable of oxidizing light alkanes, such as a methane monooxygenase (MMO), a butane monooxygenase (BMO), a propane monooxygenase (PMO), an ammonia monooxygenase (AMO), an alkane hydroxylase, or a P450 enzyme.

[0135] An enzyme with monooxygenase activity may comprise multiple components. In certain embodiments, a nucleic acid molecule encoding a polypeptide with monooxygenase activity (capable of oxidizing light alkanes) may comprise polynucleotides encoding a gene cluster or operon for an enzyme with methane monooxygenase activity, or for a single subunit that constitutes the active site for the enzyme. By way of example, where an enzyme with methane monooxygenase activity is pMMO, a nucleic acid may comprise polynucleotides comprising apmoCAB gene cluster or a pmoA gene (.beta. subunit). In another example, where an enzyme with methane monooxygenase activity is sMMO, a nucleic acid may comprise polynucleotides comprising a mmoXYZ gene cluster or a mmoX gene (.alpha. subunit).

[0136] The introduction of an exogenous nucleic acid encoding a polypeptide capable of oxidizing light alkanes, such as a monooxygenase, can confer upon non-naturally occurring microorganisms provided herein the capability of converting light alkanes into their corresponding alcohols (e.g., converting ethane, propane, and butane into their corresponding alcohols, ethanol, propanol, and butanol, respectively), or converting alkenes into their corresponding epoxides (e.g., converting ethylene, propylene, butene, and butadiene into their corresponding epoxides ethylene oxide, propylene oxide, butene oxide, and butadiene 1,2 oxide respectively).

[0137] In certain embodiments, methane is converted into methanol, ethane is converted into ethanol, propane is converted into propanol, butane is converted into butanol, pentane is converted into pentanol, or any combination thereof. In further embodiments, butane is converted into butanol and the butanol is comprised substantially of n-butanol (i.e n-butanol comprises at least 50% or more of the butanol product). In still further embodiments, propane is converted into propanol and the propanol is comprised substantially of n-propanol (i.e n-propanol comprises at least 50% or more of the propanol product).

[0138] In certain embodiments, recombinant C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms, or a cell lysate thereof, of this disclosure may be capable of converting ethylene, propylene, butene, butadiene into their corresponding epoxides, ethylene oxide, propylene oxide, butene oxide, and butadiene 1,2 oxide respectively.

[0139] In certain embodiments, provided are recombinant C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms, or a cell lysate thereof, of this disclosure capable of converting a mixed alkane gas into a mixed alcohol composition. A mixed alkane gas may be wet (unprocessed) natural gas or a partially separated derivative thereof (e.g., natural gas liquids separated from wet natural gas during processing). Natural gas liquids may include ethane, propane, butane, or a combination thereof. In certain embodiments, provided are non-naturally occurring C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms capable of converting light alkanes (i.e., any combination of two or more alkanes selected from methane, ethane, propane, butane, pentane, or any combination thereof) into their corresponding alcohols, which produces a mixed alcohol composition.

[0140] In certain embodiments, provided are recombinant C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms, or a cell lysate thereof, of this disclosure capable of converting a mixed alkene gas into a mixed epoxide product. A mixed alkene gas may be a gas stream from a petroleum cracker or a partially separated derivative thereof. In certain embodiments, provided are recombinant microorganisms of this disclosure capable of converting light alkenes (i.e., any combination of two or more alkenes selected from ethylene, propylene, butene, butadiene, or any combination thereof) into their corresponding epoxides.

[0141] By way of background, alcohol or epoxide products, including methanol, produced by enzymes with monooxygenase or hyrdroxylase activity may be oxidized further into unwanted products by endogenous alcohol dehydrogenases (see, e.g., Anthony and Zatman, Biochem. J. 96:808, 1965; Lu et al., J. Am. Chem. Soc. 132:15451, 2010). Recombinant microorganisms provided herein may exhibit poor yields due to downstream metabolism of methanol, other alcohol products (e.g., ethanol, propanol, and butanol), or epoxides. By inactivating at least one alcohol dehydrogenase (e.g., methanol dehydrogenase), reduction of alcohol or epoxide product loss and improvement of product yield may be achieved.

[0142] In certain embodiments, a recombinant microorganism or cell lysate thereof of this disclosure is capable of using a reducing agent to convert the light alkane gas to an alcohol composition. In one embodiment, the reducing agent is hydrogen (H.sub.2) gas. For example, an alkane monooxygenase, alkene monooxygenase, alkane hydroxylases, or combination thereof expressed in the recombinant microorganism or cell lysate thereof is capable of directly using H.sub.2 as a reducing agent to convert light alkane gas to an alcohol composition. In certain embodiments, an alkane monooxygenase is pMMO, sMMO, AMO, pBMO, sBMO, sPMO, PMO:P450, P450, or any combination thereof. In certain embodiments, the alkane monooxygenase is a methane monooxygenase, such as a pMMO, sMMO, or P450. In certain embodiments, the chemical or environmental stress is a temperature at least 60.degree. C., a pH of at least 9, or a pH of no more than 5. In certain embodiments, the alcohol dehydrogenase is inactivated by the chemical or environmental stress, such as a temperature at least 60.degree. C., a pH of at least 9, or a pH of no more than 5, or the alcohol dehydrogenase is inactivated by genetic modification. In certain embodiments, the at least one inactivated alcohol dehydrogenase comprises a methanol dehydrogenase.

[0143] Any one of the aforementioned C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms comprising an exogenous hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: quinone oxidoreductase, sulfide:flavocytochrome-c oxidoreductase, sulfur dioxygenase, sulfur oxygenase, cysteine synthase, O-acetylhomoserine sulfhydrylase, methionine synthase (cobalamin dependent or independent), or homocysteine methyltransferase; an exogenous nucleic acid molecule that encodes a polypeptide capable of oxidizing light alkanes (e.g., monooxygenase or hydroxylase) or a cell lysate thereof, or both; may further comprise at least one endogenous protein function that is attenuated, inhibited, substantially reduced or functionally deleted, such as alcohol dehydrogenase (e.g., methanol dehydrogenase activity).

[0144] In certain embodiments, at least one alcohol dehydrogenase that is deactivated or reduced in activity comprises a methanol dehydrogenase (MIDH). In certain embodiments, alcohol dehydrogenase activity is reduced, inhibited or knocked-out by genetic modification or by a chemical or environmental stress (e.g., a temperature of at least 60.degree. C., a pH of at least 9, or a pH no more than 5).

[0145] As used herein, an alcohol dehydrogenase refers to any enzyme that catalyzes the reversible conversion of alcohols into their corresponding aldehydes or ketones with the reduction of NAD.sup.+ to NADH. An alcohol dehydrogenase is inactivated if it possesses less than 25% activity as compared to a wild type or reference enzyme or possesses less than 25% activity during or after exposure to a chemical or environmental stress as compared to normal conditions. For example, an inactivated ADH (e.g., genetically inactivated) may possess 24%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less activity as compared to a wild type ADH. In another example, an inactivated ADH may possess 24%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less activity during exposure to a chemical or environmental stress (e.g., heat of at least 60.degree. C.) as compared to in the absence of a chemical or environmental stress (e.g., at normal temperature).

[0146] In certain embodiments, provided are C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms or a cell lysate thereof in which two, three, four, or more alcohol dehydrogenases are attenuated, inhibited, inactivated, or functionally deleted. As an example, ADH sequences that may be inactivated in Methylosinus trichosporium OB3b, Methylococcus capsulatus str. Bath, and Methylomicrobium alcaliphilum are provided in FIG. 5 of U.S. Provisional Patent Application No. 61/714,123, which sequences are incorporated herein in their entirety.

[0147] In certain embodiments, a nucleic acid molecule encoding a polypeptide capable of metabolizing sulfur, a nucleic acid molecule encoding a polypeptide capable of oxidizing light alkanes, or both, may encode polypeptides that naturally possess a desired "stability" or "stable activity" in the presence of a chemical or environmental stress, which may be used to generate a recombinant C.sub.1 metabolizing microorganism, a recombinant C.sub.1 metabolizing non-photosynthetic microorganism, or a cell lysate thereof of this disclosure. In further embodiments, an enzyme may inherently have chemical or environmental stress stability, e.g., thermophilic, alkaliphilic, or acidophilic polypeptides may be used. In some embodiments, a substantial amount or most of the endogenous enzymes of the C.sub.1 metabolizing microorganism are inactivated by a chemical or environmental stress, except for the exogenous polypeptides with sulfur oxidizing activity, alkane oxidizing activity, or both.

[0148] An environmental or chemical stress refers to a condition that can affect the ability of a microorganism to metabolize normally, survive, or affect the ability of a protein or enzyme to function. Environmental stress conditions include temperature extremes (heat or cold), light availability, water availability, or oxygen concentration. Chemical stress conditions include increased metal concentration, pH stress (high acidity or alkalinity), increased salt concentration, exposure to chemicals, and low nutrient availability. By way of example, an environment stress may be a temperature of at least 40.degree. C., at least 45.degree. C., at least 50.degree. C., at least 55.degree. C., at least 60.degree. C., at least 65.degree. C., at least 70.degree. C., at least 75.degree. C., at least 80.degree. C., at least 85.degree. C., at least 90.degree. C., or at least 95.degree. C. In another example a chemical stress may be a pH of at least 8, at least 8.5, at least 9, at least 9.2, at least 9.4, at least 9.6, at least 9.8, at least 10 or a pH no more than 6, no more than 5.5, no more than 5, no more than 4.8, no more than 4.6, no more than 4.4, no more than 4.2, no more than 4. In certain embodiments, a chemical or environmental stress is a temperature of at least 60.degree. C., a pH of at least 9, or a pH of no more than 5.

[0149] A polypeptide with enzyme activity that is stable in the presence of a chemical or environmental stress refers to a polypeptide that retains substantial activity (e.g., ability to oxidize sulfur, alkanes, or both) during exposure to a chemical or environmental stress (i.e., retains at least 25% catalytic activity under the stress condition as compared to in the absence of the stress condition), which stability may be inherent or may be genetically engineered. A polypeptide with enzyme activity that is stable in the presence of a chemical or environmental stress may retain at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% activity under the stress condition as compared to in the absence of the stress condition, or for genetically modified, as compared to a wild-type or reference enzyme exposed to the same stress condition (i.e., wild type or reference enzyme retains less than 25% catalytic activity during exposure to a stress condition than in the absence of the stress condition).

[0150] Exemplary pMMO amino acid sequences from thermostable methanotrophic bacteria that may be used are provided in FIG. 3 of U.S. Provisional Patent Application No. 61/714,123, which sequences are incorporated herein in their entirety. Exemplary AMO amino acid sequences from halotolerant bacteria and from highly stress resistant bacteria are provided in FIG. 4 of U.S. Provisional Patent Application No. 61/714,123, which sequences are incorporated herein in their entirety. Exemplary reference pMMO and AMO amino acid sequences that may be genetically engineered are provided in FIGS. 2 and 4 of U.S. Provisional Patent Application No. 61/714,123, which sequences are incorporated herein in their entirety.

[0151] In certain further aspects, for example, in addition to converting contaminants in tainted gas into non-toxic and non-polluting molecules or utilizing gas and contaminant molecules as a source of carbon and energy, a recombinant C.sub.1 metabolizing microorganism of the present disclosure may further comprise an exogenous nucleic acid molecule encoding a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or any combination thereof.

[0152] In certain embodiments, any one of the aforementioned C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms comprising an exogenous nucleic acid encoding hydrogen sulfide:NADP.sup.+oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: quinone oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfur dioxygenase, or any combination thereof, may further comprise a second exogenous nucleic acid molecule encoding a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof. In some embodiments, the recombinant fatty acid converting enzyme of a C.sub.1 metabolizing microorganism (e.g., non-natural methanotroph bacteria) is a fatty acyl-CoA reductase (FAR) for converting a gas feedstock (e.g., natural gas) into C.sub.8 to C.sub.24 fatty acid derivatives, such as fatty alcohol. In various embodiments, a recombinant C.sub.1 metabolizing microorganism expresses or over expresses a nucleic acid molecule that encodes a FAR enzyme. In certain embodiments, a FAR enzyme may be endogenous to the C.sub.1 metabolizing microorganism or a FAR enzyme may be heterologous to the C.sub.1 metabolizing microorganism.

[0153] In certain embodiments, any one of the aforementioned C.sub.1 metabolizing microorganisms or C.sub.1 metabolizing non-photosynthetic microorganisms comprising an exogenous nucleic acid encoding sulfur oxygenase, may further comprise a second exogenous nucleic acid molecule encoding a fatty acid converting enzyme capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof. In some embodiments, the recombinant fatty acid converting enzyme of a C.sub.1 metabolizing microorganism (e.g., non-natural methanotroph bacteria) is a fatty acyl-CoA reductase (FAR) for converting a gas feedstock (e.g., natural gas) into C.sub.8 to C.sub.24 fatty acid derivatives, such as fatty alcohol. In various embodiments, a recombinant C.sub.1 metabolizing microorganism expresses or over expresses a nucleic acid molecule that encodes a FAR enzyme. In certain embodiments, a FAR enzyme may be endogenous to the C.sub.1 metabolizing microorganism or a FAR enzyme may be heterologous to the C.sub.1 metabolizing microorganism.

[0154] In further embodiments, the present disclosure provides a non-natural methanotroph contains a fatty acid converting enzyme that is an acyl-CoA dependent fatty acyl-CoA reductase, such as acrl, FAR, CER4 (Genbank Accession No. JN315781.1), or Maqu 2220, capable of forming a fatty alcohol. In certain embodiments, the non-natural methanotroph contains a fatty acid converting enzyme that is an acyl-CoA dependent fatty acyl-CoA reductase capable of forming a fatty aldehyde, such as acrl. In some embodiments, the process will result in the production of fatty alcohols comprising C.sub.8, C.sub.10, C.sub.12, C.sub.14, C.sub.16, C.sub.18, C.sub.20, C.sub.22 or C.sub.24 carbons in length.

[0155] In any of the aforementioned recombinant C.sub.1 metabolizing microorganisms capable of producing fatty acid derivatives (e.g., fatty alcohols), a C.sub.1 metabolizing microorganism (e.g., non-natural methanotroph bacteria) further comprises a recombinant nucleic acid molecule encoding a thioesterase, such as a tesA lacking a leader sequence, UcFatB, or BTE. In certain embodiments, the endogenous thioesterase activity is reduced, minimal or abolished as compared to unaltered endogenous thioesterase activity.

[0156] In any of the aforementioned recombinant C.sub.1 metabolizing microorganisms capable of producing fatty acid derivatives (e.g., fatty alcohols), a C.sub.1 metabolizing microorganism (e.g., non-natural methanotroph bacteria) further comprises a recombinant nucleic acid molecule encoding an acyl-CoA synthetase, such as FadD, yng 1, or FAA2. In certain embodiments, the endogenous acyl-CoA synthetase activity is reduced, minimal or abolished as compared to unaltered endogenous acyl-CoA synthetase activity.

[0157] In still further embodiments, the present disclosure provides a C.sub.1 metabolizing microorganism (e.g., non-natural methanotroph bacteria) having a recombinant nucleic acid encoding hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: quinone oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfur dioxygenase, or any combination thereof; a recombinant nucleic acid molecule encoding a heterologous acyl-CoA dependent fatty acyl-CoA reductase, a recombinant nucleic acid molecule encoding a heterologous thioesterase, and a recombinant nucleic acid molecule encoding a heterologous acyl-CoA synthetase, wherein the C.sub.1 metabolizing microorganism is capable of oxidizing sulfur and converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty alcohol. In certain embodiments, a fatty acyl-CoA reductase is over-expressed as compared to the expression level of the native fatty acyl-CoA reductase. In certain embodiments, an acyl-CoA dependent fatty acyl-CoA reductase capable of forming a fatty aldehyde, fatty alcohol, or both is acrl, or the acyl-CoA independent fatty acyl-CoA reductase capable of forming a fatty alcohol is FAR, CER4, or Maqu 2220. In certain embodiments, the acyl-CoA synthetase is FadD, yng1, or FAA2.

[0158] In still further embodiments, the present disclosure provides a C.sub.1 metabolizing microorganism (e.g., non-natural methanotroph bacteria) having a recombinant nucleic acid encoding sulfur oxygenase; a recombinant nucleic acid molecule encoding a heterologous acyl-CoA dependent fatty acyl-CoA reductase, a recombinant nucleic acid molecule encoding a heterologous thioesterase, and a recombinant nucleic acid molecule encoding a heterologous acyl-CoA synthetase, wherein the C.sub.1 metabolizing microorganism is capable of oxidizing sulfur and converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty alcohol. In certain embodiments, a fatty acyl-CoA reductase is over-expressed as compared to the expression level of the native fatty acyl-CoA reductase. In certain embodiments, an acyl-CoA dependent fatty acyl-CoA reductase capable of forming a fatty aldehyde, fatty alcohol, or both is acrl, or the acyl-CoA independent fatty acyl-CoA reductase capable of forming a fatty alcohol is FAR, CER4, or Maqu_2220. In certain embodiments, the acyl-CoA synthetase is FadD, yng1, or FAA2.

[0159] In yet further embodiments, there is provided a C.sub.1 metabolizing microorganism (e.g., non-natural methanotroph bacteria) having a recombinant nucleic acid molecule encoding a heterologous acyl-CoA independent fatty acyl-CoA reductase, and a recombinant nucleic acid molecule encoding a heterologous thioesterase, wherein the methanotroph is capable of converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty alcohol. In certain embodiments, the fatty acyl-CoA reductase is over-expressed in the non-natural methanotroph as compared to the expression level of the native fatty acyl-CoA reductase.

[0160] In certain embodiments, recombinant C.sub.1 metabolizing microorganisms capable of producing fatty acid derivatives (e.g., fatty alcohols) will comprise a heterologous nucleic acid molecule encoding a carboxylic acid reductase (CAR). In some embodiments, recombinant microorganisms will additionally comprise one or more heterologous nucleic acid molecules selected from an acyl-ACP thioesterase (TE), ketoreductase/alcohol dehydrogenase (ADH), or phosphopantetheinyl transferase (PPTase), as further described herein.

[0161] Intracellular expression of a carboxylic acid reductase of this disclosure will lead to production not only of fatty aldehyde but also the corresponding fatty alcohol, which is due to alcohol dehydrogenase activity within a recombinant host cell. In some embodiments, the process will result in the production of fatty alcohols comprising C.sub.8, C.sub.10, C.sub.12, C.sub.14, C.sub.16, C.sub.18, C.sub.20, C.sub.22 or C.sub.24 carbons in length.

[0162] In even further embodiments, there is provided a C.sub.1 metabolizing microorganism or non-natural methanotroph having a recombinant nucleic acid encoding hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: quinone oxidoreductase, sulfide:flavocytochrome-c oxidoreductase, sulfur dioxygenase, or any combination thereof; a recombinant nucleic acid molecule encoding a carboxylic acid reductase; a recombinant nucleic acid molecule encoding a phosphopantetheinyl transferase; and a recombinant nucleic acid molecule encoding an alcohol dehydrogenase; wherein the C.sub.1 metabolizing microorganism or methanotroph is capable of oxidizing sulfur and converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty alcohol.

[0163] In even further embodiments, there is provided a C.sub.1 metabolizing microorganism or non-natural methanotroph having a recombinant nucleic acid encoding sulfur oxygenase; a recombinant nucleic acid molecule encoding a carboxylic acid reductase; a recombinant nucleic acid molecule encoding a phosphopantetheinyl transferase; and a recombinant nucleic acid molecule encoding an alcohol dehydrogenase; wherein the C.sub.1 metabolizing microorganism or methanotroph is capable of oxidizing sulfur and converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 fatty alcohol.

[0164] In other aspects, this disclosure provides any of the aforementioned C.sub.1 metabolizing microorganisms or non-natural methanotrophs that further comprise a recombinant nucleic acid molecule encoding a P450 enzyme, monooxygenase, or hydroxylase enzyme to produce an w-hydroxy fatty acid. In certain embodiments, the endogenous alcohol dehydrogenase activity is inhibited as compared to unaltered endogenous alcohol dehydrogenase activity. In other embodiments, the endogenous alcohol dehydrogenase activity is increased or elevated as compared to unaltered endogenous alcohol dehydrogenase activity to produce dicarboxylic acid.

[0165] In still further embodiments, there is provided a C.sub.1 metabolizing microorganism or non-natural methanotroph having a recombinant nucleic acid molecule encoding a heterologous fatty acyl-CoA reductase; a recombinant nucleic acid molecule encoding a heterologous thioesterase; and a recombinant nucleic acid molecule encoding a heterologous P450, monooxygenase or hydroxylase, wherein the native alcohol dehydrogenase is inhibited, and wherein the C.sub.1 metabolizing microorganism or methanotroph is capable of converting a C.sub.1 substrate into a C.sub.8-C.sub.24 w-hydroxy fatty acid.

[0166] In still further embodiments, there is provided a C.sub.1 metabolizing microorganism or non-natural methanotroph having a recombinant nucleic acid encoding hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: quinone oxidoreductase, sulfide:flavocytochrome-c oxidoreductase, sulfur dioxygenase, or any combination thereof; a recombinant nucleic acid molecule encoding a heterologous fatty acyl-CoA reductase, and a recombinant nucleic acid molecule encoding a heterologous thioesterase, wherein the C.sub.1 metabolizing microorganism or methanotroph is over-expressing native alcohol dehydrogenase as compared to the normal expression level of native alcohol dehydrogenase, transformed with a recombinant nucleic acid molecule encoding a heterologous alcohol dehydrogenase, or both, and wherein the C.sub.1 metabolizing microorganism or methanotroph is capable of oxidizing sulfur and converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 dicarboxylic acid alcohol.

[0167] In still further embodiments, there is provided a C.sub.1 metabolizing microorganism or non-natural methanotroph having a recombinant nucleic acid encoding sulfur oxygenase; a recombinant nucleic acid molecule encoding a heterologous fatty acyl-CoA reductase, and a recombinant nucleic acid molecule encoding a heterologous thioesterase, wherein the C.sub.1 metabolizing microorganism or methanotroph is over-expressing native alcohol dehydrogenase as compared to the normal expression level of native alcohol dehydrogenase, transformed with a recombinant nucleic acid molecule encoding a heterologous alcohol dehydrogenase, or both, and wherein the C.sub.1 metabolizing microorganism or methanotroph is capable of oxidizing sulfur and converting a gas (e.g., natural gas) into a C.sub.8-C.sub.24 dicarboxylic acid alcohol.

[0168] In any of the aforementioned C.sub.1 metabolizing microorganisms or non-natural methanotrophs, a fatty alcohol is produced comprising one or more of C.sub.8-C.sub.14 or C.sub.10-C.sub.16 or C.sub.12-C.sub.14 or C.sub.14-C.sub.18 or C.sub.14-C.sub.24 fatty alcohols. In certain embodiments, the C.sub.1 metabolizing microorganism or non-natural methanotroph produces fatty alcohol comprising C.sub.10 to C.sub.18 fatty alcohol and the C.sub.1o to C.sub.18 fatty alcohols comprise at least 70% of the total fatty alcohol. In further embodiments, the C.sub.1 metabolizing microorganism or non-natural methanotroph produces fatty alcohol comprising a branched chain fatty alcohol.

[0169] In further embodiments, a fatty acid derivative is a saturated or unsaturated surfactant product having a carbon chain length of about 8 to about 24 carbon atoms, about 8 to about 18 carbon atoms, about 8 to about 14 carbon atoms, about 10 to about 18 carbon atoms, or about 12 to about 16 carbon atoms. In another example, the surfactant product has a carbon chain length of about 10 to about 14 carbon atoms, or about 12 to about 14 carbon atoms.

[0170] In yet other embodiments, a fatty acid derivative contains a carbon chain of about 8 to 24 carbon atoms, about 8 to 18 carbon atoms, about 10 to 18 carbon atoms, about 10 to 16 carbon atoms, about 12 to 16 carbon atoms, about 12 to 14 carbon atoms, about 14 to 24 carbon atoms, about 14 to 18 carbon atoms, about 8 to 16 carbon atoms, or about 8 to 14 carbon atoms. In alternative embodiments, a fatty acid derivative contains a carbon chain less than about 20 carbon atoms, less than about 18 carbon atoms, less than about 16 carbon atoms, less than about 14 carbon atoms, or less than about 12 carbon atoms. In more embodiments, a fatty ester product is a saturated or unsaturated fatty ester product having a carbon atom content between 8 and 24 carbon atoms. In further embodiments, a fatty ester product has a carbon atom content between 8 and 14 carbon atoms. In other embodiments, a fatty ester product has a carbon content of 14 and 20 carbons. In yet other embodiments, a fatty ester is the methyl ester of C.sub.18:1. In still further embodiments, a fatty ester is the ethyl ester of C.sub.16:1. In further embodiments, a fatty ester is a methyl ester of C.sub.16:1. In yet other embodiments, a fatty ester is octadecyl ester of octanol.

[0171] In certain other aspects, a recombinant C.sub.1 metabolizing microorganism or a non-natural methanotroph of the present disclosure is capable of converting gas (e.g., light alkanes such as methane, ethane, propane, butane) and associated contaminants (e.g., H.sub.2S, CO.sub.2) into biological material, wherein an oil composition can be extracted from the biological material, or the biological material can be used as animal feed or fertilizer. In certain embodiments, for example, in addition to converting contaminants in tainted gas into non-toxic and non-polluting molecules or utilizing gas and contaminant molecules as a source of carbon and energy, a recombinant C.sub.1 metabolizing microorganism or a non-natural methanotroph of the present disclosure may further comprise an exogenous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, capable of converting a gas (e.g., natural gas) into an oil composition.

[0172] In further embodiments, there is provided a C.sub.1 metabolizing microorganism or non-natural methanotroph having a recombinant nucleic acid molecule encoding hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: quinone oxidoreductase, sulfide:flavocytochrome-c oxidoreductase, sulfur dioxygenase, or any combination thereof; and a recombinant nucleic acid molecule encoding a fatty acid producing enzyme, a recombinant nucleic acid molecule encoding a formaldehyde assimilation enzyme, or a combination thereof; wherein the C.sub.1 metabolizing microorganism or methanotroph is capable of oxidizing sulfur and converting a gas (e.g., natural gas) into an oil composition.

[0173] In further embodiments, there is provided a C.sub.1 metabolizing microorganism or non-natural methanotroph having a recombinant nucleic acid molecule encoding sulfur oxygenase; and a recombinant nucleic acid molecule encoding a fatty acid producing enzyme, a recombinant nucleic acid molecule encoding a formaldehyde assimilation enzyme, or a combination thereof; wherein the C.sub.1 metabolizing microorganism or methanotroph is capable of oxidizing sulfur and converting a gas (e.g., natural gas) into an oil composition.

[0174] In still further embodiments, a recombinant C.sub.1 metabolizing microorganism or a non-natural methanotroph of the present disclosure may have one or more improved properties (e.g., higher growth rate, ability to grow in high pH, improved utilization of nutrients, temperature stability, increased biomaterial yield). In related embodiments, a product such as an oil composition (e.g., fatty acids, triglycerides, phospholipids, isoprenes, terpenes, PHA) is recovered from the recombinant C.sub.1 metabolizing microorganism or non-natural methanotroph, and optionally an oil composition is refined to produce plastic prescursors or one or more fuels, such as jet fuel, diesel fuel, gasoline, or a combination thereof. In still further embodiments, a recombinant C.sub.1 metabolizing microorganism or a non-natural methanotroph of the present disclosure may produce an oil composition and an alcohol (such as methanol, ethanol, propanol, or longer chain fatty alcohols), wherein the oil composition is reacted with an alcohol (e.g., in an esterification plant) to generate biodiesel.

[0175] In yet further embodiments, an oil composition is derived or extracted from cell membrane of the C.sub.1 metabolizing non-photosynthetic microorganism (e.g., methylotroph, methanotroph) or may be recovered from a culture supernatant if secreted or excreted, or a combination thereof. Extraction of an oil composition may be accomplished using various different solvents (e.g., a polar solvent, a non-polar solvent, a neutral solvent, an acidic solvent, a basic solvent, hexane, or a combination thereof), such as hexane or acidic methanol or chloroform/methanol mix, in extraction methods known in the art.

[0176] In certain embodiments, the present disclosure provides a recombinant C.sub.1 metabolizing microorganism comprising a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing an S substrate, wherein the recombinant microorganism is capable of scrubbing sulfur containing compounds from sour gas to produce sweet gas when all the light alkanes are not fully consumed or converted.

[0177] In any of the aforementioned embodiments, a plasmid containing one or more of the aforementioned genes, all under the control of a constitutive or otherwise controllable promoter, can be used. Several additional different modifications can be made as described herein, either in combination or individually, to a C.sub.1 metabolizing microorganism or a C.sub.1 metabolizing non-photosynthetic microorganism or to any of the exogenous nucleic acid molecules introduced into the microorganism or any combination changes to microorganism and recombinant nucleic acid molecules to produce high-value molecules (e.g., alcohols, fatty acid derivatives, amino acids), biological materials (e.g., animal feed, fertilizer, oil composition), sweetened gas, or any combination thereof.

[0178] In any of the aforementioned recombinant C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria), the recombinant microorganism is converting natural gas, unconventional natural gas, casinghead gas, or vapor above a confined sour hydrocarbon, and is capable of producing an alcohol composition, such as methanol, ethanol, propanol, butanol, or a combination thereof.

[0179] In some embodiments, a variant sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide :ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, cysteine synthase, O-acetylhomoserine sulfhydrylase, methionine synthase (cobalamin dependent or independent), homocysteine methyltransferase, sulfur dioxygenase, sulfur oxygenase, or sulfite oxidase may encompass one or more amino acid substitutions, including variants having one or more conservative substitutions. In certain embodiments, conservatively substituted variants of a sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide :ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, hydrogen sulfide S-acetyltransferase, cysteine synthase, sulfur dioxygenase, sulfur oxygenase, or sulfite oxidase will include substitutions of a small percentage, such as less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the amino acids of a sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, hydrogen sulfide S-acetyltransferase, sulfur dioxygenase, sulfur oxygenase, or sulfite oxidase polypeptide sequence, respectively.

[0180] In any of the aforementioned recombinant C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) capable of metabolizing an S substrate as encompassed by the present disclosure, the amount of alcohol produced ranges from about 1 mg/L to about 0.5 g/L to about 1 g/L to about 2 g/L to about 5 g/L to about 10 g/L to about 50 g/L to about 100 g/L to about 500 g/L. In certain other embodiments, a C.sub.1 substrate feedstock for a C.sub.1 metabolizing microorganism or non-natural methanotroph as described herein is a light alkane gas mixture, natural gas, unconventional natural gas, syngas, casinghead gas, wellhead condensate, refinery gas, pyrolysis gas, ventilation (air) stream, or vapor above a confined sour hydrocarbon. In certain embodiments, a C.sub.1 metabolizing microorganism or non-natural methanotroph is capable of converting alkane gas mixture, natural gas, unconventional natural gas, syngas, casinghead gas, wellhead condensate, refinery gas, pyrolysis gas, ventilation (air) stream, or vapor above a confined sour hydrocarbon into their corresponding alcohols or other compositions.

[0181] For example, the recombinant microorganisms of this disclosure are capable of converting a mixture of light alkanes (i.e., any combination of two or more alkanes selected from methane, ethane, propane, butane, pentane, or any combination thereof) into their corresponding alcohols. In further embodiments, ethane, propane, and butane are converted into their corresponding alcohols, ethanol, propanol, and butanol, respectively, or converting ethylene, propylene, butene, and butadiene into their corresponding epoxides. In yet further embodiments, butanol comprises substantially of n-butanol (i.e., n-butanol comprises at least 50% or more of the butanol product). In still further embodiments, propanol comprises substantially of n-propanol (i.e., n-propanol comprises at least 50% or more of the propanol product). In further embodiments, recombinant microorganisms of this disclosure may be capable of converting ethylene, propylene, butene, butadiene into their corresponding epoxides, ethylene oxide, propylene oxide, butene oxide, and butadiene 1,2 oxide respectively. In certain embodiments, provided are recombinant microorganisms of this disclosure capable of converting a mixed alkene gas from, for example, a petroleum cracker or a partially separated derivative thereof, into a mixed epoxide product.

[0182] Any of the aforementioned C.sub.1 metabolizing microorganisms or non-natural methanotroph bacteria may also have undergone strain adaptation under selective conditions to produce variants with improved properties for metabolizing a gas and any associated contaminants, before or after introduction of the recombinant nucleic acid molecules. Improved properties may include increased growth rate, yield of desired products (e.g., increased sulfite oxidase activity or production, desulfurized gas, light alkanes oxidized to alcohols), or tolerance to process or culture contaminants. In particular embodiments, a high growth variant C.sub.1 metabolizing microorganism or methanotroph comprises a host bacteria capable of growing on a light alkane gas or methane feedstock as a primary carbon and energy source and that possesses a faster exponential phase growth rate (i.e., shorter doubling time) than its parent, reference, or wild-type bacteria (see, e.g., U.S. Pat. No. 6,689,601).

[0183] Each of the recombinant microorganisms of this disclosure may be grown as an isolated culture, with a heterologous organism that may aid with growth, or one or more of these bacteria may be combined to generate a mixed culture. In still further embodiments, C.sub.1 metabolizing microorganisms of this disclosure are obligate C.sub.1 metabolizing microorganisms capable of utilizing (e.g., oxidizing or assimilating) an S substrate.

Culture Methods

[0184] In certain embodiments, methods described herein use recombinant microorganisms or cell lysates thereof immobilized on, within, or behind a solid matrix. In further embodiments, the non-naturally occurring microorganisms, cell lysates or cell-free extracts thereof are in a substantially non-aqueous state (e.g., lyophilized). Recombinant microorganisms, cell lysates or cell-free fractions thereof are temporarily or permanently attached on, within, or behind a solid matrix within a bioreactor. Nutrients, substrates, and other required factors are supplied to the solid matrices so that the cells may catalyze desired reactions. Recombinant microorganisms may grow on the surface of a solid matrix (e.g., as a biofilm). Recombinant microorganisms, cell lysates or cell-free fractions derived thereof may be attached on the surface or within a solid matrix without cellular growth or in a non-living state. Exemplary solid matrix supports for microorganisms include polypropylene rings, ceramic bio-rings, ceramic saddles, fibrous supports (e.g., membrane), porous glass beads, polymer beads, charcoal, activated carbon, dried silica gel, particulate alumina, Ottawa sand, clay, polyurethane cell support sheets, and fluidized bed particle carrier (e.g., sand, granular-activated carbon, diatomaceous earth, calcium alginate gel beads).

[0185] A variety of culture methodologies may be used for recombinant methanotrophic bacteria described herein. For example, methanotrophic bacteria may be grown by batch culture or continuous culture methodologies. In certain embodiments, the cultures are grown in a controlled culture unit, such as a fermenter, bioreactor, hollow fiber membrane bioreactor, or the like.

[0186] A classical batch culturing method is a closed system where the composition of the media is set at the beginning of the culture and not subject to external alterations during the culture process. Thus, at the beginning of the culturing process, the media is inoculated with the desired C.sub.1 metabolizing microorganism (e.g., methanotroph) and growth or metabolic activity is permitted to occur without adding anything to the system. Typically, however, a "batch" culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures, cells moderate through a static lag phase to a high growth logarithmic phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in logarithmic growth phase are often responsible for the bulk production of end product or intermediate in some systems. Stationary or post-exponential phase production can be obtained in other systems.

[0187] The Fed-Batch system is a variation on the standard batch system. Fed-Batch culture processes comprise a typical batch system with the modification that the substrate is added in increments as the culture progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measureable factors, such as pH, dissolved oxygen, and the partial pressure of waste gases such as CO.sub.2. Batch and Fed-Batch culturing methods are common and known in the art (see, e.g., Thomas D. Brock, Biotechnology: A Textbook of Industrial Microbiology, 2.sup.nd Ed. (1989) Sinauer Associates, Inc., Sunderland, Mass.; Deshpande, Appl. Biochem. Biotechnol. 36:227, 1992).

[0188] Continuous cultures are "open" systems where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in logarithmic phase growth. Alternatively, continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added and valuable products, by-products, and waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials.

[0189] Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limited nutrient, such as the carbon source or nitrogen level, at a fixed rate and allow all other parameters to modulate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing the rate of product formation, are well known in the art, and a variety of methods are detailed by Brock, supra.

[0190] Liquid phase bioreactors (e.g., stirred tank, packed bed, one liquid phase, two liquid phase, hollow fiber membrane) are well known in the art and may be used for growth of non-naturally occurring microorganisms and biocatalysis.

[0191] By using gas phase bioreactors, substrates for biocatalysis or bioremediation are absorbed from a gas by non-naturally occurring microorganisms, cell lysates or cell-free fractions thereof, rather than from a liquid. Use of gas phase bioreactors with microorganisms is known in the art (e.g., U.S. Pat. Nos. 2,793,096; 4,999,302; 5,585,266; 5,079,168; and 6,143,556; U.S. Statutory Invention Registration H1430; U.S. Patent Application Publication No. 2003/0032170; Emerging Technologies in Hazardous Waste Management III, 1993, eds. Tedder and Pohland, pp 411-428). Exemplary gas phase bioreactors include single pass system, closed loop pumping system, and fluidized bed reactor. By utilizing gas phase bioreactors, methane or other gaseous substrates is readily available for biocatalysis by polypeptides with monooxygenase or hydroxylase activity. Furthermore, distillation of an alcohol product from aqueous solution, which represents a significant cost in liquid phase bioreactors, may be bypassed in gas phase bioreactors. In preferred embodiments, methods for desulfurizing a gas or converting a gas into an alcohol composition are performed in gas phase bioreactors. In further embodiments, methods for desulfurizing a gas or converting a gas into an alcohol composition are performed in fluidized bed reactors. In a fluidized bed reactor, a fluid (i.e., gas or liquid) is passed upward through particle bed carriers, usually sand, granular-activated carbon, or diatomaceous earth, on which microorganisms can attach and grow. The fluid velocity is such that particle bed carriers and attached microorganisms are suspended (i.e., bed fluidization). The microorganisms attached to the particle bed carriers freely circulate in the fluid, allowing for effective mass transfer of substrates in the fluid to the microorganisms and increased microbial growth. Exemplary fluidized bed reactors include plug-flow reactors and completely mixed reactors. Uses of fluidized bed reactors with microbial biofilms are known in the art (e.g., Pfluger et al., Bioresource Technol. 102:9919, 2011; Fennell et al., Biotechnol, Bioengin. 40:1218, 1992; Ruggeri et al., Water Sci. Technol. 29:347, 1994; U.S. Pat. Nos. 4,032,407; 4,009, 098; 4,009,105; and 3,846,289).

[0192] Methanotrophic bacteria described in the present disclosure may be grown as an isolated pure culture, with a heterologous non-methanotrophic microorganism(s) that may aid with growth, or with one or more different strains or species of methanotrophic bacteria may be combined to generate a mixed culture.

Methods for Treating and Converting Gas

[0193] In other aspects, as described herein, there are provided methods for treating a gas by culturing a recombinant C.sub.1 metabolizing microorganism with a tainted gas feedstock for a time sufficient for the recombinant microorganism to metabolize unwanted contaminants from the tainted gas and convert the gas into compounds of interest, wherein the tainted feedstock comprises a C.sub.1 substrate and an S substrate and the recombinant C.sub.1 metabolizing microorganism comprises an exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate. In certain embodiments, the recombinant C.sub.1 metabolizing microorganism assimilates or oxidizes each substrate. The assimilation or oxidation can be partial, substantial, or complete.

[0194] In further aspects, as described herein, there are provided methods for treating a gas by culturing a recombinant C.sub.1 metabolizing microorganism with a sulfur-containing gas feedstock for a time sufficient to metabolize an S substrate, wherein the recombinant C.sub.1 metabolizing microorganism comprises an exogenous nucleic acid molecule encoding a sulfide converting enzyme.

[0195] In yet further aspects, as described herein, there are provided methods for treating a gas by culturing a recombinant C.sub.1 metabolizing microorganism with an acid or sour gas feedstock for a time sufficient to recover sweetened gas, wherein the recombinant C.sub.1 metabolizing microorganism comprises an exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing sulfide.

[0196] In still further aspects, as described herein, there are provided methods for treating a gas by culturing a recombinant C.sub.1 metabolizing microorganism with a tainted gas feedstock for a time sufficient for the recombinant microorganism to in part decontaminate the gas and in part convert the gas to biomass, wherein the recombinant C.sub.1 metabolizing microorganism comprises an exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing sulfide.

[0197] In any of the aforementioned methods for using recombinant C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) to produce compounds of interest (or even some treated gas) as disclosed in the present disclosure, the polypeptide capable of oxidizing sulfur is a hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, sulfur dioxygenase, or any combination thereof, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity and that are optionally stable in the presence of chemical or environmental stress. In certain embodiments, the S substrate is oxidized to a sulfate.

[0198] In any of the aforementioned methods for using recombinant C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) to produce compounds of interest (or even some treated gas) as disclosed in the present disclosure, the polypeptide capable of oxidizing sulfur is a sulfur oxygenase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity and that are optionally stable in the presence of chemical or environmental stress. In certain embodiments, the S substrate is oxidized to a sulfide or a sulfate.

[0199] In certain embodiments, the polypeptide capable of oxidizing an S substrate is a sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, and sulfur dioxygenase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase. In further embodiments, the polypeptide capable of oxidizing an S substrate is hydrogen sulfide:NADP.sup.+ oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase. In further embodiments, the polypeptide capable of oxidizing an S substrate is hydrogen sulfide:ferredoxin oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase. In further embodiments, the polypeptide capable of oxidizing an S substrate is sulfide:flavocytochrome-c oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase. In further embodiments, the polypeptide capable of oxidizing an S substrate is sulfide:quinone oxidoreductase, and optionally expresses an exogenous sulfite oxidase, has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase, or is expressing a sulfide:flavocytochrome-c oxidoreductase encoded by an exogenous nucleic acid molecule. In further embodiments, the polypeptide capable of oxidizing an S substrate is sulfur dioxygenase, and optionally expresses an exogenous sulfite oxidase, has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase, or is expressing a sulfide:flavocytochrome-c oxidoreductase encoded by an exogenous nucleic acid molecule, or is expressing a sulfide:quinone oxidoreductase encoded by an exogenous nucleic acid molecule.

[0200] In certain embodiments, the polypeptide capable of oxidizing an S substrate is a sulfur oxygenase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase.

[0201] In any of the aforementioned methods, the C.sub.1 substrate, the S substrate, or both are converted into a biological material, such as animal feed, a fertilizer or an oil composition.

[0202] In any of the aforementioned methods, a C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) having a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate further comprises a second exogenous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, wherein the recombinant C.sub.1 metabolizing microorganism converts the C.sub.1 substrate into an oil composition. In certain embodiments, the oil composition is substantially located in the cell membrane of the C.sub.1 metabolizing microorganism. In some embodiments, the method further comprises the step of obtaining the oil composition by extraction. In certain embodiments, the method further comprises the step of refining the extracted oil composition into a fuel, wherein the fuel comprises jet fuel, diesel fuel, paraffinic kerosene, gasoline, or a combination thereof.

[0203] In any of the aforementioned methods for using recombinant C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) to produce treated gas or convert light alkanes to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the method further comprises a second recombinant C.sub.1 metabolizing microorganism or cell lysate thereof, wherein the second recombinant microorganism or cell lysate thereof (e.g., methanotrophic bacterium) comprises an exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing light alkanes, wherein the second recombinant microorganism or cell lysate thereof (e.g., methanotrophic bacterium) oxidizes the C.sub.1 substrate into an alcohol composition. In certain embodiments, the culturing is performed in the presence of a reducing agent, such as hydrogen gas (H.sub.2) or formate, and optionally air or oxygen.

[0204] Alternatively, in any of the aforementioned methods for using recombinant C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) to decontaminate gas or to partially, substantially, or fully convert the gas to a mixed alcohol composition as disclosed in the present disclosure, the method comprises a recombinant C.sub.1 metabolizing microorganism further comprising a second exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing light alkanes, wherein the recombinant C.sub.1 metabolizing microorganism or cell lysate thereof oxidizes the C.sub.1 substrate into an alcohol composition. In certain embodiments, the polypeptide capable of oxidizing light alkanes, such as an alkane monooxygenase, alkene monooxygenase or alkane hydroxylase, is capable of directly using H.sub.2 as a reducing agent to convert light alkane gas to an alcohol composition. In further embodiments, the polypeptide capable of oxidizing light alkanes is a monooxygenase, such as pMMO, sMMO, AMO, pBMO, sBMO, sPMO, PMO:P450, P450, or any combination thereof. In further embodiments, the method is performed under a chemical or environmental stress, such as a temperature at least 60.degree. C., a pH of at least 9, or a pH of no more than 5. In further embodiments, the alcohol dehydrogenase is inactivated by the chemical or environmental stress, such as a temperature at least 60.degree. C., a pH of at least 9, or a pH of no more than 5.

[0205] In other embodiments, at least one alcohol dehydrogenase is inactivated, such as by genetic modification. In certain embodiments, the at least one alcohol dehydrogenase comprises methanol dehydrogenase. In further embodiments, the recombinant C.sub.1 metabolizing microorganism or cell lysate thereof is immobilized on a solid matrix in a substantially non-aqueous state.

[0206] In any of the aforementioned methods, a C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) having a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate further comprises a second exogenous nucleic acid molecule encoding a fatty acid converting enzyme capable of converting a C.sub.1 substrate into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a hydroxy fatty acid, a dicarboxylic acid, or any combination thereof. In certain embodiments, the fatty acid converting enzyme is a fatty acyl-CoA reductase, such as FAR, CER4, or Maqu 2220, capable of forming a fatty alcohol. In some embodiments, the fatty acid converting enzyme is a fatty acyl-CoA reductase, such as acr1, capable of forming a fatty aldehyde. In some embodiments, the fatty acid converting enzyme is a carboxylic acid reductase.

[0207] In any of the aforementioned embodiments comprising a fatty acid converting enzyme, the recombinant C.sub.1 metabolizing microorganism further comprises an exogenous nucleic acid molecule encoding a thioesterase, such as a tesA lacking a signal peptide, UcFatB or BTE. In some embodiments, endogenous thioesterase activity is reduced, minimal or abolished as compared to unaltered endogenous thioesterase activity. In any of these embodiments, the recombinant C.sub.1 metabolizing microorganism further comprises an exogenous nucleic acid molecule encoding an acyl-CoA synthetase, such as FadD, yng1, or FAA2. In some embodiments, endogenous acyl-CoA synthetase activity is reduced, minimal or abolished as compared to unaltered endogenous acyl-CoA synthetase activity.

[0208] In any of the aforementioned embodiments comprising a fatty acid converting enzyme and another exogenous nucleic acid molecule encoding a thioesterase or acyl-CoA synthetase, the recombinant C.sub.1 metabolizing microorganism further comprises a recombinant nucleic acid molecule encoding a monooxygenase or hydroxylase to produce w-hydroxy fatty acid. In certain embodiments, endogenous alcohol dehydrogenase activity is reduced, minimal or abolished as compared to unaltered endogenous alcohol dehydrogenase activity.

[0209] In any of the aforementioned embodiments comprising a fatty acid converting enzyme and one or more other exogenous nucleic acid molecules, endogenous alcohol dehydrogenase activity is increased or elevated as compared to unaltered endogenous alcohol dehydrogenase activity to produce dicarboxylic acid.

[0210] In any of the aforementioned methods, the C.sub.1 metabolizing microorganisms can be cultured in a controlled culturing unit, such as a fermenter or bioreactor. In further embodiments, the bioreactor is a gas phase bioreactor or a fluidized bed reactor.

[0211] In any of the aforementioned methods for using recombinant C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) to produce treated gas or convert light alkanes to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the gas feedstock is a light alkane gas, natural gas, unconventional natural gas, syngas, casinghead gas, wellhead condensate, refinery gas, pyrolysis gas, ventilation (air) stream, or any combination thereof. In certain embodiments, the tainted gas feedstock is an acid gas or a sour gas.

[0212] In any of the aforementioned methods for using recombinant C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) to produce treated gas or convert light alkanes to other products (e.g., alcohol, epoxide, biomass) as disclosed in the present disclosure, the C.sub.1 metabolizing microorganism being cultured is Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocysti s, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, Pseudomonas, Candida, Yarrowia, Hansenula, Pichia, Torulopsis, or Rhodotorula. In further embodiments, C.sub.1 metabolizing microorganism being cultured is bacteria, such as a methanotroph or methylotroph.

[0213] The recombinant microorganism may be a Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402), Methylosinus trichosporium OB3b (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris, Methylocella palustris (ATCC 700799), Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystis bryophila, Methylocapsa aurea KYG, Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylomicrobium alcaliphilum, or a combination thereof. In certain related embodiments, the recombinant microorganism is a Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402), or Methylomicrobium alcaliphilum.

[0214] In further embodiments, the C.sub.1 metabolizing microorganism or bacteria can metabolize natural gas, unconventional natural gas, or syngas. In certain embodiments, the syngas metabolizing bacteria include Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium woodii, Clostridium neopropanologen, or a combination thereof.

[0215] In certain other embodiments, the metabolizing microorganism is an obligate C.sub.1 metabolizing microorganism. In certain other embodiments, the metabolizing microorganism is a facultative C.sub.1 metabolizing microorganism. In certain embodiments, the culture comprises a C.sub.1 metabolizing microorganism that is a methanotroph and the culture further comprises one or more heterologous bacteria.

[0216] In certain embodiments, methods for converting light alkane gas into a composition of interest as provided herein produce at least about or up to 1 liter (L), at least about or up to 10 L, at least about or up to 100 L, at least about or up to 1000 L, at least about or up to 10000 L, or at least about or up to 50000 L compound(s) of interest/day.

[0217] In any of the aforementioned methods, the C.sub.1 metabolizing microorganism is an obligate C.sub.1 metabolizing microorganism.

Systems for Treating and Converting Gas

[0218] Substantial amounts of natural gas containing undesirable components (such as acid and sour gas) can be produced at natural gas wells, oil wells (e.g., as associated gas), and from natural gas storage reservoirs, for example, infected with hydrogen sulfide producing bacteria. Hydrogen sulfide and other sulfhydryl compounds can be found in natural gas, in refinery gases, pyrolysis gas, ventilation (air) streams, or in vapor spaces above confined hydrogen sulfide containing hydrocarbons (such as storage tanks or barges). The compositions, methods and systems of this disclosure can be used to treat and convert gas in any of these settings.

[0219] Natural gas has a wide range of acid gas concentrations, ranging from parts per million to 50 volume percent or higher, depending on the source. Acid gases are corrosive in the presence of water (H.sub.2S and CO.sub.2), toxic (H.sub.2S), or lack heating value (CO.sub.2), so salable gas must be sweetened to contain no more than, depending on regulations or agreements, a quarter grain H.sub.2S per 100 standard cubic feet (4 parts per million) and to have a heating value of no less than 920 to 980 Btu/SCF. The most widely used processes to sweeten natural gas entail the use of alkanolamines; the two most common are monoethanolamine (MEA) and diethanolamine (DEA). As simple gas sweetening system involve introducing acid or sour gas into the bottom of an absorber where the gas flows up the tower countercurrent of an aqueous (lean) amine stream, which fed through the top of the tower. Within the tower, the acid or sour gas is absorbed by the amine (referred to as rich amine). From the absorber, the rich amine is directed to the top of a stripping tower where a drop in pressure and application of heat strips the solvent of the sour or acid gas. The once again lean amine is circulated back to the absorber for sweetening. But, amine gas sweetening plants can experience operating difficulties including foaming, failure to meet sweet gas specification, high solvent losses, corrosion, fouling of equipment, and contamination of the amine solution. Often one operating difficulty is the cause of another, although not all plants experience the same problems or to the same degree.

[0220] The compositions, methods and systems of the instant disclosure solve many of these problems, although the compositions, methods and systems of the instant disclosure can be used with the amine systems currently in operation.

[0221] In certain aspects, there is provided a system for treating a gas comprising a source of gas comprising a C.sub.1 substrate and an S substrate and a bioreactor comprising a recombinant C.sub.1 metabolizing microorganism, wherein the recombinant microorganism comprises an exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate, wherein a connector disposed between the gas source and the bioreactor is present to allow flow of gas into the bioreactor and the recombinant microorganism oxidizes or assimilates each substrate. In certain embodiments, the source of gas is first treated through an amine system, and the H.sub.2S, CO.sub.2, or both released from the rich amine are fed into the bioreactor. In further embodiments, the bioreactor is a gas bioreactor, such as a fluidized bed reactor, and the recombinant C.sub.1 metabolizing microorganism in contact with a solid matrix in the bioreactor. In further embodiments, the solid matrix comprises a polypropylene, ceramic, glass, charcoal, sand, activated carbon, or diatomaceous earth support. In further embodiments, the recombinant microorganism is a whole cell or a cell lysate thereof that is immobilized on the solid matrix and in a substantially non-aqueous state.

[0222] In other aspects, there is provided a system for recovering stranded gas or oil, comprising a mechanism for recovering oil or gas from an underground formation, wherein the gas comprises a C.sub.1 substrate and an S substrate and the mechanism for recovering comprises a well, a mechanism for oxidizing or assimilating at least a portion of each substrate from the recovered gas, the mechanism for oxidizing or assimilating comprising a bioreactor, wherein the bioreactor comprises a recombinant C.sub.1 metabolizing microorganism comprising an exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate, and a mechanism for recovering the bioremediated stranded oil from the underground formation, wherein the mechanism for recovering comprises a well. In certain embodiments, the source of gas is first treated through an amine system, and the H.sub.2S, CO.sub.2, or both released from the rich amine are fed into the bioreactor. In further embodiments, the bioreactor is a gas bioreactor, such as a fluidized bed reactor, and the recombinant C.sub.1 metabolizing microorganism in contact with a solid matrix in the bioreactor. In further embodiments, the solid matrix comprises a polypropylene, ceramic, glass, charcoal, sand, activated carbon, or diatomaceous earth support. In further embodiments, the recombinant microorganism is a whole cell or a cell lysate thereof that is immobilized on the solid matrix and in a substantially non-aqueous state.

[0223] The recovery of oil or gas with a sulfur compound from an underground formation may be accomplished by any known method. Suitable methods include subsea production, surface production, fracking, primary, secondary, or tertiary production. The selection of the method used to recover the oil or gas from an underground formation is not critical. For example, oil or gas with a sulfur compound may be recovered from a formation into a well, and flow through the well and flowline to a facility. In other instances, enhanced oil recovery, with the use of an agent such as steam, water, a surfactant, a polymer flood, or a miscible agent, may be used to increase the flow of oil or gas from the formation.

[0224] In any of the aforementioned systems, the C.sub.1 substrate, the S substrate, or both are converted into a biological material, such as animal feed, a fertilizer or an oil composition.

[0225] In any of the aforementioned systems, a C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) having a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate further comprises a second exogenous nucleic acid molecule encoding a fatty acid producing enzyme, a formaldehyde assimilation enzyme, or a combination thereof, wherein the recombinant C.sub.1 metabolizing microorganism converts the C.sub.1 substrate into an oil composition. In certain embodiments, the oil composition is substantially located in the cell membrane of the C.sub.1 metabolizing microorganism. In some embodiments, the method further comprises the step of obtaining the oil composition by extraction. In certain embodiments, the method further comprises the step of refining the extracted oil composition into a fuel, wherein the fuel comprises jet fuel, diesel fuel, paraffinic kerosene, gasoline, or a combination thereof.

[0226] In any of the aforementioned systems for treating gas or converting gas to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the polypeptide capable of capable of metabolizing the S substrate is a hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide:quinone oxidoreductase, sulfur dioxygenase, or any combination thereof, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity. In certain embodiments, the polypeptide capable of metabolizing an S substrate is a sulfide:flavocytochrome-c oxidoreductase, sulfide:quinone oxidoreductase, and sulfur dioxygenase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase. In further embodiments, the polypeptide capable of metabolizing an S substrate is hydrogen sulfide:NADP.sup.+ oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase. In further embodiments, the polypeptide capable of metabolizing an S substrate is hydrogen sulfide:ferredoxin oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase. In further embodiments, the polypeptide capable of metabolizing an S substrate is sulfide:flavocytochrome-c oxidoreductase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase. In further embodiments, the polypeptide capable of metabolizing an S substrate is sulfide:quinone oxidoreductase, and optionally expresses an exogenous sulfite oxidase, has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase, or is expressing a sulfide:flavocytochrome-c oxidoreductase encoded by an exogenous nucleic acid molecule. In further embodiments, the polypeptide capable of metabolizing an S substrate is sulfur dioxygenase, and optionally expresses an exogenous sulfite oxidase, has increased endogenous sulfite oxidase activity or has increased endogenous sulfide:quinone oxidoreductase, or is expressing a sulfide:flavocytochrome-c oxidoreductase encoded by an exogenous nucleic acid molecule, or is expressing a sulfide:quinone oxidoreductase encoded by an exogenous nucleic acid molecule.

[0227] In any of the aforementioned systems for treating gas or converting gas to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the polypeptide capable of capable of metabolizing the S substrate is a sulfur oxygenase, and optionally expresses an exogenous sulfite oxidase or has increased endogenous sulfite oxidase activity.

[0228] In any of the aforementioned systems for treating gas or converting gas to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the recombinant C.sub.1 metabolizing microorganism further comprises a second exogenous nucleic acid molecule encoding a polypeptide oxidizing light alkanes (e.g., alkyl monooxygenase or hydroxylase) that is optionally stable in the presence of chemical or environmental stress.

[0229] Alternatively, any of the aforementioned systems for treating gas or converting gas to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the bioreactor further comprises a second recombinant C.sub.1 metabolizing microorganism comprising a second exogenous nucleic acid molecule encoding a polypeptide capable of oxidizing light alkanes that is optionally stable in the presence of chemical or environmental stress. In certain embodiments, the recombinant microorganism or a cell lysate thereof partially converts the gas into a mixed alcohol composition. In further embodiments, the system further comprises a chemical or environmental control unit capable of maintaining a chemical or environmental stress condition in the bioreactor, such as a temperature at least 60.degree. C., a pH of at least 9, or a pH of no more than 5.

[0230] In any of the aforementioned systems, a C.sub.1 metabolizing microorganisms (e.g., non-natural methanotroph bacteria) having a first exogenous nucleic acid molecule encoding a polypeptide capable of metabolizing the S substrate further comprises a second exogenous nucleic acid molecule encoding a fatty acid converting enzyme capable of converting a C.sub.1 substrate into a C.sub.8-C.sub.24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, a hydroxy fatty acid, a dicarboxylic acid, or any combination thereof. In certain embodiments, the fatty acid converting enzyme is a fatty acyl-CoA reductase, such as FAR, CER4, or Maqu_2220, capable of forming a fatty alcohol. In some embodiments, the fatty acid converting enzyme is a fatty acyl-CoA reductase, such as acrl, capable of forming a fatty aldehyde. In some embodiments, the fatty acid converting enzyme is a carboxylic acid reductase.

[0231] In any of the aforementioned embodiments comprising a fatty acid converting enzyme, the recombinant C.sub.1 metabolizing microorganism further comprises an exogenous nucleic acid molecule encoding a thioesterase, such as a tesA lacking a signal peptide, UcFatB or BTE. In some embodiments, endogenous thioesterase activity is reduced, minimal or abolished as compared to unaltered endogenous thioesterase activity. In any of these embodiments, the recombinant C.sub.1 metabolizing microorganism further comprises an exogenous nucleic acid molecule encoding an acyl-CoA synthetase, such as FadD, yng1, or FAA2. In some embodiments, endogenous acyl-CoA synthetase activity is reduced, minimal or abolished as compared to unaltered endogenous acyl-CoA synthetase activity.

[0232] In any of the aforementioned embodiments comprising a fatty acid converting enzyme and another exogenous nucleic acid molecule encoding a thioesterase or acyl-CoA synthetase, the recombinant C.sub.1 metabolizing microorganism further comprises a recombinant nucleic acid molecule encoding a monooxygenase or hydroxylase to produce w-hydroxy fatty acid. In certain embodiments, endogenous alcohol dehydrogenase activity is reduced, minimal or abolished as compared to unaltered endogenous alcohol dehydrogenase activity.

[0233] In any of the aforementioned embodiments comprising a fatty acid converting enzyme and one or more other exogenous nucleic acid molecules, endogenous alcohol dehydrogenase activity is increased or elevated as compared to unaltered endogenous alcohol dehydrogenase activity to produce dicarboxylic acid.

[0234] In any of the aforementioned systems for treating gas or converting gas to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the recombinant C.sub.1 metabolizing microorganism or cell lysate thereof is capable of using H.sub.2 as a reducing agent to convert light alkane gas to an alcohol composition. In certain embodiments, the system further comprises a reducing agent source connected to the bioreactor, such as hydrogen gas (H.sub.2) gas. In further embodiments, the polypeptide having monooxygenase activity, such as an alkane monooxygenase, alkene monooxygenases or alkane hydroxylase, is capable of directly using H.sub.2 as a reducing agent to convert light alkane gas to an alcohol composition. In further embodiments, the polypeptide having monooxygenase activity is an alkane monooxygenase, such as pMMO, sMMO, AMO, pBMO, sBMO, sPMO, PMO:P450, P450, or any combination thereof. In certain embodiments, the polypeptide having monooxygenase activity is a methane monooxygenase, such as pMMO, sMMO, P450, or any combination thereof. In further embodiments, the system further comprises a source of air or oxygen connected to the bioreactor. In further embodiments, the alcohol dehydrogenase is inactivated by the chemical or environmental stress, such as a temperature at least 60.degree. C., a pH of at least 9, or a pH of no more than 5. In other embodiments, the alcohol dehydrogenase is inactivated by genetic modification. In certain embodiments, the at least one alcohol dehydrogenase comprises methanol dehydrogenase. In further embodiments, the recombinant C.sub.1 metabolizing microorganism or cell lysate thereof is immobilized on a solid matrix in a substantially non-aqueous state.

[0235] In any of the aforementioned systems for treating gas or converting gas to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the gas source is a light alkane gas mixture, natural gas, unconventional natural gas, syngas, casinghead gas, wellhead condensate, refinery gas, pyrolysis gas, ventilation (air) stream, or vapor above a confined sour hydrocarbon or a combination thereof. In certain embodiments, the source of gas is from an oil refinery, oil well, or natural gas well. In further embodiments, the gas comprises methane and the corresponding alcohol composition comprises methanol. In further embodiments, the gas comprises ethane and the corresponding alcohol composition comprises ethanol. In further embodiments, the gas comprises propane and the corresponding alcohol composition comprises propanol, n-propanol, or a combination thereof. In further embodiments, the gas comprises butane and the corresponding alcohol composition comprises butanol, n-butanol, or a combination thereof.

[0236] In any of the aforementioned systems for treating gas or converting gas to other products (e.g., alcohol, oil, biomass) as disclosed in the present disclosure, the system further comprises a collection unit for collecting the alcohol composition, such as a condenser. In further embodiments, the system further comprises a distillation unit for separating the alcohol composition from water byproduct. In further embodiments, the system further comprises a recycling unit for recycling unconverted gas back into the bioreactor. In further embodiments, the system further comprises a pipeline for transporting the sweet gas. In further embodiments, the system further comprises a refrigeration unit for liquefying the sweet gas.

[0237] In any of the aforementioned systems for treating gas or converting gas to other products (e.g., alcohol, epoxide, biomass) as disclosed in the present disclosure, the C.sub.1 metabolizing microorganism being cultured is Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, Pseudomonas, Candida, Yarrowia, Hansenula, Pichia, Torulopsis, or Rhodotorula. In further embodiments, C.sub.1 metabolizing microorganism being cultured is bacteria, such as a methanotroph or methylotroph.

[0238] The recombinant microorganism may be a Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402), Methylosinus trichosporium OB3b (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris, Methylocella palustris (ATCC 700799), Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystis bryophila, Methylocapsa aurea KYG, Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylomicrobium alcaliphilum, or a combination thereof. In certain related embodiments, the recombinant microorganism is a Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402), or Methylomicrobium alcaliphilum.

[0239] In further embodiments, the C.sub.1 metabolizing microorganism or bacteria can metabolize natural gas, unconventional natural gas, or syngas. In certain embodiments, the syngas metabolizing bacteria include Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium woodii, Clostridium neopropanologen, or a combination thereof.

[0240] In certain other embodiments, the metabolizing microorganism is an obligate C.sub.1 metabolizing microorganism. In certain other embodiments, the metabolizing microorganism is a facultative C.sub.1 metabolizing microorganism. In certain embodiments, the culture comprises a C.sub.1 metabolizing microorganism that is a methanotroph and the culture further comprises one or more heterologous bacteria.

[0241] The various embodiments described above can be combined to provide further embodiments. All of the patent and non-patent publications referred to in this specification or listed in the Application Data Sheet, including the disclosure of U.S. provisional application No. 61/928,349, filed Jan. 16, 2014, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide further embodiments.

[0242] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

EXAMPLES

Example 1

Conversion of Contaminated Gas Into Animal Feed Using a Methylotroph

[0243] Host cells (Methylococcus capsulatus Bath) are engineered to possess an exogenous hydrogen sulfide:ferredoxin oxidoreductase (sir) gene to enable conversion of contaminated gas into biomass. A nucleic acid sequence encoding the SIR protein from Cyanidioschyzon merolae 10D (SEQ ID NO.:31) is codon optimized for introduction into M. capsulatus Bath. The sir nucleic acid molecule is cloned into an expression vector (encoding kanamycin resistance) for conjugation intoM. capsulatus Bath based on the methods reported by Ali and Murrell (Microbiology 155:761, 2009).

[0244] Briefly, a mobilizable plasmid containing a gene of interest (e.g., sir) operatively linked to a methanol dehydrogenase promoter (for constitutive expression), and encoding kanamycin resistance is first transformed into E. coli S17-1 using standard electroporation methods. Transformation is confirmed by selection of kanamycin-resistant colonies on Luria-Bertani (LB)-agar containing 30 .mu.g/mL kanamycin. Transformed colonies are inoculated into LB media containing 30 .mu.g/mL kanamycin and shaken overnight at 37.degree. C. A 10 mL aliquot of the overnight culture is then collected on a sterile 47 mm nitrocellulose filter (0.2 mm pore size). The E. coli donor cells are washed on the filter with 50 mL sterile Higgins minimal nitrate salts medium (NSM; Cornish et al., J. Gen. Microbiol. 130:2565, 1984; Park et al., Biotechnol. Bioeng. 38:423, 1991)to remove residual media and antibiotic.

[0245] In parallel, a sample of the M. capsulatus Bath (NCIMB 11132) recipient strain is inoculated into 100 mL serum bottles containing 20-50 mL NSM media. The headspace of the bottles is then flushed with a 1:1 mixture of oxygen and methane, and the bottles are sealed with butyl rubber septa and crimped. The bottles are shaken continuously in a 45.degree. C. incubator until reaching an OD.sub.600 of approximately 0.3. The M. capsulatus Bath cells are then collected on the same filter as the E. coli donor strain. The filter is again washed with 50 mL of sterile NSM media. The filter is placed cell-side up on an NSM agar plate containing 0.2% yeast extract and incubated for 24 h at 37.degree. C. in the presence of a 1:1 mixture of methane and air. After 24 h, cells are re-suspended in 10 mL sterile NSM medium before being concentrated by centrifugation. The harvested cells are re-suspended in 1 mL sterile NSM media. 100 .mu.L aliquots of the re-suspended cells are spread onto NSM agar plates containing 10 .mu.g/mL kanamycin.

[0246] The plates are incubated at 45.degree. C. in sealed chambers containing a 1:1 mixture of methane and air. The gas mixture is replenished every 2 days until colonies form, typically after 7-14 days. Colonies are streaked onto NSM plates containing kanamycin to confirm kanamycin resistance and to further isolate transformed methanotroph cells from residual E. coli donor cells.

[0247] The presence of sir expression or SIR function is verified by one or more of (1) PCR and sequencing, (2) Western blot analysis, or (3) assaying for SIR activity. For example, to verify transfer, plasmid DNA is isolated and subjected to PCR using the Illustra PuReTaq Ready-To-Go.TM. PCR Beads (GE Healthcare) under standard conditions (95.degree. C. for 5 min; 32 cycles of 95.degree. C. for 30 s, 50.degree. C. for 30 s, and 72.degree. C. for 1 min; 72.degree. C. for 10 min). As a further control, 1 .mu.l of each of the isolated plasmids is transformed into E. coli XL1-Blue MRF' Kan (Stratagene, La Jolla, Calif.), and plasmids are isolated to verify the presence of the sir insert by restriction endonuclease digests.

[0248] The recombinant M. capsulatus Bath are cultured at 42.degree. C. in serum bottles containing NSM or MM-W1 medium (0.8 mM MgSO.sub.4*7H.sub.2O, 10 mMNaNO.sub.3, 0.14 mM CaCl.sub.2, 1.2 mM NaHCO.sub.3, 2.35 mM KH.sub.2PO.sub.4, 3.4 mM K.sub.2HPO.sub.4, 20.7 .mu.M Na.sub.2MoO.sub.4*2H.sub.2O, 1 .mu.M CuSO.sub.4*5H.sub.2O, 10 .mu.M Fe.sup.III-Na-EDTA, and 1 mL per liter of trace metals solution (containing, per liter 500 mg FeSO.sub.4*7H.sub.2O, 400 mg ZnSO.sub.4*7H.sub.2O, 20 mg MnCl.sub.2*7H.sub.2O, 50 mg CoCl.sub.2*6H.sub.2O, 10 mg NiCl.sub.2*6H.sub.2O, 15 mg H.sub.3BO.sub.3, 250 mg EDTA)). The headspace composition is adjusted to a 1:1 volume of pure methane:air, or a 1:1 volume of contaminated natural gas:air. The bottles are shaken at a rate of 200-250 rpm. The growth of the non-recombinant control strains are compared to the SIR expressing strains.

Sequence CWU 1

1

10811635DNAArtificial SequenceCodon optimized Gordonia sp. propane monooxygenase hydroxylase large subunit sequence 1atgagccgcc agtcccttac caaagcccat gccaagatca ccgaactgtc ctgggagccg 60acgttcgcaa cgcccgcgac tcgcttcggc accgactaca ccttcgaaaa ggccccgaag 120aaagaccccc tgaaacagat aatgcgctcc tacttcccga tggaggaaga aaaggacaat 180cgcgtgtacg gcgcgatgga cggcgctatc cggggtaata tgttccgcca ggtgcaagag 240cgctggctgg agtggcagaa actcttcctt tcgattatcc cgttccctga gatctcggcg 300gcacgggcca tgcccatggc catcgacgcc gtgccgaatc ccgagatcca taatggcctc 360gcggtccaga tgatcgatga ggtccgccat agcacgattc agatgaatct gaagaagctg 420tatatgaaca actacatcga tccggccggt ttcgatatca ccgagaaggc cttcgcgaat 480aactatgccg ggaccatcgg caggcaattc ggtgagggct tcatcaccgg ggacgcgatc 540accgcggcca atatctacct gaccgtggtg gcggaaacgg ccttcaccaa tactctcttt 600gtcgccatgc ccgacgaagc ggcagcgaat ggcgactacc tcctgccgac cgtgttccac 660agcgtccagt cggatgagtc gcggcatatc agcaacggct attcgatcct cctcatggcc 720ctggcggacg agcggaatcg gccgctcttg gagcgggatc tccgctacgc gtggtggaac 780aaccattgcg tggtggacgc tgccattggc acgttcatcg agtatggcac caaggatcgg 840cgcaaggacc gcgagagtta cgccgagatg tggcggcgct ggatatacga cgactattat 900cgctcctatc tgctgcctct ggagaagtat ggcctcacca tcccgcacga cctcgtcgag 960gaggcctgga acaggatcgt cgataagcat tacgtgcatg aagtggcgcg gttcttcgcc 1020acgggctggc cggtcaacta ctggcgcatc gacgcgatga ccgacaccga cttcgagtgg 1080ttcgaagaga agtatcccgg ctggtacaac aagttcggca agtggtggga gaactacaat 1140cgccttgcgt atccggggaa gaacaagccg atcgcgttcg aggacgttga ctacgagtat 1200ccgcaccggt gctggacctg tatggtccca tgcctgatcc gcgaggacat ggttaccgat 1260aaagtcgacg gccagtggcg cacctattgc tcggaaacgt gcgcttggac ggacaaagtc 1320gcatttcggc cggagtatga gggcaggccc acgcccaaca tgggacggct caccggattc 1380cgcgagtggg agactctgca tcacggcaaa gacttggctg atatcatcac ggacctcggt 1440tacgtccgcg acgacggcaa gaccctgatt ccgcaacccc atctcgacct ggaccccaag 1500aaaatgtgga ccctcgacga tgtgcgcggc atcccgtttg gctcgccgaa cgtcgcgctg 1560aatgaaatgt ccgacgatga gcgcgaggcg cacatcgccg cgtacatggc gaacaaaaac 1620ggtgccgtca cggtc 163521104DNAArtificial SequenceCodon optimized Gordonia sp. propane monooxygenase hydroxylase small subunit sequence 2atgagtgccc cagcccaacc ccgcgaacgc agtttcccct ccattgagtt caccgacgca 60gaagccgacg cccgcgagtt cccgtcgtcg cgctcccgca agtataacta ctatcagccg 120agtaagaaac gggccacgat ctatgaagat gtgacggtcg acgtccagcc tgatccggag 180cgccatctca cccagggctg gatctacggc ttcggcgacg gcccgggcgg ctaccccaaa 240gagtggactt cggcccagag ctcgaactgg catcagtttc tcgatccgaa cgaggagtgg 300gagcagagca tctaccgcaa taactcggca gtcgtgcacc aagtcgatct ctgcctgcag 360aacgcgaagc gcgcgagggc ctatgacggc tggaatagcg cgtggctcaa gttcatcgag 420aggaatctgg gcgcgtggat gcacgccgag tcgggtatgg gcctccacgt gttcacctcc 480atccagcggt ccgcgccgac gaatatgatc aataatgccg tctgcgtcaa tgccgcgcat 540aagctgcggt tcgcccaaga cctcgcgctg ttcaaccttg acttgtccga ggccgaggaa 600gcgttcgacg gttccgcgca taaagaggtg tggcagtccg caccggagtg gcagccgacc 660cgcgaggccg tcgagcgcct gaccgcgatc ggcgactggg ctgagctgct gttctgctcg 720aacatcgttt tcgagcaact ggtcggcagc ctcttccgca gcgagcttgt gatgcaagtg 780gctgcccgga acggcgatta tatcacgccg accatcgtcg gcaccggtga gtatgactac 840gatcgggacc tgaactactc ccgggctctg ttccagatgc ttgcgcgcga cgagaaacat 900gggatagaca atcgcaagct gttctcgcgc tggatgagcg agtggtttcc cggagcgagc 960acgcgggctc gggggctcca gccgatctgg tcccagccgg cagacaagag cgtgaccttc 1020tcgtcgtcgc tggagcatgc caagaccaag ttcgccgacg tgctcgcggc gattgacgtg 1080gacatccccg aagaactgaa caag 110431038DNAArtificial SequenceCodon optimized Gordonia sp. propane monooxygenase reductase sequence 3atggcagaca cccacaagat tagcttcgag ccagtggata tagagatgga agttggcgaa 60gatgaaacga ttctcgatgc ggcgttccgg caagagtcca cgtcgtgcac cgctgccgcc 120cggccgctgt tcggttgcaa gtcgtacatg cttgagggcg acgtgcagat ggacgactac 180tcgaccttcg cgtgcaacga cgccgaggaa gccgagggct acgtgcttct ctgccgcacc 240tatgcctaca gcgactgcga gatcgagctg ctcaatttcg acgaggacga gctcctgggc 300ggagccccga tccaagatgt gacgaccaaa gtcgcggcca tcgagcccat gaccccggac 360atcgtgtcgc tcaagctcga cgtcgtggag ccggagtccg tcgagtttaa gtccggccag 420tacttcgacc tgttcatccc gggcaccgag gacaagcgca gcttctccat cgcgacgacc 480cccgctaccc cggaccggct cgaattcctc atcaaaaagt acccgggggg actgttcgcg 540ggcatgttga ctgatggcct gagtgtcggg caagaaatca agctgaatgg gccctatggt 600agttgcaccc tccgcaatgg ccatgtgctg ccgatcgtcg ccatcggtgg cggcgccggc 660atggccccgt tgctcagcct cctgaggcat atctcggaaa ccggcctcaa tcgcccggtc 720cgcttctatt atggtgcgcg gaccgcagcc gacctgttcc tgctggatga gatcgcgacg 780ctgggcgaga aaattgatga cttctcgttc accgcctgcc tgtccgagtc cacggacaac 840gcgcctgagg gcgtcaccgt gatcggcggc aacgtgacgg acatcgtcaa cgataacgaa 900gcggaccttg cccgcaccga ggtgtacttt tgtgccccgc ccccgatggt cgacgcggca 960ctggcgctgg cggagcagca tagcgtcccc cacgaccaga tcttctatga caagttcacg 1020tcgcccgctt tcgacagc 10384333DNAArtificial SequenceCodon optimized Gordonia sp. propane monooxygenase coupling protein sequence 4atgcagtttg gagcggatac ggaatttagt aacatgtgcg gtgtgaccct gatgaatacc 60cctataggcc gggttgtcgc cgacgtcatg ggcgctaaag acggcgtgga actcaccgag 120tatccgtcga tgatccgcgt ggacggcgtc aaccgccttg atttcgacta cgacgagctg 180accgacgcgc tcggtcaaga cttcgacggg tccattttcg aggagatcag cagcacgcac 240tacggccgca tggtccatct ggacgataag accatcctct tcgcctcgcc cgaggatgcg 300gcagagttca tcggcttcga cctgacggcc tcc 33351704DNAArtificial SequenceCodon optimized Thauera butanivorans BmoG sequence 5atgatctccc tgaactgcaa gaaaacgacc actggcctca ccgcccatct cgccctcgtc 60cgcggcatga aagccctcgc cgagctcgtc ggcaccacgc tgggcccgca gggtcgccat 120gtgatgctcg cgcatcgcgc aggcttggca ccgcacgtgt cgaaagacgg cgttgaggtc 180gctcgccatt tgtccctgcc cgactccgag gaagaactcg gcgttcggct gctccgcaat 240gctgccgtgg ccgtgagtga gtcgttcggc gacggcacca gtaccgcgac ggtgtttacc 300gccgacctcg ccgtgcgcgc tctgaagctg attggtgccg gggccgacac gcttgaagtc 360cgcaggggtc tgggcctcgc ggcgtatgcc gcgctggtcg cgctgaacga catggcgagg 420cgcgcagacc ggggcatgct caccgccgtc gcgcaaaccg cggcaaatgg cgatcggcgg 480gtcgccgatc tcctcgtcga ggccttcgag cgcgtgggag cggaaggcac gatcgaggtc 540gagatgggca actccgtcga ggatgtcctc gaagttgcgc agggctcgta cttcgacacg 600gttccgctcg tgaccgcgct gctgccgccg acgggccagg tcgagtttgc gaggcctctg 660atcctgttcc attgcgacgc catcgagact gcggacgaga tattgccggc gcttgagctc 720gcgcgctcct cgcggcgccc gctgctgatc ttggcggaca gcgtcggcat cgatgtggaa 780acgctgctcg tgcgcaatca gaacgagggc accctcgcgg tcgcagtggt gcgggcaccc 840atgtatggcg atacgcggcg cgaggccctc ctggacctca cctccaagtt cggcgggacg 900gcgttcggac gcgagggctt cgtcgagttc gcgctgcgca gcctgggctc cctgtccgag 960ggcgacctcg gccaggccga cgaagcgatc cttgaggccg acggtgtcac ccttcgcggt 1020gcgggcaaca accccagcgc gcttgaggat aggatcgcgc tggtgcgcgc cgagctggac 1080cggggtgacg tgagcgtggg cgactcgccg agcgccaagc tcgactacat tgaaaagcgc 1140aaggagcgcc tgaagctgtt ggctgcgggc agcgccaaac tccacatcgg cggaccgacc 1200gacgtcgaga tcaagacccg cctgccgctg gcggagaatg cccaccgggc tctgctggcc 1260gcagcgaaat ccggcgtgct gcccggtggc ggcgtcgcga tgattcgcgc ggcggagaag 1320gtccaacagg agatgggacg gcttgagggt gacgtcgcct cgggggcttc catcttcctg 1380cagtcgctgg acacgccgat ccggtggatc gcccggaatg ccgggctgag gccagacgag 1440gtcctcgcgc gcaccctggc gaacgagtcg gacttctacg gcttgaacgc gatgaccgga 1500cggtacggtg acctcgccga agatggcgtc ctggacgccc tcgacatggt caccgatgtg 1560atccgcgtgg cagtctcggt cgtggggagc atgctcggcg tcggtgccct ggtgacccgc 1620gctagcccga agcccgcacc cgagcgcttc aagggcaccg agcgcgtgca tgacaagctc 1680atgcgcgagg gcggcttcga tgag 170461092DNAArtificial SequenceCodon optimized Thauera butanivorans butane monooxygenase reductase sequence 6atgttgatgc agcagtataa gatagtggca aggtttgagg atggcgtgac ttacgagtac 60gattgtgggg aagatgagaa tctgctggca gcggcgctcc ggcaaaatgt ccggctgctc 120tgccagtgcc gcaaagcgtt ctgcgggtcc tgcaaggccc tgtgctccga gggtgactac 180gaattgggcg accacatcaa cgtccaggtc cttcccccgg acgaggagga ggacggcgtc 240gttgtgacgt gcgacacgtt cccgcggtcc gacctcgtgc tcgaattccc ctataccagc 300gatcggctcg gcacggtgac ggccaccgag gccaagacct ccgtcgtgtc ggtggagcgc 360ctgagcagca ccgtgtatcg cctggtgctg caggccctgg acgccgaggg catgcccgca 420cgcttcgact tcgtcccggg acaatatgtc gagatttcga ccgccgacag cctggaaacc 480cgggcgttca gtctcgcgaa cctcccgaat gacgccggct tgcttgagtt cctgatccgg 540ctggtgccgg gcggctacta cgctgcgtac ctggagcagc gggctgcggc tggccagacc 600atcaacgtga agggtccgtt tggcgagttc gtcctgcgcg agcatgagct cgtcgaagat 660ttcacccttc cggcggacag cccggcgcgc ggtggcacga tcgccttcct ggcgggctcg 720accggcctcg cccctctcgc gtcgatgctc cgcgagcttg gccgccgcgg attcaatggc 780gagtgccatc tcttcttcgg catgcaagac accgccacca tgttctatga gaaagaactc 840cgcgacatca agcgcacgct ccccggtctg accctgcatc tcgccctgat ggttcccagt 900gcggagtggg agggctaccg gggcaatgcc gtcgcggcct tcaaggagca cttcgcggcc 960tcctcgcaga ttcccgagaa cgtctatctg tgcggcccgg gcccaatgat cgcagcggcc 1020ctgggggcgt gccgcgagct gggtatcccg gacaacaggg tccatcgcga agagttcgtg 1080gcgtccggtg gc 10927504DNAArtificial SequenceCodon optimized Thauera butanivorans butane monooxygenase hydroxylase BMOH gamma sequence 7atgagcaaac aagtttggta taacacgccc gtgcgcgatg agtggattga gaaaataacg 60gctatcagga ctgcccgcga gggcaccgac atgctggcgc gcttccgggc tgagcacacg 120ggtccggacc gcaccaccta tgacctcaag aaagaataca attggatcga gtcccggatc 180gagatgcgcg tgtcgcagct gcatgccgag gcgacggcct cggatgagga cctcctgacc 240aagaccatcg acggccgctg cgcgaaggaa gtcgccgcag agtggctcaa aaaagccgcg 300gacatcgatt gccattacga aatggaacgg ttgtgcgtgg ccttccgcaa ggcctgcaag 360ccgccgatga tgccgatcaa cttcttcgca ccggcggaga aagaacttgt cgcgaagctc 420atgaagctgc gggcgcccac ctacctcacg acctccctgg acgagctgcg cgaggcgcgg 480ggcgtgacca tgatcagcgt ccag 5048261DNAArtificial SequenceCodon optimized Thauera butanivorans butane monooxygenase hypothetical assembly protein sequence 8atgaaagaag cccccgccat ccctgatctg cccggactcc cagaaaccgt cggtgagccc 60acccttgtcc tcgaagagga cggcttccgg gtgttcgcga ccgagctgac gattatgtgg 120cgctgggaca tctacaacgg cgacgcccat gtgcacaccg gctgcgcaca gcatccggag 180tcgtgcgttg tggcggctcg gtccaagatc cgcttcctgc gcaggccgac ggtcgcgatg 240ctcctggggg gcgagggcca a 2619411DNAArtificial SequenceCodon optimized Thauera butanivorans butane monooxygenase regulatory protein sequence 9atgagcaatg ttaacgcata ccatgctgga accaatggga aagagggcca agatttcatc 60gacgacttcc tgagcgaaga gaactccgcg ctcccgacgt cggaagccgt ggtgctggcg 120ctcatgaaaa ccgaagagat cgacgccgtg gtggacgaaa tgatcaagcc gcagatggag 180gacaacccca ccatagcggt cgaggaccgg ggtggctact ggtggatcaa ggccaacggc 240aagatcgtca tcgattgcga tgaagccacc gagctgctcg gcaaaaagta caccgtctat 300gacctcctgg tcaatgtgtc gacgacggtc ggtcgcgcga tgaccctggg caatcagttc 360attatcacca acgagttgct tggcctggag actaaggtcg agtccgtgta t 411101173DNAArtificial SequenceCodon optimized Thauera butanivorans butane monooxygenase hydroxylase BMOH beta subunit sequence 10atgtccacca acatttttac ccgcggaatg gtcgatccag aaaggcaagc gtgcatccaa 60gaagtcgtcc cgaaagcgcc cctcgaaacc aagcgcgatc acatcccctt cgccaagcgc 120ggctggcgca ggctcaccga gtatgaggcc gtgatgctgc acgcgcagaa ttcgctcgac 180gccgtcccgg gctcccaaga ggtcggcgag gtcgtgcaga agtggccggg tggacgcccg 240aactatggcg tcgagagtac cgctgccctg agctccaatt ggttccattt ccgcgatccc 300agcaaacgct ggttcatgcc gtatgtgaag caaaagaacg aggagggcca gacggccgag 360cgggccatga agtcgtgggc cgagggtggc gacgcagaga tgatgaacgc cgcctggcgc 420gaacacatcc tcgctcggca ttatggcgcg ttcgtgtaca atgaatatgg cctgttctcg 480gcgcatagca ccaccgtgta tggcggcctt tcggacctga tcaagacctg gatcgccgaa 540gcggcgttcg ataagaatga cgctggccag atgatccaga tgcaacgggt gctgctctcc 600aaagtgttcc cggggtttga cgcggacttg gccgaggcga aacaggcgtg gaccgaggat 660aagtcgtgga agcctgcacg cgagttcgtc gagcacatct gggccgaaac gtacgactgg 720gtcgagcaac tgtgggcgat ccatgccgtg tacgaccata tcttcggcca gtttgtcagg 780cgcgagttct tccagcgcct gggcggcatc catggggaca ctctcacccc gttcatccag 840aatcaggccc tgacctacca tcttcaggca cgcgacggtg tgacggccct ctgcttcaag 900ttcctgatcg aggacgagcc cgtttacgcc cagcacaacc gccgctacct ccgggcgtgg 960acgggtcggt atctgcccca ggtcgggcgg gcgctgaagg ccttcctcgc gatctacaaa 1020gaggtcccgg tcaagatcga cggcgtgacg tgccgcgagg gcgtgcgcgc gagcgtggag 1080cgcgtggtcg acgactgggc agcgcggttc gcggagccga ttaatttcaa gttcaaccgg 1140gctgcgttca tagacgacgt tctgtccggc tac 1173111590DNAArtificial SequenceCodon optimized Thauera butanivorans butane monooxygenase hydroxylase BMOH alpha subunit sequence 11atgtccgcca atatggccgt gaaacaagcc ctgaaagcaa atcccgtccc gagctccgtc 60gatccccaag aggtgcataa gtggctgcaa gacttcacct gggacttcaa gggcaagacc 120gcgaagtatc ccacgaaata tgaaatggac gtcaatacgc gggagcaatt caagcttacc 180gccaaagagt atgcgcgcat ggaatccatc aaagaggaac ggcagtacgg gaccctcctg 240gacggcctgg accgcctgga cgcgggcaac aaagtccatc cgaagtgggg cgaagtgatg 300aagctcgtgt cgaacttcct ggagactgga gagtacggcg caatcgccgg cagcgcgctc 360ctctgggata cggctcagag tccggagcag cgcaacggct atctggccca ggtcatcgac 420gaaattcgcc acgtcaacca gaccgcgtat gtcaactact actatggtaa gcattactat 480gacccggctg gccacaccaa catgcgccag ctccgcgcaa ttaacccact ctatccgggc 540gtgaagcgcg cctttggcga gggcttcttg gcgggtgacg ccgtggagtc gagcatcaat 600ctccagctcg tcggtgaggc ctgcttcacg aaccccctca tcgtgagcct caccgagtgg 660gccgctgcca acggcgatga gatcacgccc acggttttcc tgtcgatcga gactgacgag 720ctgcggcaca tggcgaacgg gtaccagacc atcgtgagca tcatgaataa tcccgaaacc 780atgaagtatc tccagaccga tctggacaat gcgttctgga cccaacataa gttcctcacg 840ccgttcgtgg gcgtggccct ggagtacggc tccaagtaca aagtcgagcc ttgggcgaag 900tcgtggaacc ggtgggtcta cgaagattgg gcgggaatct ggctgggtcg cttgcaacag 960tttggcgtca agacgcccaa gtgcctgccg gacgcgaaga aagacgccgt gtgggcgcat 1020catgacctcg cgctcctcgc gcttgcgctt tggccgctca ccggcatccg catggaactg 1080ccggactcgc tggcgatgga gtggttcgag gcgaattacc ccggttggta taaccactac 1140ggcaagatct acgaagagtg gcgggccgca ggctttgagg accctaagtc cggcttctgc 1200ggtgcgctgt ggctcatgga gaggggccac ggcattttcg tcgatcatgc ctcgggattg 1260ccgttctgcc cctccctggc gaaatccagc ataaagccgc ggttcaccga gtacaacggt 1320aagcgctacg cgttcgccga gccgtatggc gagcgccagt ggctcctgga gccggagcgg 1380tatgaattcc agaatttctt cgagcaattc gagggctggg aactgtcgga cctggttaaa 1440gccgctgggg gcgtgcgcag tgacggtaag accctcatcg cgcagccgca tctcagggac 1500accgacatgt ggacgctgga cgatctgaag cgcatcaacc tcaccatccc ggacccgatg 1560aagatcctga attggcagcc agtcgcccag 159012765DNAArtificial SequenceCodon optimized Nocardioides sp. pariculate butane monooxygenase subunit A sequence 12atgaccgttg ccaccgaatc cgtcgagact ccccaacatc cacccccgac ccgcatgata 60ggccgccgct gggatatcct gctcgtcgcc agtgcgctcc tcctggtcgc cggtgcggcc 120catctgaaca atatgctgtt cgtgggtgat tggtccttct gggtcgactg gaaagaccgc 180cagtggtggc cgctgctcac gcccgcgctt agcatcatcg tgcccgcggc gctgcagtac 240atcacctgga cccaactgcg gctcccgttc ggcgccacgc tgggagccgt ggcgctggtc 300ctcgccgaat gggtgtcgcg ctatttcagc ttcgagtggt gggcgaatat cccgctgaac 360ttcacctggc cggaaaccct ggttctggct gccgtcgtgc tcgacgtgat cctcctcatc 420acccggtcgt tcttcctcac gtcgctgttc ggcggcttga tgtggggctt tgtgttctgg 480ttcttcaact ggccggcact ggcgccgttc atgcagcctg tcgagttcca cggctacatc 540gtcaccgtgg ccgacgtgat gtccttcaac atcgtccgca cgcagacgcc ggagtatctc 600cgcattatcg aggagggcag gcttcgggcg ctggtcgaga atatcaccat ggtcgtgtcc 660ttcttcgcgg gcatgttgag cgcagcggtc tactggtttg gcctcgccat tggtaagttc 720cttgccgtgg ctccggcagg gcgcttcttc cggctgggct cggac 765131254DNAArtificial SequenceCodon optimized Nocardioides sp. particulate butane monooxygenase subunit B sequence 13atgcgcctta tgagaatctc gatgaatcct gagtccaccg gtcacctcct ccgccgcctg 60ttccgccttg ccgtcggggt tctggctctc ctggtgctcc ccgtgagtcc ggcctccgcc 120catggcgagg agtcgcagca agcgttccag cgcacgagca ccgtggtgtt ctatgacgtc 180aagttcagcg atgatacggt cgacgtgggc gagtccgtca ccatcaccgg catggtccgg 240gtgatgaaat cctggccgga ccatactctg gagccgccag agatgggcta cttgacggtg 300tcgacgcccg gcccggtgtt ctacgtccaa gagcgcgaga tgtcgggcga gttcaccccg 360cagagcgtcc gcatcgaaaa gggtgcgacc tacccgttca agctcgtgat taaggccagg 420cagccgggca cttggcacgt ccatccgggc ttcggggtcg agggcgcagg caccctggtg 480ggagcgggca aagatatcac ggtcaacgac accggggttt tcgagaacac ggtgacgctt 540gctaacggca ccaccgtgga cctggaaacc ttcggcctcg gtcgcgtcgt gacctggcat 600ctgatatcgc tcgtggtggg cctggcgtgg ctcctgttct ggctgcggcg gcccatcctc 660gacagggcga tggccatttc cgagggtcgc ggtgccacgc tcatcacgcg ctcggaccgc 720cggatcggaa tcggcttcgc ggttgtggcg ctggtcgtcg gcacgggcgg atatgcgtat 780gccgagatga cgcagtccag ttcggtgccg ctgcaagtcg tcaggaccac cccggtcccg 840ctcgctgagg aagaggttag cggtgccgtc gcacccgaga tcgagagcat ccggttcaat 900gccgaagcgg acactctgac catgaagctg cgcgtggaga ataccggtgc agcggccgtg 960cgcctgcagc gcgtccagtt tggcgatgtc gagttcgtca gcccctcgtt cgcctccgca 1020gcggaccccg acgcccaggc catgacggtg acccccgacc aggccatcga gcccgggggc 1080tcggccacct tcaccgtcga gattcagtcc gaggacctga tcgtgcggag cctcgtcccg 1140gttaacgaag cggaactccg cgtcaccggc ctcctgtttt tcgaggacga aaccggcgag 1200caagtcgtgt ccgaggtcaa tgaactgacc tcggcgatct tgcaggattt ccac 125414741DNAArtificial SequenceCodon optimized Nocardioides sp. particulate butane monooxygenase subunit C sequence 14atgctcctct ggcgctggta tcaacaagca ttcgcattca ctaaaggact ggataggacc 60ctccccgaat tcaatcagtt ctggggcacc atgtttctcg tcaacatgac cgtgttgccg 120cttctggcag gggcctggta cgtgtacctg tggtccagct cgcggaagct tgcgcctccg 180gccaatggtg ccgaggaggc cggtcgcatt tggcgcctct ggctgctggt cgcgggcttc 240accgccgccg tctactgggg cggctcctac ttcgccgagc aagacgcgtc ctggcatcag 300gtgaccatgc

gggacagcgc gttcaccccc agccacgcga tcctgttcta cggcgtgttc 360ccactcatga tctatatggc gacgggtacc tatttgtatg cgcgcacccg cctgccgcat 420ctgtacgggg gcaaggccat cccggtgtcc ttcgcgctca tgatcggcgg ctcgtcgctg 480ctggtgttcc aggtcgccat gaacgagttt ggccattcgt tctgggaagc cgaggaactc 540ttcagtgcct cgctgcactg gccgttcgtc atcttcggct atctcctggc ggccacgttc 600tcggtttggt tcgaaaccac cccgcggctg ttcgcgatag cgcgccaaga gcgcgacgcc 660ctcgtcgcgg cggagcaaca gatgaccccc gctgctcccg ctggcgagag caacacggcg 720acgacccagc cgacgagcat c 741151491DNAArtificial SequenceCodon optimized Xanthobacter autotrophicus oxygenase alpha subunit XamoA sequence 15atggcgctct tgaatcggga tgattggtat gacatagcac gcgacgttga ttggaccctt 60agctacgtgg atagggccgt ggctttccct gaggagtgga agggcgagaa agacatctgc 120ggcacggcgt gggatgactg ggacgagccg ttccgcgtga gcttccgcga atatgtcatg 180gtccagaggg ataaagaggc ctccgtgggc gcgatccgcg aggccatggt ccgcgccaag 240gcctatgaga agctcgacga cgggcataag gccacgagcc atctgcacat gggcaccatc 300accatggtcg agcacatggc tgtgacgatg cagtcccggt tcgtgcgctt cgctccgagt 360gcgcggtggc gctccctcgg cgcgttcggt atgctcgacg agactcgcca cacccagctc 420gacctccggt tctcgcatga tctgctgaat gacagcccat cgttcgactg gtcgcagagg 480gcattccata ccgacgagtg ggcagtcctg gccacccgca acctcttcga cgacatcatg 540ctgaacgccg actgcgtcga ggccgcactg gccacctcct tgacgctgga gcacggcttc 600acgaacatcc aatttgtcgc gctggcgagc gacgcgatgg aggccggtga cgtgaacttc 660agcaatctcc tttcctccat tcagaccgac gaagcgcgcc acgcgcagct cggcttcccg 720accctcgacg tcatgatgaa gcatgacccg aagcgcgctc aacagatcct ggatgtcgcc 780ttctggcgca gttatcgcat cttccaagcc gtcaccggcg tgtcgatgga ctattacacc 840cccgtcgcta agcgccagat gtcgttcaaa gagtttatgc tcgaatggat cgtgaagcat 900cacgagcgga tcctgcggga ctacggactc cagaagccgt ggtactggga caccttcgag 960aaaacgctcg accacggcca tcatgcgctg catatcggta cctggttctg gcgcccgacc 1020ttgttctggg acccgaatgg cggggtcagc cgcgaggaac ggcgctggct gaatcagaag 1080tatccgaatt gggaagagtc gtggggcgtc ctgtgggatg aaatcatttc gaacatcaac 1140gccggcaaca tcgagaaaac cctgccggaa accctgccga tgctctgcaa tgttaccaac 1200ctccccatcg gctcgcattg ggatcgcttt cacctcaagc ccgagcaact tgtgtacaag 1260ggtcgcctgt ataccttcga ctccgacgtg tcgaagtgga tcttcgagct ggaccccgag 1320cggtacgcgg gtcataccaa cgtcgtcgac cggtttatcg ggggacagat ccagccgatg 1380acgatcgaag gcgtgctgaa ttggatgggc ctgacgcccg aggtcatggg caaagacgtg 1440ttcaactacc gctgggcggg tgactacgcg gagaaccgga ttgccgccga g 149116264DNAArtificial SequenceCodon optimized Xanthobacter autotrophicus oxygenase gamma subunit XamoB sequence 16atgagcctgt tccccattgt cggtcgcttt gttggagact tcgtccctca cctcgtcgct 60gttgatactt ccgatacgat cgaccagatc gcagagaaag tcgccgtgca taccgtgggc 120cgccggttgc cgccggaccc gaccgcgacg ggctacgaag tgctgctgga cggcgaaacc 180ctcgacggcg gggcgaccct tgaggccatc atgaccaagc gcgagatgct ccccctgcaa 240tggttcgacg tgcggttcaa gaag 26417366DNAArtificial SequenceCodon optimized Xanthobacter autotrophicus ferredoxin XamoC sequence 17atgaatttgc acgcccccaa tgctgaacaa gatgacatcg aatatgttga cgtctgcgca 60gtcgacgacc tctgggatgg cgagatggac gtgttcgatg tgggcgagca cgaagtgctc 120ctcgtcaagc atgagggtcg cttccatgcg tacgacggga tttgcccgca ccagtccgtg 180agcctggtcg agggccatct gaccgaggac ggcgtgctta tctgcaaggc ccatgagtgg 240cagttctcgg tcgagggcgg acagggcatc aacccggcga acgtgtgcct gcagtcgttc 300ccgctcaaag tcgagggtgg ccgggtcctg atcggcacgg aacccctgcc gaaagagggc 360gaggcg 36618303DNAArtificial SequenceCodon optimized Xanthobacter autotrophicus coupling/effector protein XamoD sequence 18atgagtaacg cgacggtgga tgatatggac gagaatttgg tgggaccggt gattagggca 60ggcgatctgg cagacgccgt gatcgacgcc gtcatcgcgg acaaccccgg caaagaggtt 120cacgtcatcg agcggggtga ctacgtccgg atccataccg accgcgactg ccggctgacc 180cgcgcttcga tcgagcaagc gctcgggcgc agcttcgtcc tcgcggccat cgaagccgaa 240atgtccagct tcaagggccg catgtcgtcg tccgactccg agatgcgctg gtactataag 300agc 303191023DNAArtificial SequenceCodon optimized Xanthobacter autotrophicus oxygenase beta subunit XamoE sequence 19atgactcaac aacgccccac ccggacccgc gaacgcaaaa agacctggac tgcatttgga 60aaccttggcc gcaagccgac cgactacgaa gttgtcaccc acaacatgaa tcacacgatg 120cgcggcacgc cgctcgaact ctcgcccacc gtgcatgcca acgtctggct gaagaaaaat 180cgcgacgaga tcgcgctgaa agtcgattcg tgggacctgt tccgcgaccc tgatcgcacc 240acgtacgaca cctatgtgaa gatgcaagat gaccaggaaa cctacgtcga caatctgctc 300ctcagctaca ccggcgaggg ccgctatgac gaggagctgt cgtcccgctc cctcgacttg 360ctgagtgccg gccttacccc gacgcgctat ctgggacatg gtctgcagat gttggccgct 420tatatccagc aactcgcccc gtcggcgtac gtcggcaatt gcgccgtgtt ccagaccagc 480gacgcgctca ggcgcgtcca gcgcgtggcg tatcggaccc gccagctcgc ggatgcccac 540ccagctcggg gtttcggctc cggggaccgg gctgtctggg agaagtcgcc ggactggcag 600ccgatccgga aggccatcga ggagcttctg gtgacgttcg agtgggacaa agcgctggcc 660ggcacgaact tcgttgtcaa gccgattctg gacgagctgt tcctcaacca tctggcacgg 720ctgctgcatg tcgagggcga cgagctggac agcctggtgc tgcggaatct ccacggggac 780gcgcagcggc acgcccgctg gacggccgca ctgggtcgct tcgcggtcga gcagaacgtg 840aacaacagga ccgtgcttcg cgacgcgatc gccggctggc atgaaaccgg cgaggcagtg 900ctcgccgcgg gcgctggcat gctcgcgagc cgggcgccct ccgccgatgc ggcgaagatc 960gcggatgagg tccgcgccac cctcgcgcag ctccatgcga atgccggtct gggccatgac 1020gcc 102320981DNAArtificial SequenceCodon optimized Xanthobacter autotrophicus reductase XamoF sequence 20atgcgcttga atgatggccg ctccttctcc tgccgctccg accagactgt tctccatgcc 60gcccttgctg ccggtatcga catgccgtac gagtgcgcgt cgggcagctg cggttcctgt 120cggtgccgcc tctcgcacgg cagcgtcagt ctgctgtggc ccgaggcccc gggcctgagc 180gcgagggacc gccaaaaggg tgaccgcatc ctcgcgtgcc aatcgacgcc cagctcggac 240cttgagataa acgtccgcgc cggcgacgcg ctcctggagc cccctccacg ccgccatgcg 300gcacgcgtga ccgtgaaaga aacgctgtgc gcgagtgtga tccgcctggt gctcaatgtc 360ggcggaccga tccattttct tccgggccag ttcttcatcc tcgatctgcc gggtgcgggc 420cggcgcgcct attcggtcgc gaacctggag aacgccgcgg gtggcatcga gctcctgatt 480aagcggaaga ttggcggagc cggcaccgct gcgttgttcg atcagtgcgc cccgggtatg 540ggcctggtga tcgaagggcc gtacgggcgg gcgtatctcc gggcggactc ggccaggggc 600atcgttgcgg tcgcgggggg cagcggcctc gcgccgatgc tgtccatctt gcgcggtgcg 660ctcgcacggg gcttcggcgg cccgatggac ctctatttcg gcgtgaacac cgccgaggaa 720ctgttctgcg tgcccgagct gtccgccctg caggccgccg gggcacgggt ccatctggcc 780ctgcgggatg gtggccccgg ccccgcgggc ctgcaccgcc aggctggcct catcggcgac 840gccctcgtcg cgggagagcc ggacctcaag gcaaaagacc tgtacgtcgc cggcccggcg 900ccgatgaccg atgacatcct ggcccggacc gtgcgccagg aagccatccc cgctgaccgc 960gtgttcttcg acaggttcgt c 981211710DNAArtificial SequenceCodon optimized Escherichia coli sulfite reductase (NADPH) hemoprotein alpha subunit sequence 21atgtccgaga agcaccccgg tccccttgtt gttgaaggca aactcactga cgccgaacgc 60atgaagcatg agtccaacta tctgcgcggt accattgcgg aagatctcaa tgacggcctg 120acgggcggat ttaagggcga taacttcctg ctcattcggt ttcatggtat gtatcagcaa 180gacgacaggg acattcgggc ggagagggcc gagcaaaagc tcgaacctcg ccacgccatg 240ctcctgcgct gccggttgcc gggcggggtc atcaccacga agcaatggca ggccatcgac 300aagttcgcgg gtgagaacac catctacggc tccatccgcc tgaccaatcg ccaaaccttc 360cagttccatg gcatcctcaa gaaaaacgtc aagcccgtgc atcagatgct ccattcggtc 420ggtctggatg ccctcgcgac ggcgaacgac atgaatcgca acgtgctctg caccagcaat 480ccctacgaga gtcagctcca cgctgaggcg tatgagtggg cgaaaaagat cagcgagcac 540ttgctcccgc gcacgcgcgc gtacgccgag atctggctcg atcaggaaaa agtcgccacc 600accgatgagg agccgatcct tggccagacc tatctgcccc gcaagttcaa gaccaccgtg 660gtgatcccac cgcagaatga catcgatctc cacgccaatg acatgaactt cgtggcgatc 720gccgagaatg gtaagctcgt cggcttcaac ctcctcgtcg gcggggggct gtcgatagag 780catggcaaca agaaaacgta cgcccgcacc gcgtccgagt tcggttatct gcccctggaa 840cataccctgg ccgtcgctga ggcagtcgtc acgacgcagc gggactgggg caaccgcacc 900gaccgcaaaa acgccaagac gaaatacacc ctggagcgcg tcggcgtgga gactttcaaa 960gccgaggtcg agaggcgcgc tgggatcaag ttcgagccga tccggccgta cgagttcacg 1020ggtcggggcg atcggatcgg ctgggtgaaa ggcatcgacg acaattggca cctgacgctg 1080ttcatcgaaa atggccgcat cttggactat ccggcacggc cgctcaagac cggactgctt 1140gagatcgcca agatccataa gggcgatttc cgcatcaccg cgaatcagaa cctgatcatt 1200gcgggcgtgc cagagtcgga gaaagcgaag atcgaaaaga ttgcaaaaga atcgggcctg 1260atgaacgccg tcaccccgca gcgcgagaat tccatggcgt gcgtgagctt cccgacgtgc 1320ccgctcgcga tggccgaggc cgagcggttc ctcccgtcgt tcatcgacaa catcgacaat 1380ctcatggcga agcacggcgt gtcggacgag catatcgtca tgcgcgtgac cggttgccct 1440aatggctgtg gtcgcgctat gctggctgag gtcggccttg tcggcaaggc cccgggccgg 1500tacaacttgc atctgggcgg caaccgcatt gggacccgca tcccgcgcat gtacaaggag 1560aacatcaccg agcccgagat cctggccagc ctggacgaac tgatcggccg gtgggccaag 1620gaacgcgagg ccggagaggg cttcggcgac ttcaccgtcc gggcgggcat aatccgcccg 1680gtcctggacc cggcgaggga cctctgggac 1710221797DNAArtificial SequenceCodon optimized Escherichia coli sulfite reductase (NADPH) flavoprotein beta subunit sequence 22atgaccacgc aagtcccgcc ctccgccctc ctgcccctga accccgagca actcgcacgc 60ctccaagccg cgaccaccga cctgacgccg acgcagctcg cgtgggtgag cggctacttc 120tggggagtgc tcaatcagca gccggcagct ctggcggcga ccccggcacc cgcagccgag 180atgccgggca tcaccattat ctcggcgtcg cagacgggta atgccaggcg cgtcgccgag 240gccctccggg acgacctcct cgccgccaaa ctgaatgtga agctcgtcaa cgctggggac 300tataagttca aacagatcgc gtccgagaag cttctcatcg tggtcacctc gacccagggc 360gagggcgagc cgcctgaaga ggctgttgcg ctgcacaagt tcctcttctc gaagaaagcc 420cccaaactgg aaaacaccgc ttttgcggtg ttctccctgg gggattcgag ctatgagttc 480ttttgccaga gcggcaaaga ctttgatagc aagcttgccg agctgggtgg ggagcgcctg 540ctggaccgcg tcgatgccga cgtggagtac caagccgcgg cgagtgagtg gcgcgccagg 600gtcgtcgacg ccctgaagtc gcgggctccg gtcgcggcgc cgtcccagtc cgtggcgacg 660ggcgcagtga atgaaatcca caccagcccg tatagcaagg acgcgcctct ggtcgcgtcc 720ttgagcgtta accaaaagat caccggccgg aactccgaga aagatgtccg gcacattgag 780atcgacctcg gcgactcggg catgcgctac cagccgggtg atgccctggg cgtctggtac 840cagaacgacc ccgcgttggt caaggaactg gtcgagctcc tgtggctcaa gggcgatgag 900cccgtgaccg tcgagggcaa gaccctcccc ctcaacgagg ccctgcagtg gcatttcgag 960ctgacggtga acactgcgaa cattgtggag aattacgcga ccctcacccg cagcgagact 1020ctcctgccgc tggtcggtga caaggccaag ctccagcatt acgcggcgac gaccccgatc 1080gtggacatgg tccgcttcag tcccgcccaa ctcgatgccg aggcgctgat caatttgctt 1140cggcccctca ccccacgcct gtactcgata gcgagctcgc aggcagaggt tgagaacgaa 1200gtgcatgtga cggtcggagt cgtccggtac gacgtcgagg gtagggctcg cgctggcgga 1260gcgagttcct tcctggctga ccgggtggaa gaggagggcg aggtgcgcgt gttcatcgag 1320cacaatgaca atttccggct cccggcgaac ccggaaaccc cggtgatcat gatcggccca 1380ggcacgggca tcgccccgtt ccgcgccttc atgcaacaac gggcagcgga cgaagcgccg 1440ggcaagaact ggctgttctt cggcaatccg catttcaccg aagatttcct gtaccaagtg 1500gagtggcagc ggtatgtcaa ggacggcgtg ctgacccgca tcgatctggc gtggtcgcgg 1560gaccagaaag aaaaagtcta tgtccaggac aaactgcgcg agcagggtgc cgagctttgg 1620cgctggatca acgacggtgc ccatatctat gtttgcgggg acgccaatcg catggccaag 1680gacgtggaac aggccctcct tgaggtcatc gccgagttcg gcggcatgga caccgaagcc 1740gcggacgagt tcctgagcga gttgcgcgtg gagcgccgct atcagcgcga cgtctat 1797231659DNAArtificial SequenceCodon optimized Rhodobacter capsulatus sulfite reductase (NADPH) hemoprotein subunit beta sequence 23atgtatgagt atagcgattt cgatgaagct tttgttagga atagggttgc acagtttagg 60gaccaagtgg cgaggcgcct cgacggctcg ctcaccgagg aagagttccg gcctctccgc 120ctcatgaacg gcctctatct ccagctgcat gcctacatgc tccgcgtcgc catcccctac 180ggcacgctga gctccaacca gatgcgcgcc cttgccgacg tggcggaccg cttcgaccgc 240gggtacggtc atttcacgac ccggcagaac atccagttca attggatcaa gctgaccgac 300accccggata tcctggagcg gctggcggac gatggcctcc atgcgatcca gaccagcggc 360aactgcattc gcaacgtcac gaccgacgcg ttcgcgggtg cggcggcgga cgagatcgag 420gacccacgcc cgtacgccga gctcatccgg cagtggtcgt cggatcacgc agagttccaa 480ttcctcccgc gcaagttcaa gatcgcaatc acgggctcgc ccgaggaccg ggcagcgatc 540cgggcccacg acgtcggcct gcagttgatc cagcgcggtg gagagactgg cttccgcgtg 600cttgtgggtg gcggcctggg ccggaccccg atgctcgctc ccgagcttcg cgacttcctc 660ccgaaagccg acctcctccc gtatctggaa gcgatcctcg ccgcgtataa cttgattggg 720cggcgggaca ataagtacaa agcgcgcatc aagattaccg tgttcgaaac cggcatcgag 780ccgttccgcg acctggtgga gcaagagttc gagcggatac gccctcagtt caccggcgcg 840gaccaggccc tgcttgccga gatcacgccc cacttcgcgc tccccgacct ggtcgcgaaa 900gatcccgcgc cgttcgctgc ggcccaggtg accgacccgg cattcgccgc gtgggtcaag 960cactccgtga ccgaccataa acggccggat cacgccgtcg tgaccatttc cgtcaagacc 1020ccgggagagg aaccgggcga cgtgagtgcc gcgcaaatgc gcgcagtggc cgacctggcc 1080gatctgcatg ggtacggcga gctgcggatc agccacatgc agaacatcgt cctgccccat 1140gtcgctcgcg ccgacctccc ggctctccac gccgctctgc gcaaggttgg cctcgccgct 1200gcgaacgtcg gtctgatctc ggatatgatc gcgtgtcccg gcatggacta ctgcgcgctt 1260gcgaccgcca ggtcgatccc gctcgcgcaa gagatcgccc agcatttcga aaccctgggc 1320ctggtcgaaa cgataggccc gctgcccttg aagatctccg gctgcatcaa tgcctgcggc 1380catcatcatc tgggcgcgat cgggattctg ggcctggatc gcgcgggggc cgagaattac 1440cagatcaccc tgggtggcgc cgagggccca gaggcagcga tcggagagaa aatgggtccg 1500ggcttcgctt atgacgccgt ggtcccggcc atcgagcgcc tggtccgggc gtacctcacg 1560ctgcggctgt ccgagggcga gactttcctc gcggccttgc atcggctcgg tcgcgagccg 1620tttcgcgcag ccctgtatga cgaagcgcaa gacgccgcc 1659242205DNAArtificial SequenceCodon optimized Rhodobacter capsulatus sulfite reductase (NADPH) flavoprotein subunit alpha sequence 24atgctccgct tccttcaccg ctggcctggc ctcctcgctg ccctcctcgt ccttgtcctc 60gcgttgtccg gttccgccct gagcctgtat ccggccctgg agcggctcgc ggtgccgcag 120gcagagtcga cccttactgt ggccgacctc gccgctcgcg tggcctcggc gcatcccggc 180ctggagcaaa tccgcagggc accgagtgga cgcgtcgtcg cgtggtggtt cgagggtggc 240cggccggggg cggccgtcat agaccccgcc accggcgcgg accttggcag cccggacccg 300cttccgggca gccgcttcct gacggatctc caccgcgagc tgttcgcggg cgatacgggt 360cgcctggtgg cagcggctgg agccgccctg atgctggcgc tcgccatctc gggtgcctgg 420ctggttgctc ggcgcatggg cgggtggagt cgctggttcg gccgcacccg gggtccgttc 480gccggtcgcc tgcatgtcga gctcgcccgc tttgcggtcg gcggcttgat cctctcgtcc 540ctgaccgcgc tctggatgac cgcgtccatg ttctccctgc tccccgacgg tgccgccgag 600cccgctcccg ccctggcgac ggcgaccctc ccgcggttgc cgtacgacca gattccggcg 660ctggcgaaca ctccggcagc ggccctgcgc gagctgtccc tgccgagccc ggacgatccc 720accgacacgt tcaagctcgt taccgagggt ggcgcagccc tgatcgaccc cggcaccgga 780gccatgctgt cgagtgccac gccgggattc ttcgagaaag cgaccgagat catggtgatg 840ctgcacaccg ggcagggcgc gtcggccctg ggcctgctgc tcggcctcat gtcgctgtcc 900gtgccagcgc ttgcgctcac cggtgcccag cattggtggg cgggcctgcg gtcgaatcgg 960cggattcggc gcaacgccag ggcgcagctg gcggaaaccg tggtcctggt ggcctccgag 1020ggtgggacca cgtggggctt cgcgcgcacg ctgcacgacg gcctgactgc cgctgggcaa 1080aaagtccata ccgcgcctct ggcgagcttc gaccccgcca ggtatgccag ggcacgccgc 1140ttcctcattc tggccgccac gtatggcgag ggcgaggcgc cgacggcagc gaaagccgtc 1200ctcgaccgca ttgcggcgct cacctcggca ccggctgccc cgctggccat actgggcttt 1260ggggatcgca ccttcccgca attctgcggc ttcgcggagt ccctgcgcgc cgcagccgca 1320gccatcggct gggagagcct catgccgatg gccaccgtcg accgccagag cgcccaagac 1380ttcgcgcgct ggtcgaggga tctgggtgcc gtcctcggcc tcccgctgga tctcacgcat 1440ctccccgaac gcccaaagac cacggctctt accctcatca gtcggcggga ccatggcgcc 1500gaggtccagg cgcccacctc gatcctgcgc ttcgaggttc cgcaggcgac gctctggcag 1560cgcctgaccg gccagggctt tgcgcggttt gaagcgggcg acctcatcgg catcctgccc 1620aagggctcgg accttccgcg cttctactcg ctggcgagca gcgctcgcga tggcttcctg 1680gaaatctgcg tgcgcaggca cccgggtggc ctgtgctccg gccagctcac cgacctcacc 1740ccaggcgcca cggtcgcggg cttcgtgcgc cgcaaccccg ccttccggcc tcaaaagggt 1800cggaaaccgg tcatcctcat cggagccggc accggggtgg ggcccctggc aggcttcctc 1860cgcgcgaatc ggcgccaccg gccgatgcat ttgtatttcg gcgcccgcgc accgcagtcg 1920gacttgctgt acgaagccga gctgcgggat tggcaggccg cggggcaact cagccgcttg 1980accaccgcgt tctcccggca tggccccaag acctatgtgc aagacgcgct ccgcgcagac 2040gcgcccgagc tggcacggct gatcggtgct ggcgcccaga tcatggtgtg tggcggacgc 2100gacatggccg ctgcggtgcg ggacgcgctg gccgaaatcc tcgtcccgat cggccagacc 2160ccagcctccc tgaaggccga gggccgctac gctgaggacg tgtac 2205251695DNAArtificial SequenceCodon optimized Shewanella putrefaciens sulfite reductase (NADPH) beta subunit sequence 25atgagtgagc aaaaactggc cctgaatgaa tatcttaaaa ccgacagcga ctatctgagg 60ggtaccatca aggaaggcct ggacagctcg gtcaccgggt ccttctccga tggcgaccaa 120cagctgatca agttccacgg cttctaccag caagacgatc gcgacctccg caacgagcgg 180aaggagcaga aattggagcc gctctactcc ttcatgctgc gcgctcgcgt ccccggcggc 240atctgctcgc cgcagcagtg gctcggcgtc gacaaaatcg cgagtacgct tacgtcgtcg 300aactcgatcc ggctcacgac ccggcagacc ttccagtatc atggtatccc caagcgcaac 360ctcaagacca tcattcaaga tctcgaccgc caagcgctgg acagcatcgc ggcctgcggc 420gacgtcaatc gcaatgtgat gtgtaacccg aacccggtcg agtcgaagct gcacgagcaa 480gcctacgcag tggcgaaaaa gctctccgac catctcctgc cgcatacccg cgcttacgcg 540gaaatctggc tggatgagga gaagctcctg accaccgagg acgaaacggt cgagcccgtg 600tacggcaaga cctatcttcc gcgcaagttt aagatggccg tggcagtgcc acccgacaac 660gacgtcgatg tgtataccaa cgacctcggc ttcatcgccg tggccgagaa cggtgaactc 720gtgggcttca acctgaccgc gggtggcggc atgggatcca ctcacggcga ggttgaaacg 780ttccctcggc tcgcggatga ctttgggttc atcaagaccg aggacgtcat gaaattcgcc 840gaggctgtca tgaccgtgca gcgcgactgg ggcaaccggt ccaatcggaa gcgcagccgc 900cttaagtaca ccatagtcga ccatggctac gagaagttta aggcagaggt cgaggcgcgc 960gcaggcgtga agttcgagcc gaagcgcgag gtcgtgatcg gcgacagggg tgaccgctac 1020ggctgggttg agggcgtgga cggcaagtgg cacctgacgc tgttcattga gtccgggcgg 1080atcaaagatc tgccgggaca gacgctgcag acgggcctgc gcgagatcgc gaagatccat 1140aagggcgact tccgcatgac ctccaatcag aacatgatca tcgcgggtgt ggccgccgag 1200gataaggcca cgatcgaagg gctcgccagg aaacacggct tgctcggtca ggttctgacc 1260caaacccgcg

gacatagcat cgcgtgcgtc gcgctgccca cgtgccccct ggcgatggcc 1320gaggccgagc ggtacttccc cgagttcatc gaccatatcg acgcgctcca ggcgaaacat 1380ggcatcagcg agcaagccat tgtggtccgg atgaccgggt gcccgaacgg ctgcgctcgg 1440ccattcgccg cagagatcgg cctcgtgggc aaggccccgg gacggtataa tctgtatctt 1500ggtgcgtcgt tcgagggtac ccgcctgaat aagatgcatc gcgagaacat acaggaagcc 1560gacatcctcg cggaactcga taccttgttc ggccgctacg ccgtggagcg cgacgcgggc 1620gaaaccttcg gcaatttcac cgtccgggtc ggcgtcgtca aagccgtcat tgacgccgcc 1680aaagacttcc acggc 1695261797DNAArtificial SequenceCodon optimized Shewanella putrefaciens sulfite reductase (NADPH) alpha subunit sequence 26atgctcctga aagaacttag ctccctcgcc tccccgctgt cccaaggcca agtcgaaaag 60ctcaaacagc tcaccagcga gctgagcgcc gtgcagctcg cctgggtgag cggctacctc 120gccgctaccg cgaatgccgg tcagctggcc cccgtcgcgc aggcgcagac ggcgcagacc 180gtgaccatcc tctatggtag ccagacgggc aatggccggg gtgtggccaa ggcactggcg 240gacaaggccc aagcgcaggg ttatgccgtg aacctggcct ccatgggcga gtacaatgtg 300cggcagttga agcaagaggc cgtcctgctg ctcgtcgtga gcacgcatgg cgagggtgag 360gctccggacg acgcaatcga gcttcataag ttcctggcgt cgaagcgcgc gcctaagctt 420gacaatctcc actattcggt gctggccctg ggggatagct cctacgagtt cttctgccag 480accggcaaag acttcgacac ccggctggcg gcccttgggg cgaagagtct cctgccgctc 540atcgagtgcg acgtggacta tgaagctgcg gcagggcagt ggcatgccga tgtgctggaa 600gctgtcaagc cgctgatcga aacgtcctcc gcctcggtcg tgtcgatcgg cacggcgaaa 660gcgatcggcg agagtgagtt caccaagcaa aacccctatt cggccgaggt cctcgtttcg 720cagaagatca ccgggcgggg cagtgatcgc gacgtccgcc atgtcgagat tgacctgggc 780gactcgggac tgacctacca ggctggcgac gccctgggcg tgtggttttc gaataacgaa 840gctctggtcg aggaaatcct gacggcgctg tcgctgtcgg gcgatgagca agtcgtcgtg 900gagaaagagt cgttgaccct gaagcaggcc ctcgtggaca agaaagagct cacccagctg 960tatcccgggt tggtcaaggc ctgggcggag ctgtccggca gcgcagagct cctcgccctg 1020tccgaggaca aggaacaagt taggcacttc atcctcaagc atcagttcgc ggacctcgtc 1080acccagtatc cgctgtcgaa taacagcgtg acgctcaatg ccgcgaagtt gcttgagctg 1140ctcaggccac tcacgcctcg cctctacagc atagcgagct cccaatccga ggtcgaaacc 1200gaggtgcatc tcaccgttgc cctggtggaa gatgagcgcc atggcgctgc gcgcttcggg 1260ggagcgagcc acttcctcgc gtccgcgcag gaaggcaccc aggtcaaggt ctacgtggag 1320cccaacaagc acttccggct gccggagaac ccggagactc cggtcattat gatcggtccc 1380ggcacgggcg tcgcgccgtt ccgcgccttc atgcaagagc gcgtggccca gggaatccag 1440ggtgactcct ggttgttctt cggcaatccg catttcgagc aagactttct gtaccagacc 1500gagtggcaac agtacctgaa aaacggcgac ctgtcgcgga tcgacgtggc cttctcgcgc 1560gatcaggcgc ataagattta cgtccagcac cgcatcaagg accagggcca ggccctgtgg 1620cagtggctcc aaaacggcgc ccacatctac atctgcggcg acgcagagcg gatggcaaaa 1680gacgtgcacc aggcactgat cgaggtcgcg gttgaggttg gcggcctgaa caccgaagcg 1740gccgaggcgt acttcgagac tctccgctcc gataagcgct accagaaaga tgtctat 1797271815DNAArtificial SequenceCodon optimized Bacillus subtilis sulfite reductase [NADPH] flavoprotein, alpha-component sequence 27atgcagctcc aagtgatgaa ctcccccttc aaccaagaac aagccgagct cctgaatagg 60ctcctcccga cccttaccga gagccagaaa atctggctgt ccggctatct gtcggcgcag 120agcgtgagtg cgcaagaggc cgcgggcacg cccgcggcgg ccgtgagcgc cgaggcaccc 180gctcccgccg tgtcgaagga agtcactgtc ctttacggct cgcaaaccgg caacgcacaa 240gggctggcag agaacgccgg aaagcagctt gagcagtccg gtttccaggt caccgtttcg 300tccatgagcg acttcaagcc caaccagctg aaaaaagtca ccaatttgct gatcgtcgtg 360tcgacccacg gtgagggcga gccgcctgac aatgccctct ccttccatga gttcctgcat 420ggtcgcaggg ctccgaaact ggaagatctc cggttctccg tcctggcgct cggagactcg 480tcctatgagt tcttttgcca aaccggcaaa gagttcgacc agaggcttga ggagctgggc 540ggcaagcgca ttagtccgcg ggtggattgc gacctcgact atgacgagcc ggcggcggag 600tggctggaag gcgtcctgaa gggcctgaat gaggccggtg ggggctccgc cgctcccgcg 660ccggccgcgg ccagccagac cggcgagtcc agctatagtc gcacgaaccc gtttcgggcc 720gaggtcctgg agaacctcaa cttgaatggg cgcggcagca ataaagaaac gcgccatgtc 780gagctctccc tcgaaggctc gggcctgacc tatgagcctg gcgactcgtt gggcgtctat 840cccgagaacg acccagagct cgttgagctc ctgctgaagg agatgaactg ggatccggaa 900gagatcgtga ccctgaataa acagggcgat gtccgcccct tgaaagaggc actcatctcc 960cattacgaga tcaccgtgct taccaagccg ctcctggagc aagcggcgca gctcacgggc 1020aacgacgaac tccgcgagct cctcgcccca ggcaatgagg aaaacgtgaa ggcctacatc 1080gagggtcgcg atctcctgga cctcgtgcgg gactacggcc cgttctcggt ttcggcgcaa 1140gaattcgtgt cgatcctccg gaagatgccg gcccgcctgt actcgatcgc gagcagcctc 1200tcggcgaacc ccgatgaggt ccacctgacc attggagcgg tccgctatga cgcccatggc 1260cgggagcgca agggcgtgtg ctccatcctg tgcgctgagc gcctccagcc gggtgatacg 1320ctgccggtct acgtgcagca taatcagaat ttcaagctgc cgaaggaccc ggaaacgccg 1380ataatcatgg tcggcccggg caccggtgtg gcgccgttcc gctcgttcat gcaagagcgc 1440gaggaaacgg gcgctgaggg taaggcctgg atgttcttcg gcgaccagca cttcgtcacc 1500gacttcctgt accagaccga gtggcagaac tggctcaagg acggcgtgct gactaaaatg 1560gacgtcgcct tcagccgcga caccgaagaa aaagtctacg tccagcatcg gatgctggag 1620cacagcgcgg aactgttcga gtggttgcag gagggcgccg cggtgtacat ctgtggtgac 1680gagaagcaca tggcccatga tgttcacaac acgctccttg agatcatcga gaaagagggc 1740aatatgtcgc gcgaggaggc cgaggcgtat ctggcagata tgcagcagca gaaacggtac 1800cagcgcgacg tgtac 1815281713DNAArtificial SequenceCodon optimized Bacillus subtilis sulfite reductase (NADPH) hemoprotein, beta-component sequence 28atggtgacca aaattctcaa agctcccgat ggctccccct ccgacgttga acgcatcaaa 60aaagaatcgg attatctgcg gggtacgctc aaagaggtga tgctcgaccg catcagtgcc 120ggcattccgg acgacgacaa ccgcctgatg aagcatcacg gatcgtactt gcaggacgac 180cgcgacctcc gcaacgagcg ccagaagcaa aagcttgagc cggcgtatca gttcatgctg 240cgcgtgcgga tgcctggcgg ggtgtcgacc ccggagcagt ggctcgtcat ggacgatctc 300agccagaaat acgggaatgg cactctgaag ctgaccaccc gggagacttt ccagatgcac 360ggcatcctga agtggaatat gaaaaagacc attcagacca tccactcggc actgctcgac 420accatcgcgg cgtgcggcga cgtcaaccgc aacgtgatgt gtgcgtccaa cccctatcag 480agtgagatcc attccgaggt gtacgagtgg agcaaaaagc ttagcgacga cctcctcccg 540cgcacccggg cgtatcacga aatctggctc gacgaggagc gcgtggcagg caccccggaa 600gaagaggtgg agcccatgta cggcccgctg tatctccccc gcaagttcaa gattggcatc 660gccgtcccgc cgtccaacga catcgacgtg ttcagccaag acctgggctt catcgcgatc 720gtcgaggatg gcaagctgat cggcttcaat gtcgcgatcg gtggcggcat gggcatgacg 780catggcgaca cggcgacgta tccgcagctg gccaaggtta tcggtttctg caggccagag 840caaatgtacg atgtcgcgga aaagaccatc accatccaac gcgactatgg aaatcgcagc 900gtgcggaaga acgcgcgctt caagtatacg gtcgatcggc tcggcttgga gaatgtcaag 960gaagaattgg agaacaggct gggctggtcg cttgaggaag ccaagccgta ccatttcgac 1020cataatggcg atcgctacgg gtgggtcgag ggcatagagg acaaatggca tttcacgctg 1080ttcgtcgagg gtggccggat cacggattac gatgactaca agctgatgac gggtctgcgc 1140gagatcgcca aggtccatac cggcgagttc cggctgaccg cgaaccagaa cctcatgatt 1200gcgaatgtgt cctcggacaa gaaagaagag atcagcgccc tgatcgagca gtatgggctc 1260accgatggca agcactattc cgccctccgc cggtccagca tggcatgcgt cgcgctcccg 1320acgtgcggcc tggcgatggc cgaggccgag cggtacctcc ccacgctgct cgacaagatc 1380gaagagatca tcgacgagaa cggcctgcgc gaccaagaga tcaccattcg catgaccggg 1440tgcccgaacg gctgcgccag gcatgccctg ggagagatcg gctttatcgg caaggctccg 1500ggcaagtaca atatgtatct cggagcagcg ttcgacggct cgcgcctgtc gaaaatgtac 1560cgggagaaca taggtgaggc cgacatcctg tccgagctcc gcatccttct gtcgcggtac 1620gcgaaggagc gcgaggaggg cgagcatttc ggcgacttcg tcatccgggc tggtatcatt 1680aaagccacca ccgatgggac caattttcat gac 1713291764DNAArtificial SequenceCodon optimized Acidithiobacillus ferrooxidans sulfite reductase (NADPH) flavoprotein subunit alpha sequence 29atggaactca ttcgccaatc cgactttctc ctggacccac gcaaacaaga agatctccgc 60cgctttgccg agggcatgac gcgcgagcaa cttctctggt ccagcggcta cctcaccggc 120ttcggggagt ccgctccggc gtccaagatc caggaggaca taggcgagaa aatcaccatc 180ctgttcggca cggaaacggg aaatgcgaag cgcctggccg agctgctcgc ggcgcgggcg 240caggccatgg gcgtgcagac cagcatccaa gacatgctca cctacggccg ggcgcagctg 300aggcgcgacc gcgtcatcgt gcttatcgtg agcacccacg gcgacggcga gccaccggac 360agcgcccgga tgctgctggc gagcctgacc gatggccccg tccccgacct ccatggctcc 420cggttcgcca tcctcgcgct gggcgacgcg tcctacccga agttctgcca ggccggcaaa 480gcgttcgaca tcgcccttgc gtcggctggc gccgagcggc tgttgcctcg ggtggactgc 540gacgtcgact atgagcgcga cgccatgtat tggatggagc aagtcctcgg tgcactgacg 600accggcaagt cgtcccctgc cgtccccttc cccgctccgg tgcccaaaca gggttacagc 660agccacgcga cctttcccgc ggtcctgttg ggtaaggtca acctgagtgg acgcggcagt 720gaccgcgagg tgtggcatct ggaactggat ctcgacggct cgggcctcca ctatgctccg 780ggggacatag tttccgtggc cccctcgaat ccgccgcaac tggtcgaaga actcctggat 840cgcctggagc tcgatcacaa ggcatcggtt cgcacccgcc agggcgagat gccgctggtc 900gaggccctgg ctgcgcatta cgagattact cgcattacgt ggccgttcct ggaaaggtac 960gcacggctga gcgacgccaa ggcactgcag agcgccatcg cgggccgcga cgttaacggc 1020ctggacacct ggactgacgg gcgcgaagtg atcgacattg tgggccagta cccggtgaag 1080ggcctcagtg cgcagtcgtt cgcggactgc cttcggccgc tcccgccccg ccgctattcg 1140atcgcatcgt cgctcctcgc cgtcccggga gaggtccatc tcaccgtcgc ggccatccgc 1200tattcctccc atggccgcga gcggctgggc gtggcctcga cgttcctcgc agaccgcgtc 1260gccatcggca ggcccgtccc gatcttcatc gagcccaacg ccgagttccg gctcccggaa 1320gatagcggac aggccatgat catgatcggt gcgggcaccg gggtcgcgcc gttccggtcg 1380ttcctgcaag agcgcgaggc actcggggca gctggcaaca attggctgtt cttcggcgac 1440cggcatttca gcaccgattt cctctaccag cgcgagtggc tgcggtggct gcgcgatggc 1500aggctgacgc gcctcgatgt ggcgttctcg cgggaccaag agcggaagat ctacgtgcag 1560gatcgcctgc gggagagggc cggcgacgtg ttcgcctggc ttgaggaggg tgcggccgtt 1620tacgtctgcg gtgccgaggc catgggtcgc gcggtccatc agtccctggt ggacatcgtc 1680cagtccgcgg gtcgcaccca ggtccaagcc gaggaataca tcctggagtt gaaacagacg 1740ggccggtatc accgcgacgt gtat 1764301689DNAArtificial SequenceCodon optimized Acidithiobacillus ferrooxidans sulfite reductase (NADPH) hemoprotein subunit beta sequence 30atgagcataa acgacaaggc acttagcgat gtggagagga taaaagcgga aagtcaagga 60ttgcggggca ccctgcgcga gtcgctccac aacccggtca cgggtgccct ggccgaagat 120gacgtccagg tcatcaagtt ccatggcatc tatcaacagg actatcgcga cctccgggcg 180gagcgccacc aacagaaact ggagcccctc taccagttca tggcccggct ccgcctgccg 240ggtggcgtcc tgagcggagc gcagtggctg gcgttgggcg atatcgcgcg cacctatgga 300aatgcctccc ttcggatcac ctcgcggcag agcatccagt tccacggcct gctgaaaccg 360catctccggc cggtgctgca ggccctcgac cgcgctctcc tcgacaccgt gtccgcctgc 420ggcgacgtca accggaatgt catcgcgtcg tccgcacccc agatcagtgc cttccacgcc 480gaggcgtatg gctgggccca aaagatcgca gagcatctgc tgccgcagag ccatgcatac 540catgagatct ggctcggcgg ccaacagatt accgcgcctg aggaagatct tctgtacggc 600tcgacctatc tgccgcgcaa attcaagatc gcgatcgccg ttcccccgca caatgacgtc 660gacgtcctga cccaggacct cggcttcatt gcgatccacg aagagggtcg cctcgccggc 720ttcaatgtct gcgtcggcgg cggcctcggc cgctcccata acaagcctga cacctattcg 780cgcctggccg atgtgtgcgg gttctgtgcg ccgggtcaag tgctggcgat cgccgaggct 840gtgctgatca cgcagcgcga ccacggcgac cgcagcaacc gctcccatgc gaggctcaag 900tataccgtgg accggatggg actcgaccgc ttcatggaag aggtgcagca gcgcacgggc 960ttctcgctgg cgccgccacg ccccttccat tttgaaacct ccggcgaccg cttcggctgg 1020ctggagaacg atgacggcac cgcgtgcctc accctgttca ttccgggggg gcgggtggcg 1080gacggcgata tcccgctgct ctcgggtctg gacgcgctcg cgaggctgca tcatggcgag 1140atccgcctca cgtgcaacca gaatctgctt atcgcgggca tctccccagc cgagaggccc 1200gtggttgaga ctgtcctggc cgagtacggc ctcaaccggc tcctgaacct cgcgcccgtg 1260cgcgcccatg cgatggcctg cgtcgccctc ccgacgtgcc ccctcgcgat ggcagaggcc 1320gagcgctact tgccggtgtt tctcgaccgg attgaagccc tcctcgccga ggtcggcctt 1380gagggcgagg ccctgaccgt gcggatgacg ggttgcccga atggctgcgc tcgcccgtac 1440ctggccgaga tcgggctcgt gggcaaggct cccggcctgt acgacctgta cctgggtggc 1500gaccgcaccg gcatgcgcct gaacgcgctc taccgcgagg cacttgacga agaggccctg 1560ctggatgcgc tgcggccctt gttgaagcgc ttcgcagggc agcggtgggc gggtgaaacc 1620ttcggcgact tcgtcaggcg ccaagacctc ctgcccccgg acccgggtct gccgcacacg 1680ggtcgccgg 1689312037DNAArtificial SequenceCodon optimized Cyanidioschyzon merolae sulfite reductase, ferredoxin dependent sequence 31atgatgtttg ttacctacgc taaaccactt gtcggagccc gccgcggact tgcacctacc 60ggaagcgctg cccccggcgt gtaccccctc accgaagtgc tgctgcgcga taggctccgg 120cggcagcgcc agtgccgcac cgcccgccgc aacatcatcg cgaatcttag cagtgagcag 180agccgcaaga aacataccgt cgtgccgatt accacccgga agcatatcga agaggccatc 240cgcgacggca ccttggacca actgaagctt aacccctacg agctgcccaa gctgaactcg 300gactatctcc gccacccgct gatggaagaa ctgggcaacg accaaatctt catttccgac 360gactgcatcg gcctgatcaa gttccacggc ggctatctgc aagacaatcg cgaccagcgg 420gtccgcggtg agcttaaaaa gtatcagttc atgctgaggc tgaaaatgcc cgcaggcgag 480tgccctccgt cgctctatac gaccctcgac gacatctcgg agacttatgg caataagacg 540ctgcgcctca ccacccgctc gtcgttccaa attcatggca tccacaagag caacctcaag 600accgtcgtgc agtccatcgt gcgggccggg ggcggcctct acggcgcgtc cggcgactgc 660tcgcgcaatg tcattgcgcc cccggctccg ttcgtcgacg ccgcatacgc ccaggccagg 720catgtcgcgc ggatggtggc cgagctgttc gccatccaga gccatgcctt cgctgacctc 780tggctggacg gtgagctggc ggcctccatc gagtactgga agaaagagtt ggatatggac 840gaggttcgcc ggctgatgac cgaggacaat ggtcgcggcc aggtcctgca agactcggtg 900gagccgctgt acggcaagct ctatttgccg cgcaagttca aggttggcgt cacggtgccg 960ggcgacaact cgatagacat atacacgcac gacatcggca tcgtcgtgtt ctgcgacgcc 1020cagggccagc tcgaaggcgc gaatatcctc gtcgggggcg gcatgggccg gacgcacaac 1080aaagaggaga ctttcgcgcg ggcggcggac ccgctgggct atgtgccggc ggcggccctg 1140tatgataccc tgaaggccat cctcgctgcc cagcgcgacc acgggaaccg ggcggtccgg 1200accaacgccc gcatgaagta tctcgtccat cgcctgggca tcgaccgctt tcgcgaactc 1260gtgaagtcgt acatggtcgg cggcggctcg gccctggaga gcattcggtc catgccgccg 1320tggaccttcc aggactacct cggctggcgc gagcagggcg atgggcgctg gttcttcggc 1380ctctatgtcc agaatggcag gattaaagac gagctcaaaa aagcgctgcg cgcgttgacc 1440gaccggttca acttcccgct ggtgtgcacg ccccagcaga atctgctgat tacccaggtg 1500cccgcaaccg ctcgcccgga tgtggaaacg ctcctggcct cgtttggtgt ggaaacggcc 1560gccagcgcgc tcgatccgtt gatgcgcgac gcgatggcct gcccggctct cccgctgtgt 1620ccgccagcaa tcaccgaggc agagcgcgtc atgccccggt atgtgcagcg cgtgcgggag 1680ctcctctcca aggtcggcat cagtccgcat gcgtccttcg tcatgcggat gaccgggtgc 1740ccaaatggtt gcacccgccc gtacatggcc gaactgggct tcgtgggcag tggcccgaat 1800tgcacgtacc aagtctggct gggtgggtcg cctatgcaga cgcgcctcgc gtggccgtac 1860atcgataggg tcaccgatga tcaagtggag cgcgtccttg agcccgtttt cgtgttctgg 1920aaaagcgcgc gcgagccgga cgagtcgttc ggcgacttct gcgaccgggt gggaaaggcc 1980cagcttgagg cgtatgcgca acggtattgg gatggtgtcc ccgctgcgcc tgtgtcg 2037321986DNAArtificial SequenceCodon optimized Oscillatoria nigro-viridis sulfite reductase (ferredoxin) sequence 32atgatcacct cctccactag caccccggtt gcccgcaaac ccagcaaaag cgaaggcctg 60aaagagcgct ccaactacct ccgggagcca gtcgccaccg agctcctgca agaaaccacg 120cacttcaccg aggacggcat ccagatcctg aagttccatg gctcctacca gcaagacaac 180cgggacaatc gcgtgaaggg ccaagagaaa gactaccagt tcatgctgcg gacccgcaat 240ccgggcggat tcacgccgcc tcagctgtat ctggccctgg ataagctgtc cgaggaatat 300ggtaaccaca ccatccgcgt gaccacccgc cagggcttcc agctccatgg cgtgctgaaa 360aagaacttga aggccgtgtt cagttcgatc atcaagaaca tgggctcgac gcttggggcg 420tgcggcgacc tgaaccggaa cgtcatggcg ccgccggctc cctacaagaa taggcccgag 480tacaaatacg cactgcagta tgcgaataat gtcgcggact tgctgacgcc ccaaacggga 540gcgtattacg agatctggct ggatggcgag aaagccatca gcgcagagga ggacccagca 600gtgaaagctg cgcgccagaa gaatggcaat gggaccatct tttcggataa agaggagccg 660atctacggca gccattacat gccccgcaag ttcaagtgct cggtgaccgt cccgggagac 720aacagcatcg acctgtacag tcaagacctc tcgttggtcg tgattaccaa caaagctggc 780gagctccagg gttttgacgt gttcgcgggt ggcggcctcg gtcgcaccca caataaagag 840gaaacctttg cgcgcgtggc cgatgaaatt tgctacgtcg cgaaggatga cgtttacgac 900ctcgtgaagg ccatcgtggc gacgcagcgg gactatggtg accgcacgga ccgcaggcat 960gctcgcctca agtacctgat caacgacaag ggcgtgcagt ggttccgcga gaaagtcgcg 1020gagtatttcg gcaagccact cgaagcgttc aagcccctgc cggagtggaa gtatttcgac 1080ttccttggct ggcatgacca gggtgatgga aagctgttcg tcggtatctc ggtggataat 1140ggccggatta aggacgaggg ctccttccag ctgaaaaccg cgctccgcga gatcgtgcag 1200aagtataacc tcccggtgct cgccaccccg catcaaaacg tcctcattta cgatatttcc 1260ccggacctca agcaagagat ccagggcatc ctcgaccgct gcggtatcca gcgcgaaacc 1320gccatcgacc cgctcgtgcg ctatgccatg gcgtgtccgg ccatgcccac gtgcggcctc 1380gccataaccg agtccgagcg cgtcatccct tcgatcctgg agcgcatccg ggctctcctg 1440accaaggtcg gccttgaaga tgagcatctg gtcgtccgga tgaccgggtg cccgaacggc 1500tgcgcgaggc cctatatggc cgagctgggc ttcgtcgggt ccagcccgga gtcgtatcag 1560atatggctgg gcggcagccc ggaccagacg cggctggcga agccgatcga ggaaaagctc 1620cacgtcaaga atttcgaggc cttcctggag cccatcttcg tctacttcaa gcaaaagcgc 1680caactgagcg agagcttcgg gaatttctgc gaccgggtgg gcttggagtc catccgccag 1740ttcgtgacga actaccaatc ggccgacagc atgaccaccg agattaacga acttgaggtc 1800acgtcgtcga acggcgaaga gaacgaaacc gcgactgcgg gcggcggcaa ggtccggcgc 1860aggatcagcg ttcgggacga gatctataac gagctcaagg aagaggccgc acgccagggc 1920aagcccatca cccagctggc caccgaggcc atctcgacgt atctgaagaa aatcaaagag 1980gaggcg 1986332007DNAArtificial SequenceCodon optimized Pseudomonas putida sulfite reductase (ferredoxin) sequence 33atgaatgatt gtcacttgat atgcgcgaat aggttggatg atggagcagt ggtgtggttg 60gatgcgggac atgaatgggt ggagactctc cagcaggccg gcaccttcga cgcccaggca 120ctggtgagcg cgacgctggc ggcagaggct gcggtcctgg cgaaccaagt ggtcgcgccg 180accccgtgcg aggcatggct cgtggacggt cgccccgagc ccaagtccct gcgcgagcgg 240cttcgggccc ggggtcccag cgtccggtcg gaccttggga aacaagcggc tggcacccca 300ccgtcgtcca ttgcccgcat gcgcccagtc ctccccgtcg aggccggtca ggctggcgtg 360taccgctacg accgcttcga gagggaattc ctgaaagacc gggctcgcca gttcgagcag 420caagtggcgc gccgcctctc cggcgagctg gacgaggaag cgttcaaggt ctaccggctt 480atgaacggcc tgtatctgca gctgcatggc tacatgctgc gcgtggcgat cccgtacggc 540accctgagtg cgctccaact gcggcagctc gcgtacgtgg cgcacaccta tgacaagggc 600tacggccatc tgaccacccg

ccagaatatt cagttcaact ggccgaggct ggcggacacg 660cccgagatcc tcagcgtgct ggcggacgct gacttgcatt gcatccagac cagcggtaac 720tgcatccgca atgtcacgac cgaccatttc gccggggcgg ccgaggacga ggtcctggat 780ccgcgcgtcc atgcggaaat cctccgccaa tggtcgactg agcacccgga gtttacgtat 840ctcccgcgca aattcaagat tgcgatcacc ggctccccga aagatcgggc cgccgtccgc 900ttccatgaca ttggcatcct cgcgcagcgc aatgcgcagg gcgaggtcgg cttccaggtg 960tatgccggtg gcgggctggg ccgcaccccc atcgttggca cccgcgtgcg ggagtggctg 1020ccggagcgcg agctcctgcg ctatgtggaa gcgatcctcc gcgtgtacaa tgcgctgggt 1080cgccgcgaca acctgtacaa ggccaggatc aagatcctcg ttcgggagct taagcctggc 1140cgcttcatcg agatgatcga agaagagttc gcctcgctgc ccgcggatca ccaatatctc 1200gaaccggcca tcgtccaagg gatccatgcc cgcttcgtcc agccggcgtt cgaggcgctg 1260ccgggcctct gcgacagctt tctgagggca cgggccgatg acaacgcgtt cgcgagttgg 1320gtgcgcacga atacgcatcc ccacaaaaag cgcgggtata tctgcgcggt gatttccctc 1380aagccgcctg gcggcatacc gggcgacatc agcgccgagg aaatgctggc gcttgcagat 1440ctcgccgagg cctactcgct gaacgagatc cgcgtgtccc atgagcaaaa cgtcgtcctg 1500ccgcacattc ggctcgtcga tctctactcg gtgtggcagg ccctccggca ggccggactg 1560gcgacgtcga atatcgggct gctgtcggac acgatcgcct gcccaggcat ggactattgc 1620tcgcttgcca ccgctaggtc cgtgcccgtc gcgcagcgga tcgcccagcg gttcgacgcg 1680gctcggcagc aagacatcgg cgagctcaag ctcaacgtca gcggttgcat caacgcatgc 1740gcccaccacc atgtcgccca tatcggcatc ctgggcctgg acaaggccgg ccatgagaac 1800taccaaatca ccctgggcgg cagcgcagag gaggacgccg ctgtcgggac cattctcggc 1860cggtccgtcc cgttcgaaga ggttccggac atcgtcgagg ccatcgtggc tatctatctc 1920cagctgcgcg aggacgatga gcgctttctc gacacctatc gccgcgtcgg tatcgagccg 1980ttcaaagaag tcctccgcga cgcgcgg 2007341911DNAArtificial SequenceCodon optimized Anabaena cylindrica sulfite reductase (ferredoxin) sequence 34atggttaact ccgcaccctc ccccgtcagc aatcgcaaac cgtccaaagt cgaaggcatc 60aaagagaaca gcaacttcct gcgggagccc gtggccactg agatactcca ggacaccacg 120cacttttccg aggacgcgat ccagatcctg aagttccatg gttcgtacca acaggacaat 180cgggacaaca gggcgaaggg ccaggaaaaa gactaccagt tcatgttgcg gacgaagaac 240cccggtggcc tggtgcctcc gcagctgtac ttggcccttg ataagctcgc cgacgagtat 300ggtaatcaca cgctccgggc gaccacccgc cagggcttcc aagtgcacgg catcctgaag 360aaaaacctga aaagcgccat cgccacgatc gtccagaacc tgggctcgac cctcggagcc 420tgcggggaca tcaaccgcaa cgtcatggct ccgccagcgc cgctcaagaa tcggccggag 480tacgagtatg cttgggagta tgctcagaac attgccgatc tgctctcccc gcagactggc 540gcatactatg agatctggct ggacggcgag aaggccatct cggtcgagga acatcccgac 600gtcaaagccg cacgccagtc gaatggtaac gggacgattg tccatgacag cgtggagccg 660atctacggca cccattacat gccgcgcaag ttcaagatct gcgtgaccgt gcccggtgat 720aactcggtcg atctgtattc ccaagacttg acgcttgtgg tgatcaccaa taagaagggc 780gagctgcagg gcttcgacgt gttcgccggt ggcggactgg gcaggaccca taacaaagag 840gaaacgttcg cgcgggtggc ggacccgatc tgctacgtcg ggaaggacga cgtgtacaat 900ttcgtgaagg ccgtcgtcgc cacccagcgg gattacggcg accggaccga tcgccggcac 960gctcgcctga agtacttgat caacgactgg ggcgtcgaca agttccgcac ccaggtcgag 1020gagtatttcg gcaaaagtgt cgagcctttc aagcccctcc cgaagtttaa gtatcaagac 1080ttcctgggct ggaatgaaca gggcgatggg aaactgttcc tcggaatcag catcgagaat 1140ggcagggtca aggacgaggg cgcgttccag ctcaagaccg cgctgcgcga gatcgttgaa 1200aagttcaacc tcccgatacg cctcaccggc aaccagaatc tgcttttcta tgaaatcgac 1260cccgaggaca aggccgcgat tcaagaaatc ctggaccgct gcggcgttgt cgcggatccg 1320tcccaaattg ctgccctcac ccgcttcgcg atggcgtgcc cagcgctgcc gacgtgcggc 1380ctcgcgatca ccgagtcgga gcgcgcgatc ccgggcatcc tggatcggat cagggcgctc 1440ctcgacaagc tcggcctgca aaaggaccat ttcgtggtgc ggatgacggg ctgccctaat 1500ggttgtgcac gcccgtacat ggccgagctg ggttttgtgg ggagtgcgcc ggagtcctat 1560caagtctggc tgggcggcag cccggatcag acccgcctcg cccagcccat catcgagaag 1620ctccacgaca acgatatcga gagcttcctt gagccgatct tcatctattt caagaaattc 1680cgcaagggca aagagagctt cggcgacttc tgcgaccgca tgggctttga cgccattcgc 1740gagttctcgg cgacctacac gcccggtgag ccgacctcgt cgggcaaatc gcgccatcgc 1800gtgtcccttc gcgacgacgt ctatctgcat ctgaaggaaa ccgccgagaa gcaaaaccgg 1860cccatgaccg atctcgtcca cgacgcgctc gacaagtact tccagaatct c 1911352001DNAArtificial SequenceCodon optimized Halothece sp. sulfite reductase (ferredoxin) sequence 35atgatggaac tgataaccgt tataccctcc gaattgtccg ttatccagaa gtgcgctgcc 60attgagcaaa aagctgtgat ggtcgcgagc aaggccaaaa aggcatcgaa gccgagcaag 120ctcgaaggca tcaaggagaa cagcaacttc ctgcgcgagc ccctggccac cgagctcctg 180gaagatacca cgcacttctc ccaggacgcc gtgcagatcc tcaaattcca tggctcgtat 240caacaggata accgggataa tcgccaaaag ggccaagaga aagactacca gttcatgctg 300aggactcgca atccgggtgg cttcatcccg ccagagctgt atctcaccct ggacgatctg 360tccagtgagt atggcaatga aaccctccgg gtgaccaccc gccagggatt ccagctccat 420ggcatcctga agaaaaatct gaaagaaacg atcaaccgga tcgttcgcaa cctcgggtcg 480accctgggcg cgtgcggtga cctgaaccgc aatgtcatgg cgccaccggc accgttcaag 540gaccggaaag aatatcaata cgcctggcaa tatgcggaca atatcgcgga ccttctccgc 600ccgcagaccg aggcgtatta tgagatctgg ctcgacggcg agaaattcct ctccgtcgag 660gaagcgcccg aggtccaagc tgcccgcgag aggaacggga acggcacgat tttccacgag 720ggtgaggagc ccatctacgg caagtactac atgccgcgca agttcaagtg ctgcgtcacc 780gtcccgggcg acaactcgat cgacgtgtac acgcacgacg tgtccctgat cgtgatcacc 840gacgaccagg gcgagctcaa gggcttcaat gtccttgcgg ggggcggcat gggtcggacc 900cataacaaag aagagacttt cgcccggatg agtgatccga tctgctacgt ggacaaggca 960gatgtctacg atttgctcaa ggccattgtg gcaacccagc gcgactacgg cgaccgggtc 1020caacgccgcc atgcccgcat gaaatacttg ctgtacgact ggggcgtcga gaagtttcag 1080tcgaagcttg aagagtacta tggcaaaccg ctgcagccgt atcaggacct cccgcctttt 1140gagtataaag acttccttgg gtggcatgag cagggcgatg gtaagctgtt cttcggactc 1200tcggtggaaa atggccgggt gaaagacgag ggtaagttcc gcctcaagac cgccttgcgg 1260aagatcgtgg agcagtacca ggtccccatg cgcctcacgg ctaaccacga cgtgatcctg 1320tacgagatta agcctgagga ccagtcggcg atcgagaaaa tccttacgga ccatggcctg 1380atcacggacc cgaataatct ggatcatctg ctgcgctata gcatggcgtg ccccgcgctc 1440ccgacgtgtg gcctcgccat caccgagagc gagcgcgccc tgccctcgat cctcgacagg 1500gtccgcaatg tcctcaagaa gctcggcatg gctgagcaag acctcgtggt ccgcatgacg 1560ggctgcccca acggctgcgc ccggccgtat atggccgagc tcgggttcgt cggctcggcg 1620cccaaggcct atcagctctg gctgggtggc accccgaacc agaccgcgct ggctcgcccg 1680tatatggagc ggatgccgat cgacgaactg gagtcctaca tcgagcccat gctggcgttt 1740tacaaggaga agcgccaaaa ggacgagagc ttcggcgagt tctgcaaccg ggtcggattc 1800gaggccatcg aaacgtatgt caagtcgtat gagttcaagc ccaccaagac cccgagcgcg 1860ggtggcaagg gtcggcgcca tcgcatctcg gtgtacgagg gcctgcacga gcggctcaaa 1920gccgcagccg agaagcgcgg cacctccatg acccagctcg tgtccgaggc cctggagcag 1980tacctggacg acagccagag g 2001361281DNAArtificial SequenceCodon optimized Rhodobacter capsulatus sulfidequinone oxidoreductase sequence 36atggcacata tagtggtgtt gggggcgggt ttgggtggag caattatggc gtatgaactg 60agggagcaag ttaggaaaga ggataaggtc accgtcatca cgaaagaccc gatgtaccat 120ttcgtcccgt ccaacccctg ggtggctgtt ggctggcgcg accggaagga gatcaccgtg 180gacctggctc cgaccatggc acgcaagaat atcgacttta tcccggtcgc agccaagcgc 240ctccatccgg cggagaaccg cgtcgagctg gagaacggcc aaagtgtgtc gtacgaccag 300atcgtcatcg cgacgggacc cgagctggcg ttcgacgaaa ttgagggatt cggtcccgag 360ggccataccc agtcgatctg ccatatcgac catgccgagg aagcccggct ggccttcgat 420cgcttctgcg agaacccggg tccgatcctc atcggtgcgg cgcagggcgc ctcgtgcttt 480ggtccggcgt acgagttcac cttcatcctt gataccgcgc tccgcaagcg gaagattcgc 540gacaaggttc cgatgacctt cgtgacgagc gagccctacg tcggccacct gggtctggat 600ggcgtcggcg acaccaaagg gctgcttgag ggcaatctcc gcgacaagca catcaagtgg 660atgacttcca cgcggatcaa gcgcgtcgag aagggcaaga tggtcgtgga agaggtcacc 720gaggacggga ccgtgaagcc cgagaaagag ctcccgttcg gctatgcgat gatgctgcct 780gcgttccggg gcatcaaggc cctcatgggt atcgaagggc tggtcaatcc acggggcttc 840gtgatcgtcg accagcatca gcagaacccg acgttcaaaa acgtgttcgc cgtcggcgtg 900tgcgtcgcca taccgcccat tggcccgacc cccgtgccgt gcggcgtgcc gaaaaccggc 960ttcatgatcg agagcatggt cacggccacg gcccacaaca tcggccggat cgtccgcggc 1020ttcgaggccg acgaggtcgg ctcctggaat gctgtgtgtc tcgcggactt cggcgaccaa 1080ggcatcgcct tcgtggccca gccccagatc ccgcctcgca acgtcaattg gtcgtcccag 1140ggcaagtggg tgcactgggc gaaggaaggc tttgagcggt atttcatgca caagctgcgc 1200cgcggcacca gcgaaacctt ctacgagaaa gccgccatga aattcctcgg tatcgataag 1260ctcaaggctg tgaaaaaggg c 1281371308DNAArtificial SequenceCodon optimized Oscillatoria limnetica str. sulfide quinone reductase sequence 37atggcacatg ttgcagttat tggagcagga ctcgcaggac ttcccaccgc atacgaactc 60aggcatatac ttccacggca gcatcgggtg accctgatca gcgacaaacc gaactttacc 120tttaccccgt ccctgccgtg ggtggcgttc gacctgacgc ccttggagcg cgtgcaactc 180gacgtgggta agctcctcaa gggtcgcaac attgactgga tccacggtaa ggtcaaccat 240atcgaccctg agaacaagac tctggtcgcc ggcgagcaga ccctggagta tgactacgtc 300gtcgtcgcga ccggtccgga gctggccacc gacgcgatag cgggcctggg gcctgaaaac 360ggctacaccc agagtgtctg caatccccat catgcgctga tggcgaaaga ggcgtggcag 420aaattcctgc aagacccggg tcccctcgtg gtgggtgccg ttccgggcgc ctcgtgcttc 480ggccccgcct atgaattcgc cctcctcgct gactatgtcc tccgccggaa aggcatgcgc 540gaccgggtcc cgatcacctt cgtgacgccg gagccgtatg tcggccatct gggcatcggc 600ggcatggcga attcggccga gctcgtgacc gatctcctgg agaacaaggg tattcgcgtg 660ctgccgaaca ccgcggttaa ggagatccac cccgagcaca tggatctgga tagcggcgag 720cagctgccat tcaaatacgc gatgctcctg ccgccgttcc gcgggccggc attcctgcgc 780gaggcgcccg agctcaccaa tccgaagggc ttcgtgcccg tgaccaatac gtaccaacac 840cccaaatacg aaagcgtcta ctcggccgga gtcatcgtcg agatcaatcc gccggagaaa 900acgccgttgc cggtcggcgt gccgaaaacc gggcagatga ctgaggccat gggtatggct 960gcggcccata atatcgcgat caagcttggc gtcagcaagg ctaagcccgt gcagccgacg 1020ctcgaagcga tctgcatcgc cgacttcggc gacacgggca tcgtgttcgt ggctgacccg 1080gtcctgccgg accccaagac gggcactcgg cgcagggcca tcacgaagcg cggcaaatgg 1140gtttcctggt ccaagaccgc gttcgaaacc ttctttctca gcaagatgcg cttcgggctg 1200gccgtcccat ggttcgagcg gtggggcctc cggttcatgg gcctctcgct ggtcgagccg 1260ctcgacacca cccgcgaaac gggcaatcag gccttcgcgt ccaagtcg 1308381125DNAArtificial SequenceCodon optimized Acidithiobacillus ferrooxidans sulfide quinone reductase sequence 38atgactcaag tgacgataat aggggcagga tttggaggac ttacggcggt taggcatctt 60cggcggcgca tgccagatgc tgagatcacc gtgatcgcgc cccgcgccga gttcgtctac 120tacccgtcgc tgatctggat cccaacgggc ctccggcaag gtgagaatct caggatccct 180ctggaccgct tcttccaacg ccgccgcgtc cagttccatc agggccgcgt caccggcctg 240cgcgacggtg gccgcacggt gattaccgac cagggtgagg tccgcaacga cgcgctgatc 300atcgcgtccg gcggccgcgg catccggaag ctgccgggca tagaacatag cttcgccatc 360tgcgacggta tcgatgcagc agagaatatt cgggaccggc tcgcgctcat ggataagggc 420accatcgcgt ttggcttcgc gggcaatccc ctggaaccga cggccgtgcg cggtggcccg 480gtgttcgagc tgttgttcgg catcgacacc tacctccgcc aaatcgacaa gcgcgggcag 540atcgagctcg tctttttcaa ccccatgact gaaccgggca accggctggg accgaaagcc 600gtcgagggcc tgctggcgga aatgcagcgc cgggacatcc gcacccatct tggccacaag 660atcagcggct tctccgtcaa caaagtcatg accgagggcg gcgacattgc cgccgatctc 720atcctcttca tgccgggcat gacgggtccc gactgggcgg ccgactcggg cctgcccctg 780agtgctggcg gcttcttcca gtccgatctg cactgcaccg tgccggacca ccccggtgtg 840ttcgtgattg gggacggtgg ctcgtacgcg ggcagcccgg attggctgcc caaacagggc 900cacatggccg atctgcaggc cgggaccgcg gtccataacc tcctgctcca tctgcagggc 960aaggccgctg ataacacctt ccgcagcgag ctcatctgca tcgtcgacac cctcgactcc 1020ggcatcatgg tgtatcggtc cccgaatcac gcgagcatcc tgccgaattc gctctggcat 1080gccgctaaag tcgccttcga gtggcgctat ttgctccatt accgg 1125391290DNAArtificial SequenceCodon optimized Aquifex aeolicus sulfide- quinone reductase sequence 39atggcgaaac atgtggtggt gattggtggg ggagtggggg ggatagcaac ggcgtacaat 60ttgcggaatc tgatgccgga cctcaagatc accctcatct cggatcgccc gtatttcggc 120ttcacccctg cattcccgca cctggcgatg ggctggcgca agttcgaaga tatttcggtc 180ccgctggcac cgctcctgcc caagtttaac attgagttta tcaatgaaaa ggccgagtcc 240atcgacccgg acgcgaacac ggtgaccacg cagtcgggca agaaaattga gtacgactac 300ctcgtgatcg ccaccggccc aaagctcgtg ttcggtgccg agggccaaga agagaatagc 360acctccatct gcacggccga gcatgcgctg gagactcaga agaagctgca agagctctac 420gctaatccgg gaccggtcgt catcggcgcc atcccgggcg tgtcgtgctt cggtccggct 480tatgagttcg cgctgatgct tcactatgag ctgaagaaac ggggcatccg ctacaaagtc 540ccgatgacct ttatcacgag tgagccgtat ctcggccatt tcggcgtggg cgggatcggc 600gcctccaagc gcctggtcga ggacctcttc gcggagcgga atatcgactg gatcgccaac 660gttgccgtca aggccatcga gccggacaaa gtgatctacg aagatttgaa cggcaatacc 720catgaggtgc cagcgaagtt caccatgttc atgcccagct tccagggacc cgaagtcgtg 780gcgtcggcgg gtgacaaagt tgccaatccc gcgaacaaaa tggtgatcgt gaaccgctgc 840ttccagaatc cgacctataa gaacattttc ggcgtcggtg tcgtcaccgc gataccgccc 900atcgagaaaa cgccgatccc gaccggcgtg cccaagaccg gtatgatgat cgaacagatg 960gcgatggccg tcgcacataa catcgtcaac gacatccgca acaaccccga caagtatgca 1020ccccgcctga gcgcgatctg catcgccgac ttcggcgagg atgcgggctt tttcttcgcc 1080gacccggtca tccctccgcg ggagcgcgtc atcacgaaga tgggcaaatg ggcccactac 1140ttcaagaccg ccttcgagaa gtatttcctc tggaaagtca ggaacggcaa catcgcgccg 1200tccttcgagg aaaaggtcct tgagatcttc ctcaaggtcc acccgatcga gctgtgcaag 1260gactgcgagg gcgctcccgg cagccgctgt 1290401320DNAArtificial SequenceCodon optimized Halothece sp. sulfide-quinone oxidoreductase sequence 40atggcacata tagttattgt tggtggggga tttgggggat tgagtgcggc gtatgaattg 60aaacaccttc ttcatggcaa gcacaagatc acgctgatct cggacgaaac caccttcacg 120ttcatcccga gcctgccgtg ggtcgcgttc aatctgaggc gcctcgaaga tgtgcaactg 180ccgctggccc cactgctggc tcggcagggc atcaactggc agcatggccg cgtgaccggc 240ctggacccta atcagaaacg ggtgagcgtg ggcgaggaca tcaccttcga ctacgactac 300ctcgtcatca ccacgggcgc ttccctggcg tatcatctca tgtccggcct cggccccgag 360gaggggtata cccagagcgt ctgcaacgcc catcatgccg agatggcacg ggacgcgtgg 420gatgagttcc ttgagaatcc ggggccgctg ttggtgggtg ccgtgcctgg cgcttcgtgt 480atgggccccg cctatgagtt cgccctcctg gcggactatg ccctgcggca ggagggcaag 540cgcgaccaag tgccgatcac gttcatttcg ccggagccgt acctgggcca cctcggcatc 600ggcggcatgg ccaactccgg gaagctcgtc acggaactga tgaagcagcg caatatcgac 660tgggtggaga acgcagagat agccgagatc aaagaggatc acgtcaagct taccgatggc 720cgcgagttcc cgttcaacta ctccatgttc ctcccgccgt tccgcggtgc gcagttcctc 780aaagaggtcc cgggtctgac cgacgaaaag ggctttctcc cggtcctcga cacgtaccag 840catcccgact acccctcgat ctatagcgcg ggagtcatca cccaactcgc tgcccccgag 900gaaaccgagg tgcccctggg agcgccaaag actggccaaa tgaccgagtc gatggcgatg 960gccgtcgcgc acaatatcgc ccgcgagctg ggcgagatca atgcgcgccc cgtgaagccg 1020agcctggaag cgatttgcat ggccgatttc ggcgacaccg gtatcatctt catcgcagcc 1080cccgtcgtcc cggatccctc cgtcggccat cggcgccacg cgaccgccct caggggcctg 1140tgggtgaact gggccaaaaa cgcgttcgag tggtatttcc tcgcgaagat gcgctggggc 1200accgccgtgc cgtggttcga aaagctcggt ctgtacctcc tgcggctcac gctggtcacg 1260ccgatttccg agactccgac ccagcagaaa gacctcacct cgatcaaggg tttctgctgc 1320411293DNAArtificial SequenceCodon optimized Allochromatium vinosum flavocytochrome c flavoprotein subunit precursor sequence 41atgactttga ataggcgcga ttttatcaag acgagcgggg cagcggtggc agcggtgggt 60atacttggat tcccccatct ggccttcggc gcgggccgca aggtcgtggt cgtgggtggc 120ggcaccggtg gggccacggc agcgaagtat atcaagctcg ccgatccgtc catcgaagtg 180accctcatcg agccgaacac cgactactac acgtgctatc tgtcgaacga ggtcattggg 240ggcgacagga aactggagtc gatcaaacat ggctatgatg gactccgggc gcatggcatc 300caggtcgtgc acgactccgc gaccggcatc gaccccgata agaaactcgt gaaaaccgcc 360ggcggcgccg agttcggcta cgaccgctgc gtggtggccc ccggcatcga gctcatttac 420gacaagatcg agggctattc ggaagaggct gcggcgaagc tgccgcatgc gtggaaggcc 480ggtgagcaga cggcgatcct ccggaaacag cttgaggaca tggcagacgg cggcaccgtg 540gtgatcgccc caccggcggc tccgttccgc tgcccgcctg gcccctacga gcgggcgtcc 600caggtcgcct attacctgaa ggcccacaag ccgaaatcga aagtcatcat cctggatagc 660tcgcaaacct tcagtaagca aagtcagttc agcaagggct gggagcgcct gtatggcttc 720ggcaccgaga atgcgatgat agagtggcac cccggtccgg atagcgccgt cgtcaaggtc 780gatggcggag agatgatggt tgaaacggca ttcggtgacg aattcaaggc cgacgtgatc 840aacctgatcc caccccaacg ggctggtaag atcgcccaga tcgctggact gaccaacgac 900gccggctggt gtccggtcga catcaagacc ttcgagagct cgatccataa gggtatccat 960gtcatcggcg acgcctgcat cgcgaacccc atgccgaaat ccggctactc cgcgaattcg 1020caaggtaaag tcgcggcggc ggccgtggtt gcgctcctca agggcgagga acccggcacg 1080ccgtcgtatc tcaatacgtg ctactcgatc ctggcaccgg cgtatggcat ttccgtcgcc 1140gccatttacc gccccaacgc cgacggcagc gcgatcgagt ccgtgccgga ctccggcggc 1200gtcaccccgg tcgacgctcc ggactgggtg ctggagcgcg aggtccagta tgcctactcc 1260tggtacaata acatcgtcca tgacaccttc ggc 129342597DNAArtificial SequenceCodon optimized Allochromatium vinosum flavocytochrome c heme subunit sequence 42atgacccagt ccactcctcg cctcatgctc gccgcctccg tcctcgctct tggcctcgcc 60tccaatgccg gtgcagagcc gacggccgag atgctgacga ataactgcgc gggttgccac 120ggcacccatg gcaactcggt gggacccgcg agcccgtcga ttgcgcagat ggacccgatg 180gtgttcgttg aggtcatgga gggcttcaag agtggcgaga tcgccagcac catcatgggt 240cgcatcgcca agggctattc gaccgccgat ttcgagaaaa tggccggcta ctttaaacag 300caaacctacc agccggcgaa gcaatcgttc gacaccgcgc tggcggacac gggcgcaaag 360ctgcatgaca agtactgcga gaagtgccac gtcgagggcg ggaagcccct ggcggacgag 420gaggattatc atatcctggc agggcagtgg accccctact tgcaatatgc gatgtccgac 480ttccgggaag aacggcgccc gatggaaaag aaaatggcga gcaagctgcg cgagctgctc 540aaagctgagg gcgatgcggg cctggacgcc ctcttcgcct tctatgcgag ccagcag 597431290DNAArtificial SequenceCodon optimized Chlorobium limicola Flavocytochrome c sulphide dehydrogenase flavin-binding sequence 43atgagtcaaa agttcagtcg gcgggatttt aacaagttgt tggttagtgg ggttgcggga 60agcgcgttcg ggatcttcgg cgcagtcagg ccggcttacg ctgcgcagaa ccggatcgtg 120gtcattggtg gcgggttcgg cggagcgtcg gcggcaaagt acctccgcaa actggacccg 180agcctgtccg tcaccctcgt cgagccgaag gccacgttct acacgtgccc gtttagcaat 240tgggtgctcg gcggactgaa gaacatggaa gatatcgcgc agacctacac cgtgctgaag 300aacaagtatg

gcgtgaatgt catcgccgac tatgcctcct ccatcgacgc agcgaagggc 360accgtcacgc tgaagtccgg caaagtcctg aactatgacc gccttattgt gagccccggt 420atcgacttca agtggaacac gatcgaaggc tattcggagt ccgtgtcgaa tacgaagatg 480ccccatgcgt acgaggccgg tccgcagact gtgctgctgc ataagcaact cctcgcgatg 540aatgacggcg gcaccgtgct catttgccct cccgccaacc ccttccgctg tccgccaggg 600ccctatgagc gcgcctccct ggtggctcac tacctcaaag aaaagaaacc gaagtcgaag 660attatcatcc tcgatccgaa ggacaagttc tcgaagcaag gcctgttcaa aaagggttgg 720gagaaactct accccggcat gatcgagtgg cgctccgtgg ccaccggtgg gaaaatctcg 780aaagttgatg cggccaccat gaccgtgacc accgacttcg gcgtcgagaa aggcgacgtg 840atcaatatca taccgccgca acaggctggt aagatcgcgg tcgatgcggg cctgaccgac 900gcctcgggct ggtgcccggt caacccgatc accttcgagt cgaccatcca tcctggcatc 960cacgtgatcg gcgacgcctg cattgctggc gccatgccga agtccggctt cgccgcgagc 1020tcgcagggta aagtcgtggc ggcctccatc atccgcctct gccagggcaa ggtccccgcg 1080ccgcccagcc tggtcaatac gtgctatagc ctgataggtc cgggctatgg cgtgagcgtc 1140gccggagtct acaagctgac ctcggcaggc atcgtcgaga tcccgggctc gggtggcctc 1200acgccaatgg acgcggacga tgaccatctt aacgaagagg cgaccttcgc ccggggctgg 1260tacaacaata tcgtccaaga catctggggc 129044369DNAArtificial SequenceCodon optimized Chlorobium limicola cytochrome c subunit of flavocytochrome c sulfide dehydrogenase sequence 44atgctcggtc ttgttttcac tgtcgtcccc ctcttccacg ctggctccac cgtcatggcc 60gctgacgccc ccgccccggc aaccgtggcg gcccctgcgc cgaccccggc aatggatccg 120gccaagatgc gcgagcgcgg gcagatcctg gccctgagtt gctcgggctg ccatggcacc 180gacggcaaat cgtcgtcgat tatgccgtcc atctacggca agacgaccgg ctatatcgag 240agcgcgctgc tggacttcaa gagcggagcg cggatgagca ccgtgatggg tcgccatgcg 300aagggctata cgcccgagga aatccacctc atcgcggagt acttcggcaa tttgtccaag 360aaaaagaac 369451290DNAArtificial SequenceCodon optimized Chlorobium tepidum sulfide dehydrogenase, flavoprotein subunit sequence 45atgggaaata ctattagtcg ccgcaccttc aatcgcctcc tgatctccgg acttgctggt 60tcctcgctgc ttatgtcggg cggacccctc atggccagcg ccccaaaggc ccatgtcgtg 120gtcatcggcg gcgggttcgg tggcgcgacc gtcgcccgct acctccggca gctggacccg 180tcgatcagcg tcaccctcgt ggagccgaag aaagttttcc acacgtgtcc gatgagcaat 240tgggtcatcg gtggcctctt ctccatgcag aacacggcgc atacgtatca cgccctgcgg 300tcccggtacg gtgtcgaggt tgtccaggaa atggccaccg gcattgaccc cgtcaagaaa 360accgtgaagc tcaagggcgg ccggatgctc tcgtatgata ggctcgtcgt gtcgcccggt 420gtggacttca tctgggacgc gattgagggc tacagccgcg atgtcgccga gagctccatg 480ccctatgcgt gggaggctgg cccgcagacg ctgctcctgc gcaggcagct gttggggatg 540aaagacggcg agaacgtcat aatctgcgca ccgaagaatc cattccgctg cccggcggcg 600ccttacgagc gcgcctcgct catcgcctac tatctcaaaa agtcgaagcc caagagtaag 660gtcatcatcc tggacgacaa ggaagtgttc acgaaacaag acctgtttat gcttggctgg 720gatcggctct atcgcggcaa gattgagtgg cgctccgcga gcgccggcgg caaagtcgag 780cgcctggacc cggccaagat gaccgtggct accgagttcg gcgacgagaa aggcggcgtg 840atcaacgtga taccgcccca gaaagccggc cggatcgcgg tggaaaccgg cctcgccgat 900accagcggat ggtgccccgt caatccggcc aacttcgaga gcctgcaaca tccgggcatc 960catgtcatcg gtgacgcggc cttggtgggc acgatgccga agagtggcac cgcagccaac 1020acccaggcta aggccctggc ggcgtggctc gtggcgagct tcggcggggg caatgccggg 1080gagcacgacc tggcgtccct gtgctactcg ctgctggcgc cgggctacgc catctcggtc 1140gccggtggct atatccagtc gccggaaggc atcaaagaca acccggacac cgtgcatctc 1200acgtccatgg aagcgacgac cgcgcagctg gcaggcgagg cagagcaagc gctgcaatgg 1260taccataaca tctcgcagga cacctggggg 129046330DNAArtificial SequenceCodon optimized Chlorobium tepidum TLS cytochrome subunit of sulfide dehydrogenasesequence 46atgctcgctg ccgcccctct ccttctcgct agtggaaatg gatttgcaac taccggaccc 60gccgccaaac ccgccgtgaa gcccgtcacc gagtcgcgcg gtgagatctt gagcctgtcg 120tgcgcgggct gccatggcac cgacggcaat tcgtcctcgg tgatcccgtc catctacggc 180aagtccccgg agtacatcga aacggcgctg atcgacttca aaaacggctc ccgcaccagc 240accgtcatgg gtcggcacgc gaagggctat acgggcgaag aaatccacct gattgcggag 300tatttcggga acctgagcaa aaagaaccat 330471257DNAArtificial SequenceCodon optimized Thiobacillus denitrificans sulfide dehydrogenase (flavocytochrome), flavoprotein subunit sequence 47atgcatctcg atcgccgcga cttcctgaaa ctttccgctg ccactgccct cgctgccctt 60cccggttgtg cctccctgtc cgggaccgcc cggccgcgcg tggtcgtggt gggcgcaggc 120ttcggtggcg ccacgtgcgc gaagtacctc cgccgctggg gccctgccct cgacgtgacc 180ctcatcgagc cgaacgaaag gttcgtgagc tgcccgatct cgaactgggt gttgggtggc 240ctgcggtcca tggatgacat cacgcatggc tatggtggcc tggcgaggca cggaatcacc 300ctcatccgcg acagcgtcgt cgcgatcgac ccggataccc gcacgctgcg cacggcccag 360ggcctccaga tcgggtacga gcggctggtg ctggcccctg gggttgagct cctcaccgat 420agcgtgcgcg gcttcgccga tgcagaggcc gcaggccggg ttgtgcatgc gtggaaggct 480ggcgcccaaa ccgcgctgct ccggcgccag ttggaagcga tgccggacgg gggcaccttt 540atcgtcagca ttccggctgc cccataccgc tgcccgccgg gaccttatga gcgcgcgtgc 600ctggtcgccc attacttcaa acagcggaag ccgcggtcga aaatcatcgt cctggacgcg 660aatccggaca ttgtgtcgaa gaaacccctg ttcaccgacg cgtggaatac cctctatccg 720ggcatgattg actatcgccc caactcgccc gcactggtcg tcgacgccgc caagatgact 780gtctcgaccg acttcgagga cgtcaggggc gacgtcctca atatcgtgcc acggcagcgc 840gcggccgcgg tctgcgacct cgttggcgca cggaacgacg gcaacaagac ctggtgcacc 900gtggatttcg cgaccttcga gtccaccgcg gctcccggcg tccacatcat aggcgactcc 960atggcgtccc cgctgccgcg cagcggccac atggcgacca atcaagccaa ggtctgcgcg 1020ggagccatcg tggatctgct ggcggaccgc gcaccggatc cggcaccggt gatcgcgaac 1080acgtgctact cggcgacgag cgacagtacg gctggctacg tcgcccatgt gtaccggctc 1140gtccccggca agggctatgt cgcggccccc gagggtggcg cgaccacgac cggtgacgcg 1200cgcaatttcc gctatgccgc ctcctgggcg aagaacatct gggccgagat gctgagt 125748294DNAArtificial SequenceCodon optimized Thiobacillus denitrificans sulfide dehydrogenase (flavocytochrome), cytochrome c subunit sequence 48atgaccccca gttccgccgt cgcctcctgc ctcctccttg ccttgtccgg tttcgccgtc 60gccgcagatc gccacacgct gaccatcgcg gcgacgtgca tgtcgtgcca tggcccggat 120gggcgcagcc tgggcgaaat cccgaggctg gacggactgt cgcgcaccga gttcgtgacc 180gcgctccggg acttccggag cggcgctcgg cgcgcgacga tcatgcagcg ccaagcgtcg 240ggctataccg acgccgagat tgacgcgctc ggcgactact tcgcgaccct gaag 294491290DNAArtificial SequenceCodon optimized Thiocystis violascens NAD(FAD)-dependent dehydrogenase sequence 49atgaaactta gtaggaggga ttttgtgaag gttagtgggg cagcaacggc ggtgggattg 60ttcggttttc cgtatctggc cctgggcgca acccaaaaag tcgtcgtgat cgggggcggc 120accggtggcg ccacggcagc taagtatctg aagctcgccg actccagcat cgacgtgacg 180ctcatcgagc cgaacgaagt gtactatacc tgttacctgt ccaacgaagt cattggcggc 240gagcggaagc ttgagtcgct ccgccagacg tacgacgggc tgaaagcgca tggcgtcaaa 300gtcgtgcatg actccgccac gggaattgat cccgacaaaa agaccgtcaa gaccgcgggt 360ggcaccgagt atagctatga ccgctgcatc gttgctccgg gcatcgagct gctctacgag 420aaaatagacg ggtactcgga agcggcggcg gagactctgc cccacgcctg gaaggctggc 480gagcagaccc ggattctgcg caagcaattg gaagatatga aagacggcgg caccgtcatc 540attgcggccc cgcccgcgcc gttccggtgc ccgccaggcc cctatgagcg cgccagccag 600atcgcccatt acctgaaggc ccataagccc aagagcaagg tcatcatcct ggacaacagc 660caaaagttct cgaaacaagc gcagttcacc aagggttggg aaaccctcta cggtttcggt 720acggacaatg cgctgataga gtggcgcccg ggcccggacg ctgcggtcgt gaaggttgat 780gcgggccaga tgctcgcgga gactaacttc ggcgacgaga tcaaagccga cgtcatcaat 840gtgatccctc cgcagcgggc cggctcgatc gcgcagaccg caggcctcgc caatgagtcc 900ggctggtgcc cggtcgacgt gaaaaccttc gagtcgaagc tccacaaggg catccatgtc 960atcggcgacg cctgcatcgc caccgagatg ccgaagtccg gatactcggc gaactcgcag 1020ggcaaggtcg ccgcggccgc cgtggtggcc ctgctcaagg gcgaggagcc gggtacgccg 1080tcgtatctga acacgtgcta cagcatcatc ggtcccgcgt acggcatctc cgtcgcaggg 1140gtctaccgcc tgtcggaaga tggcgcaacg atcgccagcg tgcccgacag cggtggcgtg 1200accccggtgg acgcgcccga ttgggctctt gcgcgcgaag tcgagtatgc gtattcctgg 1260tacaacaata tcgtccacga catcttcggc 129050597DNAArtificial SequenceCodon optimized Thiocystis violascens cytochrome c553 sequence 50atggcacgca aaattcttca aactactctt ctcaccggcg cattggcact tggcgcatcc 60tccggagcat gggcagaggc gaccggggcc atgctggcca actcgtgcgc tggctgccat 120ggcacccacg gtaactccgt cggtccggcc agtcccagca tcgcggccat ggaccccgtc 180gtgttcgtcg aaaccatgga agagttcaaa aatggcgaaa cgtactcgac catcatgggc 240cgcatcgcga agggttacag caccggcgag ttcgagaaaa tggcggagta tttccacgcg 300caaacctacc agccggcgaa gcaaagcttc gacacggccc tcgccgataa gggcgccaag 360ctgcacgaca agtattgcga gaagtgtcat gctgagggcg ggaagccgct cgtggatgaa 420gaggactata atatcctggc gggccagtgg ctcccgtacc tccagtacgc gatggaggac 480ttccgggcgg accggcgcga aatggagaag aaaatgcgca ccaagctgaa cgagctgctg 540aaagccgagg gcgaggatgg catcgccgcc gtgaacgctt tttatgcctc gcagcag 59751924DNAArtificial SequenceCodon optimized Acidianus tengchongensis sulfur oxygenase/reductase sequence 51atgcccaagc cctatatcgc tatcaacatg gccgacctca aaaatgaacc gaaaacgttc 60gagatgttct ccgcggtggg ccccaaggtc tgcatggtca cggcgaggca tccgggcttc 120gtgggcttcc agaatcatgt gcagatcggg gtgctcccgt ttggagagcg gttcggtggc 180gccaagatgg acatgacgaa agaatcgtcg actgtccggg tcctccagta caccatgtgg 240aaggattgga aagaccacga agagatgcat cgccaaaact ggtcctacct cttccgcctg 300tgctacagct gcgccagcca aatggtttgg ggcccatggg agccgattta tgagatcaag 360tacgcggata tgccgatcaa taccgagatg accgacttca ccgccgtggt gggtaaaaag 420ttcgccgagg gcaagccgct ggaaattccc gtcatctcgc agccctacgg caagcgcgtc 480gtcgcgttcg gagagcacac cgtgattccg ggcaaggaga aacagttcga ggacgcgatc 540atcaagacgc ttgagatgtt caagcgcgca ccgggtttcc tcggcgcgat gctgttgaag 600gagatcggcg tgagcggcat cggctcgttc cagtttgggt ccaagggctt ccatcagctg 660ctggagagtc cgggctccct ggagccggat ccgaacaatg tcatgtatca agtgcccgag 720gccaagccca cccctccgca gtacatcgtc catgttgagt gggccaacct ggacgcactc 780cagttcggta tgggtcgcgt cctcctcagc ccggagtatc gcgaagtgca cgacgaagcg 840ctggacaccc tgatctatgg cccgtatatc cggataatca acccggtcat ggaaggcacc 900ttctggcgcg agtaccttaa cgag 92452948DNAArtificial SequenceCodon optimized Sulfolobus metallicus sulfur oxygenase-reductase sequence 52atgccgaagc cgtatgtcgc gattaaccaa gtcatcgtca aaaatgaacc gaaaacgttc 60gagatgttcc agagcgtggg ccccaaggtc tgcatgacga ctgcccggca taagggcttt 120gtgggcttcc agaaccatat cgagattggc gttgtcccga tggggacccg ctacggtgcg 180gcgaagatgg acatgctgaa agagagctcg accatgggcc tctaccagta caccatgtgg 240aaagactgga aagaccacga agagatgcat aagcaaaatt ggtcgagcct cttccgcctc 300tgctattcgt gcatgagtca agtcgtgtgg ggtccctggg agccgctgta cgaaatcacg 360atggccgaca tgcctctcaa caccgagatg accgatttta cggtgatggt cggacaaaag 420ttcgcgtcgg gtgacgccct ctccttgccg cccatctccc agccgtatgg caagcgcgtc 480gtgacctacg gcgagcatgt cgtgaaggaa ggcatggaga aagagttcga ggaaaccctc 540agtcgcctgc tgccgatgtt caagcgcgca cccggtttcc tgggctacat ggtcctgaag 600gaaatcggcg cgtccccgct gggctcgctg cagctttcgg cgaagtcctg gcatcagctg 660ctggagtccg ccaacggcat ggacgtgccc gatccaaatg gcaacttcag cccggaacag 720gcccggaaca agccgcagaa atatgttgtg cacatggagt ggtcgaatac cgatgccgcc 780cagttcggcc tcggacgcgt gttccttagc ccggagtacc gcgagataca cgaccagatc 840gtggacacgc tcatctatgg gccctatatc aggatcctca atccggtgat ggagggctcc 900ttctggcggg agtacctcaa cgaggtcaat ctgcaaaagg ctacctgg 94853933DNAArtificial SequenceCodon optimized Acidithiobacillus caldus sulfur oxygenase reductase sequence 53atggataaga atccgatcgt cgccatcaat cagtccaaag ttgtgaaccg cccagagagt 60tttgccacca tgatgaaggt cgggcccaag gtctgcataa ccaccgcgtc ccatccgggt 120ttcctgggat tcgagcaact cctccagacc ggcatgcatc ccatggccgg tcgctacggc 180gggggcgccg tggacatgcg ggacactatc aacccgatgg cgatgtatca gtatacggtg 240tggcaagatg tgaagtcgca tgaagagatg catcacgaca acttcaaaga aatctatgaa 300ctgtgcggca gctgcctgga catggtcatc gaagggccct gggagccgta ctatgagatt 360gtgcgctcgg acctcccgcg catcatgggc atgaccgatg ttccggcgca actgggcgca 420gcgttcgcag cccagaagcc cgtgtccaag gtcgcccttg ctagccagcg gtgcatcgcc 480ctgggcgacc attgggtgag cgacggccat gagaaagatt tcgagaaagg cgctgtggcg 540accctgacgt ggatgaaaga aaacatcccg ggtatggtcg gctggatgat cctcaagcaa 600ttcggagtct ccgccattgg ctcgttccag ttggaccccg agggcatgat gaaagcgacg 660ctgggcgcca acccgcctgc gtacgcgacc aaccacggca ccgcgatccc cgacaagccg 720cagatcccgg gtcagaggcc gacgcagtac ctcgtgcaca tggagtggga gtcgcccgag 780atggcccaca tgggcatcgg ctacgccatg gtcgactacg agctccgcca gatccacaat 840catggcgtcc ttgcgcatct ggaccggggt ccgtactatc tgttcttcgc gccgatgatg 900gaacagggcc agtggcgccg gaagctcgtc ctg 93354918DNAArtificial SequenceCodon optimized Sulfobacillus thermosulfidooxidans sulfur oxidation protein sequence 54atgccacgcc cctacatcgc aatcaatgac gccaaagttg tcaatgccga gtccagcttt 60caagccttcc aacaggtcgg gcccaaggtc tgcatggtga ccgcgaatca tccgggcttc 120gtcggcttcc agaaccatgt gcagatcggc gtgttcccca tgggtggccg ctacggcgga 180gcgaaaatgg atatgcatga ggaacttaac ccgatcggca tccggcagta caccatgtgg 240aagcgctggg aagatcacga ggagatgcat taccaacagt tcgacagcat cttccggctg 300tgctcctcct gcctgggcat ggtcgtggaa ggcccgtggg aagatatgta tgagatcatt 360tcgtcggacc tccccgaagt cattgcgatg accgacgttc cgtcgaagct cggagccgcg 420ttcatggccg gtcagcagcc tgccccggtg gccatgccgt atggccagcg cgtgatcgcg 480gggtcggacc actacataat cccgggcagg gaacaagagt tcgagactgc catcaccgag 540ctgatgaaaa tgttccaaaa ggcaccgggg ttcctgggct acatggtgct caagcaaatt 600ggcgcgagtg cgatcggttc cttccagctg cagcccgagg gcatccatca ggctttgcag 660accctcggcg acaatccccc gaagaataaa gagggcaact ttaagctgat cgaggctaag 720aaaacgccga ccaagtatct ggtccacatg gagtggtcgg acctcaacag cgcgatgttc 780ggcatctccc gcgtcgtgat caacggtcgc tatcgggccc agcatgacaa ggtccttgcg 840acggtgctgc agggcccgta tgtgacgctg tggagcccga tgatggagga cacctcgtgg 900cgcgagtatc tcaacgag 91855570PRTEscherichia coli 55Met Ser Glu Lys His Pro Gly Pro Leu Val Val Glu Gly Lys Leu Thr1 5 10 15Asp Ala Glu Arg Met Lys His Glu Ser Asn Tyr Leu Arg Gly Thr Ile 20 25 30Ala Glu Asp Leu Asn Asp Gly Leu Thr Gly Gly Phe Lys Gly Asp Asn 35 40 45Phe Leu Leu Ile Arg Phe His Gly Met Tyr Gln Gln Asp Asp Arg Asp 50 55 60Ile Arg Ala Glu Arg Ala Glu Gln Lys Leu Glu Pro Arg His Ala Met65 70 75 80Leu Leu Arg Cys Arg Leu Pro Gly Gly Val Ile Thr Thr Lys Gln Trp 85 90 95Gln Ala Ile Asp Lys Phe Ala Gly Glu Asn Thr Ile Tyr Gly Ser Ile 100 105 110Arg Leu Thr Asn Arg Gln Thr Phe Gln Phe His Gly Ile Leu Lys Lys 115 120 125Asn Val Lys Pro Val His Gln Met Leu His Ser Val Gly Leu Asp Ala 130 135 140Leu Ala Thr Ala Asn Asp Met Asn Arg Asn Val Leu Cys Thr Ser Asn145 150 155 160Pro Tyr Glu Ser Gln Leu His Ala Glu Ala Tyr Glu Trp Ala Lys Lys 165 170 175Ile Ser Glu His Leu Leu Pro Arg Thr Arg Ala Tyr Ala Glu Ile Trp 180 185 190Leu Asp Gln Glu Lys Val Ala Thr Thr Asp Glu Glu Pro Ile Leu Gly 195 200 205Gln Thr Tyr Leu Pro Arg Lys Phe Lys Thr Thr Val Val Ile Pro Pro 210 215 220Gln Asn Asp Ile Asp Leu His Ala Asn Asp Met Asn Phe Val Ala Ile225 230 235 240Ala Glu Asn Gly Lys Leu Val Gly Phe Asn Leu Leu Val Gly Gly Gly 245 250 255Leu Ser Ile Glu His Gly Asn Lys Lys Thr Tyr Ala Arg Thr Ala Ser 260 265 270Glu Phe Gly Tyr Leu Pro Leu Glu His Thr Leu Ala Val Ala Glu Ala 275 280 285Val Val Thr Thr Gln Arg Asp Trp Gly Asn Arg Thr Asp Arg Lys Asn 290 295 300Ala Lys Thr Lys Tyr Thr Leu Glu Arg Val Gly Val Glu Thr Phe Lys305 310 315 320Ala Glu Val Glu Arg Arg Ala Gly Ile Lys Phe Glu Pro Ile Arg Pro 325 330 335Tyr Glu Phe Thr Gly Arg Gly Asp Arg Ile Gly Trp Val Lys Gly Ile 340 345 350Asp Asp Asn Trp His Leu Thr Leu Phe Ile Glu Asn Gly Arg Ile Leu 355 360 365Asp Tyr Pro Ala Arg Pro Leu Lys Thr Gly Leu Leu Glu Ile Ala Lys 370 375 380Ile His Lys Gly Asp Phe Arg Ile Thr Ala Asn Gln Asn Leu Ile Ile385 390 395 400Ala Gly Val Pro Glu Ser Glu Lys Ala Lys Ile Glu Lys Ile Ala Lys 405 410 415Glu Ser Gly Leu Met Asn Ala Val Thr Pro Gln Arg Glu Asn Ser Met 420 425 430Ala Cys Val Ser Phe Pro Thr Cys Pro Leu Ala Met Ala Glu Ala Glu 435 440 445Arg Phe Leu Pro Ser Phe Ile Asp Asn Ile Asp Asn Leu Met Ala Lys 450 455 460His Gly Val Ser Asp Glu His Ile Val Met Arg Val Thr Gly Cys Pro465 470 475 480Asn Gly Cys Gly Arg Ala Met Leu Ala Glu Val Gly Leu Val Gly Lys 485 490 495Ala Pro Gly Arg Tyr Asn Leu His Leu Gly Gly Asn Arg Ile Gly Thr 500 505 510Arg Ile Pro Arg Met Tyr Lys Glu Asn Ile Thr Glu Pro Glu Ile Leu 515 520 525Ala Ser Leu Asp Glu Leu Ile Gly Arg Trp Ala Lys Glu Arg Glu Ala 530 535 540Gly Glu Gly Phe Gly Asp Phe Thr Val Arg

Ala Gly Ile Ile Arg Pro545 550 555 560Val Leu Asp Pro Ala Arg Asp Leu Trp Asp 565 57056599PRTEscherichia coli 56Met Thr Thr Gln Val Pro Pro Ser Ala Leu Leu Pro Leu Asn Pro Glu1 5 10 15Gln Leu Ala Arg Leu Gln Ala Ala Thr Thr Asp Leu Thr Pro Thr Gln 20 25 30Leu Ala Trp Val Ser Gly Tyr Phe Trp Gly Val Leu Asn Gln Gln Pro 35 40 45Ala Ala Leu Ala Ala Thr Pro Ala Pro Ala Ala Glu Met Pro Gly Ile 50 55 60Thr Ile Ile Ser Ala Ser Gln Thr Gly Asn Ala Arg Arg Val Ala Glu65 70 75 80Ala Leu Arg Asp Asp Leu Leu Ala Ala Lys Leu Asn Val Lys Leu Val 85 90 95Asn Ala Gly Asp Tyr Lys Phe Lys Gln Ile Ala Ser Glu Lys Leu Leu 100 105 110Ile Val Val Thr Ser Thr Gln Gly Glu Gly Glu Pro Pro Glu Glu Ala 115 120 125Val Ala Leu His Lys Phe Leu Phe Ser Lys Lys Ala Pro Lys Leu Glu 130 135 140Asn Thr Ala Phe Ala Val Phe Ser Leu Gly Asp Ser Ser Tyr Glu Phe145 150 155 160Phe Cys Gln Ser Gly Lys Asp Phe Asp Ser Lys Leu Ala Glu Leu Gly 165 170 175Gly Glu Arg Leu Leu Asp Arg Val Asp Ala Asp Val Glu Tyr Gln Ala 180 185 190Ala Ala Ser Glu Trp Arg Ala Arg Val Val Asp Ala Leu Lys Ser Arg 195 200 205Ala Pro Val Ala Ala Pro Ser Gln Ser Val Ala Thr Gly Ala Val Asn 210 215 220Glu Ile His Thr Ser Pro Tyr Ser Lys Asp Ala Pro Leu Val Ala Ser225 230 235 240Leu Ser Val Asn Gln Lys Ile Thr Gly Arg Asn Ser Glu Lys Asp Val 245 250 255Arg His Ile Glu Ile Asp Leu Gly Asp Ser Gly Met Arg Tyr Gln Pro 260 265 270Gly Asp Ala Leu Gly Val Trp Tyr Gln Asn Asp Pro Ala Leu Val Lys 275 280 285Glu Leu Val Glu Leu Leu Trp Leu Lys Gly Asp Glu Pro Val Thr Val 290 295 300Glu Gly Lys Thr Leu Pro Leu Asn Glu Ala Leu Gln Trp His Phe Glu305 310 315 320Leu Thr Val Asn Thr Ala Asn Ile Val Glu Asn Tyr Ala Thr Leu Thr 325 330 335Arg Ser Glu Thr Leu Leu Pro Leu Val Gly Asp Lys Ala Lys Leu Gln 340 345 350His Tyr Ala Ala Thr Thr Pro Ile Val Asp Met Val Arg Phe Ser Pro 355 360 365Ala Gln Leu Asp Ala Glu Ala Leu Ile Asn Leu Leu Arg Pro Leu Thr 370 375 380Pro Arg Leu Tyr Ser Ile Ala Ser Ser Gln Ala Glu Val Glu Asn Glu385 390 395 400Val His Val Thr Val Gly Val Val Arg Tyr Asp Val Glu Gly Arg Ala 405 410 415Arg Ala Gly Gly Ala Ser Ser Phe Leu Ala Asp Arg Val Glu Glu Glu 420 425 430Gly Glu Val Arg Val Phe Ile Glu His Asn Asp Asn Phe Arg Leu Pro 435 440 445Ala Asn Pro Glu Thr Pro Val Ile Met Ile Gly Pro Gly Thr Gly Ile 450 455 460Ala Pro Phe Arg Ala Phe Met Gln Gln Arg Ala Ala Asp Glu Ala Pro465 470 475 480Gly Lys Asn Trp Leu Phe Phe Gly Asn Pro His Phe Thr Glu Asp Phe 485 490 495Leu Tyr Gln Val Glu Trp Gln Arg Tyr Val Lys Asp Gly Val Leu Thr 500 505 510Arg Ile Asp Leu Ala Trp Ser Arg Asp Gln Lys Glu Lys Val Tyr Val 515 520 525Gln Asp Lys Leu Arg Glu Gln Gly Ala Glu Leu Trp Arg Trp Ile Asn 530 535 540Asp Gly Ala His Ile Tyr Val Cys Gly Asp Ala Asn Arg Met Ala Lys545 550 555 560Asp Val Glu Gln Ala Leu Leu Glu Val Ile Ala Glu Phe Gly Gly Met 565 570 575Asp Thr Glu Ala Ala Asp Glu Phe Leu Ser Glu Leu Arg Val Glu Arg 580 585 590Arg Tyr Gln Arg Asp Val Tyr 59557553PRTRhodobacter capsulatus 57Met Tyr Glu Tyr Ser Asp Phe Asp Glu Ala Phe Val Arg Asn Arg Val1 5 10 15Ala Gln Phe Arg Asp Gln Val Ala Arg Arg Leu Asp Gly Ser Leu Thr 20 25 30Glu Glu Glu Phe Arg Pro Leu Arg Leu Met Asn Gly Leu Tyr Leu Gln 35 40 45Leu His Ala Tyr Met Leu Arg Val Ala Ile Pro Tyr Gly Thr Leu Ser 50 55 60Ser Asn Gln Met Arg Ala Leu Ala Asp Val Ala Asp Arg Phe Asp Arg65 70 75 80Gly Tyr Gly His Phe Thr Thr Arg Gln Asn Ile Gln Phe Asn Trp Ile 85 90 95Lys Leu Thr Asp Thr Pro Asp Ile Leu Glu Arg Leu Ala Asp Asp Gly 100 105 110Leu His Ala Ile Gln Thr Ser Gly Asn Cys Ile Arg Asn Val Thr Thr 115 120 125Asp Ala Phe Ala Gly Ala Ala Ala Asp Glu Ile Glu Asp Pro Arg Pro 130 135 140Tyr Ala Glu Leu Ile Arg Gln Trp Ser Ser Asp His Ala Glu Phe Gln145 150 155 160Phe Leu Pro Arg Lys Phe Lys Ile Ala Ile Thr Gly Ser Pro Glu Asp 165 170 175Arg Ala Ala Ile Arg Ala His Asp Val Gly Leu Gln Leu Ile Gln Arg 180 185 190Gly Gly Glu Thr Gly Phe Arg Val Leu Val Gly Gly Gly Leu Gly Arg 195 200 205Thr Pro Met Leu Ala Pro Glu Leu Arg Asp Phe Leu Pro Lys Ala Asp 210 215 220Leu Leu Pro Tyr Leu Glu Ala Ile Leu Ala Ala Tyr Asn Leu Ile Gly225 230 235 240Arg Arg Asp Asn Lys Tyr Lys Ala Arg Ile Lys Ile Thr Val Phe Glu 245 250 255Thr Gly Ile Glu Pro Phe Arg Asp Leu Val Glu Gln Glu Phe Glu Arg 260 265 270Ile Arg Pro Gln Phe Thr Gly Ala Asp Gln Ala Leu Leu Ala Glu Ile 275 280 285Thr Pro His Phe Ala Leu Pro Asp Leu Val Ala Lys Asp Pro Ala Pro 290 295 300Phe Ala Ala Ala Gln Val Thr Asp Pro Ala Phe Ala Ala Trp Val Lys305 310 315 320His Ser Val Thr Asp His Lys Arg Pro Asp His Ala Val Val Thr Ile 325 330 335Ser Val Lys Thr Pro Gly Glu Glu Pro Gly Asp Val Ser Ala Ala Gln 340 345 350Met Arg Ala Val Ala Asp Leu Ala Asp Leu His Gly Tyr Gly Glu Leu 355 360 365Arg Ile Ser His Met Gln Asn Ile Val Leu Pro His Val Ala Arg Ala 370 375 380Asp Leu Pro Ala Leu His Ala Ala Leu Arg Lys Val Gly Leu Ala Ala385 390 395 400Ala Asn Val Gly Leu Ile Ser Asp Met Ile Ala Cys Pro Gly Met Asp 405 410 415Tyr Cys Ala Leu Ala Thr Ala Arg Ser Ile Pro Leu Ala Gln Glu Ile 420 425 430Ala Gln His Phe Glu Thr Leu Gly Leu Val Glu Thr Ile Gly Pro Leu 435 440 445Pro Leu Lys Ile Ser Gly Cys Ile Asn Ala Cys Gly His His His Leu 450 455 460Gly Ala Ile Gly Ile Leu Gly Leu Asp Arg Ala Gly Ala Glu Asn Tyr465 470 475 480Gln Ile Thr Leu Gly Gly Ala Glu Gly Pro Glu Ala Ala Ile Gly Glu 485 490 495Lys Met Gly Pro Gly Phe Ala Tyr Asp Ala Val Val Pro Ala Ile Glu 500 505 510Arg Leu Val Arg Ala Tyr Leu Thr Leu Arg Leu Ser Glu Gly Glu Thr 515 520 525Phe Leu Ala Ala Leu His Arg Leu Gly Arg Glu Pro Phe Arg Ala Ala 530 535 540Leu Tyr Asp Glu Ala Gln Asp Ala Ala545 55058735PRTRhodobacter capsulatus 58Met Leu Arg Phe Leu His Arg Trp Pro Gly Leu Leu Ala Ala Leu Leu1 5 10 15Val Leu Val Leu Ala Leu Ser Gly Ser Ala Leu Ser Leu Tyr Pro Ala 20 25 30Leu Glu Arg Leu Ala Val Pro Gln Ala Glu Ser Thr Leu Thr Val Ala 35 40 45Asp Leu Ala Ala Arg Val Ala Ser Ala His Pro Gly Leu Glu Gln Ile 50 55 60Arg Arg Ala Pro Ser Gly Arg Val Val Ala Trp Trp Phe Glu Gly Gly65 70 75 80Arg Pro Gly Ala Ala Val Ile Asp Pro Ala Thr Gly Ala Asp Leu Gly 85 90 95Ser Pro Asp Pro Leu Pro Gly Ser Arg Phe Leu Thr Asp Leu His Arg 100 105 110Glu Leu Phe Ala Gly Asp Thr Gly Arg Leu Val Ala Ala Ala Gly Ala 115 120 125Ala Leu Met Leu Ala Leu Ala Ile Ser Gly Ala Trp Leu Val Ala Arg 130 135 140Arg Met Gly Gly Trp Ser Arg Trp Phe Gly Arg Thr Arg Gly Pro Phe145 150 155 160Ala Gly Arg Leu His Val Glu Leu Ala Arg Phe Ala Val Gly Gly Leu 165 170 175Ile Leu Ser Ser Leu Thr Ala Leu Trp Met Thr Ala Ser Met Phe Ser 180 185 190Leu Leu Pro Asp Gly Ala Ala Glu Pro Ala Pro Ala Leu Ala Thr Ala 195 200 205Thr Leu Pro Arg Leu Pro Tyr Asp Gln Ile Pro Ala Leu Ala Asn Thr 210 215 220Pro Ala Ala Ala Leu Arg Glu Leu Ser Leu Pro Ser Pro Asp Asp Pro225 230 235 240Thr Asp Thr Phe Lys Leu Val Thr Glu Gly Gly Ala Ala Leu Ile Asp 245 250 255Pro Gly Thr Gly Ala Met Leu Ser Ser Ala Thr Pro Gly Phe Phe Glu 260 265 270Lys Ala Thr Glu Ile Met Val Met Leu His Thr Gly Gln Gly Ala Ser 275 280 285Ala Leu Gly Leu Leu Leu Gly Leu Met Ser Leu Ser Val Pro Ala Leu 290 295 300Ala Leu Thr Gly Ala Gln His Trp Trp Ala Gly Leu Arg Ser Asn Arg305 310 315 320Arg Ile Arg Arg Asn Ala Arg Ala Gln Leu Ala Glu Thr Val Val Leu 325 330 335Val Ala Ser Glu Gly Gly Thr Thr Trp Gly Phe Ala Arg Thr Leu His 340 345 350Asp Gly Leu Thr Ala Ala Gly Gln Lys Val His Thr Ala Pro Leu Ala 355 360 365Ser Phe Asp Pro Ala Arg Tyr Ala Arg Ala Arg Arg Phe Leu Ile Leu 370 375 380Ala Ala Thr Tyr Gly Glu Gly Glu Ala Pro Thr Ala Ala Lys Ala Val385 390 395 400Leu Asp Arg Ile Ala Ala Leu Thr Ser Ala Pro Ala Ala Pro Leu Ala 405 410 415Ile Leu Gly Phe Gly Asp Arg Thr Phe Pro Gln Phe Cys Gly Phe Ala 420 425 430Glu Ser Leu Arg Ala Ala Ala Ala Ala Ile Gly Trp Glu Ser Leu Met 435 440 445Pro Met Ala Thr Val Asp Arg Gln Ser Ala Gln Asp Phe Ala Arg Trp 450 455 460Ser Arg Asp Leu Gly Ala Val Leu Gly Leu Pro Leu Asp Leu Thr His465 470 475 480Leu Pro Glu Arg Pro Lys Thr Thr Ala Leu Thr Leu Ile Ser Arg Arg 485 490 495Asp His Gly Ala Glu Val Gln Ala Pro Thr Ser Ile Leu Arg Phe Glu 500 505 510Val Pro Gln Ala Thr Leu Trp Gln Arg Leu Thr Gly Gln Gly Phe Ala 515 520 525Arg Phe Glu Ala Gly Asp Leu Ile Gly Ile Leu Pro Lys Gly Ser Asp 530 535 540Leu Pro Arg Phe Tyr Ser Leu Ala Ser Ser Ala Arg Asp Gly Phe Leu545 550 555 560Glu Ile Cys Val Arg Arg His Pro Gly Gly Leu Cys Ser Gly Gln Leu 565 570 575Thr Asp Leu Thr Pro Gly Ala Thr Val Ala Gly Phe Val Arg Arg Asn 580 585 590Pro Ala Phe Arg Pro Gln Lys Gly Arg Lys Pro Val Ile Leu Ile Gly 595 600 605Ala Gly Thr Gly Val Gly Pro Leu Ala Gly Phe Leu Arg Ala Asn Arg 610 615 620Arg His Arg Pro Met His Leu Tyr Phe Gly Ala Arg Ala Pro Gln Ser625 630 635 640Asp Leu Leu Tyr Glu Ala Glu Leu Arg Asp Trp Gln Ala Ala Gly Gln 645 650 655Leu Ser Arg Leu Thr Thr Ala Phe Ser Arg His Gly Pro Lys Thr Tyr 660 665 670Val Gln Asp Ala Leu Arg Ala Asp Ala Pro Glu Leu Ala Arg Leu Ile 675 680 685Gly Ala Gly Ala Gln Ile Met Val Cys Gly Gly Arg Asp Met Ala Ala 690 695 700Ala Val Arg Asp Ala Leu Ala Glu Ile Leu Val Pro Ile Gly Gln Thr705 710 715 720Pro Ala Ser Leu Lys Ala Glu Gly Arg Tyr Ala Glu Asp Val Tyr 725 730 73559565PRTShewanella putrefaciens 59Met Ser Glu Gln Lys Leu Ala Leu Asn Glu Tyr Leu Lys Thr Asp Ser1 5 10 15Asp Tyr Leu Arg Gly Thr Ile Lys Glu Gly Leu Asp Ser Ser Val Thr 20 25 30Gly Ser Phe Ser Asp Gly Asp Gln Gln Leu Ile Lys Phe His Gly Phe 35 40 45Tyr Gln Gln Asp Asp Arg Asp Leu Arg Asn Glu Arg Lys Glu Gln Lys 50 55 60Leu Glu Pro Leu Tyr Ser Phe Met Leu Arg Ala Arg Val Pro Gly Gly65 70 75 80Ile Cys Ser Pro Gln Gln Trp Leu Gly Val Asp Lys Ile Ala Ser Thr 85 90 95Leu Thr Ser Ser Asn Ser Ile Arg Leu Thr Thr Arg Gln Thr Phe Gln 100 105 110Tyr His Gly Ile Pro Lys Arg Asn Leu Lys Thr Ile Ile Gln Asp Leu 115 120 125Asp Arg Gln Ala Leu Asp Ser Ile Ala Ala Cys Gly Asp Val Asn Arg 130 135 140Asn Val Met Cys Asn Pro Asn Pro Val Glu Ser Lys Leu His Glu Gln145 150 155 160Ala Tyr Ala Val Ala Lys Lys Leu Ser Asp His Leu Leu Pro His Thr 165 170 175Arg Ala Tyr Ala Glu Ile Trp Leu Asp Glu Glu Lys Leu Leu Thr Thr 180 185 190Glu Asp Glu Thr Val Glu Pro Val Tyr Gly Lys Thr Tyr Leu Pro Arg 195 200 205Lys Phe Lys Met Ala Val Ala Val Pro Pro Asp Asn Asp Val Asp Val 210 215 220Tyr Thr Asn Asp Leu Gly Phe Ile Ala Val Ala Glu Asn Gly Glu Leu225 230 235 240Val Gly Phe Asn Leu Thr Ala Gly Gly Gly Met Gly Ser Thr His Gly 245 250 255Glu Val Glu Thr Phe Pro Arg Leu Ala Asp Asp Phe Gly Phe Ile Lys 260 265 270Thr Glu Asp Val Met Lys Phe Ala Glu Ala Val Met Thr Val Gln Arg 275 280 285Asp Trp Gly Asn Arg Ser Asn Arg Lys Arg Ser Arg Leu Lys Tyr Thr 290 295 300Ile Val Asp His Gly Tyr Glu Lys Phe Lys Ala Glu Val Glu Ala Arg305 310 315 320Ala Gly Val Lys Phe Glu Pro Lys Arg Glu Val Val Ile Gly Asp Arg 325 330 335Gly Asp Arg Tyr Gly Trp Val Glu Gly Val Asp Gly Lys Trp His Leu 340 345 350Thr Leu Phe Ile Glu Ser Gly Arg Ile Lys Asp Leu Pro Gly Gln Thr 355 360 365Leu Gln Thr Gly Leu Arg Glu Ile Ala Lys Ile His Lys Gly Asp Phe 370 375 380Arg Met Thr Ser Asn Gln Asn Met Ile Ile Ala Gly Val Ala Ala Glu385 390 395 400Asp Lys Ala Thr Ile Glu Gly Leu Ala Arg Lys His Gly Leu Leu Gly 405 410 415Gln Val Leu Thr Gln Thr Arg Gly His Ser Ile Ala Cys Val Ala Leu 420 425 430Pro Thr Cys Pro Leu Ala Met Ala Glu Ala Glu Arg Tyr Phe Pro Glu 435 440 445Phe Ile Asp His Ile Asp Ala Leu Gln Ala Lys His Gly Ile Ser Glu 450 455 460Gln Ala Ile Val Val Arg Met Thr Gly Cys Pro Asn Gly Cys Ala Arg465 470 475 480Pro Phe Ala Ala Glu Ile Gly Leu Val Gly Lys Ala Pro Gly Arg Tyr 485 490 495Asn Leu Tyr Leu Gly Ala Ser Phe Glu Gly Thr Arg Leu Asn Lys Met 500 505 510His Arg Glu Asn Ile Gln Glu Ala Asp Ile Leu Ala Glu Leu Asp Thr 515 520 525Leu Phe Gly Arg Tyr Ala Val Glu Arg Asp Ala Gly Glu Thr Phe Gly 530 535 540Asn Phe Thr Val Arg Val Gly Val Val Lys Ala Val

Ile Asp Ala Ala545 550 555 560Lys Asp Phe His Gly 56560599PRTShewanella putrefaciens 60Met Leu Leu Lys Glu Leu Ser Ser Leu Ala Ser Pro Leu Ser Gln Gly1 5 10 15Gln Val Glu Lys Leu Lys Gln Leu Thr Ser Glu Leu Ser Ala Val Gln 20 25 30Leu Ala Trp Val Ser Gly Tyr Leu Ala Ala Thr Ala Asn Ala Gly Gln 35 40 45Leu Ala Pro Val Ala Gln Ala Gln Thr Ala Gln Thr Val Thr Ile Leu 50 55 60Tyr Gly Ser Gln Thr Gly Asn Gly Arg Gly Val Ala Lys Ala Leu Ala65 70 75 80Asp Lys Ala Gln Ala Gln Gly Tyr Ala Val Asn Leu Ala Ser Met Gly 85 90 95Glu Tyr Asn Val Arg Gln Leu Lys Gln Glu Ala Val Leu Leu Leu Val 100 105 110Val Ser Thr His Gly Glu Gly Glu Ala Pro Asp Asp Ala Ile Glu Leu 115 120 125His Lys Phe Leu Ala Ser Lys Arg Ala Pro Lys Leu Asp Asn Leu His 130 135 140Tyr Ser Val Leu Ala Leu Gly Asp Ser Ser Tyr Glu Phe Phe Cys Gln145 150 155 160Thr Gly Lys Asp Phe Asp Thr Arg Leu Ala Ala Leu Gly Ala Lys Ser 165 170 175Leu Leu Pro Leu Ile Glu Cys Asp Val Asp Tyr Glu Ala Ala Ala Gly 180 185 190Gln Trp His Ala Asp Val Leu Glu Ala Val Lys Pro Leu Ile Glu Thr 195 200 205Ser Ser Ala Ser Val Val Ser Ile Gly Thr Ala Lys Ala Ile Gly Glu 210 215 220Ser Glu Phe Thr Lys Gln Asn Pro Tyr Ser Ala Glu Val Leu Val Ser225 230 235 240Gln Lys Ile Thr Gly Arg Gly Ser Asp Arg Asp Val Arg His Val Glu 245 250 255Ile Asp Leu Gly Asp Ser Gly Leu Thr Tyr Gln Ala Gly Asp Ala Leu 260 265 270Gly Val Trp Phe Ser Asn Asn Glu Ala Leu Val Glu Glu Ile Leu Thr 275 280 285Ala Leu Ser Leu Ser Gly Asp Glu Gln Val Val Val Glu Lys Glu Ser 290 295 300Leu Thr Leu Lys Gln Ala Leu Val Asp Lys Lys Glu Leu Thr Gln Leu305 310 315 320Tyr Pro Gly Leu Val Lys Ala Trp Ala Glu Leu Ser Gly Ser Ala Glu 325 330 335Leu Leu Ala Leu Ser Glu Asp Lys Glu Gln Val Arg His Phe Ile Leu 340 345 350Lys His Gln Phe Ala Asp Leu Val Thr Gln Tyr Pro Leu Ser Asn Asn 355 360 365Ser Val Thr Leu Asn Ala Ala Lys Leu Leu Glu Leu Leu Arg Pro Leu 370 375 380Thr Pro Arg Leu Tyr Ser Ile Ala Ser Ser Gln Ser Glu Val Glu Thr385 390 395 400Glu Val His Leu Thr Val Ala Leu Val Glu Asp Glu Arg His Gly Ala 405 410 415Ala Arg Phe Gly Gly Ala Ser His Phe Leu Ala Ser Ala Gln Glu Gly 420 425 430Thr Gln Val Lys Val Tyr Val Glu Pro Asn Lys His Phe Arg Leu Pro 435 440 445Glu Asn Pro Glu Thr Pro Val Ile Met Ile Gly Pro Gly Thr Gly Val 450 455 460Ala Pro Phe Arg Ala Phe Met Gln Glu Arg Val Ala Gln Gly Ile Gln465 470 475 480Gly Asp Ser Trp Leu Phe Phe Gly Asn Pro His Phe Glu Gln Asp Phe 485 490 495Leu Tyr Gln Thr Glu Trp Gln Gln Tyr Leu Lys Asn Gly Asp Leu Ser 500 505 510Arg Ile Asp Val Ala Phe Ser Arg Asp Gln Ala His Lys Ile Tyr Val 515 520 525Gln His Arg Ile Lys Asp Gln Gly Gln Ala Leu Trp Gln Trp Leu Gln 530 535 540Asn Gly Ala His Ile Tyr Ile Cys Gly Asp Ala Glu Arg Met Ala Lys545 550 555 560Asp Val His Gln Ala Leu Ile Glu Val Ala Val Glu Val Gly Gly Leu 565 570 575Asn Thr Glu Ala Ala Glu Ala Tyr Phe Glu Thr Leu Arg Ser Asp Lys 580 585 590Arg Tyr Gln Lys Asp Val Tyr 59561605PRTBacillus subtilis 61Met Gln Leu Gln Val Met Asn Ser Pro Phe Asn Gln Glu Gln Ala Glu1 5 10 15Leu Leu Asn Arg Leu Leu Pro Thr Leu Thr Glu Ser Gln Lys Ile Trp 20 25 30Leu Ser Gly Tyr Leu Ser Ala Gln Ser Val Ser Ala Gln Glu Ala Ala 35 40 45Gly Thr Pro Ala Ala Ala Val Ser Ala Glu Ala Pro Ala Pro Ala Val 50 55 60Ser Lys Glu Val Thr Val Leu Tyr Gly Ser Gln Thr Gly Asn Ala Gln65 70 75 80Gly Leu Ala Glu Asn Ala Gly Lys Gln Leu Glu Gln Ser Gly Phe Gln 85 90 95Val Thr Val Ser Ser Met Ser Asp Phe Lys Pro Asn Gln Leu Lys Lys 100 105 110Val Thr Asn Leu Leu Ile Val Val Ser Thr His Gly Glu Gly Glu Pro 115 120 125Pro Asp Asn Ala Leu Ser Phe His Glu Phe Leu His Gly Arg Arg Ala 130 135 140Pro Lys Leu Glu Asp Leu Arg Phe Ser Val Leu Ala Leu Gly Asp Ser145 150 155 160Ser Tyr Glu Phe Phe Cys Gln Thr Gly Lys Glu Phe Asp Gln Arg Leu 165 170 175Glu Glu Leu Gly Gly Lys Arg Ile Ser Pro Arg Val Asp Cys Asp Leu 180 185 190Asp Tyr Asp Glu Pro Ala Ala Glu Trp Leu Glu Gly Val Leu Lys Gly 195 200 205Leu Asn Glu Ala Gly Gly Gly Ser Ala Ala Pro Ala Pro Ala Ala Ala 210 215 220Ser Gln Thr Gly Glu Ser Ser Tyr Ser Arg Thr Asn Pro Phe Arg Ala225 230 235 240Glu Val Leu Glu Asn Leu Asn Leu Asn Gly Arg Gly Ser Asn Lys Glu 245 250 255Thr Arg His Val Glu Leu Ser Leu Glu Gly Ser Gly Leu Thr Tyr Glu 260 265 270Pro Gly Asp Ser Leu Gly Val Tyr Pro Glu Asn Asp Pro Glu Leu Val 275 280 285Glu Leu Leu Leu Lys Glu Met Asn Trp Asp Pro Glu Glu Ile Val Thr 290 295 300Leu Asn Lys Gln Gly Asp Val Arg Pro Leu Lys Glu Ala Leu Ile Ser305 310 315 320His Tyr Glu Ile Thr Val Leu Thr Lys Pro Leu Leu Glu Gln Ala Ala 325 330 335Gln Leu Thr Gly Asn Asp Glu Leu Arg Glu Leu Leu Ala Pro Gly Asn 340 345 350Glu Glu Asn Val Lys Ala Tyr Ile Glu Gly Arg Asp Leu Leu Asp Leu 355 360 365Val Arg Asp Tyr Gly Pro Phe Ser Val Ser Ala Gln Glu Phe Val Ser 370 375 380Ile Leu Arg Lys Met Pro Ala Arg Leu Tyr Ser Ile Ala Ser Ser Leu385 390 395 400Ser Ala Asn Pro Asp Glu Val His Leu Thr Ile Gly Ala Val Arg Tyr 405 410 415Asp Ala His Gly Arg Glu Arg Lys Gly Val Cys Ser Ile Leu Cys Ala 420 425 430Glu Arg Leu Gln Pro Gly Asp Thr Leu Pro Val Tyr Val Gln His Asn 435 440 445Gln Asn Phe Lys Leu Pro Lys Asp Pro Glu Thr Pro Ile Ile Met Val 450 455 460Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Ser Phe Met Gln Glu Arg465 470 475 480Glu Glu Thr Gly Ala Glu Gly Lys Ala Trp Met Phe Phe Gly Asp Gln 485 490 495His Phe Val Thr Asp Phe Leu Tyr Gln Thr Glu Trp Gln Asn Trp Leu 500 505 510Lys Asp Gly Val Leu Thr Lys Met Asp Val Ala Phe Ser Arg Asp Thr 515 520 525Glu Glu Lys Val Tyr Val Gln His Arg Met Leu Glu His Ser Ala Glu 530 535 540Leu Phe Glu Trp Leu Gln Glu Gly Ala Ala Val Tyr Ile Cys Gly Asp545 550 555 560Glu Lys His Met Ala His Asp Val His Asn Thr Leu Leu Glu Ile Ile 565 570 575Glu Lys Glu Gly Asn Met Ser Arg Glu Glu Ala Glu Ala Tyr Leu Ala 580 585 590Asp Met Gln Gln Gln Lys Arg Tyr Gln Arg Asp Val Tyr 595 600 60562571PRTBacillus subtilis 62Met Val Thr Lys Ile Leu Lys Ala Pro Asp Gly Ser Pro Ser Asp Val1 5 10 15Glu Arg Ile Lys Lys Glu Ser Asp Tyr Leu Arg Gly Thr Leu Lys Glu 20 25 30Val Met Leu Asp Arg Ile Ser Ala Gly Ile Pro Asp Asp Asp Asn Arg 35 40 45Leu Met Lys His His Gly Ser Tyr Leu Gln Asp Asp Arg Asp Leu Arg 50 55 60Asn Glu Arg Gln Lys Gln Lys Leu Glu Pro Ala Tyr Gln Phe Met Leu65 70 75 80Arg Val Arg Met Pro Gly Gly Val Ser Thr Pro Glu Gln Trp Leu Val 85 90 95Met Asp Asp Leu Ser Gln Lys Tyr Gly Asn Gly Thr Leu Lys Leu Thr 100 105 110Thr Arg Glu Thr Phe Gln Met His Gly Ile Leu Lys Trp Asn Met Lys 115 120 125Lys Thr Ile Gln Thr Ile His Ser Ala Leu Leu Asp Thr Ile Ala Ala 130 135 140Cys Gly Asp Val Asn Arg Asn Val Met Cys Ala Ser Asn Pro Tyr Gln145 150 155 160Ser Glu Ile His Ser Glu Val Tyr Glu Trp Ser Lys Lys Leu Ser Asp 165 170 175Asp Leu Leu Pro Arg Thr Arg Ala Tyr His Glu Ile Trp Leu Asp Glu 180 185 190Glu Arg Val Ala Gly Thr Pro Glu Glu Glu Val Glu Pro Met Tyr Gly 195 200 205Pro Leu Tyr Leu Pro Arg Lys Phe Lys Ile Gly Ile Ala Val Pro Pro 210 215 220Ser Asn Asp Ile Asp Val Phe Ser Gln Asp Leu Gly Phe Ile Ala Ile225 230 235 240Val Glu Asp Gly Lys Leu Ile Gly Phe Asn Val Ala Ile Gly Gly Gly 245 250 255Met Gly Met Thr His Gly Asp Thr Ala Thr Tyr Pro Gln Leu Ala Lys 260 265 270Val Ile Gly Phe Cys Arg Pro Glu Gln Met Tyr Asp Val Ala Glu Lys 275 280 285Thr Ile Thr Ile Gln Arg Asp Tyr Gly Asn Arg Ser Val Arg Lys Asn 290 295 300Ala Arg Phe Lys Tyr Thr Val Asp Arg Leu Gly Leu Glu Asn Val Lys305 310 315 320Glu Glu Leu Glu Asn Arg Leu Gly Trp Ser Leu Glu Glu Ala Lys Pro 325 330 335Tyr His Phe Asp His Asn Gly Asp Arg Tyr Gly Trp Val Glu Gly Ile 340 345 350Glu Asp Lys Trp His Phe Thr Leu Phe Val Glu Gly Gly Arg Ile Thr 355 360 365Asp Tyr Asp Asp Tyr Lys Leu Met Thr Gly Leu Arg Glu Ile Ala Lys 370 375 380Val His Thr Gly Glu Phe Arg Leu Thr Ala Asn Gln Asn Leu Met Ile385 390 395 400Ala Asn Val Ser Ser Asp Lys Lys Glu Glu Ile Ser Ala Leu Ile Glu 405 410 415Gln Tyr Gly Leu Thr Asp Gly Lys His Tyr Ser Ala Leu Arg Arg Ser 420 425 430Ser Met Ala Cys Val Ala Leu Pro Thr Cys Gly Leu Ala Met Ala Glu 435 440 445Ala Glu Arg Tyr Leu Pro Thr Leu Leu Asp Lys Ile Glu Glu Ile Ile 450 455 460Asp Glu Asn Gly Leu Arg Asp Gln Glu Ile Thr Ile Arg Met Thr Gly465 470 475 480Cys Pro Asn Gly Cys Ala Arg His Ala Leu Gly Glu Ile Gly Phe Ile 485 490 495Gly Lys Ala Pro Gly Lys Tyr Asn Met Tyr Leu Gly Ala Ala Phe Asp 500 505 510Gly Ser Arg Leu Ser Lys Met Tyr Arg Glu Asn Ile Gly Glu Ala Asp 515 520 525Ile Leu Ser Glu Leu Arg Ile Leu Leu Ser Arg Tyr Ala Lys Glu Arg 530 535 540Glu Glu Gly Glu His Phe Gly Asp Phe Val Ile Arg Ala Gly Ile Ile545 550 555 560Lys Ala Thr Thr Asp Gly Thr Asn Phe His Asp 565 57063588PRTAcidithiobacillus ferrooxidans 63Met Glu Leu Ile Arg Gln Ser Asp Phe Leu Leu Asp Pro Arg Lys Gln1 5 10 15Glu Asp Leu Arg Arg Phe Ala Glu Gly Met Thr Arg Glu Gln Leu Leu 20 25 30Trp Ser Ser Gly Tyr Leu Thr Gly Phe Gly Glu Ser Ala Pro Ala Ser 35 40 45Lys Ile Gln Glu Asp Ile Gly Glu Lys Ile Thr Ile Leu Phe Gly Thr 50 55 60Glu Thr Gly Asn Ala Lys Arg Leu Ala Glu Leu Leu Ala Ala Arg Ala65 70 75 80Gln Ala Met Gly Val Gln Thr Ser Ile Gln Asp Met Leu Thr Tyr Gly 85 90 95Arg Ala Gln Leu Arg Arg Asp Arg Val Ile Val Leu Ile Val Ser Thr 100 105 110His Gly Asp Gly Glu Pro Pro Asp Ser Ala Arg Met Leu Leu Ala Ser 115 120 125Leu Thr Asp Gly Pro Val Pro Asp Leu His Gly Ser Arg Phe Ala Ile 130 135 140Leu Ala Leu Gly Asp Ala Ser Tyr Pro Lys Phe Cys Gln Ala Gly Lys145 150 155 160Ala Phe Asp Ile Ala Leu Ala Ser Ala Gly Ala Glu Arg Leu Leu Pro 165 170 175Arg Val Asp Cys Asp Val Asp Tyr Glu Arg Asp Ala Met Tyr Trp Met 180 185 190Glu Gln Val Leu Gly Ala Leu Thr Thr Gly Lys Ser Ser Pro Ala Val 195 200 205Pro Phe Pro Ala Pro Val Pro Lys Gln Gly Tyr Ser Ser His Ala Thr 210 215 220Phe Pro Ala Val Leu Leu Gly Lys Val Asn Leu Ser Gly Arg Gly Ser225 230 235 240Asp Arg Glu Val Trp His Leu Glu Leu Asp Leu Asp Gly Ser Gly Leu 245 250 255His Tyr Ala Pro Gly Asp Ile Val Ser Val Ala Pro Ser Asn Pro Pro 260 265 270Gln Leu Val Glu Glu Leu Leu Asp Arg Leu Glu Leu Asp His Lys Ala 275 280 285Ser Val Arg Thr Arg Gln Gly Glu Met Pro Leu Val Glu Ala Leu Ala 290 295 300Ala His Tyr Glu Ile Thr Arg Ile Thr Trp Pro Phe Leu Glu Arg Tyr305 310 315 320Ala Arg Leu Ser Asp Ala Lys Ala Leu Gln Ser Ala Ile Ala Gly Arg 325 330 335Asp Val Asn Gly Leu Asp Thr Trp Thr Asp Gly Arg Glu Val Ile Asp 340 345 350Ile Val Gly Gln Tyr Pro Val Lys Gly Leu Ser Ala Gln Ser Phe Ala 355 360 365Asp Cys Leu Arg Pro Leu Pro Pro Arg Arg Tyr Ser Ile Ala Ser Ser 370 375 380Leu Leu Ala Val Pro Gly Glu Val His Leu Thr Val Ala Ala Ile Arg385 390 395 400Tyr Ser Ser His Gly Arg Glu Arg Leu Gly Val Ala Ser Thr Phe Leu 405 410 415Ala Asp Arg Val Ala Ile Gly Arg Pro Val Pro Ile Phe Ile Glu Pro 420 425 430Asn Ala Glu Phe Arg Leu Pro Glu Asp Ser Gly Gln Ala Met Ile Met 435 440 445Ile Gly Ala Gly Thr Gly Val Ala Pro Phe Arg Ser Phe Leu Gln Glu 450 455 460Arg Glu Ala Leu Gly Ala Ala Gly Asn Asn Trp Leu Phe Phe Gly Asp465 470 475 480Arg His Phe Ser Thr Asp Phe Leu Tyr Gln Arg Glu Trp Leu Arg Trp 485 490 495Leu Arg Asp Gly Arg Leu Thr Arg Leu Asp Val Ala Phe Ser Arg Asp 500 505 510Gln Glu Arg Lys Ile Tyr Val Gln Asp Arg Leu Arg Glu Arg Ala Gly 515 520 525Asp Val Phe Ala Trp Leu Glu Glu Gly Ala Ala Val Tyr Val Cys Gly 530 535 540Ala Glu Ala Met Gly Arg Ala Val His Gln Ser Leu Val Asp Ile Val545 550 555 560Gln Ser Ala Gly Arg Thr Gln Val Gln Ala Glu Glu Tyr Ile Leu Glu 565 570 575Leu Lys Gln Thr Gly Arg Tyr His Arg Asp Val Tyr 580 58564563PRTAcidithiobacillus ferrooxidans 64Met Ser Ile Asn Asp Lys Ala Leu Ser Asp Val Glu Arg Ile Lys Ala1 5 10 15Glu Ser Gln Gly Leu Arg Gly Thr Leu Arg Glu Ser Leu His Asn Pro 20 25 30Val Thr Gly Ala Leu Ala Glu Asp Asp Val Gln Val Ile Lys Phe His 35 40 45Gly Ile Tyr Gln Gln Asp Tyr Arg Asp Leu Arg Ala Glu Arg His Gln 50 55 60Gln Lys Leu Glu Pro Leu Tyr Gln Phe Met Ala Arg Leu Arg Leu Pro65

70 75 80Gly Gly Val Leu Ser Gly Ala Gln Trp Leu Ala Leu Gly Asp Ile Ala 85 90 95Arg Thr Tyr Gly Asn Ala Ser Leu Arg Ile Thr Ser Arg Gln Ser Ile 100 105 110Gln Phe His Gly Leu Leu Lys Pro His Leu Arg Pro Val Leu Gln Ala 115 120 125Leu Asp Arg Ala Leu Leu Asp Thr Val Ser Ala Cys Gly Asp Val Asn 130 135 140Arg Asn Val Ile Ala Ser Ser Ala Pro Gln Ile Ser Ala Phe His Ala145 150 155 160Glu Ala Tyr Gly Trp Ala Gln Lys Ile Ala Glu His Leu Leu Pro Gln 165 170 175Ser His Ala Tyr His Glu Ile Trp Leu Gly Gly Gln Gln Ile Thr Ala 180 185 190Pro Glu Glu Asp Leu Leu Tyr Gly Ser Thr Tyr Leu Pro Arg Lys Phe 195 200 205Lys Ile Ala Ile Ala Val Pro Pro His Asn Asp Val Asp Val Leu Thr 210 215 220Gln Asp Leu Gly Phe Ile Ala Ile His Glu Glu Gly Arg Leu Ala Gly225 230 235 240Phe Asn Val Cys Val Gly Gly Gly Leu Gly Arg Ser His Asn Lys Pro 245 250 255Asp Thr Tyr Ser Arg Leu Ala Asp Val Cys Gly Phe Cys Ala Pro Gly 260 265 270Gln Val Leu Ala Ile Ala Glu Ala Val Leu Ile Thr Gln Arg Asp His 275 280 285Gly Asp Arg Ser Asn Arg Ser His Ala Arg Leu Lys Tyr Thr Val Asp 290 295 300Arg Met Gly Leu Asp Arg Phe Met Glu Glu Val Gln Gln Arg Thr Gly305 310 315 320Phe Ser Leu Ala Pro Pro Arg Pro Phe His Phe Glu Thr Ser Gly Asp 325 330 335Arg Phe Gly Trp Leu Glu Asn Asp Asp Gly Thr Ala Cys Leu Thr Leu 340 345 350Phe Ile Pro Gly Gly Arg Val Ala Asp Gly Asp Ile Pro Leu Leu Ser 355 360 365Gly Leu Asp Ala Leu Ala Arg Leu His His Gly Glu Ile Arg Leu Thr 370 375 380Cys Asn Gln Asn Leu Leu Ile Ala Gly Ile Ser Pro Ala Glu Arg Pro385 390 395 400Val Val Glu Thr Val Leu Ala Glu Tyr Gly Leu Asn Arg Leu Leu Asn 405 410 415Leu Ala Pro Val Arg Ala His Ala Met Ala Cys Val Ala Leu Pro Thr 420 425 430Cys Pro Leu Ala Met Ala Glu Ala Glu Arg Tyr Leu Pro Val Phe Leu 435 440 445Asp Arg Ile Glu Ala Leu Leu Ala Glu Val Gly Leu Glu Gly Glu Ala 450 455 460Leu Thr Val Arg Met Thr Gly Cys Pro Asn Gly Cys Ala Arg Pro Tyr465 470 475 480Leu Ala Glu Ile Gly Leu Val Gly Lys Ala Pro Gly Leu Tyr Asp Leu 485 490 495Tyr Leu Gly Gly Asp Arg Thr Gly Met Arg Leu Asn Ala Leu Tyr Arg 500 505 510Glu Ala Leu Asp Glu Glu Ala Leu Leu Asp Ala Leu Arg Pro Leu Leu 515 520 525Lys Arg Phe Ala Gly Gln Arg Trp Ala Gly Glu Thr Phe Gly Asp Phe 530 535 540Val Arg Arg Gln Asp Leu Leu Pro Pro Asp Pro Gly Leu Pro His Thr545 550 555 560Gly Arg Arg65679PRTCyanidioschyzon merolae 65Met Met Phe Val Thr Tyr Ala Lys Pro Leu Val Gly Ala Arg Arg Gly1 5 10 15Leu Ala Pro Thr Gly Ser Ala Ala Pro Gly Val Tyr Pro Leu Thr Glu 20 25 30Val Leu Leu Arg Asp Arg Leu Arg Arg Gln Arg Gln Cys Arg Thr Ala 35 40 45Arg Arg Asn Ile Ile Ala Asn Leu Ser Ser Glu Gln Ser Arg Lys Lys 50 55 60His Thr Val Val Pro Ile Thr Thr Arg Lys His Ile Glu Glu Ala Ile65 70 75 80Arg Asp Gly Thr Leu Asp Gln Leu Lys Leu Asn Pro Tyr Glu Leu Pro 85 90 95Lys Leu Asn Ser Asp Tyr Leu Arg His Pro Leu Met Glu Glu Leu Gly 100 105 110Asn Asp Gln Ile Phe Ile Ser Asp Asp Cys Ile Gly Leu Ile Lys Phe 115 120 125His Gly Gly Tyr Leu Gln Asp Asn Arg Asp Gln Arg Val Arg Gly Glu 130 135 140Leu Lys Lys Tyr Gln Phe Met Leu Arg Leu Lys Met Pro Ala Gly Glu145 150 155 160Cys Pro Pro Ser Leu Tyr Thr Thr Leu Asp Asp Ile Ser Glu Thr Tyr 165 170 175Gly Asn Lys Thr Leu Arg Leu Thr Thr Arg Ser Ser Phe Gln Ile His 180 185 190Gly Ile His Lys Ser Asn Leu Lys Thr Val Val Gln Ser Ile Val Arg 195 200 205Ala Gly Gly Gly Leu Tyr Gly Ala Ser Gly Asp Cys Ser Arg Asn Val 210 215 220Ile Ala Pro Pro Ala Pro Phe Val Asp Ala Ala Tyr Ala Gln Ala Arg225 230 235 240His Val Ala Arg Met Val Ala Glu Leu Phe Ala Ile Gln Ser His Ala 245 250 255Phe Ala Asp Leu Trp Leu Asp Gly Glu Leu Ala Ala Ser Ile Glu Tyr 260 265 270Trp Lys Lys Glu Leu Asp Met Asp Glu Val Arg Arg Leu Met Thr Glu 275 280 285Asp Asn Gly Arg Gly Gln Val Leu Gln Asp Ser Val Glu Pro Leu Tyr 290 295 300Gly Lys Leu Tyr Leu Pro Arg Lys Phe Lys Val Gly Val Thr Val Pro305 310 315 320Gly Asp Asn Ser Ile Asp Ile Tyr Thr His Asp Ile Gly Ile Val Val 325 330 335Phe Cys Asp Ala Gln Gly Gln Leu Glu Gly Ala Asn Ile Leu Val Gly 340 345 350Gly Gly Met Gly Arg Thr His Asn Lys Glu Glu Thr Phe Ala Arg Ala 355 360 365Ala Asp Pro Leu Gly Tyr Val Pro Ala Ala Ala Leu Tyr Asp Thr Leu 370 375 380Lys Ala Ile Leu Ala Ala Gln Arg Asp His Gly Asn Arg Ala Val Arg385 390 395 400Thr Asn Ala Arg Met Lys Tyr Leu Val His Arg Leu Gly Ile Asp Arg 405 410 415Phe Arg Glu Leu Val Lys Ser Tyr Met Val Gly Gly Gly Ser Ala Leu 420 425 430Glu Ser Ile Arg Ser Met Pro Pro Trp Thr Phe Gln Asp Tyr Leu Gly 435 440 445Trp Arg Glu Gln Gly Asp Gly Arg Trp Phe Phe Gly Leu Tyr Val Gln 450 455 460Asn Gly Arg Ile Lys Asp Glu Leu Lys Lys Ala Leu Arg Ala Leu Thr465 470 475 480Asp Arg Phe Asn Phe Pro Leu Val Cys Thr Pro Gln Gln Asn Leu Leu 485 490 495Ile Thr Gln Val Pro Ala Thr Ala Arg Pro Asp Val Glu Thr Leu Leu 500 505 510Ala Ser Phe Gly Val Glu Thr Ala Ala Ser Ala Leu Asp Pro Leu Met 515 520 525Arg Asp Ala Met Ala Cys Pro Ala Leu Pro Leu Cys Pro Pro Ala Ile 530 535 540Thr Glu Ala Glu Arg Val Met Pro Arg Tyr Val Gln Arg Val Arg Glu545 550 555 560Leu Leu Ser Lys Val Gly Ile Ser Pro His Ala Ser Phe Val Met Arg 565 570 575Met Thr Gly Cys Pro Asn Gly Cys Thr Arg Pro Tyr Met Ala Glu Leu 580 585 590Gly Phe Val Gly Ser Gly Pro Asn Cys Thr Tyr Gln Val Trp Leu Gly 595 600 605Gly Ser Pro Met Gln Thr Arg Leu Ala Trp Pro Tyr Ile Asp Arg Val 610 615 620Thr Asp Asp Gln Val Glu Arg Val Leu Glu Pro Val Phe Val Phe Trp625 630 635 640Lys Ser Ala Arg Glu Pro Asp Glu Ser Phe Gly Asp Phe Cys Asp Arg 645 650 655Val Gly Lys Ala Gln Leu Glu Ala Tyr Ala Gln Arg Tyr Trp Asp Gly 660 665 670Val Pro Ala Ala Pro Val Ser 67566662PRTOscillatoria nigro-viridis 66Met Ile Thr Ser Ser Thr Ser Thr Pro Val Ala Arg Lys Pro Ser Lys1 5 10 15Ser Glu Gly Leu Lys Glu Arg Ser Asn Tyr Leu Arg Glu Pro Val Ala 20 25 30Thr Glu Leu Leu Gln Glu Thr Thr His Phe Thr Glu Asp Gly Ile Gln 35 40 45Ile Leu Lys Phe His Gly Ser Tyr Gln Gln Asp Asn Arg Asp Asn Arg 50 55 60Val Lys Gly Gln Glu Lys Asp Tyr Gln Phe Met Leu Arg Thr Arg Asn65 70 75 80Pro Gly Gly Phe Thr Pro Pro Gln Leu Tyr Leu Ala Leu Asp Lys Leu 85 90 95Ser Glu Glu Tyr Gly Asn His Thr Ile Arg Val Thr Thr Arg Gln Gly 100 105 110Phe Gln Leu His Gly Val Leu Lys Lys Asn Leu Lys Ala Val Phe Ser 115 120 125Ser Ile Ile Lys Asn Met Gly Ser Thr Leu Gly Ala Cys Gly Asp Leu 130 135 140Asn Arg Asn Val Met Ala Pro Pro Ala Pro Tyr Lys Asn Arg Pro Glu145 150 155 160Tyr Lys Tyr Ala Leu Gln Tyr Ala Asn Asn Val Ala Asp Leu Leu Thr 165 170 175Pro Gln Thr Gly Ala Tyr Tyr Glu Ile Trp Leu Asp Gly Glu Lys Ala 180 185 190Ile Ser Ala Glu Glu Asp Pro Ala Val Lys Ala Ala Arg Gln Lys Asn 195 200 205Gly Asn Gly Thr Ile Phe Ser Asp Lys Glu Glu Pro Ile Tyr Gly Ser 210 215 220His Tyr Met Pro Arg Lys Phe Lys Cys Ser Val Thr Val Pro Gly Asp225 230 235 240Asn Ser Ile Asp Leu Tyr Ser Gln Asp Leu Ser Leu Val Val Ile Thr 245 250 255Asn Lys Ala Gly Glu Leu Gln Gly Phe Asp Val Phe Ala Gly Gly Gly 260 265 270Leu Gly Arg Thr His Asn Lys Glu Glu Thr Phe Ala Arg Val Ala Asp 275 280 285Glu Ile Cys Tyr Val Ala Lys Asp Asp Val Tyr Asp Leu Val Lys Ala 290 295 300Ile Val Ala Thr Gln Arg Asp Tyr Gly Asp Arg Thr Asp Arg Arg His305 310 315 320Ala Arg Leu Lys Tyr Leu Ile Asn Asp Lys Gly Val Gln Trp Phe Arg 325 330 335Glu Lys Val Ala Glu Tyr Phe Gly Lys Pro Leu Glu Ala Phe Lys Pro 340 345 350Leu Pro Glu Trp Lys Tyr Phe Asp Phe Leu Gly Trp His Asp Gln Gly 355 360 365Asp Gly Lys Leu Phe Val Gly Ile Ser Val Asp Asn Gly Arg Ile Lys 370 375 380Asp Glu Gly Ser Phe Gln Leu Lys Thr Ala Leu Arg Glu Ile Val Gln385 390 395 400Lys Tyr Asn Leu Pro Val Leu Ala Thr Pro His Gln Asn Val Leu Ile 405 410 415Tyr Asp Ile Ser Pro Asp Leu Lys Gln Glu Ile Gln Gly Ile Leu Asp 420 425 430Arg Cys Gly Ile Gln Arg Glu Thr Ala Ile Asp Pro Leu Val Arg Tyr 435 440 445Ala Met Ala Cys Pro Ala Met Pro Thr Cys Gly Leu Ala Ile Thr Glu 450 455 460Ser Glu Arg Val Ile Pro Ser Ile Leu Glu Arg Ile Arg Ala Leu Leu465 470 475 480Thr Lys Val Gly Leu Glu Asp Glu His Leu Val Val Arg Met Thr Gly 485 490 495Cys Pro Asn Gly Cys Ala Arg Pro Tyr Met Ala Glu Leu Gly Phe Val 500 505 510Gly Ser Ser Pro Glu Ser Tyr Gln Ile Trp Leu Gly Gly Ser Pro Asp 515 520 525Gln Thr Arg Leu Ala Lys Pro Ile Glu Glu Lys Leu His Val Lys Asn 530 535 540Phe Glu Ala Phe Leu Glu Pro Ile Phe Val Tyr Phe Lys Gln Lys Arg545 550 555 560Gln Leu Ser Glu Ser Phe Gly Asn Phe Cys Asp Arg Val Gly Leu Glu 565 570 575Ser Ile Arg Gln Phe Val Thr Asn Tyr Gln Ser Ala Asp Ser Met Thr 580 585 590Thr Glu Ile Asn Glu Leu Glu Val Thr Ser Ser Asn Gly Glu Glu Asn 595 600 605Glu Thr Ala Thr Ala Gly Gly Gly Lys Val Arg Arg Arg Ile Ser Val 610 615 620Arg Asp Glu Ile Tyr Asn Glu Leu Lys Glu Glu Ala Ala Arg Gln Gly625 630 635 640Lys Pro Ile Thr Gln Leu Ala Thr Glu Ala Ile Ser Thr Tyr Leu Lys 645 650 655Lys Ile Lys Glu Glu Ala 66067669PRTPseudomonas putida 67Met Asn Asp Cys His Leu Ile Cys Ala Asn Arg Leu Asp Asp Gly Ala1 5 10 15Val Val Trp Leu Asp Ala Gly His Glu Trp Val Glu Thr Leu Gln Gln 20 25 30Ala Gly Thr Phe Asp Ala Gln Ala Leu Val Ser Ala Thr Leu Ala Ala 35 40 45Glu Ala Ala Val Leu Ala Asn Gln Val Val Ala Pro Thr Pro Cys Glu 50 55 60Ala Trp Leu Val Asp Gly Arg Pro Glu Pro Lys Ser Leu Arg Glu Arg65 70 75 80Leu Arg Ala Arg Gly Pro Ser Val Arg Ser Asp Leu Gly Lys Gln Ala 85 90 95Ala Gly Thr Pro Pro Ser Ser Ile Ala Arg Met Arg Pro Val Leu Pro 100 105 110Val Glu Ala Gly Gln Ala Gly Val Tyr Arg Tyr Asp Arg Phe Glu Arg 115 120 125Glu Phe Leu Lys Asp Arg Ala Arg Gln Phe Glu Gln Gln Val Ala Arg 130 135 140Arg Leu Ser Gly Glu Leu Asp Glu Glu Ala Phe Lys Val Tyr Arg Leu145 150 155 160Met Asn Gly Leu Tyr Leu Gln Leu His Gly Tyr Met Leu Arg Val Ala 165 170 175Ile Pro Tyr Gly Thr Leu Ser Ala Leu Gln Leu Arg Gln Leu Ala Tyr 180 185 190Val Ala His Thr Tyr Asp Lys Gly Tyr Gly His Leu Thr Thr Arg Gln 195 200 205Asn Ile Gln Phe Asn Trp Pro Arg Leu Ala Asp Thr Pro Glu Ile Leu 210 215 220Ser Val Leu Ala Asp Ala Asp Leu His Cys Ile Gln Thr Ser Gly Asn225 230 235 240Cys Ile Arg Asn Val Thr Thr Asp His Phe Ala Gly Ala Ala Glu Asp 245 250 255Glu Val Leu Asp Pro Arg Val His Ala Glu Ile Leu Arg Gln Trp Ser 260 265 270Thr Glu His Pro Glu Phe Thr Tyr Leu Pro Arg Lys Phe Lys Ile Ala 275 280 285Ile Thr Gly Ser Pro Lys Asp Arg Ala Ala Val Arg Phe His Asp Ile 290 295 300Gly Ile Leu Ala Gln Arg Asn Ala Gln Gly Glu Val Gly Phe Gln Val305 310 315 320Tyr Ala Gly Gly Gly Leu Gly Arg Thr Pro Ile Val Gly Thr Arg Val 325 330 335Arg Glu Trp Leu Pro Glu Arg Glu Leu Leu Arg Tyr Val Glu Ala Ile 340 345 350Leu Arg Val Tyr Asn Ala Leu Gly Arg Arg Asp Asn Leu Tyr Lys Ala 355 360 365Arg Ile Lys Ile Leu Val Arg Glu Leu Lys Pro Gly Arg Phe Ile Glu 370 375 380Met Ile Glu Glu Glu Phe Ala Ser Leu Pro Ala Asp His Gln Tyr Leu385 390 395 400Glu Pro Ala Ile Val Gln Gly Ile His Ala Arg Phe Val Gln Pro Ala 405 410 415Phe Glu Ala Leu Pro Gly Leu Cys Asp Ser Phe Leu Arg Ala Arg Ala 420 425 430Asp Asp Asn Ala Phe Ala Ser Trp Val Arg Thr Asn Thr His Pro His 435 440 445Lys Lys Arg Gly Tyr Ile Cys Ala Val Ile Ser Leu Lys Pro Pro Gly 450 455 460Gly Ile Pro Gly Asp Ile Ser Ala Glu Glu Met Leu Ala Leu Ala Asp465 470 475 480Leu Ala Glu Ala Tyr Ser Leu Asn Glu Ile Arg Val Ser His Glu Gln 485 490 495Asn Val Val Leu Pro His Ile Arg Leu Val Asp Leu Tyr Ser Val Trp 500 505 510Gln Ala Leu Arg Gln Ala Gly Leu Ala Thr Ser Asn Ile Gly Leu Leu 515 520 525Ser Asp Thr Ile Ala Cys Pro Gly Met Asp Tyr Cys Ser Leu Ala Thr 530 535 540Ala Arg Ser Val Pro Val Ala Gln Arg Ile Ala Gln Arg Phe Asp Ala545 550 555 560Ala Arg Gln Gln Asp Ile Gly Glu Leu Lys Leu Asn Val Ser Gly Cys 565 570 575Ile Asn Ala Cys Ala His His His Val Ala His Ile Gly Ile Leu Gly 580 585 590Leu Asp Lys Ala Gly His Glu Asn Tyr Gln Ile Thr Leu Gly Gly Ser 595 600 605Ala Glu Glu Asp Ala Ala Val Gly Thr Ile Leu Gly Arg Ser Val Pro 610 615 620Phe Glu Glu Val Pro Asp Ile Val Glu Ala Ile Val Ala Ile Tyr

Leu625 630 635 640Gln Leu Arg Glu Asp Asp Glu Arg Phe Leu Asp Thr Tyr Arg Arg Val 645 650 655Gly Ile Glu Pro Phe Lys Glu Val Leu Arg Asp Ala Arg 660 66568637PRTAnabaena cylindrica 68Met Val Asn Ser Ala Pro Ser Pro Val Ser Asn Arg Lys Pro Ser Lys1 5 10 15Val Glu Gly Ile Lys Glu Asn Ser Asn Phe Leu Arg Glu Pro Val Ala 20 25 30Thr Glu Ile Leu Gln Asp Thr Thr His Phe Ser Glu Asp Ala Ile Gln 35 40 45Ile Leu Lys Phe His Gly Ser Tyr Gln Gln Asp Asn Arg Asp Asn Arg 50 55 60Ala Lys Gly Gln Glu Lys Asp Tyr Gln Phe Met Leu Arg Thr Lys Asn65 70 75 80Pro Gly Gly Leu Val Pro Pro Gln Leu Tyr Leu Ala Leu Asp Lys Leu 85 90 95Ala Asp Glu Tyr Gly Asn His Thr Leu Arg Ala Thr Thr Arg Gln Gly 100 105 110Phe Gln Val His Gly Ile Leu Lys Lys Asn Leu Lys Ser Ala Ile Ala 115 120 125Thr Ile Val Gln Asn Leu Gly Ser Thr Leu Gly Ala Cys Gly Asp Ile 130 135 140Asn Arg Asn Val Met Ala Pro Pro Ala Pro Leu Lys Asn Arg Pro Glu145 150 155 160Tyr Glu Tyr Ala Trp Glu Tyr Ala Gln Asn Ile Ala Asp Leu Leu Ser 165 170 175Pro Gln Thr Gly Ala Tyr Tyr Glu Ile Trp Leu Asp Gly Glu Lys Ala 180 185 190Ile Ser Val Glu Glu His Pro Asp Val Lys Ala Ala Arg Gln Ser Asn 195 200 205Gly Asn Gly Thr Ile Val His Asp Ser Val Glu Pro Ile Tyr Gly Thr 210 215 220His Tyr Met Pro Arg Lys Phe Lys Ile Cys Val Thr Val Pro Gly Asp225 230 235 240Asn Ser Val Asp Leu Tyr Ser Gln Asp Leu Thr Leu Val Val Ile Thr 245 250 255Asn Lys Lys Gly Glu Leu Gln Gly Phe Asp Val Phe Ala Gly Gly Gly 260 265 270Leu Gly Arg Thr His Asn Lys Glu Glu Thr Phe Ala Arg Val Ala Asp 275 280 285Pro Ile Cys Tyr Val Gly Lys Asp Asp Val Tyr Asn Phe Val Lys Ala 290 295 300Val Val Ala Thr Gln Arg Asp Tyr Gly Asp Arg Thr Asp Arg Arg His305 310 315 320Ala Arg Leu Lys Tyr Leu Ile Asn Asp Trp Gly Val Asp Lys Phe Arg 325 330 335Thr Gln Val Glu Glu Tyr Phe Gly Lys Ser Val Glu Pro Phe Lys Pro 340 345 350Leu Pro Lys Phe Lys Tyr Gln Asp Phe Leu Gly Trp Asn Glu Gln Gly 355 360 365Asp Gly Lys Leu Phe Leu Gly Ile Ser Ile Glu Asn Gly Arg Val Lys 370 375 380Asp Glu Gly Ala Phe Gln Leu Lys Thr Ala Leu Arg Glu Ile Val Glu385 390 395 400Lys Phe Asn Leu Pro Ile Arg Leu Thr Gly Asn Gln Asn Leu Leu Phe 405 410 415Tyr Glu Ile Asp Pro Glu Asp Lys Ala Ala Ile Gln Glu Ile Leu Asp 420 425 430Arg Cys Gly Val Val Ala Asp Pro Ser Gln Ile Ala Ala Leu Thr Arg 435 440 445Phe Ala Met Ala Cys Pro Ala Leu Pro Thr Cys Gly Leu Ala Ile Thr 450 455 460Glu Ser Glu Arg Ala Ile Pro Gly Ile Leu Asp Arg Ile Arg Ala Leu465 470 475 480Leu Asp Lys Leu Gly Leu Gln Lys Asp His Phe Val Val Arg Met Thr 485 490 495Gly Cys Pro Asn Gly Cys Ala Arg Pro Tyr Met Ala Glu Leu Gly Phe 500 505 510Val Gly Ser Ala Pro Glu Ser Tyr Gln Val Trp Leu Gly Gly Ser Pro 515 520 525Asp Gln Thr Arg Leu Ala Gln Pro Ile Ile Glu Lys Leu His Asp Asn 530 535 540Asp Ile Glu Ser Phe Leu Glu Pro Ile Phe Ile Tyr Phe Lys Lys Phe545 550 555 560Arg Lys Gly Lys Glu Ser Phe Gly Asp Phe Cys Asp Arg Met Gly Phe 565 570 575Asp Ala Ile Arg Glu Phe Ser Ala Thr Tyr Thr Pro Gly Glu Pro Thr 580 585 590Ser Ser Gly Lys Ser Arg His Arg Val Ser Leu Arg Asp Asp Val Tyr 595 600 605Leu His Leu Lys Glu Thr Ala Glu Lys Gln Asn Arg Pro Met Thr Asp 610 615 620Leu Val His Asp Ala Leu Asp Lys Tyr Phe Gln Asn Leu625 630 63569664PRTHalothece sp. 69Met Met Glu Leu Ile Thr Val Ile Pro Ser Glu Leu Ser Val Ile Gln1 5 10 15Lys Cys Ala Ala Ile Glu Gln Lys Ala Val Met Val Ala Ser Lys Ala 20 25 30Lys Lys Ala Ser Lys Pro Ser Lys Leu Glu Gly Ile Lys Glu Asn Ser 35 40 45Asn Phe Leu Arg Glu Pro Leu Ala Thr Glu Leu Leu Glu Asp Thr Thr 50 55 60His Phe Ser Gln Asp Ala Val Gln Ile Leu Lys Phe His Gly Ser Tyr65 70 75 80Gln Gln Asp Asn Arg Asp Asn Arg Gln Lys Gly Gln Glu Lys Asp Tyr 85 90 95Gln Phe Met Leu Arg Thr Arg Asn Pro Gly Gly Phe Ile Pro Pro Glu 100 105 110Leu Tyr Leu Thr Leu Asp Asp Leu Ser Ser Glu Tyr Gly Asn Glu Thr 115 120 125Leu Arg Val Thr Thr Arg Gln Gly Phe Gln Leu His Gly Ile Leu Lys 130 135 140Lys Asn Leu Lys Glu Thr Ile Asn Arg Ile Val Arg Asn Leu Gly Ser145 150 155 160Thr Leu Gly Ala Cys Gly Asp Leu Asn Arg Asn Val Met Ala Pro Pro 165 170 175Ala Pro Phe Lys Asp Arg Lys Glu Tyr Gln Tyr Ala Trp Gln Tyr Ala 180 185 190Asp Asn Ile Ala Asp Leu Leu Arg Pro Gln Thr Glu Ala Tyr Tyr Glu 195 200 205Ile Trp Leu Asp Gly Glu Lys Phe Leu Ser Val Glu Glu Ala Pro Glu 210 215 220Val Gln Ala Ala Arg Glu Arg Asn Gly Asn Gly Thr Ile Phe His Glu225 230 235 240Gly Glu Glu Pro Ile Tyr Gly Lys Tyr Tyr Met Pro Arg Lys Phe Lys 245 250 255Cys Cys Val Thr Val Pro Gly Asp Asn Ser Ile Asp Val Tyr Thr His 260 265 270Asp Val Ser Leu Ile Val Ile Thr Asp Asp Gln Gly Glu Leu Lys Gly 275 280 285Phe Asn Val Leu Ala Gly Gly Gly Met Gly Arg Thr His Asn Lys Glu 290 295 300Glu Thr Phe Ala Arg Met Ser Asp Pro Ile Cys Tyr Val Asp Lys Ala305 310 315 320Asp Val Tyr Asp Leu Leu Lys Ala Ile Val Ala Thr Gln Arg Asp Tyr 325 330 335Gly Asp Arg Val Gln Arg Arg His Ala Arg Met Lys Tyr Leu Leu Tyr 340 345 350Asp Trp Gly Val Glu Lys Phe Gln Ser Lys Leu Glu Glu Tyr Tyr Gly 355 360 365Lys Pro Leu Gln Pro Tyr Gln Asp Leu Pro Pro Phe Glu Tyr Lys Asp 370 375 380Phe Leu Gly Trp His Glu Gln Gly Asp Gly Lys Leu Phe Phe Gly Leu385 390 395 400Ser Val Glu Asn Gly Arg Val Lys Asp Glu Gly Lys Phe Arg Leu Lys 405 410 415Thr Ala Leu Arg Lys Ile Val Glu Gln Tyr Gln Val Pro Met Arg Leu 420 425 430Thr Ala Asn His Asp Val Ile Leu Tyr Glu Ile Lys Pro Glu Asp Gln 435 440 445Ser Ala Ile Glu Lys Ile Leu Thr Asp His Gly Leu Ile Thr Asp Pro 450 455 460Asn Asn Leu Asp His Leu Leu Arg Tyr Ser Met Ala Cys Pro Ala Leu465 470 475 480Pro Thr Cys Gly Leu Ala Ile Thr Glu Ser Glu Arg Ala Leu Pro Ser 485 490 495Ile Leu Asp Arg Val Arg Asn Val Leu Lys Lys Leu Gly Met Ala Glu 500 505 510Gln Asp Leu Val Val Arg Met Thr Gly Cys Pro Asn Gly Cys Ala Arg 515 520 525Pro Tyr Met Ala Glu Leu Gly Phe Val Gly Ser Ala Pro Lys Ala Tyr 530 535 540Gln Leu Trp Leu Gly Gly Thr Pro Asn Gln Thr Ala Leu Ala Arg Pro545 550 555 560Tyr Met Glu Arg Met Pro Ile Asp Glu Leu Glu Ser Tyr Ile Glu Pro 565 570 575Met Leu Ala Phe Tyr Lys Glu Lys Arg Gln Lys Asp Glu Ser Phe Gly 580 585 590Glu Phe Cys Asn Arg Val Gly Phe Glu Ala Ile Glu Thr Tyr Val Lys 595 600 605Ser Tyr Glu Phe Lys Pro Thr Lys Thr Pro Ser Ala Gly Gly Lys Gly 610 615 620Arg Arg His Arg Ile Ser Val Tyr Glu Gly Leu His Glu Arg Leu Lys625 630 635 640Ala Ala Ala Glu Lys Arg Gly Thr Ser Met Thr Gln Leu Val Ser Glu 645 650 655Ala Leu Glu Gln Tyr Leu Asp Asp 66070427PRTRhodobacter capsulatus 70Met Ala His Ile Val Val Leu Gly Ala Gly Leu Gly Gly Ala Ile Met1 5 10 15Ala Tyr Glu Leu Arg Glu Gln Val Arg Lys Glu Asp Lys Val Thr Val 20 25 30Ile Thr Lys Asp Pro Met Tyr His Phe Val Pro Ser Asn Pro Trp Val 35 40 45Ala Val Gly Trp Arg Asp Arg Lys Glu Ile Thr Val Asp Leu Ala Pro 50 55 60Thr Met Ala Arg Lys Asn Ile Asp Phe Ile Pro Val Ala Ala Lys Arg65 70 75 80Leu His Pro Ala Glu Asn Arg Val Glu Leu Glu Asn Gly Gln Ser Val 85 90 95Ser Tyr Asp Gln Ile Val Ile Ala Thr Gly Pro Glu Leu Ala Phe Asp 100 105 110Glu Ile Glu Gly Phe Gly Pro Glu Gly His Thr Gln Ser Ile Cys His 115 120 125Ile Asp His Ala Glu Glu Ala Arg Leu Ala Phe Asp Arg Phe Cys Glu 130 135 140Asn Pro Gly Pro Ile Leu Ile Gly Ala Ala Gln Gly Ala Ser Cys Phe145 150 155 160Gly Pro Ala Tyr Glu Phe Thr Phe Ile Leu Asp Thr Ala Leu Arg Lys 165 170 175Arg Lys Ile Arg Asp Lys Val Pro Met Thr Phe Val Thr Ser Glu Pro 180 185 190Tyr Val Gly His Leu Gly Leu Asp Gly Val Gly Asp Thr Lys Gly Leu 195 200 205Leu Glu Gly Asn Leu Arg Asp Lys His Ile Lys Trp Met Thr Ser Thr 210 215 220Arg Ile Lys Arg Val Glu Lys Gly Lys Met Val Val Glu Glu Val Thr225 230 235 240Glu Asp Gly Thr Val Lys Pro Glu Lys Glu Leu Pro Phe Gly Tyr Ala 245 250 255Met Met Leu Pro Ala Phe Arg Gly Ile Lys Ala Leu Met Gly Ile Glu 260 265 270Gly Leu Val Asn Pro Arg Gly Phe Val Ile Val Asp Gln His Gln Gln 275 280 285Asn Pro Thr Phe Lys Asn Val Phe Ala Val Gly Val Cys Val Ala Ile 290 295 300Pro Pro Ile Gly Pro Thr Pro Val Pro Cys Gly Val Pro Lys Thr Gly305 310 315 320Phe Met Ile Glu Ser Met Val Thr Ala Thr Ala His Asn Ile Gly Arg 325 330 335Ile Val Arg Gly Phe Glu Ala Asp Glu Val Gly Ser Trp Asn Ala Val 340 345 350Cys Leu Ala Asp Phe Gly Asp Gln Gly Ile Ala Phe Val Ala Gln Pro 355 360 365Gln Ile Pro Pro Arg Asn Val Asn Trp Ser Ser Gln Gly Lys Trp Val 370 375 380His Trp Ala Lys Glu Gly Phe Glu Arg Tyr Phe Met His Lys Leu Arg385 390 395 400Arg Gly Thr Ser Glu Thr Phe Tyr Glu Lys Ala Ala Met Lys Phe Leu 405 410 415Gly Ile Asp Lys Leu Lys Ala Val Lys Lys Gly 420 42571436PRTOscillatoria limnetica 71Met Ala His Val Ala Val Ile Gly Ala Gly Leu Ala Gly Leu Pro Thr1 5 10 15Ala Tyr Glu Leu Arg His Ile Leu Pro Arg Gln His Arg Val Thr Leu 20 25 30Ile Ser Asp Lys Pro Asn Phe Thr Phe Thr Pro Ser Leu Pro Trp Val 35 40 45Ala Phe Asp Leu Thr Pro Leu Glu Arg Val Gln Leu Asp Val Gly Lys 50 55 60Leu Leu Lys Gly Arg Asn Ile Asp Trp Ile His Gly Lys Val Asn His65 70 75 80Ile Asp Pro Glu Asn Lys Thr Leu Val Ala Gly Glu Gln Thr Leu Glu 85 90 95Tyr Asp Tyr Val Val Val Ala Thr Gly Pro Glu Leu Ala Thr Asp Ala 100 105 110Ile Ala Gly Leu Gly Pro Glu Asn Gly Tyr Thr Gln Ser Val Cys Asn 115 120 125Pro His His Ala Leu Met Ala Lys Glu Ala Trp Gln Lys Phe Leu Gln 130 135 140Asp Pro Gly Pro Leu Val Val Gly Ala Val Pro Gly Ala Ser Cys Phe145 150 155 160Gly Pro Ala Tyr Glu Phe Ala Leu Leu Ala Asp Tyr Val Leu Arg Arg 165 170 175Lys Gly Met Arg Asp Arg Val Pro Ile Thr Phe Val Thr Pro Glu Pro 180 185 190Tyr Val Gly His Leu Gly Ile Gly Gly Met Ala Asn Ser Ala Glu Leu 195 200 205Val Thr Asp Leu Leu Glu Asn Lys Gly Ile Arg Val Leu Pro Asn Thr 210 215 220Ala Val Lys Glu Ile His Pro Glu His Met Asp Leu Asp Ser Gly Glu225 230 235 240Gln Leu Pro Phe Lys Tyr Ala Met Leu Leu Pro Pro Phe Arg Gly Pro 245 250 255Ala Phe Leu Arg Glu Ala Pro Glu Leu Thr Asn Pro Lys Gly Phe Val 260 265 270Pro Val Thr Asn Thr Tyr Gln His Pro Lys Tyr Glu Ser Val Tyr Ser 275 280 285Ala Gly Val Ile Val Glu Ile Asn Pro Pro Glu Lys Thr Pro Leu Pro 290 295 300Val Gly Val Pro Lys Thr Gly Gln Met Thr Glu Ala Met Gly Met Ala305 310 315 320Ala Ala His Asn Ile Ala Ile Lys Leu Gly Val Ser Lys Ala Lys Pro 325 330 335Val Gln Pro Thr Leu Glu Ala Ile Cys Ile Ala Asp Phe Gly Asp Thr 340 345 350Gly Ile Val Phe Val Ala Asp Pro Val Leu Pro Asp Pro Lys Thr Gly 355 360 365Thr Arg Arg Arg Ala Ile Thr Lys Arg Gly Lys Trp Val Ser Trp Ser 370 375 380Lys Thr Ala Phe Glu Thr Phe Phe Leu Ser Lys Met Arg Phe Gly Leu385 390 395 400Ala Val Pro Trp Phe Glu Arg Trp Gly Leu Arg Phe Met Gly Leu Ser 405 410 415Leu Val Glu Pro Leu Asp Thr Thr Arg Glu Thr Gly Asn Gln Ala Phe 420 425 430Ala Ser Lys Ser 43572375PRTAcidithiobacillus ferrooxidans 72Met Thr Gln Val Thr Ile Ile Gly Ala Gly Phe Gly Gly Leu Thr Ala1 5 10 15Val Arg His Leu Arg Arg Arg Met Pro Asp Ala Glu Ile Thr Val Ile 20 25 30Ala Pro Arg Ala Glu Phe Val Tyr Tyr Pro Ser Leu Ile Trp Ile Pro 35 40 45Thr Gly Leu Arg Gln Gly Glu Asn Leu Arg Ile Pro Leu Asp Arg Phe 50 55 60Phe Gln Arg Arg Arg Val Gln Phe His Gln Gly Arg Val Thr Gly Leu65 70 75 80Arg Asp Gly Gly Arg Thr Val Ile Thr Asp Gln Gly Glu Val Arg Asn 85 90 95Asp Ala Leu Ile Ile Ala Ser Gly Gly Arg Gly Ile Arg Lys Leu Pro 100 105 110Gly Ile Glu His Ser Phe Ala Ile Cys Asp Gly Ile Asp Ala Ala Glu 115 120 125Asn Ile Arg Asp Arg Leu Ala Leu Met Asp Lys Gly Thr Ile Ala Phe 130 135 140Gly Phe Ala Gly Asn Pro Leu Glu Pro Thr Ala Val Arg Gly Gly Pro145 150 155 160Val Phe Glu Leu Leu Phe Gly Ile Asp Thr Tyr Leu Arg Gln Ile Asp 165 170 175Lys Arg Gly Gln Ile Glu Leu Val Phe Phe Asn Pro Met Thr Glu Pro 180 185 190Gly Asn Arg Leu Gly Pro Lys Ala Val Glu Gly Leu Leu Ala Glu Met 195 200 205Gln Arg Arg Asp Ile Arg Thr His Leu Gly His Lys Ile Ser Gly Phe 210 215 220Ser Val Asn Lys Val Met Thr Glu Gly Gly Asp Ile Ala Ala Asp Leu225 230 235 240Ile Leu Phe Met Pro Gly Met Thr Gly Pro Asp Trp Ala Ala Asp Ser

245 250 255Gly Leu Pro Leu Ser Ala Gly Gly Phe Phe Gln Ser Asp Leu His Cys 260 265 270Thr Val Pro Asp His Pro Gly Val Phe Val Ile Gly Asp Gly Gly Ser 275 280 285Tyr Ala Gly Ser Pro Asp Trp Leu Pro Lys Gln Gly His Met Ala Asp 290 295 300Leu Gln Ala Gly Thr Ala Val His Asn Leu Leu Leu His Leu Gln Gly305 310 315 320Lys Ala Ala Asp Asn Thr Phe Arg Ser Glu Leu Ile Cys Ile Val Asp 325 330 335Thr Leu Asp Ser Gly Ile Met Val Tyr Arg Ser Pro Asn His Ala Ser 340 345 350Ile Leu Pro Asn Ser Leu Trp His Ala Ala Lys Val Ala Phe Glu Trp 355 360 365Arg Tyr Leu Leu His Tyr Arg 370 37573430PRTAquifex aeolicus 73Met Ala Lys His Val Val Val Ile Gly Gly Gly Val Gly Gly Ile Ala1 5 10 15Thr Ala Tyr Asn Leu Arg Asn Leu Met Pro Asp Leu Lys Ile Thr Leu 20 25 30Ile Ser Asp Arg Pro Tyr Phe Gly Phe Thr Pro Ala Phe Pro His Leu 35 40 45Ala Met Gly Trp Arg Lys Phe Glu Asp Ile Ser Val Pro Leu Ala Pro 50 55 60Leu Leu Pro Lys Phe Asn Ile Glu Phe Ile Asn Glu Lys Ala Glu Ser65 70 75 80Ile Asp Pro Asp Ala Asn Thr Val Thr Thr Gln Ser Gly Lys Lys Ile 85 90 95Glu Tyr Asp Tyr Leu Val Ile Ala Thr Gly Pro Lys Leu Val Phe Gly 100 105 110Ala Glu Gly Gln Glu Glu Asn Ser Thr Ser Ile Cys Thr Ala Glu His 115 120 125Ala Leu Glu Thr Gln Lys Lys Leu Gln Glu Leu Tyr Ala Asn Pro Gly 130 135 140Pro Val Val Ile Gly Ala Ile Pro Gly Val Ser Cys Phe Gly Pro Ala145 150 155 160Tyr Glu Phe Ala Leu Met Leu His Tyr Glu Leu Lys Lys Arg Gly Ile 165 170 175Arg Tyr Lys Val Pro Met Thr Phe Ile Thr Ser Glu Pro Tyr Leu Gly 180 185 190His Phe Gly Val Gly Gly Ile Gly Ala Ser Lys Arg Leu Val Glu Asp 195 200 205Leu Phe Ala Glu Arg Asn Ile Asp Trp Ile Ala Asn Val Ala Val Lys 210 215 220Ala Ile Glu Pro Asp Lys Val Ile Tyr Glu Asp Leu Asn Gly Asn Thr225 230 235 240His Glu Val Pro Ala Lys Phe Thr Met Phe Met Pro Ser Phe Gln Gly 245 250 255Pro Glu Val Val Ala Ser Ala Gly Asp Lys Val Ala Asn Pro Ala Asn 260 265 270Lys Met Val Ile Val Asn Arg Cys Phe Gln Asn Pro Thr Tyr Lys Asn 275 280 285Ile Phe Gly Val Gly Val Val Thr Ala Ile Pro Pro Ile Glu Lys Thr 290 295 300Pro Ile Pro Thr Gly Val Pro Lys Thr Gly Met Met Ile Glu Gln Met305 310 315 320Ala Met Ala Val Ala His Asn Ile Val Asn Asp Ile Arg Asn Asn Pro 325 330 335Asp Lys Tyr Ala Pro Arg Leu Ser Ala Ile Cys Ile Ala Asp Phe Gly 340 345 350Glu Asp Ala Gly Phe Phe Phe Ala Asp Pro Val Ile Pro Pro Arg Glu 355 360 365Arg Val Ile Thr Lys Met Gly Lys Trp Ala His Tyr Phe Lys Thr Ala 370 375 380Phe Glu Lys Tyr Phe Leu Trp Lys Val Arg Asn Gly Asn Ile Ala Pro385 390 395 400Ser Phe Glu Glu Lys Val Leu Glu Ile Phe Leu Lys Val His Pro Ile 405 410 415Glu Leu Cys Lys Asp Cys Glu Gly Ala Pro Gly Ser Arg Cys 420 425 43074437PRTHalothece sp. 74Met Ala His Ile Val Ile Val Gly Gly Gly Phe Gly Gly Leu Ser Ala1 5 10 15Ala Tyr Glu Leu Lys His Leu Leu His Gly Lys His Lys Ile Thr Leu 20 25 30Ile Ser Asp Glu Thr Thr Phe Thr Phe Ile Pro Ser Leu Pro Trp Val 35 40 45Ala Phe Asn Leu Arg Arg Leu Glu Asp Val Gln Leu Pro Leu Ala Pro 50 55 60Leu Leu Ala Arg Gln Gly Ile Asn Trp Gln His Gly Arg Val Thr Gly65 70 75 80Leu Asp Pro Asn Gln Lys Arg Val Ser Val Gly Glu Asp Ile Thr Phe 85 90 95Asp Tyr Asp Tyr Leu Val Ile Thr Thr Gly Ala Ser Leu Ala Tyr His 100 105 110Leu Met Ser Gly Leu Gly Pro Glu Glu Gly Tyr Thr Gln Ser Val Cys 115 120 125Asn Ala His His Ala Glu Met Ala Arg Asp Ala Trp Asp Glu Phe Leu 130 135 140Glu Asn Pro Gly Pro Leu Leu Val Gly Ala Val Pro Gly Ala Ser Cys145 150 155 160Met Gly Pro Ala Tyr Glu Phe Ala Leu Leu Ala Asp Tyr Ala Leu Arg 165 170 175Gln Glu Gly Lys Arg Asp Gln Val Pro Ile Thr Phe Ile Ser Pro Glu 180 185 190Pro Tyr Leu Gly His Leu Gly Ile Gly Gly Met Ala Asn Ser Gly Lys 195 200 205Leu Val Thr Glu Leu Met Lys Gln Arg Asn Ile Asp Trp Val Glu Asn 210 215 220Ala Glu Ile Ala Glu Ile Lys Glu Asp His Val Lys Leu Thr Asp Gly225 230 235 240Arg Glu Phe Pro Phe Asn Tyr Ser Met Phe Leu Pro Pro Phe Arg Gly 245 250 255Ala Gln Phe Leu Lys Glu Val Pro Gly Leu Thr Asp Glu Lys Gly Phe 260 265 270Leu Pro Val Leu Asp Thr Tyr Gln His Pro Asp Tyr Pro Ser Ile Tyr 275 280 285Ser Ala Gly Val Ile Thr Gln Leu Ala Ala Pro Glu Glu Thr Glu Val 290 295 300Pro Leu Gly Ala Pro Lys Thr Gly Gln Met Thr Glu Ser Met Ala Met305 310 315 320Ala Val Ala His Asn Ile Ala Arg Glu Leu Gly Glu Ile Asn Ala Arg 325 330 335Pro Val Lys Pro Ser Leu Glu Ala Ile Cys Met Ala Asp Phe Gly Asp 340 345 350Thr Gly Ile Ile Phe Ile Ala Ala Pro Val Val Pro Asp Pro Ser Val 355 360 365Gly His Arg Arg His Ala Thr Ala Leu Arg Gly Leu Trp Val Asn Trp 370 375 380Ala Lys Asn Ala Phe Glu Trp Tyr Phe Leu Ala Lys Met Arg Trp Gly385 390 395 400Thr Ala Val Pro Trp Phe Glu Lys Leu Gly Leu Tyr Leu Leu Arg Leu 405 410 415Thr Leu Val Thr Pro Ile Ser Glu Thr Pro Thr Gln Gln Lys Asp Leu 420 425 430Thr Ser Ile Lys Gly 43575431PRTAllochromatium vinosum 75Met Thr Leu Asn Arg Arg Asp Phe Ile Lys Thr Ser Gly Ala Ala Val1 5 10 15Ala Ala Val Gly Ile Leu Gly Phe Pro His Leu Ala Phe Gly Ala Gly 20 25 30Arg Lys Val Val Val Val Gly Gly Gly Thr Gly Gly Ala Thr Ala Ala 35 40 45Lys Tyr Ile Lys Leu Ala Asp Pro Ser Ile Glu Val Thr Leu Ile Glu 50 55 60Pro Asn Thr Asp Tyr Tyr Thr Cys Tyr Leu Ser Asn Glu Val Ile Gly65 70 75 80Gly Asp Arg Lys Leu Glu Ser Ile Lys His Gly Tyr Asp Gly Leu Arg 85 90 95Ala His Gly Ile Gln Val Val His Asp Ser Ala Thr Gly Ile Asp Pro 100 105 110Asp Lys Lys Leu Val Lys Thr Ala Gly Gly Ala Glu Phe Gly Tyr Asp 115 120 125Arg Cys Val Val Ala Pro Gly Ile Glu Leu Ile Tyr Asp Lys Ile Glu 130 135 140Gly Tyr Ser Glu Glu Ala Ala Ala Lys Leu Pro His Ala Trp Lys Ala145 150 155 160Gly Glu Gln Thr Ala Ile Leu Arg Lys Gln Leu Glu Asp Met Ala Asp 165 170 175Gly Gly Thr Val Val Ile Ala Pro Pro Ala Ala Pro Phe Arg Cys Pro 180 185 190Pro Gly Pro Tyr Glu Arg Ala Ser Gln Val Ala Tyr Tyr Leu Lys Ala 195 200 205His Lys Pro Lys Ser Lys Val Ile Ile Leu Asp Ser Ser Gln Thr Phe 210 215 220Ser Lys Gln Ser Gln Phe Ser Lys Gly Trp Glu Arg Leu Tyr Gly Phe225 230 235 240Gly Thr Glu Asn Ala Met Ile Glu Trp His Pro Gly Pro Asp Ser Ala 245 250 255Val Val Lys Val Asp Gly Gly Glu Met Met Val Glu Thr Ala Phe Gly 260 265 270Asp Glu Phe Lys Ala Asp Val Ile Asn Leu Ile Pro Pro Gln Arg Ala 275 280 285Gly Lys Ile Ala Gln Ile Ala Gly Leu Thr Asn Asp Ala Gly Trp Cys 290 295 300Pro Val Asp Ile Lys Thr Phe Glu Ser Ser Ile His Lys Gly Ile His305 310 315 320Val Ile Gly Asp Ala Cys Ile Ala Asn Pro Met Pro Lys Ser Gly Tyr 325 330 335Ser Ala Asn Ser Gln Gly Lys Val Ala Ala Ala Ala Val Val Ala Leu 340 345 350Leu Lys Gly Glu Glu Pro Gly Thr Pro Ser Tyr Leu Asn Thr Cys Tyr 355 360 365Ser Ile Leu Ala Pro Ala Tyr Gly Ile Ser Val Ala Ala Ile Tyr Arg 370 375 380Pro Asn Ala Asp Gly Ser Ala Ile Glu Ser Val Pro Asp Ser Gly Gly385 390 395 400Val Thr Pro Val Asp Ala Pro Asp Trp Val Leu Glu Arg Glu Val Gln 405 410 415Tyr Ala Tyr Ser Trp Tyr Asn Asn Ile Val His Asp Thr Phe Gly 420 425 43076199PRTAllochromatium vinosum 76Met Thr Gln Ser Thr Pro Arg Leu Met Leu Ala Ala Ser Val Leu Ala1 5 10 15Leu Gly Leu Ala Ser Asn Ala Gly Ala Glu Pro Thr Ala Glu Met Leu 20 25 30Thr Asn Asn Cys Ala Gly Cys His Gly Thr His Gly Asn Ser Val Gly 35 40 45Pro Ala Ser Pro Ser Ile Ala Gln Met Asp Pro Met Val Phe Val Glu 50 55 60Val Met Glu Gly Phe Lys Ser Gly Glu Ile Ala Ser Thr Ile Met Gly65 70 75 80Arg Ile Ala Lys Gly Tyr Ser Thr Ala Asp Phe Glu Lys Met Ala Gly 85 90 95Tyr Phe Lys Gln Gln Thr Tyr Gln Pro Ala Lys Gln Ser Phe Asp Thr 100 105 110Ala Leu Ala Asp Thr Gly Ala Lys Leu His Asp Lys Tyr Cys Glu Lys 115 120 125Cys His Val Glu Gly Gly Lys Pro Leu Ala Asp Glu Glu Asp Tyr His 130 135 140Ile Leu Ala Gly Gln Trp Thr Pro Tyr Leu Gln Tyr Ala Met Ser Asp145 150 155 160Phe Arg Glu Glu Arg Arg Pro Met Glu Lys Lys Met Ala Ser Lys Leu 165 170 175Arg Glu Leu Leu Lys Ala Glu Gly Asp Ala Gly Leu Asp Ala Leu Phe 180 185 190Ala Phe Tyr Ala Ser Gln Gln 19577430PRTChlorobium limicola 77Met Ser Gln Lys Phe Ser Arg Arg Asp Phe Asn Lys Leu Leu Val Ser1 5 10 15Gly Val Ala Gly Ser Ala Phe Gly Ile Phe Gly Ala Val Arg Pro Ala 20 25 30Tyr Ala Ala Gln Asn Arg Ile Val Val Ile Gly Gly Gly Phe Gly Gly 35 40 45Ala Ser Ala Ala Lys Tyr Leu Arg Lys Leu Asp Pro Ser Leu Ser Val 50 55 60Thr Leu Val Glu Pro Lys Ala Thr Phe Tyr Thr Cys Pro Phe Ser Asn65 70 75 80Trp Val Leu Gly Gly Leu Lys Asn Met Glu Asp Ile Ala Gln Thr Tyr 85 90 95Thr Val Leu Lys Asn Lys Tyr Gly Val Asn Val Ile Ala Asp Tyr Ala 100 105 110Ser Ser Ile Asp Ala Ala Lys Gly Thr Val Thr Leu Lys Ser Gly Lys 115 120 125Val Leu Asn Tyr Asp Arg Leu Ile Val Ser Pro Gly Ile Asp Phe Lys 130 135 140Trp Asn Thr Ile Glu Gly Tyr Ser Glu Ser Val Ser Asn Thr Lys Met145 150 155 160Pro His Ala Tyr Glu Ala Gly Pro Gln Thr Val Leu Leu His Lys Gln 165 170 175Leu Leu Ala Met Asn Asp Gly Gly Thr Val Leu Ile Cys Pro Pro Ala 180 185 190Asn Pro Phe Arg Cys Pro Pro Gly Pro Tyr Glu Arg Ala Ser Leu Val 195 200 205Ala His Tyr Leu Lys Glu Lys Lys Pro Lys Ser Lys Ile Ile Ile Leu 210 215 220Asp Pro Lys Asp Lys Phe Ser Lys Gln Gly Leu Phe Lys Lys Gly Trp225 230 235 240Glu Lys Leu Tyr Pro Gly Met Ile Glu Trp Arg Ser Val Ala Thr Gly 245 250 255Gly Lys Ile Ser Lys Val Asp Ala Ala Thr Met Thr Val Thr Thr Asp 260 265 270Phe Gly Val Glu Lys Gly Asp Val Ile Asn Ile Ile Pro Pro Gln Gln 275 280 285Ala Gly Lys Ile Ala Val Asp Ala Gly Leu Thr Asp Ala Ser Gly Trp 290 295 300Cys Pro Val Asn Pro Ile Thr Phe Glu Ser Thr Ile His Pro Gly Ile305 310 315 320His Val Ile Gly Asp Ala Cys Ile Ala Gly Ala Met Pro Lys Ser Gly 325 330 335Phe Ala Ala Ser Ser Gln Gly Lys Val Val Ala Ala Ser Ile Ile Arg 340 345 350Leu Cys Gln Gly Lys Val Pro Ala Pro Pro Ser Leu Val Asn Thr Cys 355 360 365Tyr Ser Leu Ile Gly Pro Gly Tyr Gly Val Ser Val Ala Gly Val Tyr 370 375 380Lys Leu Thr Ser Ala Gly Ile Val Glu Ile Pro Gly Ser Gly Gly Leu385 390 395 400Thr Pro Met Asp Ala Asp Asp Asp His Leu Asn Glu Glu Ala Thr Phe 405 410 415Ala Arg Gly Trp Tyr Asn Asn Ile Val Gln Asp Ile Trp Gly 420 425 43078123PRTChlorobium limicola 78Met Leu Gly Leu Val Phe Thr Val Val Pro Leu Phe His Ala Gly Ser1 5 10 15Thr Val Met Ala Ala Asp Ala Pro Ala Pro Ala Thr Val Ala Ala Pro 20 25 30Ala Pro Thr Pro Ala Met Asp Pro Ala Lys Met Arg Glu Arg Gly Gln 35 40 45Ile Leu Ala Leu Ser Cys Ser Gly Cys His Gly Thr Asp Gly Lys Ser 50 55 60Ser Ser Ile Met Pro Ser Ile Tyr Gly Lys Thr Thr Gly Tyr Ile Glu65 70 75 80Ser Ala Leu Leu Asp Phe Lys Ser Gly Ala Arg Met Ser Thr Val Met 85 90 95Gly Arg His Ala Lys Gly Tyr Thr Pro Glu Glu Ile His Leu Ile Ala 100 105 110Glu Tyr Phe Gly Asn Leu Ser Lys Lys Lys Asn 115 12079430PRTChlorobium tepidum 79Met Gly Asn Thr Ile Ser Arg Arg Thr Phe Asn Arg Leu Leu Ile Ser1 5 10 15Gly Leu Ala Gly Ser Ser Leu Leu Met Ser Gly Gly Pro Leu Met Ala 20 25 30Ser Ala Pro Lys Ala His Val Val Val Ile Gly Gly Gly Phe Gly Gly 35 40 45Ala Thr Val Ala Arg Tyr Leu Arg Gln Leu Asp Pro Ser Ile Ser Val 50 55 60Thr Leu Val Glu Pro Lys Lys Val Phe His Thr Cys Pro Met Ser Asn65 70 75 80Trp Val Ile Gly Gly Leu Phe Ser Met Gln Asn Thr Ala His Thr Tyr 85 90 95His Ala Leu Arg Ser Arg Tyr Gly Val Glu Val Val Gln Glu Met Ala 100 105 110Thr Gly Ile Asp Pro Val Lys Lys Thr Val Lys Leu Lys Gly Gly Arg 115 120 125Met Leu Ser Tyr Asp Arg Leu Val Val Ser Pro Gly Val Asp Phe Ile 130 135 140Trp Asp Ala Ile Glu Gly Tyr Ser Arg Asp Val Ala Glu Ser Ser Met145 150 155 160Pro Tyr Ala Trp Glu Ala Gly Pro Gln Thr Leu Leu Leu Arg Arg Gln 165 170 175Leu Leu Gly Met Lys Asp Gly Glu Asn Val Ile Ile Cys Ala Pro Lys 180 185 190Asn Pro Phe Arg Cys Pro Ala Ala Pro Tyr Glu Arg Ala Ser Leu Ile 195 200 205Ala Tyr Tyr Leu Lys Lys Ser Lys Pro Lys Ser Lys Val Ile Ile Leu 210 215 220Asp Asp Lys Glu Val Phe Thr Lys Gln Asp Leu Phe Met Leu Gly Trp225 230 235 240Asp Arg Leu Tyr Arg Gly Lys Ile Glu Trp Arg Ser Ala Ser Ala Gly 245 250 255Gly Lys Val Glu Arg Leu Asp Pro Ala Lys Met Thr Val Ala Thr Glu 260 265 270Phe Gly Asp Glu Lys

Gly Gly Val Ile Asn Val Ile Pro Pro Gln Lys 275 280 285Ala Gly Arg Ile Ala Val Glu Thr Gly Leu Ala Asp Thr Ser Gly Trp 290 295 300Cys Pro Val Asn Pro Ala Asn Phe Glu Ser Leu Gln His Pro Gly Ile305 310 315 320His Val Ile Gly Asp Ala Ala Leu Val Gly Thr Met Pro Lys Ser Gly 325 330 335Thr Ala Ala Asn Thr Gln Ala Lys Ala Leu Ala Ala Trp Leu Val Ala 340 345 350Ser Phe Gly Gly Gly Asn Ala Gly Glu His Asp Leu Ala Ser Leu Cys 355 360 365Tyr Ser Leu Leu Ala Pro Gly Tyr Ala Ile Ser Val Ala Gly Gly Tyr 370 375 380Ile Gln Ser Pro Glu Gly Ile Lys Asp Asn Pro Asp Thr Val His Leu385 390 395 400Thr Ser Met Glu Ala Thr Thr Ala Gln Leu Ala Gly Glu Ala Glu Gln 405 410 415Ala Leu Gln Trp Tyr His Asn Ile Ser Gln Asp Thr Trp Gly 420 425 43080110PRTChlorobium tepidum 80Met Leu Ala Ala Ala Pro Leu Leu Leu Ala Ser Gly Asn Gly Phe Ala1 5 10 15Thr Thr Gly Pro Ala Ala Lys Pro Ala Val Lys Pro Val Thr Glu Ser 20 25 30Arg Gly Glu Ile Leu Ser Leu Ser Cys Ala Gly Cys His Gly Thr Asp 35 40 45Gly Asn Ser Ser Ser Val Ile Pro Ser Ile Tyr Gly Lys Ser Pro Glu 50 55 60Tyr Ile Glu Thr Ala Leu Ile Asp Phe Lys Asn Gly Ser Arg Thr Ser65 70 75 80Thr Val Met Gly Arg His Ala Lys Gly Tyr Thr Gly Glu Glu Ile His 85 90 95Leu Ile Ala Glu Tyr Phe Gly Asn Leu Ser Lys Lys Asn His 100 105 11081419PRTThiobacillus denitrificans 81Met His Leu Asp Arg Arg Asp Phe Leu Lys Leu Ser Ala Ala Thr Ala1 5 10 15Leu Ala Ala Leu Pro Gly Cys Ala Ser Leu Ser Gly Thr Ala Arg Pro 20 25 30Arg Val Val Val Val Gly Ala Gly Phe Gly Gly Ala Thr Cys Ala Lys 35 40 45Tyr Leu Arg Arg Trp Gly Pro Ala Leu Asp Val Thr Leu Ile Glu Pro 50 55 60Asn Glu Arg Phe Val Ser Cys Pro Ile Ser Asn Trp Val Leu Gly Gly65 70 75 80Leu Arg Ser Met Asp Asp Ile Thr His Gly Tyr Gly Gly Leu Ala Arg 85 90 95His Gly Ile Thr Leu Ile Arg Asp Ser Val Val Ala Ile Asp Pro Asp 100 105 110Thr Arg Thr Leu Arg Thr Ala Gln Gly Leu Gln Ile Gly Tyr Glu Arg 115 120 125Leu Val Leu Ala Pro Gly Val Glu Leu Leu Thr Asp Ser Val Arg Gly 130 135 140Phe Ala Asp Ala Glu Ala Ala Gly Arg Val Val His Ala Trp Lys Ala145 150 155 160Gly Ala Gln Thr Ala Leu Leu Arg Arg Gln Leu Glu Ala Met Pro Asp 165 170 175Gly Gly Thr Phe Ile Val Ser Ile Pro Ala Ala Pro Tyr Arg Cys Pro 180 185 190Pro Gly Pro Tyr Glu Arg Ala Cys Leu Val Ala His Tyr Phe Lys Gln 195 200 205Arg Lys Pro Arg Ser Lys Ile Ile Val Leu Asp Ala Asn Pro Asp Ile 210 215 220Val Ser Lys Lys Pro Leu Phe Thr Asp Ala Trp Asn Thr Leu Tyr Pro225 230 235 240Gly Met Ile Asp Tyr Arg Pro Asn Ser Pro Ala Leu Val Val Asp Ala 245 250 255Ala Lys Met Thr Val Ser Thr Asp Phe Glu Asp Val Arg Gly Asp Val 260 265 270Leu Asn Ile Val Pro Arg Gln Arg Ala Ala Ala Val Cys Asp Leu Val 275 280 285Gly Ala Arg Asn Asp Gly Asn Lys Thr Trp Cys Thr Val Asp Phe Ala 290 295 300Thr Phe Glu Ser Thr Ala Ala Pro Gly Val His Ile Ile Gly Asp Ser305 310 315 320Met Ala Ser Pro Leu Pro Arg Ser Gly His Met Ala Thr Asn Gln Ala 325 330 335Lys Val Cys Ala Gly Ala Ile Val Asp Leu Leu Ala Asp Arg Ala Pro 340 345 350Asp Pro Ala Pro Val Ile Ala Asn Thr Cys Tyr Ser Ala Thr Ser Asp 355 360 365Ser Thr Ala Gly Tyr Val Ala His Val Tyr Arg Leu Val Pro Gly Lys 370 375 380Gly Tyr Val Ala Ala Pro Glu Gly Gly Ala Thr Thr Thr Gly Asp Ala385 390 395 400Arg Asn Phe Arg Tyr Ala Ala Ser Trp Ala Lys Asn Ile Trp Ala Glu 405 410 415Met Leu Ser8298PRTThiobacillus denitrificans 82Met Thr Pro Ser Ser Ala Val Ala Ser Cys Leu Leu Leu Ala Leu Ser1 5 10 15Gly Phe Ala Val Ala Ala Asp Arg His Thr Leu Thr Ile Ala Ala Thr 20 25 30Cys Met Ser Cys His Gly Pro Asp Gly Arg Ser Leu Gly Glu Ile Pro 35 40 45Arg Leu Asp Gly Leu Ser Arg Thr Glu Phe Val Thr Ala Leu Arg Asp 50 55 60Phe Arg Ser Gly Ala Arg Arg Ala Thr Ile Met Gln Arg Gln Ala Ser65 70 75 80Gly Tyr Thr Asp Ala Glu Ile Asp Ala Leu Gly Asp Tyr Phe Ala Thr 85 90 95Leu Lys83430PRTThiocystis violascens 83Met Lys Leu Ser Arg Arg Asp Phe Val Lys Val Ser Gly Ala Ala Thr1 5 10 15Ala Val Gly Leu Phe Gly Phe Pro Tyr Leu Ala Leu Gly Ala Thr Gln 20 25 30Lys Val Val Val Ile Gly Gly Gly Thr Gly Gly Ala Thr Ala Ala Lys 35 40 45Tyr Leu Lys Leu Ala Asp Ser Ser Ile Asp Val Thr Leu Ile Glu Pro 50 55 60Asn Glu Val Tyr Tyr Thr Cys Tyr Leu Ser Asn Glu Val Ile Gly Gly65 70 75 80Glu Arg Lys Leu Glu Ser Leu Arg Gln Thr Tyr Asp Gly Leu Lys Ala 85 90 95His Gly Val Lys Val Val His Asp Ser Ala Thr Gly Ile Asp Pro Asp 100 105 110Lys Lys Thr Val Lys Thr Ala Gly Gly Thr Glu Tyr Ser Tyr Asp Arg 115 120 125Cys Ile Val Ala Pro Gly Ile Glu Leu Leu Tyr Glu Lys Ile Asp Gly 130 135 140Tyr Ser Glu Ala Ala Ala Glu Thr Leu Pro His Ala Trp Lys Ala Gly145 150 155 160Glu Gln Thr Arg Ile Leu Arg Lys Gln Leu Glu Asp Met Lys Asp Gly 165 170 175Gly Thr Val Ile Ile Ala Ala Pro Pro Ala Pro Phe Arg Cys Pro Pro 180 185 190Gly Pro Tyr Glu Arg Ala Ser Gln Ile Ala His Tyr Leu Lys Ala His 195 200 205Lys Pro Lys Ser Lys Val Ile Ile Leu Asp Asn Ser Gln Lys Phe Ser 210 215 220Lys Gln Ala Gln Phe Thr Lys Gly Trp Glu Thr Leu Tyr Gly Phe Gly225 230 235 240Thr Asp Asn Ala Leu Ile Glu Trp Arg Pro Gly Pro Asp Ala Ala Val 245 250 255Val Lys Val Asp Ala Gly Gln Met Leu Ala Glu Thr Asn Phe Gly Asp 260 265 270Glu Ile Lys Ala Asp Val Ile Asn Val Ile Pro Pro Gln Arg Ala Gly 275 280 285Ser Ile Ala Gln Thr Ala Gly Leu Ala Asn Glu Ser Gly Trp Cys Pro 290 295 300Val Asp Val Lys Thr Phe Glu Ser Lys Leu His Lys Gly Ile His Val305 310 315 320Ile Gly Asp Ala Cys Ile Ala Thr Glu Met Pro Lys Ser Gly Tyr Ser 325 330 335Ala Asn Ser Gln Gly Lys Val Ala Ala Ala Ala Val Val Ala Leu Leu 340 345 350Lys Gly Glu Glu Pro Gly Thr Pro Ser Tyr Leu Asn Thr Cys Tyr Ser 355 360 365Ile Ile Gly Pro Ala Tyr Gly Ile Ser Val Ala Gly Val Tyr Arg Leu 370 375 380Ser Glu Asp Gly Ala Thr Ile Ala Ser Val Pro Asp Ser Gly Gly Val385 390 395 400Thr Pro Val Asp Ala Pro Asp Trp Ala Leu Ala Arg Glu Val Glu Tyr 405 410 415Ala Tyr Ser Trp Tyr Asn Asn Ile Val His Asp Ile Phe Gly 420 425 43084199PRTThiocystis violascens 84Met Ala Arg Lys Ile Leu Gln Thr Thr Leu Leu Thr Gly Ala Leu Ala1 5 10 15Leu Gly Ala Ser Ser Gly Ala Trp Ala Glu Ala Thr Gly Ala Met Leu 20 25 30Ala Asn Ser Cys Ala Gly Cys His Gly Thr His Gly Asn Ser Val Gly 35 40 45Pro Ala Ser Pro Ser Ile Ala Ala Met Asp Pro Val Val Phe Val Glu 50 55 60Thr Met Glu Glu Phe Lys Asn Gly Glu Thr Tyr Ser Thr Ile Met Gly65 70 75 80Arg Ile Ala Lys Gly Tyr Ser Thr Gly Glu Phe Glu Lys Met Ala Glu 85 90 95Tyr Phe His Ala Gln Thr Tyr Gln Pro Ala Lys Gln Ser Phe Asp Thr 100 105 110Ala Leu Ala Asp Lys Gly Ala Lys Leu His Asp Lys Tyr Cys Glu Lys 115 120 125Cys His Ala Glu Gly Gly Lys Pro Leu Val Asp Glu Glu Asp Tyr Asn 130 135 140Ile Leu Ala Gly Gln Trp Leu Pro Tyr Leu Gln Tyr Ala Met Glu Asp145 150 155 160Phe Arg Ala Asp Arg Arg Glu Met Glu Lys Lys Met Arg Thr Lys Leu 165 170 175Asn Glu Leu Leu Lys Ala Glu Gly Glu Asp Gly Ile Ala Ala Val Asn 180 185 190Ala Phe Tyr Ala Ser Gln Gln 19585308PRTAcidianus tengchongensis 85Met Pro Lys Pro Tyr Ile Ala Ile Asn Met Ala Asp Leu Lys Asn Glu1 5 10 15Pro Lys Thr Phe Glu Met Phe Ser Ala Val Gly Pro Lys Val Cys Met 20 25 30Val Thr Ala Arg His Pro Gly Phe Val Gly Phe Gln Asn His Val Gln 35 40 45Ile Gly Val Leu Pro Phe Gly Glu Arg Phe Gly Gly Ala Lys Met Asp 50 55 60Met Thr Lys Glu Ser Ser Thr Val Arg Val Leu Gln Tyr Thr Met Trp65 70 75 80Lys Asp Trp Lys Asp His Glu Glu Met His Arg Gln Asn Trp Ser Tyr 85 90 95Leu Phe Arg Leu Cys Tyr Ser Cys Ala Ser Gln Met Val Trp Gly Pro 100 105 110Trp Glu Pro Ile Tyr Glu Ile Lys Tyr Ala Asp Met Pro Ile Asn Thr 115 120 125Glu Met Thr Asp Phe Thr Ala Val Val Gly Lys Lys Phe Ala Glu Gly 130 135 140Lys Pro Leu Glu Ile Pro Val Ile Ser Gln Pro Tyr Gly Lys Arg Val145 150 155 160Val Ala Phe Gly Glu His Thr Val Ile Pro Gly Lys Glu Lys Gln Phe 165 170 175Glu Asp Ala Ile Ile Lys Thr Leu Glu Met Phe Lys Arg Ala Pro Gly 180 185 190Phe Leu Gly Ala Met Leu Leu Lys Glu Ile Gly Val Ser Gly Ile Gly 195 200 205Ser Phe Gln Phe Gly Ser Lys Gly Phe His Gln Leu Leu Glu Ser Pro 210 215 220Gly Ser Leu Glu Pro Asp Pro Asn Asn Val Met Tyr Gln Val Pro Glu225 230 235 240Ala Lys Pro Thr Pro Pro Gln Tyr Ile Val His Val Glu Trp Ala Asn 245 250 255Leu Asp Ala Leu Gln Phe Gly Met Gly Arg Val Leu Leu Ser Pro Glu 260 265 270Tyr Arg Glu Val His Asp Glu Ala Leu Asp Thr Leu Ile Tyr Gly Pro 275 280 285Tyr Ile Arg Ile Ile Asn Pro Val Met Glu Gly Thr Phe Trp Arg Glu 290 295 300Tyr Leu Asn Glu30586316PRTSulfolobus metallicus 86Met Pro Lys Pro Tyr Val Ala Ile Asn Gln Val Ile Val Lys Asn Glu1 5 10 15Pro Lys Thr Phe Glu Met Phe Gln Ser Val Gly Pro Lys Val Cys Met 20 25 30Thr Thr Ala Arg His Lys Gly Phe Val Gly Phe Gln Asn His Ile Glu 35 40 45Ile Gly Val Val Pro Met Gly Thr Arg Tyr Gly Ala Ala Lys Met Asp 50 55 60Met Leu Lys Glu Ser Ser Thr Met Gly Leu Tyr Gln Tyr Thr Met Trp65 70 75 80Lys Asp Trp Lys Asp His Glu Glu Met His Lys Gln Asn Trp Ser Ser 85 90 95Leu Phe Arg Leu Cys Tyr Ser Cys Met Ser Gln Val Val Trp Gly Pro 100 105 110Trp Glu Pro Leu Tyr Glu Ile Thr Met Ala Asp Met Pro Leu Asn Thr 115 120 125Glu Met Thr Asp Phe Thr Val Met Val Gly Gln Lys Phe Ala Ser Gly 130 135 140Asp Ala Leu Ser Leu Pro Pro Ile Ser Gln Pro Tyr Gly Lys Arg Val145 150 155 160Val Thr Tyr Gly Glu His Val Val Lys Glu Gly Met Glu Lys Glu Phe 165 170 175Glu Glu Thr Leu Ser Arg Leu Leu Pro Met Phe Lys Arg Ala Pro Gly 180 185 190Phe Leu Gly Tyr Met Val Leu Lys Glu Ile Gly Ala Ser Pro Leu Gly 195 200 205Ser Leu Gln Leu Ser Ala Lys Ser Trp His Gln Leu Leu Glu Ser Ala 210 215 220Asn Gly Met Asp Val Pro Asp Pro Asn Gly Asn Phe Ser Pro Glu Gln225 230 235 240Ala Arg Asn Lys Pro Gln Lys Tyr Val Val His Met Glu Trp Ser Asn 245 250 255Thr Asp Ala Ala Gln Phe Gly Leu Gly Arg Val Phe Leu Ser Pro Glu 260 265 270Tyr Arg Glu Ile His Asp Gln Ile Val Asp Thr Leu Ile Tyr Gly Pro 275 280 285Tyr Ile Arg Ile Leu Asn Pro Val Met Glu Gly Ser Phe Trp Arg Glu 290 295 300Tyr Leu Asn Glu Val Asn Leu Gln Lys Ala Thr Trp305 310 31587311PRTAcidithiobacillus caldus 87Met Asp Lys Asn Pro Ile Val Ala Ile Asn Gln Ser Lys Val Val Asn1 5 10 15Arg Pro Glu Ser Phe Ala Thr Met Met Lys Val Gly Pro Lys Val Cys 20 25 30Ile Thr Thr Ala Ser His Pro Gly Phe Leu Gly Phe Glu Gln Leu Leu 35 40 45Gln Thr Gly Met His Pro Met Ala Gly Arg Tyr Gly Gly Gly Ala Val 50 55 60Asp Met Arg Asp Thr Ile Asn Pro Met Ala Met Tyr Gln Tyr Thr Val65 70 75 80Trp Gln Asp Val Lys Ser His Glu Glu Met His His Asp Asn Phe Lys 85 90 95Glu Ile Tyr Glu Leu Cys Gly Ser Cys Leu Asp Met Val Ile Glu Gly 100 105 110Pro Trp Glu Pro Tyr Tyr Glu Ile Val Arg Ser Asp Leu Pro Arg Ile 115 120 125Met Gly Met Thr Asp Val Pro Ala Gln Leu Gly Ala Ala Phe Ala Ala 130 135 140Gln Lys Pro Val Ser Lys Val Ala Leu Ala Ser Gln Arg Cys Ile Ala145 150 155 160Leu Gly Asp His Trp Val Ser Asp Gly His Glu Lys Asp Phe Glu Lys 165 170 175Gly Ala Val Ala Thr Leu Thr Trp Met Lys Glu Asn Ile Pro Gly Met 180 185 190Val Gly Trp Met Ile Leu Lys Gln Phe Gly Val Ser Ala Ile Gly Ser 195 200 205Phe Gln Leu Asp Pro Glu Gly Met Met Lys Ala Thr Leu Gly Ala Asn 210 215 220Pro Pro Ala Tyr Ala Thr Asn His Gly Thr Ala Ile Pro Asp Lys Pro225 230 235 240Gln Ile Pro Gly Gln Arg Pro Thr Gln Tyr Leu Val His Met Glu Trp 245 250 255Glu Ser Pro Glu Met Ala His Met Gly Ile Gly Tyr Ala Met Val Asp 260 265 270Tyr Glu Leu Arg Gln Ile His Asn His Gly Val Leu Ala His Leu Asp 275 280 285Arg Gly Pro Tyr Tyr Leu Phe Phe Ala Pro Met Met Glu Gln Gly Gln 290 295 300Trp Arg Arg Lys Leu Val Leu305 31088306PRTSulfobacillus thermosulfidooxidans 88Met Pro Arg Pro Tyr Ile Ala Ile Asn Asp Ala Lys Val Val Asn Ala1 5 10 15Glu Ser Ser Phe Gln Ala Phe Gln Gln Val Gly Pro Lys Val Cys Met 20 25 30Val Thr Ala Asn His Pro Gly Phe Val Gly Phe Gln Asn His Val Gln 35 40 45Ile Gly Val Phe Pro Met Gly Gly Arg Tyr Gly Gly Ala Lys Met Asp 50 55 60Met His Glu Glu Leu Asn Pro Ile Gly Ile Arg Gln Tyr Thr Met Trp65 70 75 80Lys Arg Trp Glu Asp His Glu Glu Met His Tyr Gln Gln Phe Asp Ser 85

90 95Ile Phe Arg Leu Cys Ser Ser Cys Leu Gly Met Val Val Glu Gly Pro 100 105 110Trp Glu Asp Met Tyr Glu Ile Ile Ser Ser Asp Leu Pro Glu Val Ile 115 120 125Ala Met Thr Asp Val Pro Ser Lys Leu Gly Ala Ala Phe Met Ala Gly 130 135 140Gln Gln Pro Ala Pro Val Ala Met Pro Tyr Gly Gln Arg Val Ile Ala145 150 155 160Gly Ser Asp His Tyr Ile Ile Pro Gly Arg Glu Gln Glu Phe Glu Thr 165 170 175Ala Ile Thr Glu Leu Met Lys Met Phe Gln Lys Ala Pro Gly Phe Leu 180 185 190Gly Tyr Met Val Leu Lys Gln Ile Gly Ala Ser Ala Ile Gly Ser Phe 195 200 205Gln Leu Gln Pro Glu Gly Ile His Gln Ala Leu Gln Thr Leu Gly Asp 210 215 220Asn Pro Pro Lys Asn Lys Glu Gly Asn Phe Lys Leu Ile Glu Ala Lys225 230 235 240Lys Thr Pro Thr Lys Tyr Leu Val His Met Glu Trp Ser Asp Leu Asn 245 250 255Ser Ala Met Phe Gly Ile Ser Arg Val Val Ile Asn Gly Arg Tyr Arg 260 265 270Ala Gln His Asp Lys Val Leu Ala Thr Val Leu Gln Gly Pro Tyr Val 275 280 285Thr Leu Trp Ser Pro Met Met Glu Asp Thr Ser Trp Arg Glu Tyr Leu 290 295 300Asn Glu30589545PRTGordonia sp. 89Met Ser Arg Gln Ser Leu Thr Lys Ala His Ala Lys Ile Thr Glu Leu1 5 10 15Ser Trp Glu Pro Thr Phe Ala Thr Pro Ala Thr Arg Phe Gly Thr Asp 20 25 30Tyr Thr Phe Glu Lys Ala Pro Lys Lys Asp Pro Leu Lys Gln Ile Met 35 40 45Arg Ser Tyr Phe Pro Met Glu Glu Glu Lys Asp Asn Arg Val Tyr Gly 50 55 60Ala Met Asp Gly Ala Ile Arg Gly Asn Met Phe Arg Gln Val Gln Glu65 70 75 80Arg Trp Leu Glu Trp Gln Lys Leu Phe Leu Ser Ile Ile Pro Phe Pro 85 90 95Glu Ile Ser Ala Ala Arg Ala Met Pro Met Ala Ile Asp Ala Val Pro 100 105 110Asn Pro Glu Ile His Asn Gly Leu Ala Val Gln Met Ile Asp Glu Val 115 120 125Arg His Ser Thr Ile Gln Met Asn Leu Lys Lys Leu Tyr Met Asn Asn 130 135 140Tyr Ile Asp Pro Ala Gly Phe Asp Ile Thr Glu Lys Ala Phe Ala Asn145 150 155 160Asn Tyr Ala Gly Thr Ile Gly Arg Gln Phe Gly Glu Gly Phe Ile Thr 165 170 175Gly Asp Ala Ile Thr Ala Ala Asn Ile Tyr Leu Thr Val Val Ala Glu 180 185 190Thr Ala Phe Thr Asn Thr Leu Phe Val Ala Met Pro Asp Glu Ala Ala 195 200 205Ala Asn Gly Asp Tyr Leu Leu Pro Thr Val Phe His Ser Val Gln Ser 210 215 220Asp Glu Ser Arg His Ile Ser Asn Gly Tyr Ser Ile Leu Leu Met Ala225 230 235 240Leu Ala Asp Glu Arg Asn Arg Pro Leu Leu Glu Arg Asp Leu Arg Tyr 245 250 255Ala Trp Trp Asn Asn His Cys Val Val Asp Ala Ala Ile Gly Thr Phe 260 265 270Ile Glu Tyr Gly Thr Lys Asp Arg Arg Lys Asp Arg Glu Ser Tyr Ala 275 280 285Glu Met Trp Arg Arg Trp Ile Tyr Asp Asp Tyr Tyr Arg Ser Tyr Leu 290 295 300Leu Pro Leu Glu Lys Tyr Gly Leu Thr Ile Pro His Asp Leu Val Glu305 310 315 320Glu Ala Trp Asn Arg Ile Val Asp Lys His Tyr Val His Glu Val Ala 325 330 335Arg Phe Phe Ala Thr Gly Trp Pro Val Asn Tyr Trp Arg Ile Asp Ala 340 345 350Met Thr Asp Thr Asp Phe Glu Trp Phe Glu Glu Lys Tyr Pro Gly Trp 355 360 365Tyr Asn Lys Phe Gly Lys Trp Trp Glu Asn Tyr Asn Arg Leu Ala Tyr 370 375 380Pro Gly Lys Asn Lys Pro Ile Ala Phe Glu Asp Val Asp Tyr Glu Tyr385 390 395 400Pro His Arg Cys Trp Thr Cys Met Val Pro Cys Leu Ile Arg Glu Asp 405 410 415Met Val Thr Asp Lys Val Asp Gly Gln Trp Arg Thr Tyr Cys Ser Glu 420 425 430Thr Cys Ala Trp Thr Asp Lys Val Ala Phe Arg Pro Glu Tyr Glu Gly 435 440 445Arg Pro Thr Pro Asn Met Gly Arg Leu Thr Gly Phe Arg Glu Trp Glu 450 455 460Thr Leu His His Gly Lys Asp Leu Ala Asp Ile Ile Thr Asp Leu Gly465 470 475 480Tyr Val Arg Asp Asp Gly Lys Thr Leu Ile Pro Gln Pro His Leu Asp 485 490 495Leu Asp Pro Lys Lys Met Trp Thr Leu Asp Asp Val Arg Gly Ile Pro 500 505 510Phe Gly Ser Pro Asn Val Ala Leu Asn Glu Met Ser Asp Asp Glu Arg 515 520 525Glu Ala His Ile Ala Ala Tyr Met Ala Asn Lys Asn Gly Ala Val Thr 530 535 540Val54590368PRTGordonia sp. 90Met Ser Ala Pro Ala Gln Pro Arg Glu Arg Ser Phe Pro Ser Ile Glu1 5 10 15Phe Thr Asp Ala Glu Ala Asp Ala Arg Glu Phe Pro Ser Ser Arg Ser 20 25 30Arg Lys Tyr Asn Tyr Tyr Gln Pro Ser Lys Lys Arg Ala Thr Ile Tyr 35 40 45Glu Asp Val Thr Val Asp Val Gln Pro Asp Pro Glu Arg His Leu Thr 50 55 60Gln Gly Trp Ile Tyr Gly Phe Gly Asp Gly Pro Gly Gly Tyr Pro Lys65 70 75 80Glu Trp Thr Ser Ala Gln Ser Ser Asn Trp His Gln Phe Leu Asp Pro 85 90 95Asn Glu Glu Trp Glu Gln Ser Ile Tyr Arg Asn Asn Ser Ala Val Val 100 105 110His Gln Val Asp Leu Cys Leu Gln Asn Ala Lys Arg Ala Arg Ala Tyr 115 120 125Asp Gly Trp Asn Ser Ala Trp Leu Lys Phe Ile Glu Arg Asn Leu Gly 130 135 140Ala Trp Met His Ala Glu Ser Gly Met Gly Leu His Val Phe Thr Ser145 150 155 160Ile Gln Arg Ser Ala Pro Thr Asn Met Ile Asn Asn Ala Val Cys Val 165 170 175Asn Ala Ala His Lys Leu Arg Phe Ala Gln Asp Leu Ala Leu Phe Asn 180 185 190Leu Asp Leu Ser Glu Ala Glu Glu Ala Phe Asp Gly Ser Ala His Lys 195 200 205Glu Val Trp Gln Ser Ala Pro Glu Trp Gln Pro Thr Arg Glu Ala Val 210 215 220Glu Arg Leu Thr Ala Ile Gly Asp Trp Ala Glu Leu Leu Phe Cys Ser225 230 235 240Asn Ile Val Phe Glu Gln Leu Val Gly Ser Leu Phe Arg Ser Glu Leu 245 250 255Val Met Gln Val Ala Ala Arg Asn Gly Asp Tyr Ile Thr Pro Thr Ile 260 265 270Val Gly Thr Gly Glu Tyr Asp Tyr Asp Arg Asp Leu Asn Tyr Ser Arg 275 280 285Ala Leu Phe Gln Met Leu Ala Arg Asp Glu Lys His Gly Ile Asp Asn 290 295 300Arg Lys Leu Phe Ser Arg Trp Met Ser Glu Trp Phe Pro Gly Ala Ser305 310 315 320Thr Arg Ala Arg Gly Leu Gln Pro Ile Trp Ser Gln Pro Ala Asp Lys 325 330 335Ser Val Thr Phe Ser Ser Ser Leu Glu His Ala Lys Thr Lys Phe Ala 340 345 350Asp Val Leu Ala Ala Ile Asp Val Asp Ile Pro Glu Glu Leu Asn Lys 355 360 36591346PRTGordonia sp. 91Met Ala Asp Thr His Lys Ile Ser Phe Glu Pro Val Asp Ile Glu Met1 5 10 15Glu Val Gly Glu Asp Glu Thr Ile Leu Asp Ala Ala Phe Arg Gln Glu 20 25 30Ser Thr Ser Cys Thr Ala Ala Ala Arg Pro Leu Phe Gly Cys Lys Ser 35 40 45Tyr Met Leu Glu Gly Asp Val Gln Met Asp Asp Tyr Ser Thr Phe Ala 50 55 60Cys Asn Asp Ala Glu Glu Ala Glu Gly Tyr Val Leu Leu Cys Arg Thr65 70 75 80Tyr Ala Tyr Ser Asp Cys Glu Ile Glu Leu Leu Asn Phe Asp Glu Asp 85 90 95Glu Leu Leu Gly Gly Ala Pro Ile Gln Asp Val Thr Thr Lys Val Ala 100 105 110Ala Ile Glu Pro Met Thr Pro Asp Ile Val Ser Leu Lys Leu Asp Val 115 120 125Val Glu Pro Glu Ser Val Glu Phe Lys Ser Gly Gln Tyr Phe Asp Leu 130 135 140Phe Ile Pro Gly Thr Glu Asp Lys Arg Ser Phe Ser Ile Ala Thr Thr145 150 155 160Pro Ala Thr Pro Asp Arg Leu Glu Phe Leu Ile Lys Lys Tyr Pro Gly 165 170 175Gly Leu Phe Ala Gly Met Leu Thr Asp Gly Leu Ser Val Gly Gln Glu 180 185 190Ile Lys Leu Asn Gly Pro Tyr Gly Ser Cys Thr Leu Arg Asn Gly His 195 200 205Val Leu Pro Ile Val Ala Ile Gly Gly Gly Ala Gly Met Ala Pro Leu 210 215 220Leu Ser Leu Leu Arg His Ile Ser Glu Thr Gly Leu Asn Arg Pro Val225 230 235 240Arg Phe Tyr Tyr Gly Ala Arg Thr Ala Ala Asp Leu Phe Leu Leu Asp 245 250 255Glu Ile Ala Thr Leu Gly Glu Lys Ile Asp Asp Phe Ser Phe Thr Ala 260 265 270Cys Leu Ser Glu Ser Thr Asp Asn Ala Pro Glu Gly Val Thr Val Ile 275 280 285Gly Gly Asn Val Thr Asp Ile Val Asn Asp Asn Glu Ala Asp Leu Ala 290 295 300Arg Thr Glu Val Tyr Phe Cys Ala Pro Pro Pro Met Val Asp Ala Ala305 310 315 320Leu Ala Leu Ala Glu Gln His Ser Val Pro His Asp Gln Ile Phe Tyr 325 330 335Asp Lys Phe Thr Ser Pro Ala Phe Asp Ser 340 34592111PRTGordonia sp. 92Met Gln Phe Gly Ala Asp Thr Glu Phe Ser Asn Met Cys Gly Val Thr1 5 10 15Leu Met Asn Thr Pro Ile Gly Arg Val Val Ala Asp Val Met Gly Ala 20 25 30Lys Asp Gly Val Glu Leu Thr Glu Tyr Pro Ser Met Ile Arg Val Asp 35 40 45Gly Val Asn Arg Leu Asp Phe Asp Tyr Asp Glu Leu Thr Asp Ala Leu 50 55 60Gly Gln Asp Phe Asp Gly Ser Ile Phe Glu Glu Ile Ser Ser Thr His65 70 75 80Tyr Gly Arg Met Val His Leu Asp Asp Lys Thr Ile Leu Phe Ala Ser 85 90 95Pro Glu Asp Ala Ala Glu Phe Ile Gly Phe Asp Leu Thr Ala Ser 100 105 11093568PRTThauera butanivorans 93Met Ile Ser Leu Asn Cys Lys Lys Thr Thr Thr Gly Leu Thr Ala His1 5 10 15Leu Ala Leu Val Arg Gly Met Lys Ala Leu Ala Glu Leu Val Gly Thr 20 25 30Thr Leu Gly Pro Gln Gly Arg His Val Met Leu Ala His Arg Ala Gly 35 40 45Leu Ala Pro His Val Ser Lys Asp Gly Val Glu Val Ala Arg His Leu 50 55 60Ser Leu Pro Asp Ser Glu Glu Glu Leu Gly Val Arg Leu Leu Arg Asn65 70 75 80Ala Ala Val Ala Val Ser Glu Ser Phe Gly Asp Gly Thr Ser Thr Ala 85 90 95Thr Val Phe Thr Ala Asp Leu Ala Val Arg Ala Leu Lys Leu Ile Gly 100 105 110Ala Gly Ala Asp Thr Leu Glu Val Arg Arg Gly Leu Gly Leu Ala Ala 115 120 125Tyr Ala Ala Leu Val Ala Leu Asn Asp Met Ala Arg Arg Ala Asp Arg 130 135 140Gly Met Leu Thr Ala Val Ala Gln Thr Ala Ala Asn Gly Asp Arg Arg145 150 155 160Val Ala Asp Leu Leu Val Glu Ala Phe Glu Arg Val Gly Ala Glu Gly 165 170 175Thr Ile Glu Val Glu Met Gly Asn Ser Val Glu Asp Val Leu Glu Val 180 185 190Ala Gln Gly Ser Tyr Phe Asp Thr Val Pro Leu Val Thr Ala Leu Leu 195 200 205Pro Pro Thr Gly Gln Val Glu Phe Ala Arg Pro Leu Ile Leu Phe His 210 215 220Cys Asp Ala Ile Glu Thr Ala Asp Glu Ile Leu Pro Ala Leu Glu Leu225 230 235 240Ala Arg Ser Ser Arg Arg Pro Leu Leu Ile Leu Ala Asp Ser Val Gly 245 250 255Ile Asp Val Glu Thr Leu Leu Val Arg Asn Gln Asn Glu Gly Thr Leu 260 265 270Ala Val Ala Val Val Arg Ala Pro Met Tyr Gly Asp Thr Arg Arg Glu 275 280 285Ala Leu Leu Asp Leu Thr Ser Lys Phe Gly Gly Thr Ala Phe Gly Arg 290 295 300Glu Gly Phe Val Glu Phe Ala Leu Arg Ser Leu Gly Ser Leu Ser Glu305 310 315 320Gly Asp Leu Gly Gln Ala Asp Glu Ala Ile Leu Glu Ala Asp Gly Val 325 330 335Thr Leu Arg Gly Ala Gly Asn Asn Pro Ser Ala Leu Glu Asp Arg Ile 340 345 350Ala Leu Val Arg Ala Glu Leu Asp Arg Gly Asp Val Ser Val Gly Asp 355 360 365Ser Pro Ser Ala Lys Leu Asp Tyr Ile Glu Lys Arg Lys Glu Arg Leu 370 375 380Lys Leu Leu Ala Ala Gly Ser Ala Lys Leu His Ile Gly Gly Pro Thr385 390 395 400Asp Val Glu Ile Lys Thr Arg Leu Pro Leu Ala Glu Asn Ala His Arg 405 410 415Ala Leu Leu Ala Ala Ala Lys Ser Gly Val Leu Pro Gly Gly Gly Val 420 425 430Ala Met Ile Arg Ala Ala Glu Lys Val Gln Gln Glu Met Gly Arg Leu 435 440 445Glu Gly Asp Val Ala Ser Gly Ala Ser Ile Phe Leu Gln Ser Leu Asp 450 455 460Thr Pro Ile Arg Trp Ile Ala Arg Asn Ala Gly Leu Arg Pro Asp Glu465 470 475 480Val Leu Ala Arg Thr Leu Ala Asn Glu Ser Asp Phe Tyr Gly Leu Asn 485 490 495Ala Met Thr Gly Arg Tyr Gly Asp Leu Ala Glu Asp Gly Val Leu Asp 500 505 510Ala Leu Asp Met Val Thr Asp Val Ile Arg Val Ala Val Ser Val Val 515 520 525Gly Ser Met Leu Gly Val Gly Ala Leu Val Thr Arg Ala Ser Pro Lys 530 535 540Pro Ala Pro Glu Arg Phe Lys Gly Thr Glu Arg Val His Asp Lys Leu545 550 555 560Met Arg Glu Gly Gly Phe Asp Glu 56594364PRTThauera butanivorans 94Met Leu Met Gln Gln Tyr Lys Ile Val Ala Arg Phe Glu Asp Gly Val1 5 10 15Thr Tyr Glu Tyr Asp Cys Gly Glu Asp Glu Asn Leu Leu Ala Ala Ala 20 25 30Leu Arg Gln Asn Val Arg Leu Leu Cys Gln Cys Arg Lys Ala Phe Cys 35 40 45Gly Ser Cys Lys Ala Leu Cys Ser Glu Gly Asp Tyr Glu Leu Gly Asp 50 55 60His Ile Asn Val Gln Val Leu Pro Pro Asp Glu Glu Glu Asp Gly Val65 70 75 80Val Val Thr Cys Asp Thr Phe Pro Arg Ser Asp Leu Val Leu Glu Phe 85 90 95Pro Tyr Thr Ser Asp Arg Leu Gly Thr Val Thr Ala Thr Glu Ala Lys 100 105 110Thr Ser Val Val Ser Val Glu Arg Leu Ser Ser Thr Val Tyr Arg Leu 115 120 125Val Leu Gln Ala Leu Asp Ala Glu Gly Met Pro Ala Arg Phe Asp Phe 130 135 140Val Pro Gly Gln Tyr Val Glu Ile Ser Thr Ala Asp Ser Leu Glu Thr145 150 155 160Arg Ala Phe Ser Leu Ala Asn Leu Pro Asn Asp Ala Gly Leu Leu Glu 165 170 175Phe Leu Ile Arg Leu Val Pro Gly Gly Tyr Tyr Ala Ala Tyr Leu Glu 180 185 190Gln Arg Ala Ala Ala Gly Gln Thr Ile Asn Val Lys Gly Pro Phe Gly 195 200 205Glu Phe Val Leu Arg Glu His Glu Leu Val Glu Asp Phe Thr Leu Pro 210 215 220Ala Asp Ser Pro Ala Arg Gly Gly Thr Ile Ala Phe Leu Ala Gly Ser225 230 235 240Thr Gly Leu Ala Pro Leu Ala Ser Met Leu Arg Glu Leu Gly Arg Arg 245 250 255Gly Phe Asn Gly Glu Cys His Leu Phe Phe Gly Met Gln Asp Thr Ala 260 265 270Thr Met Phe Tyr Glu Lys Glu Leu Arg Asp Ile Lys Arg Thr Leu Pro 275 280 285Gly Leu Thr Leu His Leu Ala Leu Met Val Pro Ser Ala Glu Trp Glu 290 295

300Gly Tyr Arg Gly Asn Ala Val Ala Ala Phe Lys Glu His Phe Ala Ala305 310 315 320Ser Ser Gln Ile Pro Glu Asn Val Tyr Leu Cys Gly Pro Gly Pro Met 325 330 335Ile Ala Ala Ala Leu Gly Ala Cys Arg Glu Leu Gly Ile Pro Asp Asn 340 345 350Arg Val His Arg Glu Glu Phe Val Ala Ser Gly Gly 355 36095168PRTThauera butanivorans 95Met Ser Lys Gln Val Trp Tyr Asn Thr Pro Val Arg Asp Glu Trp Ile1 5 10 15Glu Lys Ile Thr Ala Ile Arg Thr Ala Arg Glu Gly Thr Asp Met Leu 20 25 30Ala Arg Phe Arg Ala Glu His Thr Gly Pro Asp Arg Thr Thr Tyr Asp 35 40 45Leu Lys Lys Glu Tyr Asn Trp Ile Glu Ser Arg Ile Glu Met Arg Val 50 55 60Ser Gln Leu His Ala Glu Ala Thr Ala Ser Asp Glu Asp Leu Leu Thr65 70 75 80Lys Thr Ile Asp Gly Arg Cys Ala Lys Glu Val Ala Ala Glu Trp Leu 85 90 95Lys Lys Ala Ala Asp Ile Asp Cys His Tyr Glu Met Glu Arg Leu Cys 100 105 110Val Ala Phe Arg Lys Ala Cys Lys Pro Pro Met Met Pro Ile Asn Phe 115 120 125Phe Ala Pro Ala Glu Lys Glu Leu Val Ala Lys Leu Met Lys Leu Arg 130 135 140Ala Pro Thr Tyr Leu Thr Thr Ser Leu Asp Glu Leu Arg Glu Ala Arg145 150 155 160Gly Val Thr Met Ile Ser Val Gln 1659687PRTThauera butanivorans 96Met Lys Glu Ala Pro Ala Ile Pro Asp Leu Pro Gly Leu Pro Glu Thr1 5 10 15Val Gly Glu Pro Thr Leu Val Leu Glu Glu Asp Gly Phe Arg Val Phe 20 25 30Ala Thr Glu Leu Thr Ile Met Trp Arg Trp Asp Ile Tyr Asn Gly Asp 35 40 45Ala His Val His Thr Gly Cys Ala Gln His Pro Glu Ser Cys Val Val 50 55 60Ala Ala Arg Ser Lys Ile Arg Phe Leu Arg Arg Pro Thr Val Ala Met65 70 75 80Leu Leu Gly Gly Glu Gly Gln 8597137PRTThauera butanivorans 97Met Ser Asn Val Asn Ala Tyr His Ala Gly Thr Asn Gly Lys Glu Gly1 5 10 15Gln Asp Phe Ile Asp Asp Phe Leu Ser Glu Glu Asn Ser Ala Leu Pro 20 25 30Thr Ser Glu Ala Val Val Leu Ala Leu Met Lys Thr Glu Glu Ile Asp 35 40 45Ala Val Val Asp Glu Met Ile Lys Pro Gln Met Glu Asp Asn Pro Thr 50 55 60Ile Ala Val Glu Asp Arg Gly Gly Tyr Trp Trp Ile Lys Ala Asn Gly65 70 75 80Lys Ile Val Ile Asp Cys Asp Glu Ala Thr Glu Leu Leu Gly Lys Lys 85 90 95Tyr Thr Val Tyr Asp Leu Leu Val Asn Val Ser Thr Thr Val Gly Arg 100 105 110Ala Met Thr Leu Gly Asn Gln Phe Ile Ile Thr Asn Glu Leu Leu Gly 115 120 125Leu Glu Thr Lys Val Glu Ser Val Tyr 130 13598391PRTThauera butanivorans 98Met Ser Thr Asn Ile Phe Thr Arg Gly Met Val Asp Pro Glu Arg Gln1 5 10 15Ala Cys Ile Gln Glu Val Val Pro Lys Ala Pro Leu Glu Thr Lys Arg 20 25 30Asp His Ile Pro Phe Ala Lys Arg Gly Trp Arg Arg Leu Thr Glu Tyr 35 40 45Glu Ala Val Met Leu His Ala Gln Asn Ser Leu Asp Ala Val Pro Gly 50 55 60Ser Gln Glu Val Gly Glu Val Val Gln Lys Trp Pro Gly Gly Arg Pro65 70 75 80Asn Tyr Gly Val Glu Ser Thr Ala Ala Leu Ser Ser Asn Trp Phe His 85 90 95Phe Arg Asp Pro Ser Lys Arg Trp Phe Met Pro Tyr Val Lys Gln Lys 100 105 110Asn Glu Glu Gly Gln Thr Ala Glu Arg Ala Met Lys Ser Trp Ala Glu 115 120 125Gly Gly Asp Ala Glu Met Met Asn Ala Ala Trp Arg Glu His Ile Leu 130 135 140Ala Arg His Tyr Gly Ala Phe Val Tyr Asn Glu Tyr Gly Leu Phe Ser145 150 155 160Ala His Ser Thr Thr Val Tyr Gly Gly Leu Ser Asp Leu Ile Lys Thr 165 170 175Trp Ile Ala Glu Ala Ala Phe Asp Lys Asn Asp Ala Gly Gln Met Ile 180 185 190Gln Met Gln Arg Val Leu Leu Ser Lys Val Phe Pro Gly Phe Asp Ala 195 200 205Asp Leu Ala Glu Ala Lys Gln Ala Trp Thr Glu Asp Lys Ser Trp Lys 210 215 220Pro Ala Arg Glu Phe Val Glu His Ile Trp Ala Glu Thr Tyr Asp Trp225 230 235 240Val Glu Gln Leu Trp Ala Ile His Ala Val Tyr Asp His Ile Phe Gly 245 250 255Gln Phe Val Arg Arg Glu Phe Phe Gln Arg Leu Gly Gly Ile His Gly 260 265 270Asp Thr Leu Thr Pro Phe Ile Gln Asn Gln Ala Leu Thr Tyr His Leu 275 280 285Gln Ala Arg Asp Gly Val Thr Ala Leu Cys Phe Lys Phe Leu Ile Glu 290 295 300Asp Glu Pro Val Tyr Ala Gln His Asn Arg Arg Tyr Leu Arg Ala Trp305 310 315 320Thr Gly Arg Tyr Leu Pro Gln Val Gly Arg Ala Leu Lys Ala Phe Leu 325 330 335Ala Ile Tyr Lys Glu Val Pro Val Lys Ile Asp Gly Val Thr Cys Arg 340 345 350Glu Gly Val Arg Ala Ser Val Glu Arg Val Val Asp Asp Trp Ala Ala 355 360 365Arg Phe Ala Glu Pro Ile Asn Phe Lys Phe Asn Arg Ala Ala Phe Ile 370 375 380Asp Asp Val Leu Ser Gly Tyr385 39099530PRTThauera butanivorans 99Met Ser Ala Asn Met Ala Val Lys Gln Ala Leu Lys Ala Asn Pro Val1 5 10 15Pro Ser Ser Val Asp Pro Gln Glu Val His Lys Trp Leu Gln Asp Phe 20 25 30Thr Trp Asp Phe Lys Gly Lys Thr Ala Lys Tyr Pro Thr Lys Tyr Glu 35 40 45Met Asp Val Asn Thr Arg Glu Gln Phe Lys Leu Thr Ala Lys Glu Tyr 50 55 60Ala Arg Met Glu Ser Ile Lys Glu Glu Arg Gln Tyr Gly Thr Leu Leu65 70 75 80Asp Gly Leu Asp Arg Leu Asp Ala Gly Asn Lys Val His Pro Lys Trp 85 90 95Gly Glu Val Met Lys Leu Val Ser Asn Phe Leu Glu Thr Gly Glu Tyr 100 105 110Gly Ala Ile Ala Gly Ser Ala Leu Leu Trp Asp Thr Ala Gln Ser Pro 115 120 125Glu Gln Arg Asn Gly Tyr Leu Ala Gln Val Ile Asp Glu Ile Arg His 130 135 140Val Asn Gln Thr Ala Tyr Val Asn Tyr Tyr Tyr Gly Lys His Tyr Tyr145 150 155 160Asp Pro Ala Gly His Thr Asn Met Arg Gln Leu Arg Ala Ile Asn Pro 165 170 175Leu Tyr Pro Gly Val Lys Arg Ala Phe Gly Glu Gly Phe Leu Ala Gly 180 185 190Asp Ala Val Glu Ser Ser Ile Asn Leu Gln Leu Val Gly Glu Ala Cys 195 200 205Phe Thr Asn Pro Leu Ile Val Ser Leu Thr Glu Trp Ala Ala Ala Asn 210 215 220Gly Asp Glu Ile Thr Pro Thr Val Phe Leu Ser Ile Glu Thr Asp Glu225 230 235 240Leu Arg His Met Ala Asn Gly Tyr Gln Thr Ile Val Ser Ile Met Asn 245 250 255Asn Pro Glu Thr Met Lys Tyr Leu Gln Thr Asp Leu Asp Asn Ala Phe 260 265 270Trp Thr Gln His Lys Phe Leu Thr Pro Phe Val Gly Val Ala Leu Glu 275 280 285Tyr Gly Ser Lys Tyr Lys Val Glu Pro Trp Ala Lys Ser Trp Asn Arg 290 295 300Trp Val Tyr Glu Asp Trp Ala Gly Ile Trp Leu Gly Arg Leu Gln Gln305 310 315 320Phe Gly Val Lys Thr Pro Lys Cys Leu Pro Asp Ala Lys Lys Asp Ala 325 330 335Val Trp Ala His His Asp Leu Ala Leu Leu Ala Leu Ala Leu Trp Pro 340 345 350Leu Thr Gly Ile Arg Met Glu Leu Pro Asp Ser Leu Ala Met Glu Trp 355 360 365Phe Glu Ala Asn Tyr Pro Gly Trp Tyr Asn His Tyr Gly Lys Ile Tyr 370 375 380Glu Glu Trp Arg Ala Ala Gly Phe Glu Asp Pro Lys Ser Gly Phe Cys385 390 395 400Gly Ala Leu Trp Leu Met Glu Arg Gly His Gly Ile Phe Val Asp His 405 410 415Ala Ser Gly Leu Pro Phe Cys Pro Ser Leu Ala Lys Ser Ser Ile Lys 420 425 430Pro Arg Phe Thr Glu Tyr Asn Gly Lys Arg Tyr Ala Phe Ala Glu Pro 435 440 445Tyr Gly Glu Arg Gln Trp Leu Leu Glu Pro Glu Arg Tyr Glu Phe Gln 450 455 460Asn Phe Phe Glu Gln Phe Glu Gly Trp Glu Leu Ser Asp Leu Val Lys465 470 475 480Ala Ala Gly Gly Val Arg Ser Asp Gly Lys Thr Leu Ile Ala Gln Pro 485 490 495His Leu Arg Asp Thr Asp Met Trp Thr Leu Asp Asp Leu Lys Arg Ile 500 505 510Asn Leu Thr Ile Pro Asp Pro Met Lys Ile Leu Asn Trp Gln Pro Val 515 520 525Ala Gln 530100255PRTNocardioides sp. 100Met Thr Val Ala Thr Glu Ser Val Glu Thr Pro Gln His Pro Pro Pro1 5 10 15Thr Arg Met Ile Gly Arg Arg Trp Asp Ile Leu Leu Val Ala Ser Ala 20 25 30Leu Leu Leu Val Ala Gly Ala Ala His Leu Asn Asn Met Leu Phe Val 35 40 45Gly Asp Trp Ser Phe Trp Val Asp Trp Lys Asp Arg Gln Trp Trp Pro 50 55 60Leu Leu Thr Pro Ala Leu Ser Ile Ile Val Pro Ala Ala Leu Gln Tyr65 70 75 80Ile Thr Trp Thr Gln Leu Arg Leu Pro Phe Gly Ala Thr Leu Gly Ala 85 90 95Val Ala Leu Val Leu Ala Glu Trp Val Ser Arg Tyr Phe Ser Phe Glu 100 105 110Trp Trp Ala Asn Ile Pro Leu Asn Phe Thr Trp Pro Glu Thr Leu Val 115 120 125Leu Ala Ala Val Val Leu Asp Val Ile Leu Leu Ile Thr Arg Ser Phe 130 135 140Phe Leu Thr Ser Leu Phe Gly Gly Leu Met Trp Gly Phe Val Phe Trp145 150 155 160Phe Phe Asn Trp Pro Ala Leu Ala Pro Phe Met Gln Pro Val Glu Phe 165 170 175His Gly Tyr Ile Val Thr Val Ala Asp Val Met Ser Phe Asn Ile Val 180 185 190Arg Thr Gln Thr Pro Glu Tyr Leu Arg Ile Ile Glu Glu Gly Arg Leu 195 200 205Arg Ala Leu Val Glu Asn Ile Thr Met Val Val Ser Phe Phe Ala Gly 210 215 220Met Leu Ser Ala Ala Val Tyr Trp Phe Gly Leu Ala Ile Gly Lys Phe225 230 235 240Leu Ala Val Ala Pro Ala Gly Arg Phe Phe Arg Leu Gly Ser Asp 245 250 255101418PRTNocardioides sp. 101Met Arg Leu Met Arg Ile Ser Met Asn Pro Glu Ser Thr Gly His Leu1 5 10 15Leu Arg Arg Leu Phe Arg Leu Ala Val Gly Val Leu Ala Leu Leu Val 20 25 30Leu Pro Val Ser Pro Ala Ser Ala His Gly Glu Glu Ser Gln Gln Ala 35 40 45Phe Gln Arg Thr Ser Thr Val Val Phe Tyr Asp Val Lys Phe Ser Asp 50 55 60Asp Thr Val Asp Val Gly Glu Ser Val Thr Ile Thr Gly Met Val Arg65 70 75 80Val Met Lys Ser Trp Pro Asp His Thr Leu Glu Pro Pro Glu Met Gly 85 90 95Tyr Leu Thr Val Ser Thr Pro Gly Pro Val Phe Tyr Val Gln Glu Arg 100 105 110Glu Met Ser Gly Glu Phe Thr Pro Gln Ser Val Arg Ile Glu Lys Gly 115 120 125Ala Thr Tyr Pro Phe Lys Leu Val Ile Lys Ala Arg Gln Pro Gly Thr 130 135 140Trp His Val His Pro Gly Phe Gly Val Glu Gly Ala Gly Thr Leu Val145 150 155 160Gly Ala Gly Lys Asp Ile Thr Val Asn Asp Thr Gly Val Phe Glu Asn 165 170 175Thr Val Thr Leu Ala Asn Gly Thr Thr Val Asp Leu Glu Thr Phe Gly 180 185 190Leu Gly Arg Val Val Thr Trp His Leu Ile Ser Leu Val Val Gly Leu 195 200 205Ala Trp Leu Leu Phe Trp Leu Arg Arg Pro Ile Leu Asp Arg Ala Met 210 215 220Ala Ile Ser Glu Gly Arg Gly Ala Thr Leu Ile Thr Arg Ser Asp Arg225 230 235 240Arg Ile Gly Ile Gly Phe Ala Val Val Ala Leu Val Val Gly Thr Gly 245 250 255Gly Tyr Ala Tyr Ala Glu Met Thr Gln Ser Ser Ser Val Pro Leu Gln 260 265 270Val Val Arg Thr Thr Pro Val Pro Leu Ala Glu Glu Glu Val Ser Gly 275 280 285Ala Val Ala Pro Glu Ile Glu Ser Ile Arg Phe Asn Ala Glu Ala Asp 290 295 300Thr Leu Thr Met Lys Leu Arg Val Glu Asn Thr Gly Ala Ala Ala Val305 310 315 320Arg Leu Gln Arg Val Gln Phe Gly Asp Val Glu Phe Val Ser Pro Ser 325 330 335Phe Ala Ser Ala Ala Asp Pro Asp Ala Gln Ala Met Thr Val Thr Pro 340 345 350Asp Gln Ala Ile Glu Pro Gly Gly Ser Ala Thr Phe Thr Val Glu Ile 355 360 365Gln Ser Glu Asp Leu Ile Val Arg Ser Leu Val Pro Val Asn Glu Ala 370 375 380Glu Leu Arg Val Thr Gly Leu Leu Phe Phe Glu Asp Glu Thr Gly Glu385 390 395 400Gln Val Val Ser Glu Val Asn Glu Leu Thr Ser Ala Ile Leu Gln Asp 405 410 415Phe His102247PRTNocardioides sp. 102Met Leu Leu Trp Arg Trp Tyr Gln Gln Ala Phe Ala Phe Thr Lys Gly1 5 10 15Leu Asp Arg Thr Leu Pro Glu Phe Asn Gln Phe Trp Gly Thr Met Phe 20 25 30Leu Val Asn Met Thr Val Leu Pro Leu Leu Ala Gly Ala Trp Tyr Val 35 40 45Tyr Leu Trp Ser Ser Ser Arg Lys Leu Ala Pro Pro Ala Asn Gly Ala 50 55 60Glu Glu Ala Gly Arg Ile Trp Arg Leu Trp Leu Leu Val Ala Gly Phe65 70 75 80Thr Ala Ala Val Tyr Trp Gly Gly Ser Tyr Phe Ala Glu Gln Asp Ala 85 90 95Ser Trp His Gln Val Thr Met Arg Asp Ser Ala Phe Thr Pro Ser His 100 105 110Ala Ile Leu Phe Tyr Gly Val Phe Pro Leu Met Ile Tyr Met Ala Thr 115 120 125Gly Thr Tyr Leu Tyr Ala Arg Thr Arg Leu Pro His Leu Tyr Gly Gly 130 135 140Lys Ala Ile Pro Val Ser Phe Ala Leu Met Ile Gly Gly Ser Ser Leu145 150 155 160Leu Val Phe Gln Val Ala Met Asn Glu Phe Gly His Ser Phe Trp Glu 165 170 175Ala Glu Glu Leu Phe Ser Ala Ser Leu His Trp Pro Phe Val Ile Phe 180 185 190Gly Tyr Leu Leu Ala Ala Thr Phe Ser Val Trp Phe Glu Thr Thr Pro 195 200 205Arg Leu Phe Ala Ile Ala Arg Gln Glu Arg Asp Ala Leu Val Ala Ala 210 215 220Glu Gln Gln Met Thr Pro Ala Ala Pro Ala Gly Glu Ser Asn Thr Ala225 230 235 240Thr Thr Gln Pro Thr Ser Ile 245103497PRTXanthobacter autotrophicus 103Met Ala Leu Leu Asn Arg Asp Asp Trp Tyr Asp Ile Ala Arg Asp Val1 5 10 15Asp Trp Thr Leu Ser Tyr Val Asp Arg Ala Val Ala Phe Pro Glu Glu 20 25 30Trp Lys Gly Glu Lys Asp Ile Cys Gly Thr Ala Trp Asp Asp Trp Asp 35 40 45Glu Pro Phe Arg Val Ser Phe Arg Glu Tyr Val Met Val Gln Arg Asp 50 55 60Lys Glu Ala Ser Val Gly Ala Ile Arg Glu Ala Met Val Arg Ala Lys65 70 75 80Ala Tyr Glu Lys Leu Asp Asp Gly His Lys Ala Thr Ser His Leu His 85 90 95Met Gly Thr Ile Thr Met Val Glu His Met Ala Val Thr Met Gln Ser 100 105 110Arg Phe Val Arg Phe Ala Pro Ser Ala Arg Trp Arg Ser Leu Gly Ala 115 120 125Phe Gly Met Leu Asp Glu Thr Arg His Thr Gln Leu Asp Leu Arg Phe 130 135 140Ser His Asp Leu Leu Asn Asp Ser Pro Ser Phe

Asp Trp Ser Gln Arg145 150 155 160Ala Phe His Thr Asp Glu Trp Ala Val Leu Ala Thr Arg Asn Leu Phe 165 170 175Asp Asp Ile Met Leu Asn Ala Asp Cys Val Glu Ala Ala Leu Ala Thr 180 185 190Ser Leu Thr Leu Glu His Gly Phe Thr Asn Ile Gln Phe Val Ala Leu 195 200 205Ala Ser Asp Ala Met Glu Ala Gly Asp Val Asn Phe Ser Asn Leu Leu 210 215 220Ser Ser Ile Gln Thr Asp Glu Ala Arg His Ala Gln Leu Gly Phe Pro225 230 235 240Thr Leu Asp Val Met Met Lys His Asp Pro Lys Arg Ala Gln Gln Ile 245 250 255Leu Asp Val Ala Phe Trp Arg Ser Tyr Arg Ile Phe Gln Ala Val Thr 260 265 270Gly Val Ser Met Asp Tyr Tyr Thr Pro Val Ala Lys Arg Gln Met Ser 275 280 285Phe Lys Glu Phe Met Leu Glu Trp Ile Val Lys His His Glu Arg Ile 290 295 300Leu Arg Asp Tyr Gly Leu Gln Lys Pro Trp Tyr Trp Asp Thr Phe Glu305 310 315 320Lys Thr Leu Asp His Gly His His Ala Leu His Ile Gly Thr Trp Phe 325 330 335Trp Arg Pro Thr Leu Phe Trp Asp Pro Asn Gly Gly Val Ser Arg Glu 340 345 350Glu Arg Arg Trp Leu Asn Gln Lys Tyr Pro Asn Trp Glu Glu Ser Trp 355 360 365Gly Val Leu Trp Asp Glu Ile Ile Ser Asn Ile Asn Ala Gly Asn Ile 370 375 380Glu Lys Thr Leu Pro Glu Thr Leu Pro Met Leu Cys Asn Val Thr Asn385 390 395 400Leu Pro Ile Gly Ser His Trp Asp Arg Phe His Leu Lys Pro Glu Gln 405 410 415Leu Val Tyr Lys Gly Arg Leu Tyr Thr Phe Asp Ser Asp Val Ser Lys 420 425 430Trp Ile Phe Glu Leu Asp Pro Glu Arg Tyr Ala Gly His Thr Asn Val 435 440 445Val Asp Arg Phe Ile Gly Gly Gln Ile Gln Pro Met Thr Ile Glu Gly 450 455 460Val Leu Asn Trp Met Gly Leu Thr Pro Glu Val Met Gly Lys Asp Val465 470 475 480Phe Asn Tyr Arg Trp Ala Gly Asp Tyr Ala Glu Asn Arg Ile Ala Ala 485 490 495Glu10488PRTXanthobacter autotrophicus 104Met Ser Leu Phe Pro Ile Val Gly Arg Phe Val Gly Asp Phe Val Pro1 5 10 15His Leu Val Ala Val Asp Thr Ser Asp Thr Ile Asp Gln Ile Ala Glu 20 25 30Lys Val Ala Val His Thr Val Gly Arg Arg Leu Pro Pro Asp Pro Thr 35 40 45Ala Thr Gly Tyr Glu Val Leu Leu Asp Gly Glu Thr Leu Asp Gly Gly 50 55 60Ala Thr Leu Glu Ala Ile Met Thr Lys Arg Glu Met Leu Pro Leu Gln65 70 75 80Trp Phe Asp Val Arg Phe Lys Lys 85105122PRTXanthobacter autotrophicus 105Met Asn Leu His Ala Pro Asn Ala Glu Gln Asp Asp Ile Glu Tyr Val1 5 10 15Asp Val Cys Ala Val Asp Asp Leu Trp Asp Gly Glu Met Asp Val Phe 20 25 30Asp Val Gly Glu His Glu Val Leu Leu Val Lys His Glu Gly Arg Phe 35 40 45His Ala Tyr Asp Gly Ile Cys Pro His Gln Ser Val Ser Leu Val Glu 50 55 60Gly His Leu Thr Glu Asp Gly Val Leu Ile Cys Lys Ala His Glu Trp65 70 75 80Gln Phe Ser Val Glu Gly Gly Gln Gly Ile Asn Pro Ala Asn Val Cys 85 90 95Leu Gln Ser Phe Pro Leu Lys Val Glu Gly Gly Arg Val Leu Ile Gly 100 105 110Thr Glu Pro Leu Pro Lys Glu Gly Glu Ala 115 120106101PRTXanthobacter autotrophicus 106Met Ser Asn Ala Thr Val Asp Asp Met Asp Glu Asn Leu Val Gly Pro1 5 10 15Val Ile Arg Ala Gly Asp Leu Ala Asp Ala Val Ile Asp Ala Val Ile 20 25 30Ala Asp Asn Pro Gly Lys Glu Val His Val Ile Glu Arg Gly Asp Tyr 35 40 45Val Arg Ile His Thr Asp Arg Asp Cys Arg Leu Thr Arg Ala Ser Ile 50 55 60Glu Gln Ala Leu Gly Arg Ser Phe Val Leu Ala Ala Ile Glu Ala Glu65 70 75 80Met Ser Ser Phe Lys Gly Arg Met Ser Ser Ser Asp Ser Glu Met Arg 85 90 95Trp Tyr Tyr Lys Ser 100107341PRTXanthobacter autotrophicus 107Met Thr Gln Gln Arg Pro Thr Arg Thr Arg Glu Arg Lys Lys Thr Trp1 5 10 15Thr Ala Phe Gly Asn Leu Gly Arg Lys Pro Thr Asp Tyr Glu Val Val 20 25 30Thr His Asn Met Asn His Thr Met Arg Gly Thr Pro Leu Glu Leu Ser 35 40 45Pro Thr Val His Ala Asn Val Trp Leu Lys Lys Asn Arg Asp Glu Ile 50 55 60Ala Leu Lys Val Asp Ser Trp Asp Leu Phe Arg Asp Pro Asp Arg Thr65 70 75 80Thr Tyr Asp Thr Tyr Val Lys Met Gln Asp Asp Gln Glu Thr Tyr Val 85 90 95Asp Asn Leu Leu Leu Ser Tyr Thr Gly Glu Gly Arg Tyr Asp Glu Glu 100 105 110Leu Ser Ser Arg Ser Leu Asp Leu Leu Ser Ala Gly Leu Thr Pro Thr 115 120 125Arg Tyr Leu Gly His Gly Leu Gln Met Leu Ala Ala Tyr Ile Gln Gln 130 135 140Leu Ala Pro Ser Ala Tyr Val Gly Asn Cys Ala Val Phe Gln Thr Ser145 150 155 160Asp Ala Leu Arg Arg Val Gln Arg Val Ala Tyr Arg Thr Arg Gln Leu 165 170 175Ala Asp Ala His Pro Ala Arg Gly Phe Gly Ser Gly Asp Arg Ala Val 180 185 190Trp Glu Lys Ser Pro Asp Trp Gln Pro Ile Arg Lys Ala Ile Glu Glu 195 200 205Leu Leu Val Thr Phe Glu Trp Asp Lys Ala Leu Ala Gly Thr Asn Phe 210 215 220Val Val Lys Pro Ile Leu Asp Glu Leu Phe Leu Asn His Leu Ala Arg225 230 235 240Leu Leu His Val Glu Gly Asp Glu Leu Asp Ser Leu Val Leu Arg Asn 245 250 255Leu His Gly Asp Ala Gln Arg His Ala Arg Trp Thr Ala Ala Leu Gly 260 265 270Arg Phe Ala Val Glu Gln Asn Val Asn Asn Arg Thr Val Leu Arg Asp 275 280 285Ala Ile Ala Gly Trp His Glu Thr Gly Glu Ala Val Leu Ala Ala Gly 290 295 300Ala Gly Met Leu Ala Ser Arg Ala Pro Ser Ala Asp Ala Ala Lys Ile305 310 315 320Ala Asp Glu Val Arg Ala Thr Leu Ala Gln Leu His Ala Asn Ala Gly 325 330 335Leu Gly His Asp Ala 340108327PRTXanthobacter autotrophicus 108Met Arg Leu Asn Asp Gly Arg Ser Phe Ser Cys Arg Ser Asp Gln Thr1 5 10 15Val Leu His Ala Ala Leu Ala Ala Gly Ile Asp Met Pro Tyr Glu Cys 20 25 30Ala Ser Gly Ser Cys Gly Ser Cys Arg Cys Arg Leu Ser His Gly Ser 35 40 45Val Ser Leu Leu Trp Pro Glu Ala Pro Gly Leu Ser Ala Arg Asp Arg 50 55 60Gln Lys Gly Asp Arg Ile Leu Ala Cys Gln Ser Thr Pro Ser Ser Asp65 70 75 80Leu Glu Ile Asn Val Arg Ala Gly Asp Ala Leu Leu Glu Pro Pro Pro 85 90 95Arg Arg His Ala Ala Arg Val Thr Val Lys Glu Thr Leu Cys Ala Ser 100 105 110Val Ile Arg Leu Val Leu Asn Val Gly Gly Pro Ile His Phe Leu Pro 115 120 125Gly Gln Phe Phe Ile Leu Asp Leu Pro Gly Ala Gly Arg Arg Ala Tyr 130 135 140Ser Val Ala Asn Leu Glu Asn Ala Ala Gly Gly Ile Glu Leu Leu Ile145 150 155 160Lys Arg Lys Ile Gly Gly Ala Gly Thr Ala Ala Leu Phe Asp Gln Cys 165 170 175Ala Pro Gly Met Gly Leu Val Ile Glu Gly Pro Tyr Gly Arg Ala Tyr 180 185 190Leu Arg Ala Asp Ser Ala Arg Gly Ile Val Ala Val Ala Gly Gly Ser 195 200 205Gly Leu Ala Pro Met Leu Ser Ile Leu Arg Gly Ala Leu Ala Arg Gly 210 215 220Phe Gly Gly Pro Met Asp Leu Tyr Phe Gly Val Asn Thr Ala Glu Glu225 230 235 240Leu Phe Cys Val Pro Glu Leu Ser Ala Leu Gln Ala Ala Gly Ala Arg 245 250 255Val His Leu Ala Leu Arg Asp Gly Gly Pro Gly Pro Ala Gly Leu His 260 265 270Arg Gln Ala Gly Leu Ile Gly Asp Ala Leu Val Ala Gly Glu Pro Asp 275 280 285Leu Lys Ala Lys Asp Leu Tyr Val Ala Gly Pro Ala Pro Met Thr Asp 290 295 300Asp Ile Leu Ala Arg Thr Val Arg Gln Glu Ala Ile Pro Ala Asp Arg305 310 315 320Val Phe Phe Asp Arg Phe Val 325

Claims

1.-125. (canceled)

126. A recombinant methanotrophic or methylotrophic bacterium, comprising: (a) a first heterologous nucleic acid molecule encoding a polypeptide capable of metabolizing an S substrate selected from: (i) a hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, sulfur dioxygenase, sulfite oxidase, or any combination thereof; (ii) a hydrogen sulfide:NADP.sup.+ oxidoreductase, sulfite oxidase, or both; (iii) a hydrogen sulfide:ferredoxin oxidoreductase, sulfite oxidase, or both; (iv) a sulfide:flavocytochrome-c oxidoreductase, sulfite oxidase, or both; (v) a sulfide:quinone oxidoreductase, sulfite oxidase, or both; and (vi) a hydrogen sulfide:NADP.sup.+ oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, or sulfide: quinone oxidoreductase, wherein endogenous sulfite oxidase activity of the bacterium is increased; and (b) a second heterologous nucleic acid molecule encoding a cysteine biosynthesis pathway, homocysteine biosynthesis pathway, or a methionine biosynthesis pathway, wherein: (i) the cysteine biosynthesis pathway comprises a cysteine synthase; (ii) the homocysteine biosynthesis pathway comprises an O-acetylhomoserine sulfhydrylase; and (iii) the methionine biosynthesis pathway comprises an O-acetylhomoserine sulfhydrylase and a methionine synthase or a homocysteine methyltransferase; wherein the recombinant methanotrophic or methylotrophic bacterium is capable of converting a feedstock comprising the S substrate and a C.sub.1 substrate into the sulfur-containing amino acid and is capable of oxidizing or assimilating an increased amount of the S substrate as compared to wild-type methanotrophic or methylotrophic bacterium.

127. The recombinant methanotrophic or methylotrophic bacterium according to claim 126, wherein the methanotrophic or methylotrophic bacterium is a methanotrophic bacterium.

128. The recombinant methanotrophic or methylotrophic bacterium according to claim 127, wherein the methanotrophic bacterium is an obligate methanotrophic bacterium.

129. The recombinant methanotrophic or methylotrophic bacterium according to claim 127, wherein the methanotrophic bacterium is Methylococcus capsulatus Bath, Methylomonas 16a, Methylosinus trichosporium OB3b, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylobacterium organophilum, Methylomonas sp. AJ-3670, Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystis bryophila, Methylocapsa aurea KYG, Methylacidiphilum infernorum, or Methylomicrobium alcaliphilum.

130. The recombinant methanotrophic or methylotrophic bacterium according to claim 127, wherein the methanotrophic bacterium is Methylococcus capsulatus Bath, Methylosinus trichosporium OB3b, Methylomonas sp. 16a, Methylomicrobium alcaliphilum, or a high growth variant thereof.

131. The recombinant methanotrophic or methylotrophic bacterium according to claim 127, wherein the recombinant methanotrophic bacterium is in a culture further comprising a heterologous bacterium.

132. The recombinant methanotrophic or methylotrophic bacterium according to claim 126, wherein the methanotrophic or methylotrophic bacterium is a methylotrophic bacterium.

133. The recombinant methanotrophic or methylotrophic bacterium according to claim 132, wherein the methylotrophic bacterium is Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, or Methylobacterium nodulans.

134. The recombinant methanotrophic or methylotrophic bacterium according to claim 126, wherein the polypeptide capable of metabolizing the S substrate: (a) is encoded by a nucleic acid comprising the polynucleotide sequence as set forth in any one of SEQ ID NOS.:21-54; (b) comprises the amino acid sequence as set forth in any one of SEQ ID NOS.:55-88; (c) is a sulfur oxygenase; or (d) is a sulfur oxygenase having an amino acid sequence that is at least 75% identical to the sequence set forth in Genbank Accession No. AAK58572.1 or ABN04222.1, or a functional fragment thereof.

135. The recombinant methanotrophic or methylotrophic bacterium according to claim 126, wherein the second heterologous nucleic acid molecule encodes the cysteine biosynthesis pathway comprising the cysteine synthase.

136. The recombinant methanotrophic or methylotrophic bacterium according to claim 126, wherein the second heterologous nucleic acid molecule encodes the homocysteine biosynthesis pathway comprising the O-acetylhomoserine sulfhydrylase.

137. The recombinant methanotrophic or methylotrophic bacterium according to claim 126, wherein the second heterologous nucleic acid molecule encodes the methionine biosynthesis pathway comprising the O-acetylhomoserine sulfhydrylase and the methionine synthase or the homocysteine methyltransferase.

138. A method for producing a sulfur-containing amino acid from a tainted gas, the method comprising culturing a recombinant methanotrophic bacterium or methylotrophic bacterium according to claim 126 with a tainted gas feedstock, wherein the tainted gas feedstock comprises methane and an S substrate; wherein the methanotrophic bacterium or methylotrophic bacterium assimilates and oxidizes the methane and the S substrate, and converts the S substrate into the sulfur-containing amino acid selected from cysteine, homocysteine, or methionine.

139. The method according to claim 138, wherein the tainted gas feedstock comprises natural gas, unconventional natural gas, casinghead gas, wellhead condensate, or any combination thereof.

140. The method according to claim 138, wherein the recombinant methanotrophic bacterium or methylotrophic bacterium is a methanotrophic bacterium selected from Methylococcus capsulatus Bath, Methylosinus trichosporium OB3b, Methylomonas sp. 16a, Methylomicrobium alcaliphilum, or a high growth variant thereof.

141. A system for producing a sulfur-containing amino acid from a tainted gas, comprising: a source of tainted gas comprising methane and an S substrate; a bioreactor comprising a recombinant methanotrophic bacterium or methylotrophic bacterium according to claim 126; wherein the recombinant methanotrophic bacterium or methylotrophic bacterium cultured in the bioreactor converts the methane and the S substrate of the tainted gas into a sulfur-containing amino acid selected from cysteine, homocysteine, or methionine.