NOVEL BACTERIA AND METHODS OF USE THEREOF

Abstract

This invention relates generally to the field of microbial fermentation of gases. It more particularly relates to a novel strain of Clostridium autoethanogenum bacteria with improved efficiency in the production of ethanol by anaerobic fermentation of substrates containing carbon monoxide (CO).

Description

FIELD OF THE INVENTION

[0001] This invention relates generally to the field of microbial fermentation of gases. It more particularly relates to a novel class of bacteria with improved efficiency in the production of ethanol by anaerobic fermentation of substrates containing carbon monoxide (CO).

BACKGROUND OF THE INVENTION

[0002] Ethanol is rapidly becoming a major hydrogen-rich liquid transport fuel around the world. Worldwide consumption of ethanol in 2005 was an estimated 12.2 billion gallons. The global market for the fuel ethanol industry has also been predicted to grow sharply in future, due to an increased interest in ethanol in Europe, Japan, the USA, and several developing nations.

[0003] For example, in the USA, ethanol is used to produce E10, a 10% mixture of ethanol in gasoline. In E10 blends the ethanol component acts as an oxygenating agent, improving the efficiency of combustion and reducing the production of air pollutants. In Brazil, ethanol satisfies approximately 30% of the transport fuel demand, as both an oxygenating agent blended in gasoline, and as a pure fuel in its own right. Also, in Europe, environmental concerns surrounding the consequences of Green House Gas (GHG) emissions have been the stimulus for the European Union (EU) to set member nations a mandated target for the consumption of sustainable transport fuels such as biomass derived ethanol.

[0004] The vast majority of fuel ethanol is produced via traditional yeast-based fermentation processes that use crop derived carbohydrates, such as sucrose extracted from sugarcane or starch extracted from grain crops, as the main carbon source. However, the cost of these carbohydrate feed stocks is influenced by their value as human food or animal feed, while the cultivation of starch or sucrose-producing crops for ethanol production is not economically sustainable in all geographies. Therefore, it is of interest to develop technologies to convert lower cost and/or more abundant carbon resources into fuel ethanol.

[0005] CO is a major, free, energy-rich by-product of the incomplete combustion of organic materials such as coal or oil and oil derived products. For example, the steel industry in Australia is reported to produce and release into the atmosphere over 500,000 tonnes of CO annually.

[0006] Catalytic processes may be used to convert gases consisting primarily of CO and/or CO and hydrogen (H2) into a variety of fuels and chemicals. Micro-organisms may also be used to convert these gases into fuels and chemicals.

[0007] The ability of micro-organisms to grow on CO as a sole carbon source was first discovered in 1903. This was later determined to be a property of organisms that use the acetyl coenzyme A (acetyl CoA) biochemical pathway of autotrophic growth (also known as the Woods-Ljungdahl pathway and the carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) pathway). A large number of anaerobic organisms including carboxydotrophic, photosynthetic, methanogenic and acetogenic organisms have been shown to metabolize CO to various end products, namely CO2, H2, methane, n-butanol, acetate and ethanol. While using CO as the sole carbon source, all such organisms produce at least two of these end products.

[0008] Anaerobic bacteria, such as those from the genus Clostridium, have been demonstrated to produce ethanol from CO, CO2 and H2 via the acetyl CoA biochemical pathway. For example, various strains of Clostridium ljungdahlii that produce ethanol from gases are described in WO 00/68407, EP 117309, U.S. Pat. Nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438. The bacterium Clostridium autoethanogenum sp is also known to produce ethanol from gases (Abrini et al., Archives of Microbiology 161, pp 345-351 (1994)).

[0009] However, ethanol production by micro-organisms by fermentation of gases is always associated with co-production of acetate and/or acetic acid. As some of the available carbon is converted into acetate/acetic acid rather than ethanol, the efficiency of production of ethanol using such fermentation processes may be less than desirable. Also, unless the acetate/acetic acid by-product can be used for some other purpose, it may pose a waste disposal problem. Acetate/acetic acid is converted to methane by micro-organisms and therefore has the potential to contribute to GHG emissions.

[0010] Microbial fermentation of CO in the presence of H2 can lead to substantially complete carbon transfer into an alcohol. However, in the absence of sufficient H2, some of the CO is converted into alcohol, while a significant portion is converted to CO2 as shown in the following equations:

6CO+3H₂O→C₂H₅OH+4CO₂

12H₂+4CO₂→2C₂H₅OH+6H₂O

[0011] The production of CO₂ represents inefficiency in overall carbon capture and if released, also has the potential to contribute to Green House Gas emissions.

[0012] WO2007/117157 describes a process that produces alcohols, particularly ethanol, by anaerobic fermentation of gases containing carbon monoxide. Acetate produced as a by-product of the fermentation process is converted into hydrogen gas and carbon dioxide gas, either or both of which may be used in the anaerobic fermentation process.

[0013] WO2008/115080 describes a process for the production of alcohol(s) in multiple fermentation stages. By-products produced as a result of anaerobic fermentation of gas(es) in a first bioreactor can be used to produce products in a second bioreactor. Furthermore, by-products of the second fermentation stage can be recycled to the first bioreactor to produce products.

[0014] WO2009/064200 describes a novel class of bacteria which has improved efficiency in the production of ethanol by anaerobic fermentation of substrates containing carbon monoxide.

[0015] It would be beneficial to provide micro-organisms that are capable of fermentation of gases containing carbon monoxide to ethanol at increased efficiency, that is micro-organisms capable of improved uptake of carbon monoxide, of producing more ethanol, and/or a greater ratio of ethanol to acetate from the same substrate, than do micro-organisms of the prior art.

[0016] It is an object of the present invention to provide a new class of bacteria which overcomes one or more of the limitations of the prior art in the conversion of gaseous sources containing CO into ethanol, or to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

[0017] In a first aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, at a specific productivity of at least about 2 g ethanol/L fermentation broth/gram of biomass/day.

[0018] In other embodiments, the bacterium is capable of producing ethanol at a specific productivity of at least about 3 g ethanol/L fermentation broth/gram of biomass/day, at least about 4 g ethanol/L fermentation broth/gram of biomass/day, at least about 5 g ethanol/L fermentation broth/gram of biomass/day, at least about 6 g ethanol/L fermentation broth/gram of biomass/day or at least about 7 g ethanol/L fermentation broth/gram of biomass/day.

[0019] In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, at a productivity of at least about 10 g ethanol/L of fermentation broth/day.

[0020] In other embodiments, the bacterium is capable of producing ethanol at a productivity of at least about 20 g ethanol/L of fermentation broth/day, at least about 30 g ethanol/L of fermentation broth/day, at least about 40 g ethanol/L of fermentation broth/day or at least about 50 g ethanol/L of fermentation broth/day, or at least about 60 g ethanol/L of fermentation broth/day, or at least about 70 g ethanol/L of fermentation broth/day.

[0021] In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium is capable of a specific uptake of CO of at least about 1.0 mMol CO/min/g of biomass.

[0022] In one embodiment the bacterium is capable of a specific uptake of CO of at least about 1.2 mMol CO/min/g biomass, at least about 1.4 mMol CO/min/g of biomass, at least about 1.6 mMol CO/min/g of biomass, at least about 1.8 mMol CO/min/g of biomass, or at least about 2.0 mMol CO/min/g of biomass. In one particular embodiment, the bacterium is capable of a specific uptake of CO of at least about 1.2 mMol CO/min/g biomass.

[0023] In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium is capable of a specific growth rate of at least about 0.8 day^-1.

[0024] In certain embodiments the bacterium is capable of a specific growth rate of at least about 1.0 day^-1, at least about 1.2 day^-1, at least about 1.4 day^-1, at least about 1.6 day^-1, at least about 1.8 day^-1 or at least about 2.0 day^-1.

[0025] In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium is capable of producing ethanol at an ethanol to acetate ratio of at least about 2:1.

[0026] In certain embodiments the bacterium is capable of producing ethanol at an ethanol to acetate ratio of at least about 3:1, of at least about 4:1, of at least about 5:1, of at least about 7:1 or of at least about 10:1.

[0027] In one embodiment, the bacterium is capable of producing ethanol with substantially no acetate.

[0028] In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium is capable of tolerating alcohol of up to about 30 g/L of fermentation broth.

[0029] In certain embodiment the bacterium is capable of tolerating alcohol of up to about 40 g/L of fermentation broth, of up to about 50 g/L of fermentation broth, of up to about 60 g/L of fermentation broth, or of up to about 70 g/L of fermentation broth.

[0030] In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium has two or more of the following characteristics:

[0031] is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, at a specific productivity of at least about 2 g of ethanol/L of fermentation broth/gram of biomass/day;

[0032] is capable of producing ethanol at a concentration of at least about 10 g ethanol/L of fermentation broth/day;

[0033] is capable of a specific uptake of CO of at least about 1.0 mMol CO/min/g of biomass;

[0034] is capable of a growth rate of at least about 1.0 g/day;

[0035] is capable of producing ethanol at an ethanol to acetate ratio of at least about 2:1; and,

[0036] is capable of tolerating alcohol of up to about 30 g/L of broth. In one embodiment, the bacteria of the invention are derived from Clostridium autoethanogenum. In a preferred embodiment, the bacterium of the invention is a strain of Clostridium autoethanogenum.

[0037] In a particular embodiment the bacterium has the defining characteristics of the Clostridium autoethanogenum strain deposited at DSMZ under the accession number DMS23693. In one embodiment the bacterium is the Clostridium autoethanogenum strain deposited at DSMZ under the accession number DMS23693.

[0038] In a further aspect the invention provides a biologically pure isolate of the Clostridium autoethanogenum strain deposited at DSMZ under the accession number DMS23693

[0039] In another aspect, the invention provides a method for the production of one or more alcohols comprising fermenting a substrate comprising CO using a bacterium as herein before described.

[0040] In one embodiment the method comprises the steps of:

[0041] (a) providing a substrate comprising CO to a bioreactor containing a culture of a bacterium of the invention; and

[0042] (b) anaerobically fermenting the culture in the bioreactor to produce one or more alcohols.

[0043] In a further aspect, the invention provides a method for reducing the total atmospheric carbon emissions from an industrial process, the method comprising:

[0044] (a) capturing CO-containing gas produced as a result of the industrial process, before the gas is released into the atmosphere; (b) the anaerobic fermentation of the CO-containing gas to produce one or more alcohols by a culture containing one or more bacterium of the invention.

[0045] In one embodiment of the method aspects, the fermentation is conducted at a temperature of about 34° C. to about 37° C. In one preferred embodiment, the fermentation is conducted at a temperature of about 34° C.

[0046] In certain embodiments of the method aspects, acetate is produced as a by-product of the fermentation. Preferably the one or more alcohols produced includes ethanol.

[0047] In particular embodiments of the method aspects, the bacterium is maintained in an aqueous culture medium.

[0048] In particular embodiments of the method aspects, the fermentation of the substrate takes place in a bioreactor.

[0049] In certain embodiments the substrate comprises at least about 25% CO by volume, at least about 30% CO by volume, at least about 40% CO by volume, at least about 50% CO by volume, at least about 65% CO by volume or at least about 70% CO by volume. In particular embodiments the substrate comprises at least about 75% CO by volume, at least about 80% CO by volume, at least about 85% CO by volume, at least about 90% CO by volume or at least about 95% CO by volume.

[0050] In one embodiment the substrate comprises about 30% or less H₂ by volume. In another embodiments, the substrate comprises about 20% or less H₂ by volume, about 15% or less H₂ by volume, about 10% or less H₂ by volume, about 5% or less H₂ by volume, about 4% or less H₂ by volume, about 3% or less H₂ by volume, about 2% or less H₂ by volume, about 1% or less H₂ by volume, or substantially no H₂.

[0051] In one embodiment the substrate comprises less than or equal to about 20% CO₂ by volume. In particular embodiments the substrate comprises less than or equal to about 15% CO₂ by volume, less than or equal to about 10% CO₂ by volume, less than or equal to about 5% CO₂ by volume or substantially no CO₂.

[0052] In certain embodiments the substrate comprising CO is a gaseous substrate containing CO.

[0053] In certain embodiments, the gaseous substrate comprises a gas obtained as a by-product of an industrial process.

[0054] In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of biomass, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing.

[0055] In one embodiment, the gaseous substrate may comprise a gas obtained from a steel mill.

[0056] In another embodiment, the gaseous substrate may comprise automobile exhaust fumes.

[0057] In certain embodiments of the method aspects the alcohol is recovered from the fermentation broth, the fermentation broth being the aqueous culture medium comprising bacterial cells and the alcohol.

[0058] In certain embodiments acetate is produced as a by-product of the fermentation.

[0059] In a further embodiment the alcohol and the acetate are recovered from the broth.

[0060] Although the invention is broadly as defined above, it is not limited thereto and also includes embodiments of which the following description provides examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The invention will now be described in detail with reference to the accompanying Figures in which:

[0062] FIG. 1: Shows the CO consumption of DSM19630 and DSM23693

[0063] FIG. 2: Shows the metabolite production of DSM19630 (FIG. 2a) and DSM23693 (FIG. 2b)

[0064] FIG. 3: Shows the optimised biomass accumulation and metabolite production of DSM23693 as described in Example 2.

[0065] FIG. 4: Shows a genetic map of new C. autoethanogenum strain LZ1561 (DSM23693) showing the variations to strain LZ1560 (DSM19630)

[0066] FIG. 5 : Seq. ID. 1: Nucleotide sequence of DNA mismatch repair protein MutS gene in strain LZ1561

[0067] FIG. 6: Seq. ID. 2: Nucleotide sequence of DNA mismatch repair protein MutS gene in strain LZ1560

[0068] FIG. 7: Seq. ID. 3: Amino Acid sequence of DNA mismatch repair protein MutS gene in strain LZ1561

[0069] FIG. 8: Seq. ID. 4: Amino Acid sequence of DNA mismatch repair protein MutS gene in strain LZ1560

[0070] FIG. 9: Seq. ID. 5: Nucleotide sequence found to be re-arranged in strains LZ561 and LZ1560

[0071] FIG. 10: Seq. ID. 6: Nucleotide sequence of putative promoter region of F₁F_O ATP synthase operon in strain LZ1561

[0072] FIG. 11: Seq. ID. 7: Nucleotide sequence of putative promoter region of F₁F_O ATP synthase operon in strain LZ1560

[0073] FIG. 12: Seq. ID. 8: Nucleotide sequence of putative promoter region of Rnf complex operon in strain LZ1561

[0074] FIG. 13: Seq. ID. 9: Nucleotide sequence of putative promoter region of Rnf complex operon in strain LZ1560

[0075] FIG. 14: Seq. ID. 10: Nucleotide sequence of putative promoter region of carbon starvation protein in strain LZ1561

[0076] FIG. 15: Seq. ID. 11: Nucleotide sequence of putative promoter region of carbon starvation protein in strain LZ1560

[0077] FIG. 16: Seq. ID. 12: Nucleotide sequence of CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit gene in strain LZ1561

[0078] FIG. 17: Seq. ID. 13: Nucleotide sequence of CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit gene in strain LZ1560

[0079] FIG. 18: Seq. ID. 14: Amino Acid sequence of CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit gene in strain LZ1561

[0080] FIG. 19: Seq. ID. 15: Amino Acid sequence of CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit gene in strain LZ1560

[0081] FIG. 20: Seq. ID. 16: Nucleotide sequence of 5,10-methylenetetrahydrofolate reductase gene in strain LZ1561

[0082] FIG. 21: Seq. ID. 17: Nucleotide sequence of 5,10-methylenetetrahydrofolate reductase gene in strain LZ1560

DETAILED DESCRIPTION OF THE INVENTION

[0083] The inventors have developed novel bacteria. The bacteria are characterised by having one or more of a number of unexpected properties (as outlined herein after), and in one preferred embodiment all of these properties. The use of these novel bacteria in anaerobic fermentation processes provides an unexpected benefit over existing strains of bacteria which may allow for an increase in the overall efficiency of a fermentation process for producing products such as ethanol and/or acetate.

[0084] Accordingly, in broad terms, in one aspect, the present invention relates to a novel bacterium and a biologically pure isolate of a bacterium with increased efficiency in an anaerobic fermentation process. In one aspect the bacterium is capable of producing an alcohol, preferably ethanol, from a substrate comprising CO.

[0085] In a further aspect, the invention relates to processes for producing an alcohol, preferably ethanol, by anaerobic fermentation of a CO-containing substrate by the bacteria of the invention.

Definitions

[0086] Unless otherwise defined, the following terms as used throughout this specification are defined as follows:

[0087] A "substrate containing CO", a "substrate comprising CO" and like terms should be understood to include any substrate in which carbon monoxide is available to bacteria for growth and/or fermentation, for example. In particular embodiments of the invention the "substrate containing CO" is gaseous. Such substrates may be referred to herein as "gaseous substrates containing CO", "gaseous substrates comprising CO" and the like.

[0088] In the description which follows, embodiments of the invention are described in terms of delivering and fermenting a "gaseous substrate containing CO". However, it should be appreciated that the gaseous substrate may be provided in alternative forms. For example, the gaseous substrate containing CO may be provided dissolved in a liquid. Essentially, a liquid is saturated with a carbon monoxide containing gas and then that liquid is added to the bioreactor. This may be achieved using standard methodology. By way of example, a microbubble dispersion generator (Hensirisak et. al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3/October, 2002) could be used. By way of further example, the gaseous substrate containing CO may be adsorbed onto a solid support. Such alternative methods are encompassed by use of the term "substrate containing CO".

[0089] The terms "increasing the efficiency", "increased efficiency" and the like, when used in relation to a fermentation process, include, but are not limited to, increasing one or more of: the rate of growth of micro-organisms catalysing the fermentation, the uptake or consumption of CO by the micro-organisms, the volume of desired product (such as alcohols) produced per volume of substrate (such as CO) consumed, the concentration of the desired product (such as alcohols) produced in the culture medium, the rate of production or level of production of the desired product, and the relative proportion of the desired product produced compared with other by-products of the fermentation.

[0090] The term "acetate" includes both acetate salt alone and a mixture of molecular or free acetic acid and acetate salt, such as the mixture of acetate salt and free acetic acid present in a fermentation broth as described herein. The ratio of molecular acetic acid to acetate in the fermentation broth is dependent upon the pH of the system.

[0091] The term "bioreactor" includes a fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact.

[0092] The term "alcohol tolerance" as used herein should be taken to refer to the level of alcohol, preferably ethanol, that a bacterium or population of bacteria will tolerate while continuing to survive, grow and/or to produce at least a level of the desired product.

[0093] Bacteria of the invention, or cultures or isolates thereof, may be described to be in an "isolated" or "biologically pure" form. These terms are intended to mean that the bacteria have been separated from an environment or one or more constituents, cellular or otherwise, which they may be associated with if found in nature or otherwise. The terms "isolated" or "biologically pure" should not be taken to indicate the extent to which the bacteria have been purified. However, in one embodiment the isolates or cultures of the bacteria contain a predominance of the bacteria of the invention.

[0094] The invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate containing CO and wherein the bacterium is capable of one or more of:

[0095] producing ethanol at a specific productivity of about 2 g ethanol/L fermentation broth/gram of biomass/day;

[0096] producing ethanol at a productivity of at least about 10 g /L of fermentation broth/day;

[0097] a specific uptake of CO of at least about 1.0 mMol CO/min/g of biomass; a specific growth rate of at least about 0.8 day^-1;

[0098] producing ethanol at an ethanol to acetate ratio of at least about 2:1; and,

[0099] tolerating alcohol of up to about 30 g/L of broth.

[0100] In a preferred embodiment, a bacterium of the invention is capable of two, three, four, or five of the above features.

[0101] In certain embodiments, the bacterium is capable of producing ethanol at a specific productivity of at least about 3 g ethanol/L fermentation broth/gram of biomass/day, at least about 4 g ethanol/L fermentation broth/gram of biomass/day, at least about 5 g ethanol/L fermentation broth/gram of biomass/day, at least about 6 g ethanol/L fermentation broth/gram of biomass/day or at least about 7 g ethanol/L fermentation broth/gram of biomass/day.

[0102] In certain embodiments, the bacterium is capable of producing ethanol at a productivity of at least about 20 g ethanol/L of fermentation broth/day, at least about 30 g ethanol/L of fermentation broth/day, at least about 40 g ethanol/L of fermentation broth/day or at least about 50 g ethanol/L of fermentation broth/day. The maximum value takes into account stoichiometry, CO uptake and ethanol stripping.

[0103] In certain embodiments the bacterium is capable of a specific uptake of CO of at least about 1.2 mMol CO/min/g biomass, at least about 1.4 mMol CO/min/g of biomass, at least about 1.6 mMol CO/min/g of biomass, at least about 1.8 mMol CO/min/g of biomass, or at least about 2.0 mMol CO/min/g of biomass. In one particular embodiment, the bacterium is capable of a specific uptake of CO of at least about 1.2 mMol CO/min/g biomass.

[0104] In certain embodiments the bacterium is capable of a specific growth rate of at least about 1.0 day^-1, at least about 1.2 day^-1, at least about 1.4 day^-1, at least about 1.6 day^-1, at least about 1.8 day^-1 or at least about 2.0 day^-1.

[0105] In certain embodiments the bacterium is capable of producing ethanol at an ethanol to acetate ratio of at least about 3:1, of at least about 4:1, of at least about 5:1, of at least about 7:1 or of at least about 10:1. In one particular embodiment, there is no net production of acetate during fermentation.

[0106] In certain embodiments the bacterium is capable of tolerating alcohol of up to about 40 g/L of fermentation broth, of up to about 50 g/L of fermentation broth, or of up to about 60 g/L of fermentation broth. In one particular embodiment, the bacterium is capable of tolerating alcohol of up to about 70 g/L of fermentation broth.

[0107] In a preferred embodiment, the bacteria of the invention are derived from Clostridium autoethanogenum. In a more preferred embodiment of the invention the bacteria are derived from Clostridium autoethanogenum strain DSM19630 (DSMZ, Germany) (described in WO2009/064200).

[0108] In a preferred embodiment, the bacterium of the invention is a strain of Clostridium autoethanogenum.

[0109] Clostridium autoethanogenum is described, for example, in Abrini et al; Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide, Arch Microbiol (1994) 161:345-351.

[0110] In certain embodiments of the invention, the bacteria have the defining characteristics of Clostridium autoethanogenum strain DSM23693 deposited at DSMZ, Germany, in accordance with the Budapest Treaty, on 7 Jun. 2010. In a particular embodiment, the bacterium is Clostridium autoethanogenum strain DSM23693.

[0111] The invention also relates to bacteria derived from the bacteria of the invention.

[0112] The bacteria of certain embodiments of the invention are capable of an increased alcohol production rate, an increased growth rate, an increased CO consumption or update rate, a higher alcohol to acid production ratio, and/or an increased tolerance to alcohol. This provides a benefit over other strains of Clostridia sp including Clostridium autoethanogenum. Therefore, use of bacteria of the present invention may increase the overall efficiency of a fermentation process for producing products such as acetate and/or ethanol.

[0113] In certain embodiments the bacteria of the invention are capable of the productivity, growth rates, alcohol to acid ratio, CO consumption and alcohol tolerance mentioned herein before at elevated levels of CO in the gaseous substrate. For example, the gaseous substrate may comprise at least about 50% CO by volume, at least about 65% CO by volume, or at least about 70% CO by volume. In certain embodiments the gaseous substrate comprises at least about 80% CO by volume, or at least about 85% CO by volume, or at least about 90% CO by volume or at least about 95% CO by volume.

[0114] Similarly the productivity, growth rates, alcohol to acid ratio, CO consumption and alcohol tolerance herein before described are achievable in certain embodiments at low to non-existent levels of H₂ in the gaseous substrate. The gaseous substrate may comprise about 30% or less H₂ by volume. In particular embodiments the gaseous substrate comprises about 20% or less H₂ by volume, about 15% or less H₂ by volume, about 10% or less H₂ by volume, about 5% or less H₂ by volume, about 4% or less H₂ by volume, about 3% or less H₂ by volume, about 2% or less H₂ by volume, about 1% or less H₂ by volume, or substantially no H₂.

[0115] In certain embodiments the bacteria of the invention are also capable of the productivity, growth rates, alcohol to acid ratio, CO consumption and alcohol tolerance mentioned herein before when supplied with gaseous substrate comprising relatively little CO₂. In one embodiment the gaseous substrate comprises less than or equal to about 20% CO₂ by volume. In certain embodiments the gaseous substrate comprises less than or equal to about 15% CO₂ by volume, less than or equal to about 10% CO₂ by volume, or less than or equal to about 5% CO₂ by volume. In one particular embodiment, the gaseous substrate comprises substantially no CO₂.

[0116] In certain embodiments a culture of a bacterium of the invention is maintained in an aqueous culture medium. Preferably the aqueous culture medium is a minimal anaerobic microbial growth medium. Suitable media are known in the art and described for example in U.S. Pat. Nos. 5,173,429 and 5,593,886 and WO 02/08438, and in Klasson et al [(1992). Bioconversion of Synthesis Gas into Liquid or Gaseous Fuels. Enz. Microb. Technol. 14:602-608.], Najafpour and Younesi [(2006). Ethanol and acetate synthesis from waste gas using batch culture of Clostridium ljungdahlii. Enzyme and Microbial Technology, Volume 38, Issues 1-2, p. 223-228] and Lewis et al [(2002). Making the connection-conversion of biomass-generated producer gas to ethanol. Abst. Bioenergy, p. 2091-2094]. In particular embodiments of the invention, the minimal anaerobic microbial growth medium is as described herein after in the Examples section.

[0117] The invention also provides methods for the production of one or more alcohols from a gaseous substrate comprising CO, the methods comprising maintaining a culture of one or more bacterial isolate of the invention in the presence of the substrate, and the anaerobic fermentation of the substrate to one or more alcohols by the one or more bacterial isolate.

[0118] The invention also provides a method for reducing the total atmospheric carbon emissions from an industrial process, the method comprising:

[0119] (a) capturing CO-containing gas produced as a result of the industrial process, before the gas is released into the atmosphere;

[0120] (b) the anaerobic fermentation of the CO-containing gas to produce one or more alcohols by a culture containing one or more bacterial isolates of the invention.

[0121] In certain embodiments of the methods of the invention, acetate is produced as a by-product of the fermentation. The alcohol produced is ethanol.

[0122] In certain embodiments, the culture is maintained in a liquid nutrient medium.

[0123] The fermentation may be carried out in any suitable bioreactor, such as a continuous stirred tank reactor (CTSR), a bubble column reactor (BCR) or a trickle bed reactor (TBR). Also, in some preferred embodiments of the invention, the bioreactor may comprise a first, growth reactor in which the micro-organisms are cultured, and a second, fermentation reactor, to which fermentation broth from the growth reactor is fed and in which most of the fermentation product (ethanol and acetate) is produced.

[0124] As described above, the carbon source for the fermentation reaction is a gaseous substrate containing CO. The gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as from automobile exhaust fumes. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing. In these embodiments, the CO-containing gas may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method. Depending on the composition of the gaseous CO-containing substrate, it may also be desirable to treat it to remove any undesired impurities, such as dust particles before introducing it to the fermentation. For example, the gaseous substrate may be filtered or scrubbed using known methods.

[0125] In addition, it is often desirable to increase the CO concentration of a substrate stream (or CO partial pressure in a gaseous substrate) and thus increase the efficiency of fermentation reactions where CO is a substrate. Increasing CO partial pressure in a gaseous substrate increases CO mass transfer into a fermentation media. The composition of gas streams used to feed a fermentation reaction can have a significant impact on the efficiency and/or costs of that reaction. For example, O2 may reduce the efficiency of an anaerobic fermentation process. Processing of unwanted or unnecessary gases in stages of a fermentation process before or after fermentation can increase the burden on such stages (e.g. where the gas stream is compressed before entering a bioreactor, unnecessary energy may be used to compress gases that are not needed in the fermentation). Accordingly, it may be desirable to treat substrate streams, particularly substrate streams derived from industrial sources, to remove unwanted components and increase the concentration of desirable components.

[0126] Substrate streams derived from an industrial source are typically variable in composition. Furthermore, substrate streams derived from industrial sources comprising high CO concentrations (such as, for example, at least 40% CO, at least 50% CO or at least 65% CO) often have a low H2 component (such as less than 20% or less than 10% or substantially 0%). As such, it is particularly desirable that micro-organisms are capable of producing products by anaerobic fermentation of substrates comprising a range of CO and H2 concentrations, particularly high CO concentrations and low H2 concentrations. The bacteria of the present invention have a surprisingly high growth rate and ethanol production rate while fermenting a substrate comprising CO (and no H2).

[0127] The presence of hydrogen in the substrate stream can lead to an improvement in efficiency of overall carbon capture and/or ethanol productivity. For example, WO02/08438 describes the production of ethanol using gas stream of various compositions. WO02/08438 reports a substrate stream comprising 63% H2, 32% CO and 5% CH4 being provided to a culture of C. ljungdahlii in a bioreactor to promote microbial growth and ethanol production. When the culture reached a steady state and microbial growth was no longer the main objective, the substrate stream was switched to 15.8% H2, 36.5% CO, 38.4% N2 and 9.3% CO2 in order to provide CO in a slight excess and promote ethanol production. This document also describes gas streams with higher and lower CO and H2 concentrations.

[0128] It will be appreciated that the processes of the present invention as described herein can be used to reduce the total atmospheric carbon emissions from industrial processes, by capturing CO-containing gases produced as a result of such processes and using them as substrates for the fermentation processes described herein.

[0129] Alternatively, in other embodiments of the invention, the CO-containing gaseous substrate may be sourced from the gasification of biomass. The process of gasification involves partial combustion of biomass in a restricted supply of air or oxygen. The resultant gas typically comprises mainly CO and H₂, with minimal volumes of CO₂, methane, ethylene and ethane. For example, biomass by-products obtained during the extraction and processing of foodstuffs such as sugar from sugarcane, or starch from maize or grains, or non-food biomass waste generated by the forestry industry may be gasified to produce a CO-containing gas suitable for use in the present invention.

[0130] It is generally preferred that the CO-containing gaseous substrate contains a major proportion of CO. In particular embodiments, the gaseous substrate comprises at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 65%, or at least about 70% to about 95% CO by volume. It is not necessary for the gaseous substrate to contain any hydrogen. The gaseous substrate also optionally contains some CO₂, such as about 1% to about 30% by volume, such as about 5% to about 10% CO₂.

[0131] It will be appreciated that for growth of the bacteria and CO-to-ethanol fermentation to occur, in addition to the CO-containing substrate gas, a suitable liquid nutrient medium will need to be fed to the bioreactor. A nutrient medium will contain vitamins and minerals sufficient to permit growth of the micro-organism used. Anaerobic media suitable for the fermentation of ethanol using CO as the sole carbon source are known in the art. For example, suitable media are described in U.S. Pat. Nos. 5,173,429 and 5,593,886 and WO 02/08438 as well as other publications referred to herein before. In one embodiment of the invention the media is as described in the Examples section herein after.

[0132] The fermentation should desirably be carried out under appropriate conditions for the CO-to-ethanol fermentation to occur. Reaction conditions that should be considered include pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to ensure that CO in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.

[0133] The optimum reaction conditions will depend partly on the particular micro-organism of the invention used. However, in general, it is preferred that the fermentation be performed at pressure higher than ambient pressure. Operating at increased pressures allows a significant increase in the rate of CO transfer from the gas phase to the liquid phase where it can be taken up by the micro-organism as a carbon source for the production of ethanol. This in turn means that the retention time (defined as the liquid volume in the bioreactor divided by the input gas flow rate) can be reduced when bioreactors are maintained at elevated pressure rather than atmospheric pressure.

[0134] Also, since a given CO-to-ethanol conversion rate is in part a function of the substrate retention time, and achieving a desired retention time in turn dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor required, and consequently the capital cost of the fermentation equipment. According to examples given in U.S. Pat. No. 5,593,886, reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure.

[0135] The benefits of conducting a gas-to-ethanol fermentation at elevated pressures have also been described elsewhere. For example, WO 02/08438 describes gas-to-ethanol fermentations performed under pressures of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369 g/l/day respectively. However, example fermentations performed using similar media and input gas compositions at atmospheric pressure were found to produce between 10 and 20 times less ethanol per litre per day.

[0136] It is also desirable that the rate of introduction of the CO-containing gaseous substrate is such as to ensure that the concentration of CO in the liquid phase does not become limiting. This is because a consequence of CO-limited conditions may be that the ethanol product is consumed by the culture.

[0137] In certain embodiments, a fermentation process according to the present invention described above will result in a fermentation broth comprising ethanol, as well as bacterial cells, in the aqueous culture medium. In preferred embodiments of the method the ethanol is recovered from the fermentation broth.

[0138] In certain embodiments, the recovering of ethanol comprises continuously removing a portion of broth and recovering the alcohol from the removed portion of the broth.

[0139] In particular embodiments the recovery of ethanol includes passing the removed portion of the broth containing ethanol through a separation unit to separate bacterial cells from the broth, to produce a cell-free alcohol-containing permeate, and returning the bacterial cells to the bioreactor.

[0140] In certain embodiments, the methods of the invention are continuous processes.

[0141] In particular embodiments, acetate is produced as a by-product of the fermentation.

[0142] In a further embodiment the ethanol and the acetate are recovered from the broth.

[0143] In certain embodiments, the recovering of ethanol and acetate comprises continuously removing a portion of the broth and recovering separately ethanol and acetate from the removed portion of the broth.

[0144] In some embodiments the recovery of ethanol and acetate includes passing the removed portion of the broth containing ethanol and acetate through a separation unit to separate bacterial cells from the ethanol and acetate, to produce a cell-free ethanol-and acetate-containing permeate, and returning the bacterial cells to the bioreactor.

[0145] In the above embodiments, the recovery of ethanol and acetate preferably includes first removing ethanol from the cell-free permeate followed by removing acetate from the cell-free permeate. Preferably the cell-free permeate is then returned to the bioreactor.

[0146] Ethanol is the preferred desired end product of the fermentation. The ethanol may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, and extractive fermentation. Distillation of ethanol from a fermentation broth yields an azeotropic mixture of ethanol and water (i.e. 95% ethanol and 5% water). Anhydrous ethanol can subsequently be obtained through the use of molecular sieve ethanol dehydration technology, which is also well known in the art. Extractive fermentation procedures involve the use of a water-miscible solvent that presents a low toxicity risk to the fermentation organism, to recover the ethanol from the dilute fermentation broth. For example, oleyl alcohol is a solvent that may be used in this type of extraction process. Oleyl alcohol is continuously introduced into a fermenter, whereupon this solvent rises forming a layer at the top of the fermenter which is continuously extracted and fed through a centrifuge. Water and cells are then readily separated from the oleyl alcohol and returned to the fermenter while the ethanol-laden solvent is fed into a flash vaporization unit. Most of the ethanol is vaporized and condensed while the oleyl alcohol is non volatile and is recovered for re-use in the fermentation.

[0147] Acetate may also be recovered from the fermentation broth using methods known in the art. Methods for the recovery of acetate are described in detail in WO2007/117157 and WO2008/115080.

[0148] In certain embodiments of the invention, ethanol and acetate are recovered from the fermentation broth by continuously removing a portion of the broth from the fermentation bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering first ethanol and then acetate from the broth. The ethanol may conveniently be recovered by distillation, and the acetate may be recovered by adsorption on activated charcoal, using the methods described above. The separated microbial cells are preferably returned to the fermentation bioreactor. The cell free permeate remaining after the ethanol and acetate have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell free permeate to replenish the nutrient medium before it is returned to the bioreactor. Also, if the pH of the broth was adjusted as described above to enhance adsorption of acetic acid to the activated charcoal, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.

Reaction Stoichiometry

[0149] Without wishing to be bound by any theory, the chemical reactions for the fermentation of CO to ethanol (a) and acetic acid (b) in the process of the present invention are believed to be as follows:

[0150] (a) 6CO+3H₂O=>CH₃CH₂OH+4CO₂

[0151] (b) 4CO+2H₂O=>1CH₃COOH+2CO₂

[0152] The invention will now be described in more detail with reference to the following non-limiting examples.

EXAMPLES

Materials And Methods

TABLE-US-00001

[0153] Solution A NH₄Ac 3.083 g MgCl₂•6H₂O 0.4 g CaCl₂•2H₂O 0.294 g KCl 0.15 g NaCl (optional) 0.12 g Distilled Water Up to 1 L Solution B Biotin 20.0 mg Folic acid 20.0 mg Pyridoxine•HCl 10.0 mg Thiamine•HCl 50.0 mg Riboflavin 50.0 mg Nicotinic acid 50.0 mg Calcium D-(*)-pantothenate 50.0 mg Vitamin B12 50.0 mg p-Aminobenzoic acid 50.0 mg Thioctic acid 50.0 mg Distilled water To 1 Litre Solution C Component mmol/L H2O FeCl₃ 0.1 CoCl₂ 0.05 NiCl₂ 0.05 H₃BO₃ 0.01 Na₂SeO₃ 0.01 Na₂MoO₄ 0.01 ZnCl₂ 0.01 MnCl2 0.01 Na2WO3 0.01

Preparation of Cr (II) Solution

[0154] A 1 L three necked flask was fitted with a gas tight inlet and outlet to allow working under inert gas and subsequent transfer of the desired product into a suitable storage flask. The flask was charged with CrCl₃.6H₂O (40 g, 0.15 mol), zinc granules [20 mesh] (18.3 g, 0.28 mol), mercury (13.55 g, 1 mL, 0.0676 mol) and 500 mL of distilled water. Following flushing with N₂ for one hour, the mixture was warmed to about 80° C. to initiate the reaction. Following two hours of stirring under a constant N₂ flow, the mixture was cooled to room temperature and continuously stirred for another 48 hours by which time the reaction mixture had turned to a deep blue solution. The solution was transferred into N₂ purged serum bottles and stored in the fridge for future use.

Bacteria

[0155] The two types of Clostridium autoethanogenum used were those deposited at the German Resource Centre for Biological Material (DSMZ) and allocated the accession numbers DSM 19630 and DSM 23693. DSM 23693 was developed from Clostridium autoethanogenum strain DSM19630 (DSMZ, Germany) via an iterative selection process.

Sampling And Analytical Procedures

[0156] Media samples were taken from the CSTR reactor at intervals over the course of each fermentation. Each time the media was sampled care was taken to ensure that no gas was allowed to enter into or escape from the reactor.

HPLC

[0157] HPLC System Agilent 1100 Series. Mobile Phase: 0.0025N Sulfuric Acid. Flow and pressure: 0.800 mL/min. Column: Alltech 10A; Catalog #9648, 150×6.5 mm, particle size 5 ρm. Temperature of column: 60° C. Detector: Refractive Index. Temperature of detector: 45° C.

Method For Sample Preparation

[0158] 400 μL of sample and 50 μL of 0.15M ZnSO₄ and 50 μL of 0.15M Ba(OH)₂ are loaded into an Eppendorf tube. The tubes are centrifuged for 10 min. at 12,000 rpm, 4° C. 200 μL of the supernatant are transferred into an HPLC vial, and 5 μL are injected into the HPLC instrument.

Headspace Analysis

[0159] Measurements were carried out on a Varian CP-4900 micro GC with two installed channels. Channel 1 was a 10 m Mol-sieve column running at 70° C., 200 kPa argon and a backflush time of 4.2 s, while channel 2 was a 10 m PPQ column running at 90° C., 150 kPa helium and no backflush. The injector temperature for both channels was 70° C. Runtimes were set to 120 s, but all peaks of interest would usually elute before 100 s.

Cell Density

[0160] Cell density was determined by counting bacterial cells in a defined aliquot of fermentation broth. Alternatively, the absorbance of the samples was measured at 600 nm (spectrophotometer) and the dry mass determined via calculation according to published procedures.

Sequencing

[0161] Genome sequencing revealed several changes in genomes of C. autoethanogenum strain LZ1560 (DSM19630) and new strain LZ1561 (DSM23693), which are likely to contribute to the improved performance.

[0162] Both strains were grown anaerobically in PETC media to an optical density (OD₆₀₀ nm) of 1 and genomic DNA was isolated from 100 ml overnight cultures. Cells were harvested by centrifugation (6,000×g, 15 min, 4° C.), washed with potassium phosphate buffer (10 mM; pH 7.5) and suspended in 1.9 ml STE buffer (50 mM Tris-HCl, 1 mM EDTA, 200 mM sucrose; pH 8.0). This suspension was treated with 300 μl lysozyme (˜100,000 U; 30 min, 37° C.) and 280 μl of a SDS solution (10% (w/v); 10 min). RNA was digested by addition of 240 μl of an EDTA solution (0.5 M; pH 8), 20 μl Tris-HCl (1 M; pH 7.5), and 10 μl RNase A (50,000 U) for 1 hour. Proteolysis was performed by addition of 100 μl Proteinase K (0.5 U) for 1-3 h at 37° C. Finally, 600 μl of sodium perchlorate (5 M) were added, followed by a phenol-chloroform extraction and an isopropanol precipitation. Purity and quantity of DNA was verified using a NanoDrop® 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) and by gel electrophoresis.

[0163] Shotgun genome sequencing was performed using a 454 GS (Roche Applied Science, Indianapolis, Ind., USA). 191,368 single reads with a total length of 44,424,523 bases were created for LZ1560 (10× coverage), while 579,545 paired-end reads with a total length of 202,591,572 by were created for LZ1561 (47.5× coverage).The reads were assembled using the Newbler package (Roche Applied Science, Indianapolis, Ind., USA) and sequences compared using Geneious (Biomatters Ltd., Auckland, NZ) with the MAUVE package (Darling et al., 2004, Genome Res. 14: 1394-1403) and by Artemis Comparison Tool (Carver et al., 2008, Bioinformatics 24:2672-6).

[0164] A total of 64 changes were found in assembled genome sequences of LZ1560 (DSM19630) and new strain LZ1561 (DSM23693) (FIG. 4). While most changes were single base variations, one 21 bp deletion (in gene encoding a putative DNA mismatch repair protein MutS; Seq. ID. 1-4) and a rearrangement event of a 15,408 by region (Seq. ID. 5) containing 11 genes (involved in nitrogen fixation, sugar metabolism, sugar transport and catabolite control) were found. From the 62 single base variations, 22 were point mutations, and 40 insertions/deletions. 18 of these variations were found in intergenic regions and 44 in coding regions. While 5 of the variations in the coding region were silent and didn't result in a change of amino acid sequence, 14 resulted in a single amino acid change and 25 in a frameshift.

[0165] Most notably were changes in positions 212,530 (putative promoter region of F₁F_O ATP synthase operon, Seq. ID. 6-7), 1,171,874 (putative promoter region of Rnf complex operon, Seq. ID. 8-9), 3,717,495 (putative promoter region of carbon starvation protein, Seq. ID. 10-11), and two variations in the Wood-Ljungdahl-gene cluster at positions 3,741,730 (CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit, Seq. ID. 12-15) and 3,748,058 (5,10-methylenetetrahydrofolate reductase gene, Seq. ID. 16-17), which can be traced back directly to growth on CO/H₂ and energy metabolism. Most other genes affected are uncharacterized genes.

Example 1

A: Batch Fermentation In CSTR

[0166] Approximately 1500 mL of solution A was transferred into a 1.5 L fermenter and sparged with nitrogen. Resazurin (1.5 mL of a 2 g/L solution) and H₃PO₄ (85% solution, 2.25 mL) was added and the pH adjusted to 5.3 using concentrated NH₄OH (aq). Nitrilotriacetic acid (0.3 ml of a 0.15M solution) was added prior to 1.5 ml of solution C. This was followed by NiCl2 (0.75 ml of 0.1M solution) and Na₂WO₃ (1.5 mL of a 0.01M solution). 15 ml of solution B was added and the solution sparged with N2 before switching to CO containing gas (50% CO; 28% N2, 2% H2, 20% CO2) at 70 mL/min. The fermenter was then inoculated with 200 ml of a Clostridium autoethanogenum 19630 culture. The fermenter was maintained at 37° C. and stirred at 300 rpm. During this experiment, Na2S solution (0.2M solution) was added at a rate of approx 0.3 ml/hour. Substrate supply was increased in response to the requirements of the microbial culture.

[0167] The bacterial culture did not proliferate in the experimental conditions used. The culture showed a 350 mM CO uptake after 48 hrs of growth (FIG. 1a and Table 2) while the doubling time of the culture was 40.8 hrs (FIG. 2a). This corresponds to a specific growth rate of 0.41 day^-1. The specific CO uptake increased during the experiment with a maximum value of 0.54 mM CO/min/g biomass. Day 1.0 specific uptake: 0.28 mM CO/min/g biomass (Table 1). Day 2.0 specific uptake: 0.54 mM CO/min/g biomass (Table 2).

B: Batch Fermentation In CSTR

[0168] Approximately 1500 mL of solution A was transferred into a 1.5 L fermenter and sparged with nitrogen. Resazurin (1.5 mL of a 2 g/L solution) and H₃PO₄ (85% solution, 2.25 mL) was added and the pH adjusted to 5.3 using concentrated NH₄OH (aq). Nitrilotriacetic acid (0.3 ml of a 0.15M solution) was added prior to 1.5 ml of solution C. Na₂WO₃ (1.5 mL of a 0.01M solution) was added. 15 ml of Solution B was added and the solution sparged with N2 before switching to CO containing gas (50% CO; 50% N2) at 60 mL/min. The fermenter was then inoculated with 180 ml of a Clostridium autoethanogenum 23693 culture. The fermenter was maintained at 37° C. and stirred at 300 rpm. During this experiment, Na2S solution (0.5M solution) was added at a rate of approx 0.12 ml/hour. Substrate supply was increased in response to the requirements of the microbial culture.

[0169] The bacterial culture proliferated in the experimental conditions used. The culture showed a 8400 mM CO uptake after 43 hrs of growth (FIG. 2b) while the doubling time of the culture was 9.6 hrs (FIG. 2b). This corresponds to a specific growth rate of 1.73 day^-1. The maximum specific CO uptake reached during the experiment was 1.17 mMol CO/min/g biomass. Day 1.0 specific uptake: 1.17 mM CO/min/g biomass (Table 1). Day 2.0 specific uptake: 1.03 mM CO/min/g biomass (Table 2). The fermentation conditions were identical or at least highly similar to the conditions used in Example 1A. The media preparation has identical components at similar concentrations while both gasses contained CO at least 50% (v/v). The similar fermentation conditions compared to the vast difference in CO uptake indicates the culture performance varied due to the improved efficiency of the developed Clostridium autoethanogenum 23693 culture compared to the parent strain Clostridium autoethanogenum 19630.

Results

TABLE-US-00002

[0170] TABLE 1 Day 1 Strain DSM19630 DSM23693 CO consump- 113 mM 3700 mM tion mM/L Ethanol 0.48 g/L 7.98 g/L Production g/L Acetate 4.58 g/L 4.06 g/L Production g/L Biomass g/L 0.29 g/L 1.83 g/L Specific uptake 0.28 CO/min/g biomass 1.17 CO/min/g biomass Specific ethanol 2.5 g/L/g biomass/day 4.3 g/L/g biomass/day production

TABLE-US-00003 TABLE 2 Day 2 Strain DSM19630 DSM23693 CO consump- 350 mM 8150 mM tion mM/L Ethanol 1.84 g/L 26.14 g/L Production g/L Acetate 4.5 g/L 3.47 g/L Production g/L Biomass g/L 0.41 g/L 5.42 g/l Specific uptake 0.54 CO/min/g biomass 1.03 CO/min/g biomass Specific ethanol 3.0 g/L/g biomass/day 6.5 g/L/g biomass/day production

Example 2

[0171] Approximately 1500 mL of solution A was transferred into a 1.5 L fermenter and sparged with nitrogen. Resazurin (1.5 mL of a 2 g/L solution) and H₃PO₄ (85% solution, 0.56 mL) was added and the pH adjusted to 5.3 using concentrated NH₄OH(aq). Solution C (1.5 mL) was added after which Na₂WO₃ (1.5 mL of a 0.01M solution) was added. 15 ml of Solution B was added and the solution sparged with N2 before switching to CO containing gas (50% CO; 50% N2) at 60 mL/min. The fermenter was then inoculated with 100 ml of a Clostridium autoethanogenum 23693 culture. The fermenter was maintained at 37° C. and stirred at 300 rpm. During this experiment, Na2S solution (0.5M solution) was added at a rate of approx 0.15 ml/hour. Substrate supply was increased in response to the requirements of the microbial culture.

[0172] The bacterial culture proliferated in the experimental conditions used. The fermentation conditions were identical or at least highly similar to the conditions used in Example 1A+B while both gasses contained CO at least 50% (v/v). The culture was grown to the stationary phase where maximum ethanol concentration was measured by HPLC (55.8 g/L).

[0173] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the scope and spirit of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practised in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, the terms "comprising", "including", "containing" etc are to be read expansively and without limitation. Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.

[0174] The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents and publications in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Sequence CWU 1

1

1712655DNAClostridium autoethanogenum 1gtggcattaa ctccaatgat gcagcagtat atggaagtaa aagaaagtta taaagactgt 60atactgtttt ttagattagg tgatttttat gaaatgtttt ttgaagatgc aaaaatagca 120tctagggaac tagaattagt acttacaggt agagattgcg gcttaaaaga aagggctcct 180atgtgtggaa taccctatca tgccgcaaat tcatatatag gaaggcttat aaacaaggga 240tataagatag ctatatgtga gcaactagaa gatccagctc tagcaaaagg tatagttaaa 300aggggcataa tcaaggttgt aactcctggg acttatacgg actctacttt tttagaagaa 360aataaaaata attatatagc gtgtttgtac atagatacca agacaaatat ttctgcattg 420tgttttgcag atgtgtccac tggagaattc aattgtacgg acactccttt taatttatct 480ataattttag atgaaatatc aaaatactct ccaagtgaac tagtgattca aggtagtata 540agtgctgatt tattgaataa aatgaaggat atttttaatg gctcattcac taaattagat 600gaaagctatt ttgcagacga aactaaaaat atattagagg atcagtttga aaattttaca 660ggagaaaatt atagtaatga aattataaaa tgctgcggct cactactaaa atatataagg 720gaaactcaaa agaatgatct atctcatata aataaatttt cctattacaa tatagtagat 780tatctcacta tagatgggaa ttccagaaga aatttagaaa ttacagaaag cttaagagaa 840aataataaaa aaggatctct cctctgggtt atagataaaa caaatacatc tatgggaggg 900agacagctta gaagatggct ggaacagccc cttataaata aggttaagat agaagaaaga 960ctggattctg tagaggaaat ttcaaataat atatcctatc atgaagatct aaaagaggct 1020ttaaaaaaca tatatgatat tgagcgatta gttggaaaaa tatcttctaa aagtgtaaac 1080gcaaaagaac taaatttttt aaaaaattct atagaaaaaa tacctgaagt aaaatccata 1140ctatccaatt ttcatacaaa attattgaag gatatgtacg aaaacttgga tgaactaaag 1200gacatatatt cacttttaga taaatctata ttagataatc ctgcaatatc tttaaaagaa 1260ggtaacctta taaaaaaggg atataacagt gacatagatg aacttaaaga aataaaggct 1320cacggtaagg agtggatagc ttctcttgag aattcagaaa gggaagttac taaaataaaa 1380tctcttaaaa taggttataa taaagtattt ggttattata ttgaggttac taaaagtaat 1440ttaagtcttg taccagaagg tagatatata agaaaacaaa ctcttacaaa tgcagaaaga 1500tacataactc ctgaattgaa agaaatggaa gataaaatat taggtctaga atatagtgtc 1560tttatagaag taagagataa aatagaaaat gaagtagaca gaatgcaaaa atccgctaaa 1620ataatttcag aagtagattg cttaagctcc cttgcaagag ttgctataga aaataattat 1680tgtaaacctg aaataacaaa ttcagataac ataattatag aagaaggtag acatcctgta 1740gtagaaaaga tgattgactc tggagaattt atatcaaacg atataaacat agatactggt 1800aaaaatcaac ttcttttaat aacagggcct aatatggcag gcaaatctac ctacatgagg 1860cagatagctt tgatcgttat aatggcccaa attggtagct ttgtaccggc aaaaaatgct 1920tctatatctg tttgtgataa gatatttaca aggataggtg catcagatga cctggcatca 1980ggaaagagta cctttatggt ggaaatgtgg gaggtttcca acatacttaa aaatgcaact 2040aacaaaagtt tgattttact ggatgaagta ggacgtggaa caagtactta cgacggcctt 2100agcatagcct ggtcagtaat agaatatata tgtaaaaaca gcaaactaaa atgtaaaacc 2160ttatttgcaa cccattatca tgaactaact aaattagaag gtaagataga tggagtcaaa 2220aattactgtg tatccgttaa agaaatggag gataatatag tttttttgag aaaaattata 2280agaggaggag ccgaccaatc ctacggcata gaagttgcaa agcttgcagg gcttccagaa 2340gaagttttaa aaagagcagg agaaatacta aatagccttg aaagtaaaaa gctaaaagaa 2400aataaatgtg ttgattctga aattgcatta gattcagagt attcaataaa tgagaaaaaa 2460gcacctctta aaaatgagga gatgattaaa gaaaaagctc ctatgcttga accaaccaga 2520caattaggat tttcagatat agaaaagaca aatttagtaa aagatattac ggatatagat 2580atactaaaca tgactcctat ggacggattt aataaacttt atgatataat aagaagagca 2640aagtccataa gataa 265522676DNAClostridium autoethanogenum 2gtggcattaa ctccaatgat gcagcagtat atggaagtaa aagaaagtta taaagactgt 60atactgtttt ttagattagg tgatttttat gaaatgtttt ttgaagatgc aaaaatagca 120tctagggaac tagaattagt acttacaggt agagattgcg gcttaaaaga aagggctcct 180atgtgtggaa taccctatca tgccgcaaat tcatatatag gaaggcttat aaacaaggga 240tataagatag ctatatgtga gcaactagaa gatccagctc tagcaaaagg tatagttaaa 300aggggcataa tcaaggttgt aactcctggg acttatacgg actctacttt tttagaagaa 360aataaaaata attatatagc gtgtttgtac atagatacca agacaaatat ttctgcattg 420tgttttgcag atgtgtccac tggagaattc aattgtacgg acactccttt taatttatct 480ataattttag atgaaatatc aaaatactct ccaagtgaac tagtgattca aggtagtata 540agtgctgatt tattgaataa aatgaaggat atttttaatg gctcattcac taaattagat 600gaaagctatt ttgcagacga aactaaaaat atattagagg atcagtttga aaattttaca 660ggagaaaatt atagtaatga aattataaaa tgctgcggct cactactaaa atatataagg 720gaaactcaaa agaatgatct atctcatata aataaatttt cctattacaa tatagtagat 780tatctcacta tagatgggaa ttccagaaga aatttagaaa ttacagaaag cttaagagaa 840aataataaaa aaggatctct cctctgggtt atagataaaa caaatacatc tatgggaggg 900agacagctta gaagatggct ggaacagccc cttataaata aggttaagat agaagaaaga 960ctggattctg tagaggaaat ttcaaataat atatcctatc atgaagatct aaaagaggct 1020ttaaaaaaca tatatgatat tgagcgatta gttggaaaaa tatcttctaa aagtgtaaac 1080gcaaaagaac taaatttttt aaaaaattct atagaaaaaa tacctgaagt aaaatccata 1140ctatccaatt ttcatacaaa attattgaag gatatgtacg aaaacttgga tgaactaaag 1200gacatatatt cacttttaga taaatctata ttagataatc ctgcaatatc tttaaaagaa 1260ggtaacctta taaaaaaggg atataacagt gacatagatg aacttaaaga aataaaggct 1320cacggtaagg agtggatagc ttctcttgag aattcagaaa gggaagttac taaaataaaa 1380tctcttaaaa taggttataa taaagtattt ggttattata ttgaggttac taaaagtaat 1440ttaagtcttg taccagaagg tagatatata agaaaacaaa ctcttacaaa tgcagaaaga 1500tacataactc ctgaattgaa agaaatggaa gataaaatat taggtgcaga ggaaaaactt 1560ataaatctag aatatagtgt ctttatagaa gtaagagata aaatagaaaa tgaagtagac 1620agaatgcaaa aatccgctaa aataatttca gaagtagatt gcttaagctc ccttgcaaga 1680gttgctatag aaaataatta ttgtaaacct gaaataacaa attcagataa cataattata 1740gaagaaggta gacatcctgt agtagaaaag atgattgact ctggagaatt tatatcaaac 1800gatataaaca tagatactgg taaaaatcaa cttcttttaa taacagggcc taatatggca 1860ggcaaatcta cctacatgag gcagatagct ttgatcgtta taatggccca aattggtagc 1920tttgtaccgg caaaaaatgc ttctatatct gtttgtgata agatatttac aaggataggt 1980gcatcagatg acctggcatc aggaaagagt acctttatgg tggaaatgtg ggaggtttcc 2040aacatactta aaaatgcaac taacaaaagt ttgattttac tggatgaagt aggacgtgga 2100acaagtactt acgacggcct tagcatagcc tggtcagtaa tagaatatat atgtaaaaac 2160agcaaactaa aatgtaaaac cttatttgca acccattatc atgaactaac taaattagaa 2220ggtaagatag atggagtcaa aaattactgt gtatccgtta aagaaatgga ggataatata 2280gtttttttga gaaaaattat aagaggagga gccgaccaat cctacggcat agaagttgca 2340aagcttgcag ggcttccaga agaagtttta aaaagagcag gagaaatact aaatagcctt 2400gaaagtaaaa agctaaaaga aaataaatgt gttgattctg aaattgcatt agattcagag 2460tattcaataa atgagaaaaa agcacctctt aaaaatgagg agatgattaa agaaaaagct 2520cctatgcttg aaccaaccag acaattagga ttttcagata tagaaaagac aaatttagta 2580aaagatatta cggatataga tatactaaac atgactccta tggacggatt taataaactt 2640tatgatataa taagaagagc aaagtccata agataa 26763884PRTClostridium autoethanogenum 3Met Ala Leu Thr Pro Met Met Gln Gln Tyr Met Glu Val Lys Glu Ser 1 5 10 15 Tyr Lys Asp Cys Ile Leu Phe Phe Arg Leu Gly Asp Phe Tyr Glu Met 20 25 30 Phe Phe Glu Asp Ala Lys Ile Ala Ser Arg Glu Leu Glu Leu Val Leu 35 40 45 Thr Gly Arg Asp Cys Gly Leu Lys Glu Arg Ala Pro Met Cys Gly Ile 50 55 60 Pro Tyr His Ala Ala Asn Ser Tyr Ile Gly Arg Leu Ile Asn Lys Gly 65 70 75 80 Tyr Lys Ile Ala Ile Cys Glu Gln Leu Glu Asp Pro Ala Leu Ala Lys 85 90 95 Gly Ile Val Lys Arg Gly Ile Ile Lys Val Val Thr Pro Gly Thr Tyr 100 105 110 Thr Asp Ser Thr Phe Leu Glu Glu Asn Lys Asn Asn Tyr Ile Ala Cys 115 120 125 Leu Tyr Ile Asp Thr Lys Thr Asn Ile Ser Ala Leu Cys Phe Ala Asp 130 135 140 Val Ser Thr Gly Glu Phe Asn Cys Thr Asp Thr Pro Phe Asn Leu Ser 145 150 155 160 Ile Ile Leu Asp Glu Ile Ser Lys Tyr Ser Pro Ser Glu Leu Val Ile 165 170 175 Gln Gly Ser Ile Ser Ala Asp Leu Leu Asn Lys Met Lys Asp Ile Phe 180 185 190 Asn Gly Ser Phe Thr Lys Leu Asp Glu Ser Tyr Phe Ala Asp Glu Thr 195 200 205 Lys Asn Ile Leu Glu Asp Gln Phe Glu Asn Phe Thr Gly Glu Asn Tyr 210 215 220 Ser Asn Glu Ile Ile Lys Cys Cys Gly Ser Leu Leu Lys Tyr Ile Arg 225 230 235 240 Glu Thr Gln Lys Asn Asp Leu Ser His Ile Asn Lys Phe Ser Tyr Tyr 245 250 255 Asn Ile Val Asp Tyr Leu Thr Ile Asp Gly Asn Ser Arg Arg Asn Leu 260 265 270 Glu Ile Thr Glu Ser Leu Arg Glu Asn Asn Lys Lys Gly Ser Leu Leu 275 280 285 Trp Val Ile Asp Lys Thr Asn Thr Ser Met Gly Gly Arg Gln Leu Arg 290 295 300 Arg Trp Leu Glu Gln Pro Leu Ile Asn Lys Val Lys Ile Glu Glu Arg 305 310 315 320 Leu Asp Ser Val Glu Glu Ile Ser Asn Asn Ile Ser Tyr His Glu Asp 325 330 335 Leu Lys Glu Ala Leu Lys Asn Ile Tyr Asp Ile Glu Arg Leu Val Gly 340 345 350 Lys Ile Ser Ser Lys Ser Val Asn Ala Lys Glu Leu Asn Phe Leu Lys 355 360 365 Asn Ser Ile Glu Lys Ile Pro Glu Val Lys Ser Ile Leu Ser Asn Phe 370 375 380 His Thr Lys Leu Leu Lys Asp Met Tyr Glu Asn Leu Asp Glu Leu Lys 385 390 395 400 Asp Ile Tyr Ser Leu Leu Asp Lys Ser Ile Leu Asp Asn Pro Ala Ile 405 410 415 Ser Leu Lys Glu Gly Asn Leu Ile Lys Lys Gly Tyr Asn Ser Asp Ile 420 425 430 Asp Glu Leu Lys Glu Ile Lys Ala His Gly Lys Glu Trp Ile Ala Ser 435 440 445 Leu Glu Asn Ser Glu Arg Glu Val Thr Lys Ile Lys Ser Leu Lys Ile 450 455 460 Gly Tyr Asn Lys Val Phe Gly Tyr Tyr Ile Glu Val Thr Lys Ser Asn 465 470 475 480 Leu Ser Leu Val Pro Glu Gly Arg Tyr Ile Arg Lys Gln Thr Leu Thr 485 490 495 Asn Ala Glu Arg Tyr Ile Thr Pro Glu Leu Lys Glu Met Glu Asp Lys 500 505 510 Ile Leu Gly Leu Glu Tyr Ser Val Phe Ile Glu Val Arg Asp Lys Ile 515 520 525 Glu Asn Glu Val Asp Arg Met Gln Lys Ser Ala Lys Ile Ile Ser Glu 530 535 540 Val Asp Cys Leu Ser Ser Leu Ala Arg Val Ala Ile Glu Asn Asn Tyr 545 550 555 560 Cys Lys Pro Glu Ile Thr Asn Ser Asp Asn Ile Ile Ile Glu Glu Gly 565 570 575 Arg His Pro Val Val Glu Lys Met Ile Asp Ser Gly Glu Phe Ile Ser 580 585 590 Asn Asp Ile Asn Ile Asp Thr Gly Lys Asn Gln Leu Leu Leu Ile Thr 595 600 605 Gly Pro Asn Met Ala Gly Lys Ser Thr Tyr Met Arg Gln Ile Ala Leu 610 615 620 Ile Val Ile Met Ala Gln Ile Gly Ser Phe Val Pro Ala Lys Asn Ala 625 630 635 640 Ser Ile Ser Val Cys Asp Lys Ile Phe Thr Arg Ile Gly Ala Ser Asp 645 650 655 Asp Leu Ala Ser Gly Lys Ser Thr Phe Met Val Glu Met Trp Glu Val 660 665 670 Ser Asn Ile Leu Lys Asn Ala Thr Asn Lys Ser Leu Ile Leu Leu Asp 675 680 685 Glu Val Gly Arg Gly Thr Ser Thr Tyr Asp Gly Leu Ser Ile Ala Trp 690 695 700 Ser Val Ile Glu Tyr Ile Cys Lys Asn Ser Lys Leu Lys Cys Lys Thr 705 710 715 720 Leu Phe Ala Thr His Tyr His Glu Leu Thr Lys Leu Glu Gly Lys Ile 725 730 735 Asp Gly Val Lys Asn Tyr Cys Val Ser Val Lys Glu Met Glu Asp Asn 740 745 750 Ile Val Phe Leu Arg Lys Ile Ile Arg Gly Gly Ala Asp Gln Ser Tyr 755 760 765 Gly Ile Glu Val Ala Lys Leu Ala Gly Leu Pro Glu Glu Val Leu Lys 770 775 780 Arg Ala Gly Glu Ile Leu Asn Ser Leu Glu Ser Lys Lys Leu Lys Glu 785 790 795 800 Asn Lys Cys Val Asp Ser Glu Ile Ala Leu Asp Ser Glu Tyr Ser Ile 805 810 815 Asn Glu Lys Lys Ala Pro Leu Lys Asn Glu Glu Met Ile Lys Glu Lys 820 825 830 Ala Pro Met Leu Glu Pro Thr Arg Gln Leu Gly Phe Ser Asp Ile Glu 835 840 845 Lys Thr Asn Leu Val Lys Asp Ile Thr Asp Ile Asp Ile Leu Asn Met 850 855 860 Thr Pro Met Asp Gly Phe Asn Lys Leu Tyr Asp Ile Ile Arg Arg Ala 865 870 875 880 Lys Ser Ile Arg 4891PRTClostridium autoethanogenum 4Met Ala Leu Thr Pro Met Met Gln Gln Tyr Met Glu Val Lys Glu Ser 1 5 10 15 Tyr Lys Asp Cys Ile Leu Phe Phe Arg Leu Gly Asp Phe Tyr Glu Met 20 25 30 Phe Phe Glu Asp Ala Lys Ile Ala Ser Arg Glu Leu Glu Leu Val Leu 35 40 45 Thr Gly Arg Asp Cys Gly Leu Lys Glu Arg Ala Pro Met Cys Gly Ile 50 55 60 Pro Tyr His Ala Ala Asn Ser Tyr Ile Gly Arg Leu Ile Asn Lys Gly 65 70 75 80 Tyr Lys Ile Ala Ile Cys Glu Gln Leu Glu Asp Pro Ala Leu Ala Lys 85 90 95 Gly Ile Val Lys Arg Gly Ile Ile Lys Val Val Thr Pro Gly Thr Tyr 100 105 110 Thr Asp Ser Thr Phe Leu Glu Glu Asn Lys Asn Asn Tyr Ile Ala Cys 115 120 125 Leu Tyr Ile Asp Thr Lys Thr Asn Ile Ser Ala Leu Cys Phe Ala Asp 130 135 140 Val Ser Thr Gly Glu Phe Asn Cys Thr Asp Thr Pro Phe Asn Leu Ser 145 150 155 160 Ile Ile Leu Asp Glu Ile Ser Lys Tyr Ser Pro Ser Glu Leu Val Ile 165 170 175 Gln Gly Ser Ile Ser Ala Asp Leu Leu Asn Lys Met Lys Asp Ile Phe 180 185 190 Asn Gly Ser Phe Thr Lys Leu Asp Glu Ser Tyr Phe Ala Asp Glu Thr 195 200 205 Lys Asn Ile Leu Glu Asp Gln Phe Glu Asn Phe Thr Gly Glu Asn Tyr 210 215 220 Ser Asn Glu Ile Ile Lys Cys Cys Gly Ser Leu Leu Lys Tyr Ile Arg 225 230 235 240 Glu Thr Gln Lys Asn Asp Leu Ser His Ile Asn Lys Phe Ser Tyr Tyr 245 250 255 Asn Ile Val Asp Tyr Leu Thr Ile Asp Gly Asn Ser Arg Arg Asn Leu 260 265 270 Glu Ile Thr Glu Ser Leu Arg Glu Asn Asn Lys Lys Gly Ser Leu Leu 275 280 285 Trp Val Ile Asp Lys Thr Asn Thr Ser Met Gly Gly Arg Gln Leu Arg 290 295 300 Arg Trp Leu Glu Gln Pro Leu Ile Asn Lys Val Lys Ile Glu Glu Arg 305 310 315 320 Leu Asp Ser Val Glu Glu Ile Ser Asn Asn Ile Ser Tyr His Glu Asp 325 330 335 Leu Lys Glu Ala Leu Lys Asn Ile Tyr Asp Ile Glu Arg Leu Val Gly 340 345 350 Lys Ile Ser Ser Lys Ser Val Asn Ala Lys Glu Leu Asn Phe Leu Lys 355 360 365 Asn Ser Ile Glu Lys Ile Pro Glu Val Lys Ser Ile Leu Ser Asn Phe 370 375 380 His Thr Lys Leu Leu Lys Asp Met Tyr Glu Asn Leu Asp Glu Leu Lys 385 390 395 400 Asp Ile Tyr Ser Leu Leu Asp Lys Ser Ile Leu Asp Asn Pro Ala Ile 405 410 415 Ser Leu Lys Glu Gly Asn Leu Ile Lys Lys Gly Tyr Asn Ser Asp Ile 420 425 430 Asp Glu Leu Lys Glu Ile Lys Ala His Gly Lys Glu Trp Ile Ala Ser 435 440 445 Leu Glu Asn Ser Glu Arg Glu Val Thr Lys Ile Lys Ser Leu Lys Ile 450 455 460 Gly Tyr Asn Lys Val Phe Gly Tyr Tyr Ile Glu Val Thr Lys Ser Asn 465 470 475 480 Leu Ser Leu Val Pro Glu Gly Arg Tyr Ile Arg Lys Gln Thr Leu Thr 485 490 495 Asn Ala Glu Arg Tyr Ile Thr Pro Glu Leu Lys Glu Met Glu Asp Lys 500 505 510 Ile Leu Gly Ala Glu Glu Lys Leu Ile Asn Leu Glu Tyr Ser Val Phe 515 520 525 Ile Glu Val Arg Asp Lys Ile Glu Asn Glu Val Asp Arg Met Gln Lys 530 535 540 Ser Ala Lys Ile Ile Ser Glu Val Asp Cys Leu Ser Ser Leu Ala Arg 545 550 555 560 Val Ala Ile Glu Asn Asn Tyr Cys Lys Pro Glu Ile Thr Asn Ser Asp 565 570 575 Asn Ile

Ile Ile Glu Glu Gly Arg His Pro Val Val Glu Lys Met Ile 580 585 590 Asp Ser Gly Glu Phe Ile Ser Asn Asp Ile Asn Ile Asp Thr Gly Lys 595 600 605 Asn Gln Leu Leu Leu Ile Thr Gly Pro Asn Met Ala Gly Lys Ser Thr 610 615 620 Tyr Met Arg Gln Ile Ala Leu Ile Val Ile Met Ala Gln Ile Gly Ser 625 630 635 640 Phe Val Pro Ala Lys Asn Ala Ser Ile Ser Val Cys Asp Lys Ile Phe 645 650 655 Thr Arg Ile Gly Ala Ser Asp Asp Leu Ala Ser Gly Lys Ser Thr Phe 660 665 670 Met Val Glu Met Trp Glu Val Ser Asn Ile Leu Lys Asn Ala Thr Asn 675 680 685 Lys Ser Leu Ile Leu Leu Asp Glu Val Gly Arg Gly Thr Ser Thr Tyr 690 695 700 Asp Gly Leu Ser Ile Ala Trp Ser Val Ile Glu Tyr Ile Cys Lys Asn 705 710 715 720 Ser Lys Leu Lys Cys Lys Thr Leu Phe Ala Thr His Tyr His Glu Leu 725 730 735 Thr Lys Leu Glu Gly Lys Ile Asp Gly Val Lys Asn Tyr Cys Val Ser 740 745 750 Val Lys Glu Met Glu Asp Asn Ile Val Phe Leu Arg Lys Ile Ile Arg 755 760 765 Gly Gly Ala Asp Gln Ser Tyr Gly Ile Glu Val Ala Lys Leu Ala Gly 770 775 780 Leu Pro Glu Glu Val Leu Lys Arg Ala Gly Glu Ile Leu Asn Ser Leu 785 790 795 800 Glu Ser Lys Lys Leu Lys Glu Asn Lys Cys Val Asp Ser Glu Ile Ala 805 810 815 Leu Asp Ser Glu Tyr Ser Ile Asn Glu Lys Lys Ala Pro Leu Lys Asn 820 825 830 Glu Glu Met Ile Lys Glu Lys Ala Pro Met Leu Glu Pro Thr Arg Gln 835 840 845 Leu Gly Phe Ser Asp Ile Glu Lys Thr Asn Leu Val Lys Asp Ile Thr 850 855 860 Asp Ile Asp Ile Leu Asn Met Thr Pro Met Asp Gly Phe Asn Lys Leu 865 870 875 880 Tyr Asp Ile Ile Arg Arg Ala Lys Ser Ile Arg 885 890 515408DNAClostridium autoethanogenum 5taagtttttt ggccagatag ctcagtcggt agagcagagg actgaaaatc ctcgtgtccc 60tggttcgatt cctggtctgg ccaccaaaag tttcagatat aatctgaaac tttttttatt 120ttatatagaa aaaataattg aactatgtaa taattaagta taaaatataa aaatattaag 180gagaaactta tatgcaaaat aatgtgttag caattgtaaa tggaatggaa ataaaagaaa 240gcgatcttaa agaagcaatt aatagatttc cacaagataa gagaaatcaa ttaaatacag 300ctgaaggtaa gaaatattta cttaatgaga tggtattttt tgaattagca tatagttacg 360ctaaagatga aaacttagaa aaagatgatg agtatttaaa gatgctggaa tctgctaaaa 420aagaaatatt aactcaaata gctatatcta aagttatgaa taaggttaat gtaactgata 480aagaaagtca ggattattat gaagccaata aagatatgta taaaaagcct gaaagattaa 540aagcaaagca tatattagta gatagtatag aaaaagctaa gaaaatttca aaagagatct 600cagaaggtat gccttttgaa gaagctgcac aaaaatattc aacttgtcca tctaaggctc 660aaggtggcag tttaggtgaa tttgctagag gacaaatggt tccagaattt gagaacgctg 720catttagttt agatatagat gtagttagtg aacctgtaaa aactcaattt ggataccacc 780ttataaaagt ggaagagaaa atagaacctt ctatagcatc ttatgatgaa gtaaagaacg 840ctattaagaa tggattatta caggaaagac agaaatatga gtattcaaaa tttaataaag 900aattgagaag taaatataaa gttgaaatga agtaatacat attgcaataa aaatgccaag 960gtaataatgg acataggttt gtgtaaaaac aaaacctatg tccattattt tatgtaactt 1020tttgaaattt aatggaaatt tatttcaaat taaactaaaa tggtatttaa tattatagca 1080ttttctttta tttgcaggta atataatgtc accgaaattt ttataatgaa ttgcatcagg 1140taatgtctgc ggttccaaag ctattcccat attggaaact aatttttgat tttgcacttt 1200ccaagtgtca tctttaatat taagagtata aacgattaga gcattacgat ctgattcaat 1260gtctaaacgc ctacatgtat catgattaat taaagtagca atatttttac catttacatt 1320aaagggatgg tcaaagccat tgaggcccga ttcagtttgc aaatatttaa ttgcatcttc 1380caaattttta ggctgctgga aatcaaaagg tgttccagaa acttctttta aagtaccagt 1440tggtaataag gcttcattaa tttcagcata atgggatgca ttaatttgta gtgtatgacc 1500tgagaacaac tttgtaatgt cattatttaa gttgaagtat gaatgtactg taggattaaa 1560aagtgcatct ttattgctta atccactaaa agtgattaat aaatcattat tgttattaag 1620tgtatattta acctctacat ttaatgtccc aggaaatcca tcttcgtcgg gagatatagt 1680cctatgaaaa agtacactag tggcttcttc agagtcttct gtgctactat tccatataag 1740agaattgaac ccataagtgc caccatgaag tgtattttca ccttcatttt taggaacgtt 1800ataggagata cctttaattt taaaagagcc attagctatg cgaccagcta ctggtccgat 1860agctgctcct agaaacatct ttctatcttt tatataacta tctaagttgt caaagcctaa 1920taaaacgttt gaaaagttgc ctaacttatc gggtacaata atttctgtta aagtagctcc 1980gtgtgagata caggaaactt tcataccatg tttgttagtc attgtatatt tataaatttt 2040gtttcctttg aaagaaccaa aaacactttt ttctatcatt ttatatactc ctttaatatt 2100taattttatc cgatgactac ctgctctaat actcacactt ttccaagtgg gagtaaagat 2160cagctacgtc tctggataac gatttctaag catcaggcgg agttaaaaca ccgtctgatg 2220cttagaactc tgtttataag ataaaaaatt taggtatgct taattatttg tgttatgata 2280atattaaata ataagcatac ctttatataa ttaattattt tgtagtttct cttataacat 2340ctcttaaatt tgtattatga tctataataa ttgattctaa attaaccatg cgggcccaat 2400catatacttg ttcaactgtt aatcctagag taagaacagt atgatgacca ccaccagcat 2460aaatccaggc ttttacacca tcagcaaagt ttggttcagg tttccatacc atcttagcta 2520cagggagttc aggagtatcc tctgcaggtt ctacagcatt tacttcactg attatcaaac 2580gataatgtgt tccaagatct agcattgtca ttgaagtacc tttaccagta gatccattga 2640atatcaaacg tgcaggatct tcacgatcac ctattcctaa gggttttacc acaactctag 2700gtttatcaga tgcaaatgtt ggatcaacct ccaacatatg agcgcctaag atttcctcat 2760taccttcact taattcataa gtataatctt ccataaatcc tgtcttttta ttgtttgtca 2820tgatttttat aagtctactt aaagcagcag ttttccaatc accttctcct gcaaagccat 2880atccttcagc atttaaacgc tgaactgcaa gtccaggtaa ctgtttcata ccatataaat 2940cttcgaaatt tgttgtaaat gcagtatagc ctcctgcttc taagaagtga cgaagtccaa 3000tttcaatttt aatttgttct tttacttgat tctcatagaa tttagggtca ttttctccta 3060catccataat ataaatcttt ttaaattctt cataagtatc atcaatgtct ttttgggaaa 3120ctttattcat ttcagcaact aaatcaccaa taccaaaata atctactgtc catccaaact 3180gaatttgagc ttcaatctta tcaccttcag taacagcaac attacgcata ttatcaccaa 3240aacgtgcaac tttgatgtct tgacttaaga tataacctgc ggctacgttc atccaatcag 3300caatttgttt ttgaacattt tcttttttcc aatgtcctac aacaatttta ttatgtttct 3360ttaatctagc attaataaaa ccatattctc tatcaccatg agcactttga tgtaagttca 3420tataatccat atcaattgtt ttccaaggaa tatgttcgta aaattgtgtt gctagatgaa 3480gtaatggttt ttgtaataat ttagtaccag caatccacat tttagctggt gagaatgtgt 3540gcatccatgt aataacacca gctacattat cattgtagtt tacttccttc ataagttttg 3600ttatttggct cgcagaagtt gcaagagatt taaatacaat aggataaggt aatttgccac 3660tcttatttaa agcatctgca atttccatag aatgttcttt tacttctgat agtgcctctt 3720caccatataa atgttggctg cctacaataa accaaaattc catttcttta ttttttagca 3780taattgaccc tctttccttg tttttattta ttattttgac catagtatgc attttttccg 3840tgtttgcgtt taaaatgctt atctaataaa acttgatcca ttctaatatt atggggattt 3900aattgtaagg aatgatatgt aatttttgca acctcttcca atacgactgc attatgaact 3960gcatttttag gatcggttcc ccaagtaaat ggaccatgat catttacaag aactccaggt 4020atatcatttg gattaatact attgtccttg aatgttctaa caattacatc tcctgtctgc 4080ttctcataat ctgtcactat ttcatcttta gtcatctttg gtgttacagg aatatctccg 4140tagaaataat cgccatgtgt agtaccagct gcaggaatgc aaatacctgc ttgagcaaaa 4200gacacggccc aaggagaatg tgtgtgtaca atgcctaaga tgtcagggaa gtttctatat 4260aatactaagt gagttgctgt atcacttgac ggatttaaat ctccttcaat tactttccca 4320ttaagatcta caacgaccat atcacttgct ttcatttttg tatattcaac accactaggt 4380ttaataacta caagactctg actgcgatca attccactta cgttgcccca tgtaaatgtt 4440accatatggt attttggcaa catcaaatta gcctcgagca ctttttcttt taaatcttct 4500aacatgagct aaccttcttt cactaataag ataaattatc ttagttgttt ttaacttatt 4560catctagatt aaataatcta ctgcagcttg ttcaatagga attccatttt tataacgttt 4620cataaattgt tcaaaacctt taacgtcttt tgcttcaggt tttacttcat cagctacata 4680accagcaaaa actttttttg ataaaaattc tgctagcgtg gagttacttt ctttattatt 4740caaataacta gctaatagtg caattcccca tgcaccgcct tcacctgcag tttccataac 4800tgatactggt gtatttactg cagctgcaac tgcttcttgt cctacaatag gtgtcttgaa 4860taatccacca tgaccaagta acttatcaag ctttacgcct tcatctttta ggagaatgtc 4920cattccaatt ttcaaggctc ctaatgaagt aaagagatgt gctttcataa agtttggtag 4980attaaaatta ctttttggtt ttcgtacaat tagaggtcta ccttccggaa cacctgtgat 5040attttcgcct gcaaaatagt tatatgcaag aattcctcca caatcagggt cacctagaaa 5100tgcttgagta tagagtccca tataaagaag atctcttgga atgttgacat ttattgcttc 5160ggcaaattca tgaaagatat taatccatgc gtctatatca gaataaccat tatttgcatg 5220aaccattcca actaagtctc ctgtaggtgt tgttaccata tcaatctctg gatgaacatt 5280cttaagtgga ttctcaagga cgaccattgc aaaaattgat gtgcctgcag agatatttcc 5340tgtacgtggt gcaactgaat ttgtagcaac cattcctgta cctgcatctc cttcaggtgg 5400acatagtgga ataccacttt gaagatttcc acttacatca agtttattgg ctccttctgg 5460agttaagacg ccagcatttt ctccggctag taagactttt ggtagaagct tctctacatt 5520caatgaatac ttttcaactt ctggcaaatg attgaatgtt tctaacatat gtttatcgta 5580attatgagtt ttcatatcaa ttggaaacat tcctgatgca tcacaaatac ccaaaacctt 5640tttacctgtt aattcccaat gaatataacc agctaaagta gttataaaat caagcttttc 5700aatatgaggt tctttatgga gaatagcctg atataaatga gctatactcc atctctcagg 5760aatattgaag tgaaatgatt ctgttaactt tttggctgct tcctctgtca tggtattacg 5820ccatgtccta aatggaacaa gtaggtttcc ttgtttatca aatgccatat atccatgcat 5880cattgctgaa aaaccaattg aaccaatttt tgaaagtgtt acaccatatt tttcttttat 5940ctcagcaaaa agtttttgat aactatattg taatcctttc cagatttctt ctagcgaata 6000ggtccaaata ccatctctta agcttgtctc ccattcaaag ctgccactag ctaatggaga 6060aaaatcgttt ccaataagaa cagctttaat acgagtagaa ccaaattcta ttcctaatga 6120tgttttacca ttttgaattt cttttactct atccttattt atagtcaata gaattacctc 6180ccccttacaa taatagtaaa cggtttttta tgataaatat atcatttata tacgtacatg 6240tcaataaata tataaaaaaa gatacgtact tattttaaat gtatttagat aattacttat 6300taaataatta tttttagtga tgatattatc gcgtttcatt acagcgtttt aaacaaatat 6360aactactttt tcacaaacaa ttgctatatc attaaatatt aattttactt gtaaatttat 6420tagtacaaat tgctcgatga ttgtaattat tttacttttt atatttttat gctttaatat 6480aatatgatat gtattataat atgaggtgta atataataaa ctattatttt gtgcaggaga 6540taaacatgaa acataaatat gaaaaagtaa aagaagaaat tattagctgg gcagttaatg 6600aaaaatataa accacatgaa aaaattccaa cagaatcgga acttatggag ctatttaaag 6660ttagtagaca tactataagg agagcaataa gtgatctagc ggcagagaaa tacttgtata 6720gattgcaggg aagtggaata tatgtatctg attttaaaca aaatgaaatt tacttgacaa 6780acaacaagaa tgttggggtg cttacaacat atatttccaa ctatatattc cctgatataa 6840ttagaggaat tgaagataca ctatatgatg aatcatactc ccttttatta tcttctacga 6900agaataatat aatgcttgaa agcagcaatt taaaaaattt attagcacac aaaatagatg 6960gactcattgt agagcctaca aagagtgcat atcaaagtcc taacatggga tattttaata 7020atttaataga gcaagatatt ccttttatta tgataaatgc atcttattct caagttaaag 7080taccaagctt atgtgtagat gatttaaagg ggggcaatat agctgcaaag tatttaatta 7140ctttaggaca taagaatata gctggtattt tcaaggtaga tgacctacaa ggtgtgcaca 7200ggatgaatgg ttttattact ggatgtcagg aaagtaatgt attgttaagg caagataaaa 7260ttttaaccta tctgtcagag gaaacaaata cactactacc tgaaaagata aagaatgttt 7320taaaacagga aaaacgtcca acgggtatat tttgctataa tgatgaaatt gcatacatgg 7380tgttaaatat tgcatatgat ctaaaattaa aggttccaga agatttatca attattggat 7440ttgatgattc tccaatggca acaattatgg aaccgaaatt aacatcaata actcatccaa 7500aggaaaagat gggaatagat gctgctaagt taattattag attaattaat aataataatc 7560attttagtga atgtgattca atattatatg aacctgaaat tgttattaga agttctacag 7620catcaattta aatttatgca cataataaaa taaaattttc aaaatgatcc ttatgaataa 7680cataagggtc attttttgta aaaaaactaa taaaatgatc aaatttttaa gaaatacggg 7740gatttatccc tataaaaata aaataaggtt gacatttgta cgaacaaata ttattattaa 7800tttaagtaat cgctttcaaa aataaaatta aaggaggaat gttagtgtat aaatactcct 7860ggtaatgtag tggcttacga tgatgtttga aagagtacgg tgatatggtt taaaggtact 7920atcataattt attgcagatt ataaagattt ataattctct aaaaagttat attaagttat 7980attttatcta taagaaagga gattgttata aaatgaaaaa agttagttat tattatgata 8040atcattttaa gattggagat gttaatgaaa atttatatag ttcatttatt gagcatttag 8100gcagggctgt atatagtgga atttatgaac cagggcatga aaaggccgat gaagatggat 8160tcagaacaga tgctatggaa gtaataaaag atttaaaatt gggattggtt cgttaccctg 8220gcggaaattt tgtttccaat tatgattgga aagatggtat tggaccaaag gaaaacaggc 8280ctaaaagaat ggaatttgct tggtcaagtg ttgaaacaaa tcaatttgga attgatgatt 8340tttgtcgttg ggcaaaaaaa gctggtattg aaccaatgat agcagttaat ttaggaacag 8400gtagtgttaa aagtgcagct gaacttgtag aatattgtaa tcatcctggc gggacttact 8460ggagtgatct tcgtatcaaa aatggaagta aggagcctta taatataaaa tattggtgcc 8520ttggaaatga aatggaaggt acctggcaag caggtcactt atcagcagaa gactatgcaa 8580aaaaagcttg tgaagctgct aaacttatga aatgggtaga caaagatatt aaattagttg 8640cttgtggaag tagttatgaa atgcttccta cttatatgga ttgggataga attgtactta 8700aagaacttta tccttatgtt gattacatat ctactcataa ttataatatg aataccaatc 8760aaggaacgtc aaattttctt gcatcatata aacaacttga tgaccatata aaaaatacag 8820aaagagttct tgattatgta aaggcaaaaa ataaggaaga aaaagatata aaaatatgtt 8880tagatgaatg gaatgtatgg aacttccagg atataaaact tgatagtctc gacgacttac 8940agggactgac gacttttgaa gtaacttcag ctgagaaatg ggaagaagct cctgcaatct 9000tagaggaaaa atatagtctt ttagatgcac taacagttgg tggacttgca ataactttaa 9060taaataatgc tgatagagta aagattgcat gtcttgcaca attaattaat gtaatagcac 9120ctattacaac gcagagaaat ggaggagttt taaaacagtc gacttattat ccatttagta 9180tggttagtaa ttatggtaga ggaactgtac ttaaaccatc tgttaatggt gcaagctaca 9240aatgtgattt tggtgaatta cctttagtag aagcagctac tgtttatgat aaagaatctg 9300atgaaattag agtatttgca ttaaattgta accaggatga agacacagaa ttagaccttc 9360aatttaatgg atttggagat cgtaaaatct ctaagcaatt tgtattatct ggagatgact 9420tagaacttag aaatacattt gaaagtcctg ataacgttac tgtaaaggaa aaagatcttt 9480caaattgtga tggtacgaaa gttgttcttc caaagctttc ttggaatgtt ttaattataa 9540agtaatttca ataaattatg aagggagtgg gaaggtaagt ggatatgaaa gtacaagata 9600aagatgtttt taaagaaaat ttaaaattta gtgagaagtt tggttatgga tgtggtgatt 9660tagccattaa ctttacttgg gcttctttgg gaatgtttgt agtttatttc tatactgatg 9720ttgttggtat gtctgctgct attattggaa ctattatgtt gttctcacgt tgtttagatg 9780gtgtcttaga tgttataatg ggcacaattg ttgataaaac taattcaaag tatggtaaag 9840ctcgtccttg gatattatgg ggatcaattc cttttgttgt tttaacagta tcaatattta 9900tggtaccaaa cataagcact tttggaaaga tagtgtatat tgtaatatcg tataatttac 9960ttatgatagc gtttactgca attgctattc cttatggtac attaaattca ttggttactc 10020aagatcaaca tcagagagaa gtattaaatc tttttagaat gtttttggca caaataggag 10080tattaattgt tactaatctt acaatgccat tggtaaattt atttggagga aaacaacctg 10140gatgggcttt aacttattca gttttaggag tagtttctct attattattt gtttatgttt 10200ttaaaacgca aaaagaaaga gtaaaaccaa ttaaaaagga aaaaattcct ttgaagatta 10260gtctaaaggc tttatgtcaa aataaatatt ggtttatagc aactatattt tttatagttt 10320atagtattgg atatgctata aatcaaggta gtacggtata ttatgctaaa tatcttcttg 10380gtaattcttc tctagttgga ggattaacta ttgcatattt agctccagta ttagtgggat 10440tccttatgat ctcaaaagtt tatgataaat acggaaaaag aaatgctatg atcattggtt 10500caataataag cataggtggt tacttaatta caataataaa tccgtatagt ttaacagttg 10560ttatggtttc tcaaattgtt aaaggttttg gccaagcttt cttgctagga ggagtgtggg 10620cattattccc tgatactata gaatatggtg aatggaaaac aggcataaga aatgaaggat 10680tgctttatag cggaggtagt ttaggacaaa agatgggtat aggttttggt acagccataa 10740caggatggat tttagcttgg ggaggatata atggtgcaca agcagtacaa gctagttctg 10800cggtattttc aataaaagca ttgtttattc atgttccaat aataatatat gttgctcaaa 10860ttatattatt gctctgttat ggacttgaca aagagtatcc acgcattatg aaggatttac 10920aattaagaaa aagtaaaatg agtgcagaaa ataactaata aatgtacttg cagattttat 10980acaatattgt aactgacata taaaaatgaa taagtgaaat aaggttttag tttcaataaa 11040ataacaaata aataagaagt aaataatagg gagtgtcttt tctgatggtt tttttatcat 11100tagagatgat actctctata ttttttaatt tgaatgtcaa aatatgccat taaaattcaa 11160aagataataa ttaatagaaa gtaggttaat tttatgacaa aatataaaaa ttaatactga 11220taatcacaaa aaacaattga catttgtacg aacaaatatt attataaatt taagaaatcg 11280ctttcattta atggaatata agggactaat atggatacaa tgaagaccat aacagtaatt 11340acaattgtta agattagtaa taaaaattta caatataaac ttgatttttt aattaaaagg 11400agaggggatt taaaaatgat taaatttaaa tcaattagaa aaagcatgag gtcaattcta 11460ttatgtggac ttgtattagt tctaagtgta ggtctcatgg catgtggaag tacaagtact 11520tcaagttctg gtacaagcag taaaaagaaa acgatagcat ttattccacc atcacttgta 11580agcccatttt atactcaggc tgttacagga gcaaagcaag aagcagctaa agaaggattt 11640aacataaaag tattggctcc tcaaacagaa gacgatttca atggattatt aaaaatagtt 11700gaagacgtta taactcagca agttgatgct attgcaatat gtaccacaga tgataaaact 11760atggctgctg ttgtaaaaaa agcaaatgat gcaaagatac cagttattgt atttaactca 11820ttaagtccaa taaagggtgc agatgtttat gcttatgttg gatatgacca aaaacaagca 11880ggagctcagg ctgcagacta tttaggaact aaacttaaag acaagcaatt caatgttggt 11940gtattagagg gtcttcctgg tgtatttaca gacaatagaa aaggtggatt tgtaaatgaa 12000gctaaaaaat attcaaatgt aaagattgta gctacacaac cagctgattg gcagagagaa 12060aaaggcatga atgttgcaac taacctttat caagctaata aggctataaa tatgttttat 12120ggattgagtg atgaaatggc aataggtgca gcccaagctt ttaaatcagc aggggttaag 12180gatggagtta ctattggtat agatggaaat ccagctactt tagattcaat cgcacaggga 12240gaaactactg ctacaattta tacagatcca aaacaaattg gtaaagaaag tataatagat 12300tgttctaaag ctttaaaagg tgaaaaaatg gcaaataaat tagatcagac aaagactttt 12360gtagtagaca agagtaacgt tagtacttat aaagcaaaat aattctatat ttgctcttag 12420aaaatgttag ctttaacatt ttctaagagt aaaatattta gttacgtaat ttgtggcaaa 12480agggggggta atattgtcag aaactatatt gaaaatggag aacataacta aaagtttttc 12540aggagttaca gttcttaaaa attcgggaat tgaagttaaa aaaggagaag ttcatatttt 12600acttggtgaa aatggtgcag gtaaatcaac tcttatgaag atactttctg gagcatattc 12660aaaagatagt ggagacatta tactaaacgg aaacaaagta gagataaatt ctccaaaaga 12720tgctgagaaa cttggaataa gcataattta tcaggaattt aatttagttc cctatatgac 12780tgttgctgag aatatatatc ttggaagaga acctgaatcc aaggtacctg gtaaggttaa 12840tttcaaaaaa atgtacaatg atgcacaaaa gatgattgat tacttaaatg tagatatacc 12900tgtagataaa ccaataaaga atttaggaat tgcccagcaa caaatggttg agatagcaaa 12960agcactatcg

gttcattctg atatcataat aatggatgag ccaacagcgg cactgacaga 13020aaaagaaata gataatttat ttaaaataat gagaaaaatt aaatctgaag gagtttctat 13080aatatacatt tctcatagac ttgaggaatt tgctcaaatt ggtgatagag ttactgttat 13140gagagatgga gaaactgtag aaacagttaa tatcaaaggt acatctatag atgaacttat 13200aaaattaatg gtaggaagag aaattaaaga aaaatttcct aaaataaagg ttgatttagg 13260agaagaaata ttaagggtta agggattaac caaaaaagga gtttttgaaa atataaattt 13320cagtttgaga tcaggcgaaa tactaggatt ttctggtctt atgggtgcgg gaagaacaga 13380agtaatgagg gctatatttg gtatagattc atttgattct ggtgaaatat atttaaaagg 13440taaaaaagtt gaaataaatt cccctatgaa agcaataaaa aatggtatag gatttgtaac 13500ggaaaataga agagatgaag gtcttgtttt acagatgggt gtaggtcaaa atataacctt 13560agcatccctt ggcaaatata ttagtaatcc tataaaatta aatcttagaa aagaagtcaa 13620ggaaataaaa gattatatat ctaaattgtc aattaagtct tcaggatata gacaaattgc 13680aggtacactg agcggtggaa accaacaaaa gattgttata gcaaagtggc tattatctga 13740ttcaaaggtt ttaattgtgg atgaacctac tcgtggaata gatgtaggtg ctaaaataga 13800aatttataat attatgaatg atttagttaa aagtggggta ggaataatta tggtttcttc 13860agaacttcct gaagtacttg gcatgagtga tagaatatta gttatgtgta gagggaaaat 13920aactggagaa ttaaataagg atgaggctac tcaagaaaaa ataatgcatt atgcaacagg 13980aggtatagaa tgatgaatca gttagaagat actgaaaaaa agaaaaagac tagtataaat 14040gacatactgg ataagctagg ggttgtcatt gcattagttg ttttaattgt agttatggct 14100gtgttgtctc cagattttct tactgtaaaa aatgtattta acatattgca gcagattgca 14160caaattggaa taatatctgt tggtatgact tttgtaatat tacttggagg aatcgactta 14220tctgttggtt caattattgc atttacagga cttattatgg cattatgcat gaaagcagga 14280atgtctgttg tgttagctat tttagtaggt attatactag gtgcagctat tggtttctta 14340aatggaattt taatatcaaa ggttaaactc caacctttta tagctacact aggaactatg 14400actatggcta gaggacttgc atatactata acaaatggac aaccagtata ttctttctca 14460gctgggttta aaagctttgc tggatttatt ggagttgttc caattcctgc aattattatg 14520gcagtaatat ttgcattagg ttattatgtg cttaaataca caaagtttgg aagatacatg 14580tatgctattg gaggaaatag agtagcaagt aagctttcag gaataaatgt ggataaatat 14640gagatgttgg tttatacgat ttcaggaata tgctgtgcta tagctgctat aattttaaca 14700gcaaggcttg attcagcagt accagttgca ggtgatggaa acgaacttga tgctatagcg 14760gcagtagcta taggtggaac aagtatgacc ggtggagaag gcggtatagt tggaacactt 14820attggtgcct taattatggg agtaattgcg aatggaatga acctactaga tgtgcaacag 14880ggaccacaga ggtttgctaa aggtgcaatt ataattttag ctgtaggaat tgatgtaata 14940agaaagaaaa gaacttctaa ataatacttt ggataaattc acaaggtgct tattaatttt 15000tattaaaaaa gtttcagatt atatccgaaa ctttttttgt atttaaatac tatagtttta 15060tctccaatcc taaaagaaat ttgtttatta gggttattta atgatatgta ataggtatca 15120tatttattaa aaatgggtaa tatagttctc tgttagaata aattaagtat aaacatatcc 15180atgtgtatct atgtaagaaa aatggtgaat ataagtagta taaaggagaa aatgcaaatt 15240aagttataaa aataccggag taagtgaact gagttaagta taagctgtga tataataaat 15300ttcgttgtca tggagattac agaaacagat taaatcaata gttttttatt taaagaaaaa 15360taataaaacg tattgacaga tgtaatttaa aatgatatac tataaagg 154086300DNAClostridium autoethanogenum 6atctgtatat tttttcccat tttaattatt tgtactataa tattacactg agtgtattgc 60atatttaaaa aatatttggt acaattagtt agttaaataa attctaaatt gtaaattatc 120agaatcctta ttaaggaaat acatagattt aaggagaaat cataaaaagg tgtaatataa 180actggctaaa attgagcaaa aattgagcaa ttaagacttt ttgattgtat ctttttatat 240atttaaggta tataatctta tttatattgg gggaacttga tgaataaaca tattctagac 3007300DNAClostridium autoethanogenum 7atctgtatat tttttcccat tttaattatt tgtactataa tattacactg agtgtattgt 60atatttaaaa aatatttggt acaattagtt agttaaataa attctaaatt gtaaattatc 120agaatcctta ttaaggaaat acatagattt aaggagaaat cataaaaagg tgtaatataa 180actggctaaa attgagcaaa aattgagcaa ttaagacttt ttgattgtat ctttttatat 240atttaaggta tataatctta tttatattgg gggaacttga tgaataaaca tattctagac 3008300DNAClostridium autoethanogenum 8tgttaatttt ttgtgtcaat aatttttgtt atattatttt aattaaattt ttcacatgta 60taattaaaag taagatagat attctaatgt acttacttag gtagaaaaac atgtatacaa 120aattaaaaaa ctattataac acatagtatc aatattgaag gtaatactgt tcaatatcga 180tacagataaa aaaatatata atacagaaga aaaaattata aatttgtggt ataatataaa 240gtatagtaat ttaagtttaa acctcgtgaa aacgctaaca aataatagga ggtgtattat 3009300DNAClostridium autoethanogenum 9gttaattttt tgtgtcaata atttttgtta tattatttta attaaatttt tcacatgtat 60aattaaaagt aagatagata ttctaatgta cttacttagg tagaaaaaca tgtatacaaa 120attaaaaaac tattataaca catagtatca atattgaagg taatactgtt caatatcgat 180acagataaaa aaaatatata atacagaaga aaaaattata aatttgtggt ataatataaa 240gtatagtaat ttaagtttaa acctcgtgaa aacgctaaca aataatagga ggtgtattat 30010300DNAClostridium autoethanogenum 10agttggataa atagggttta tccttaaaag ttttattcac agtgtgtatg aaaaaaaatt 60cacactgtga atttttttta taaatttttc tacactgtaa aattcaatta tgttattcaa 120ctcattgtaa ataccattca catgttattt ttgaccattc gcatccgttt ttttgcactt 180tggaatattt gggtatgaaa atcaataaat tcagactatt ataaacatgt aataaaattc 240atataattaa ttattaatga actatttata attattaaaa attaaaaagg aggtttttat 30011300DNAClostridium autoethanogenum 11agttggataa atagggttta tccttaaaag ttttattcac agtgtgtata gaaaaaaaat 60tcacactgtg aatttttttt ataaattttt cacactgtaa aattcaatta tgttattcaa 120ctcattgtaa ataccattca catgttattt ttgaccattc gcatccgttt ttttgcactt 180tggaatattt gggtatgaaa atcaataaat tcagactatt ataaacatgt aataaaattc 240atataattaa ttattaatga actatttata attattaaaa attaaaaagg aggtttttat 300122127DNAClostridium autoethanogenum 12atgaatttat ttcaaactgt attcactggt tcaaagcaag ctttagcagc tgctgaaggc 60atagttaagc aagctgttga cgagaagggt agagactata aagtagcatt tcctgatact 120gaatattcat taccagtaat ttttgcagct acaggaaaaa agataactaa tgtaggagaa 180ttagaaggtg cattagatat agtaagaagt ttgatagttg aggaggaaat gcttgataag 240cttttaaatt caggacttgc aacagctgtt gcagcagaaa ttatagaagc tgcaaagtat 300gttctttccg atgctcctta tgcagaacca tgtgtaggat ttatatctga cccaataatt 360cgttctcttg gtgtaccact tgttaccgga gatataccag gtgtagcagt tatattagga 420gaatgtccag attcagaaac cgcagctaaa attataaagg attatcaatc aaaaggtctt 480ttaacatgct tagttggaaa agtaattgat caggcaatag aaggaaaagt taagatgggt 540cttgacctca gagttattcc acttggatat gatgttacat ctgtaattca cgttgtaact 600atagctataa gagctgcact tatattcgga ggaattaagg gtggtcagtt aaatgacata 660ttgaaatata cagcagaaag ggtacctgct tttgtaaatg catttggacc attaagtgaa 720cttgtagttt cagctggtgc aggagctata gcacttggat tccctgtatt aactgatcag 780gttgtaccag aagttcctac attgttgtta actcaaaaag attatgataa aatggttaaa 840acttcattag aagctagaaa tataaagata aagataactg agatcccaat tccagtttcc 900tttgcagcag catttgaagg tgaaagaata agaaagaatg atatgcttgc agagtttggt 960ggaaataaga ctaaagcttg ggaattagtt atgtgtgcag atcagggaga agttgaagat 1020cacaagatag aagttatagg accagatata gatactatag ataaggctcc tggaagaatg 1080cctcttggaa tgcttattaa agtaagtgga acaaatatgc agaaggattt tgagccagtg 1140cttgaaagaa gacttcacta cttcttaaac tatatagaag gagtaatgca tgttggtcag 1200agaaatctta cttgggtaag aataggtaag gaagcttttg aaaagggatt tagattgaaa 1260cattttggtg aagtaatata tgctaaaatg ttagatgaat ttggttcagt tgtagataaa 1320tgtgaagtaa ctataataac tgatccaggt aaggctgaag aattggaagg caaatatgct 1380gtaccaagat ataaagaaag agatgcaaga cttgaatcat tagttgatga aaaagttgat 1440actttctatt catgtaattt gtgtcaatcc tttgcacctg cacatgtatg tatagtaact 1500cctgaaagac ttggactttg cggtgcagtt tcatggcttg atgctaaagc tacacttgaa 1560ttaaatccta caggaccatg tcaggccgtt ccaaaagaag gcgtggttga tgaaaattta 1620ggtatttggg aaaaagtaaa tgaaactgtt tcaaaaattt ctcaaggtgc tgtaactagt 1680gttacattat acagtatatt acaagatcca atgacttcct gtggatgttt tgagtgtatt 1740acaggtataa tgccagaagc aaatggtgtt gtaatggtaa acagagaatt tggtgcaaca 1800actcctcttg gaatgacatt tggtgaactt gcatctatga caggtggtgg agttcagact 1860ccaggattta tgggacatgg aagacaattc atagcttcaa agaagtttat gaaaggtgaa 1920ggcggacttg gcagaatagt ttggatgcca aaagaattaa aagactttgt tgcagaaaaa 1980ttaaataaga cagcaaagga attatataat atagataatt ttgcagatat gatctgtgat 2040gaaactatag ctacagaatc tgaagaagta gtaaaattct tggaagaaaa aggtcatcct 2100gcattaaaga tggatccaat aatgtag 2127132127DNAClostridium autoethanogenum 13atgaatttat ttcaaactgt attcactggt tcaaagcaag ctttagcagc tgctgaaggc 60atagttaagc aagctgttga cgagaagggt agagactata aagtagcatt tcctgatact 120gcatattcat taccagtaat ttttgcagct acaggaaaaa agataactaa tgtaggagaa 180ttagaaggtg cattagatat agtaagaagt ttgatagttg aggaggaaat gcttgataag 240cttttaaatt caggacttgc aacagctgtt gcagcagaaa ttatagaagc tgcaaagtat 300gttctttccg atgctcctta tgcagaacca tgtgtaggat ttatatctga cccaataatt 360cgttctcttg gtgtaccact tgttaccgga gatataccag gtgtagcagt tatattagga 420gaatgtccag attcagaaac cgcagctaaa attataaagg attatcaatc aaaaggtctt 480ttaacatgct tagttggaaa agtaattgat caggcaatag aaggaaaagt taagatgggt 540cttgacctca gagttattcc acttggatat gatgttacat ctgtaattca cgttgtaact 600atagctataa gagctgcact tatattcgga ggaattaagg gtggtcagtt aaatgacata 660ttgaaatata cagcagaaag ggtacctgct tttgtaaatg catttggacc attaagtgaa 720cttgtagttt cagctggtgc aggagctata gcacttggat tccctgtatt aactgatcag 780gttgtaccag aagttcctac attgttgtta actcaaaaag attatgataa aatggttaaa 840acttcattag aagctagaaa tataaagata aagataactg agatcccaat tccagtttcc 900tttgcagcag catttgaagg tgaaagaata agaaagaatg atatgcttgc agagtttggt 960ggaaataaga ctaaagcttg ggaattagtt atgtgtgcag atcagggaga agttgaagat 1020cacaagatag aagttatagg accagatata gatactatag ataaggctcc tggaagaatg 1080cctcttggaa tgcttattaa agtaagtgga acaaatatgc agaaggattt tgagccagtg 1140cttgaaagaa gacttcacta cttcttaaac tatatagaag gagtaatgca tgttggtcag 1200agaaatctta cttgggtaag aataggtaag gaagcttttg aaaagggatt tagattgaaa 1260cattttggtg aagtaatata tgctaaaatg ttagatgaat ttggttcagt tgtagataaa 1320tgtgaagtaa ctataataac tgatccaggt aaggctgaag aattggaagg caaatatgct 1380gtaccaagat ataaagaaag agatgcaaga cttgaatcat tagttgatga aaaagttgat 1440actttctatt catgtaattt gtgtcaatcc tttgcacctg cacatgtatg tatagtaact 1500cctgaaagac ttggactttg cggtgcagtt tcatggcttg atgctaaagc tacacttgaa 1560ttaaatccta caggaccatg tcaggccgtt ccaaaagaag gcgtggttga tgaaaattta 1620ggtatttggg aaaaagtaaa tgaaactgtt tcaaaaattt ctcaaggtgc tgtaactagt 1680gttacattat acagtatatt acaagatcca atgacttcct gtggatgttt tgagtgtatt 1740acaggtataa tgccagaagc aaatggtgtt gtaatggtaa acagagaatt tggtgcaaca 1800actcctcttg gaatgacatt tggtgaactt gcatctatga caggtggtgg agttcagact 1860ccaggattta tgggacatgg aagacaattc atagcttcaa agaagtttat gaaaggtgaa 1920ggcggacttg gcagaatagt ttggatgcca aaagaattaa aagactttgt tgcagaaaaa 1980ttaaataaga cagcaaagga attatataat atagataatt ttgcagatat gatctgtgat 2040gaaactatag ctacagaatc tgaagaagta gtaaaattct tggaagaaaa aggtcatcct 2100gcattaaaga tggatccaat aatgtag 212714708PRTClostridium autoethanogenum 14Met Asn Leu Phe Gln Thr Val Phe Thr Gly Ser Lys Gln Ala Leu Ala 1 5 10 15 Ala Ala Glu Gly Ile Val Lys Gln Ala Val Asp Glu Lys Gly Arg Asp 20 25 30 Tyr Lys Val Ala Phe Pro Asp Thr Glu Tyr Ser Leu Pro Val Ile Phe 35 40 45 Ala Ala Thr Gly Lys Lys Ile Thr Asn Val Gly Glu Leu Glu Gly Ala 50 55 60 Leu Asp Ile Val Arg Ser Leu Ile Val Glu Glu Glu Met Leu Asp Lys 65 70 75 80 Leu Leu Asn Ser Gly Leu Ala Thr Ala Val Ala Ala Glu Ile Ile Glu 85 90 95 Ala Ala Lys Tyr Val Leu Ser Asp Ala Pro Tyr Ala Glu Pro Cys Val 100 105 110 Gly Phe Ile Ser Asp Pro Ile Ile Arg Ser Leu Gly Val Pro Leu Val 115 120 125 Thr Gly Asp Ile Pro Gly Val Ala Val Ile Leu Gly Glu Cys Pro Asp 130 135 140 Ser Glu Thr Ala Ala Lys Ile Ile Lys Asp Tyr Gln Ser Lys Gly Leu 145 150 155 160 Leu Thr Cys Leu Val Gly Lys Val Ile Asp Gln Ala Ile Glu Gly Lys 165 170 175 Val Lys Met Gly Leu Asp Leu Arg Val Ile Pro Leu Gly Tyr Asp Val 180 185 190 Thr Ser Val Ile His Val Val Thr Ile Ala Ile Arg Ala Ala Leu Ile 195 200 205 Phe Gly Gly Ile Lys Gly Gly Gln Leu Asn Asp Ile Leu Lys Tyr Thr 210 215 220 Ala Glu Arg Val Pro Ala Phe Val Asn Ala Phe Gly Pro Leu Ser Glu 225 230 235 240 Leu Val Val Ser Ala Gly Ala Gly Ala Ile Ala Leu Gly Phe Pro Val 245 250 255 Leu Thr Asp Gln Val Val Pro Glu Val Pro Thr Leu Leu Leu Thr Gln 260 265 270 Lys Asp Tyr Asp Lys Met Val Lys Thr Ser Leu Glu Ala Arg Asn Ile 275 280 285 Lys Ile Lys Ile Thr Glu Ile Pro Ile Pro Val Ser Phe Ala Ala Ala 290 295 300 Phe Glu Gly Glu Arg Ile Arg Lys Asn Asp Met Leu Ala Glu Phe Gly 305 310 315 320 Gly Asn Lys Thr Lys Ala Trp Glu Leu Val Met Cys Ala Asp Gln Gly 325 330 335 Glu Val Glu Asp His Lys Ile Glu Val Ile Gly Pro Asp Ile Asp Thr 340 345 350 Ile Asp Lys Ala Pro Gly Arg Met Pro Leu Gly Met Leu Ile Lys Val 355 360 365 Ser Gly Thr Asn Met Gln Lys Asp Phe Glu Pro Val Leu Glu Arg Arg 370 375 380 Leu His Tyr Phe Leu Asn Tyr Ile Glu Gly Val Met His Val Gly Gln 385 390 395 400 Arg Asn Leu Thr Trp Val Arg Ile Gly Lys Glu Ala Phe Glu Lys Gly 405 410 415 Phe Arg Leu Lys His Phe Gly Glu Val Ile Tyr Ala Lys Met Leu Asp 420 425 430 Glu Phe Gly Ser Val Val Asp Lys Cys Glu Val Thr Ile Ile Thr Asp 435 440 445 Pro Gly Lys Ala Glu Glu Leu Glu Gly Lys Tyr Ala Val Pro Arg Tyr 450 455 460 Lys Glu Arg Asp Ala Arg Leu Glu Ser Leu Val Asp Glu Lys Val Asp 465 470 475 480 Thr Phe Tyr Ser Cys Asn Leu Cys Gln Ser Phe Ala Pro Ala His Val 485 490 495 Cys Ile Val Thr Pro Glu Arg Leu Gly Leu Cys Gly Ala Val Ser Trp 500 505 510 Leu Asp Ala Lys Ala Thr Leu Glu Leu Asn Pro Thr Gly Pro Cys Gln 515 520 525 Ala Val Pro Lys Glu Gly Val Val Asp Glu Asn Leu Gly Ile Trp Glu 530 535 540 Lys Val Asn Glu Thr Val Ser Lys Ile Ser Gln Gly Ala Val Thr Ser 545 550 555 560 Val Thr Leu Tyr Ser Ile Leu Gln Asp Pro Met Thr Ser Cys Gly Cys 565 570 575 Phe Glu Cys Ile Thr Gly Ile Met Pro Glu Ala Asn Gly Val Val Met 580 585 590 Val Asn Arg Glu Phe Gly Ala Thr Thr Pro Leu Gly Met Thr Phe Gly 595 600 605 Glu Leu Ala Ser Met Thr Gly Gly Gly Val Gln Thr Pro Gly Phe Met 610 615 620 Gly His Gly Arg Gln Phe Ile Ala Ser Lys Lys Phe Met Lys Gly Glu 625 630 635 640 Gly Gly Leu Gly Arg Ile Val Trp Met Pro Lys Glu Leu Lys Asp Phe 645 650 655 Val Ala Glu Lys Leu Asn Lys Thr Ala Lys Glu Leu Tyr Asn Ile Asp 660 665 670 Asn Phe Ala Asp Met Ile Cys Asp Glu Thr Ile Ala Thr Glu Ser Glu 675 680 685 Glu Val Val Lys Phe Leu Glu Glu Lys Gly His Pro Ala Leu Lys Met 690 695 700 Asp Pro Ile Met 705 15708PRTClostridium autoethanogenum 15Met Asn Leu Phe Gln Thr Val Phe Thr Gly Ser Lys Gln Ala Leu Ala 1 5 10 15 Ala Ala Glu Gly Ile Val Lys Gln Ala Val Asp Glu Lys Gly Arg Asp 20 25 30 Tyr Lys Val Ala Phe Pro Asp Thr Ala Tyr Ser Leu Pro Val Ile Phe 35 40 45 Ala Ala Thr Gly Lys Lys Ile Thr Asn Val Gly Glu Leu Glu Gly Ala 50 55 60 Leu Asp Ile Val Arg Ser Leu Ile Val Glu Glu Glu Met Leu Asp Lys 65 70 75 80 Leu Leu Asn Ser Gly Leu Ala Thr Ala Val Ala Ala Glu Ile Ile Glu 85 90 95 Ala Ala Lys Tyr Val Leu Ser Asp Ala Pro Tyr Ala Glu Pro Cys Val 100 105 110 Gly Phe Ile Ser Asp Pro Ile Ile Arg Ser Leu Gly Val Pro Leu Val 115 120 125 Thr Gly Asp Ile Pro Gly Val Ala Val Ile Leu Gly Glu Cys Pro Asp 130 135 140 Ser Glu Thr Ala Ala Lys Ile Ile Lys Asp Tyr Gln Ser Lys Gly Leu 145 150 155 160 Leu Thr Cys Leu Val Gly Lys Val Ile Asp Gln Ala Ile Glu Gly Lys 165 170 175 Val Lys Met Gly Leu Asp Leu Arg Val Ile Pro Leu Gly Tyr Asp Val 180 185 190 Thr Ser Val Ile His Val Val Thr Ile Ala Ile Arg Ala Ala Leu Ile 195 200 205 Phe Gly Gly Ile Lys Gly Gly Gln Leu Asn Asp Ile Leu Lys Tyr Thr 210 215 220 Ala Glu Arg Val Pro Ala Phe Val Asn Ala Phe Gly Pro Leu Ser Glu 225

230 235 240 Leu Val Val Ser Ala Gly Ala Gly Ala Ile Ala Leu Gly Phe Pro Val 245 250 255 Leu Thr Asp Gln Val Val Pro Glu Val Pro Thr Leu Leu Leu Thr Gln 260 265 270 Lys Asp Tyr Asp Lys Met Val Lys Thr Ser Leu Glu Ala Arg Asn Ile 275 280 285 Lys Ile Lys Ile Thr Glu Ile Pro Ile Pro Val Ser Phe Ala Ala Ala 290 295 300 Phe Glu Gly Glu Arg Ile Arg Lys Asn Asp Met Leu Ala Glu Phe Gly 305 310 315 320 Gly Asn Lys Thr Lys Ala Trp Glu Leu Val Met Cys Ala Asp Gln Gly 325 330 335 Glu Val Glu Asp His Lys Ile Glu Val Ile Gly Pro Asp Ile Asp Thr 340 345 350 Ile Asp Lys Ala Pro Gly Arg Met Pro Leu Gly Met Leu Ile Lys Val 355 360 365 Ser Gly Thr Asn Met Gln Lys Asp Phe Glu Pro Val Leu Glu Arg Arg 370 375 380 Leu His Tyr Phe Leu Asn Tyr Ile Glu Gly Val Met His Val Gly Gln 385 390 395 400 Arg Asn Leu Thr Trp Val Arg Ile Gly Lys Glu Ala Phe Glu Lys Gly 405 410 415 Phe Arg Leu Lys His Phe Gly Glu Val Ile Tyr Ala Lys Met Leu Asp 420 425 430 Glu Phe Gly Ser Val Val Asp Lys Cys Glu Val Thr Ile Ile Thr Asp 435 440 445 Pro Gly Lys Ala Glu Glu Leu Glu Gly Lys Tyr Ala Val Pro Arg Tyr 450 455 460 Lys Glu Arg Asp Ala Arg Leu Glu Ser Leu Val Asp Glu Lys Val Asp 465 470 475 480 Thr Phe Tyr Ser Cys Asn Leu Cys Gln Ser Phe Ala Pro Ala His Val 485 490 495 Cys Ile Val Thr Pro Glu Arg Leu Gly Leu Cys Gly Ala Val Ser Trp 500 505 510 Leu Asp Ala Lys Ala Thr Leu Glu Leu Asn Pro Thr Gly Pro Cys Gln 515 520 525 Ala Val Pro Lys Glu Gly Val Val Asp Glu Asn Leu Gly Ile Trp Glu 530 535 540 Lys Val Asn Glu Thr Val Ser Lys Ile Ser Gln Gly Ala Val Thr Ser 545 550 555 560 Val Thr Leu Tyr Ser Ile Leu Gln Asp Pro Met Thr Ser Cys Gly Cys 565 570 575 Phe Glu Cys Ile Thr Gly Ile Met Pro Glu Ala Asn Gly Val Val Met 580 585 590 Val Asn Arg Glu Phe Gly Ala Thr Thr Pro Leu Gly Met Thr Phe Gly 595 600 605 Glu Leu Ala Ser Met Thr Gly Gly Gly Val Gln Thr Pro Gly Phe Met 610 615 620 Gly His Gly Arg Gln Phe Ile Ala Ser Lys Lys Phe Met Lys Gly Glu 625 630 635 640 Gly Gly Leu Gly Arg Ile Val Trp Met Pro Lys Glu Leu Lys Asp Phe 645 650 655 Val Ala Glu Lys Leu Asn Lys Thr Ala Lys Glu Leu Tyr Asn Ile Asp 660 665 670 Asn Phe Ala Asp Met Ile Cys Asp Glu Thr Ile Ala Thr Glu Ser Glu 675 680 685 Glu Val Val Lys Phe Leu Glu Glu Lys Gly His Pro Ala Leu Lys Met 690 695 700 Asp Pro Ile Met 705 16882DNAClostridium autoethanogenum 16atgagcttat tgaaggaagc ttttgaaaag ggagagtttg caattacagc tgaaatggca 60cctccaaagg gaacggatct ttctcattta attgaatgtg ccaaaaagat aaaaggaaga 120gttcagggag ttaatgtaac ggattttcag tctgctacat taaaagctac atctttagct 180acttgtaaag tattaaaaga tgcaggatta gagcctgtat ttcaaataac aggaagagat 240agaaacagaa tagcaattca aggagaattg ttatctgcag gtgtttttgg aattgaaaac 300gttttagctc ttactgggga ttatactgct acaggagatc accctggtgc aaagccagtt 360tatgatctag atagtgttgg aatattacag gtggcaagca ttttaaatgg tggaaaagac 420atgggtggaa ctgatttaaa agggaaacca gatttctttt taggggcctg tgttacacct 480agatatgatc cgttagagct tcaagttata aagatgaaga agaaaattaa agctggagct 540aaattctttc aaactcaagc tgtttatgat atggaaactt taaagaaatt caaagaagag 600actaaagctc aaggtgtaga tgctaaagtt atggtaggca taataccttt aaagtcagct 660ggtatggcta aatacatgaa taaaaacgta cctggtatat tcgtacctga tgaacttata 720gatagaatga agaatgctga ggataaagtt caagaaggca taaagatagc aggagaattt 780ataaaggccg taaaagaatc aggactttgc gatggagttc atataatggc aattggtgcg 840gaagaaaatg tgccattaat attggatgaa gcaggattat aa 88217882DNAClostridium autoethanogenum 17atgagcttat tgaaggaagc ttttgaaaag ggagagtttg caattacagc tgaaatggca 60cctccaaagg gaacggatct ttctcattta attgaatgtg ccaaaaagat aaaaggaaga 120gttcagggag ttaatgtaac ggattttcag tctgctacat taaaagctac atctttagct 180acttgtaaag tattaaaaga tgcaggatta gagcctgtat ttcaaataac aggaagagat 240agaaacagaa tagcaattca aggagaattg ttatctgcag gtgtttttgg aattgaaaat 300gttttagctc ttactgggga ttatactgct acaggagatc accctggtgc aaagccagtt 360tatgatctag atagtgttgg aatattacag gtggcaagca ttttaaatgg tggaaaagac 420atgggtggaa ctgatttaaa agggaaacca gatttctttt taggggcctg tgttacacct 480agatatgatc cgttagagct tcaagttata aagatgaaga agaaaattaa agctggagct 540aaattctttc aaactcaagc tgtttatgat atggaaactt taaagaaatt caaagaagag 600actaaagctc aaggtgtaga tgctaaagtt atggtaggca taataccttt aaagtcagct 660ggtatggcta aatacatgaa taaaaacgta cctggtatat tcgtacctga tgaacttata 720gatagaatga agaatgctga ggataaagtt caagaaggca taaagatagc aggagaattt 780ataaaggccg taaaagaatc aggactttgc gatggagttc atataatggc aattggtgcg 840gaagaaaatg tgccattaat attggatgaa gcaggattat aa 882

Claims

1-29. (canceled)

30. A biologically pure isolate of a bacterium derived from Clostridium autoethanogenum wherein the selected bacterium ferments a substrate comprising CO to products comprising ethanol and wherein the bacterium has one or more improvements compared to a parent strain, the one or more improvements being selected from the group consisting of; a. an increased production rate; b. an increased growth rate; c. an increased carbon monoxide uptake; d. an increased tolerance to alcohol; and e. a higher alcohol to acid production ratio.

31. The bacterium of claim 1 wherein the parent strain is Clostridium autoethanogenum strain deposited at the German Collection of Microorganisms and cell cultures (DSMZ) under the accession number DSM19630.

32. The selected bacterium of claim 1 wherein the bacterium is Clostridium autoethanogenum strain deposited at DSMZ under the accession number DSM23693.

33. A biologically pure isolate of Clostridium autoethanogenum having at least one DNA sequence selected from Seq. ID. 1 to Seq. ID. 17.

34. The bacterium of claim 30 wherein the production rate of the bacterium is at least 2 g ethanol/L fermentation broth/gram of biomass/day.

35. The bacterium of claim 31 wherein the production rate of the bacterium is at least 2 g ethanol/L fermentation broth/gram of biomass/day.

36. The bacterium of claim 32 wherein the production rate of the bacterium is at least 2 g ethanol/L fermentation broth/gram of biomass/day.

37. The bacterium of claim 33 wherein the bacterium ferments a substrate comprising CO to products comprising ethanol and wherein the production rate of the bacterium is at least 2 g ethanol/L fermentation broth/gram of biomass/day

38. The bacterium of claim 34 wherein the specific productivity is at least 7 g ethanol/L fermentation broth/gram of biomass/day.

39. The bacterium of claim 30 wherein the products comprise ethanol and acetate.

40. The bacterium of claims 30 wherein production rate of the bacterium is at least 10 g of ethanol/L of fermentation broth/day.

41. The bacterium of claim 40 wherein the production rate of the bacterium is at least 50 g of ethanol/L of fermentation broth/day.

42. The bacterium of claims 39 wherein the ethanol to acetate ratio is at least 2:1

43. The bacterium of any one of claim 30 wherein the bacterium has a specific uptake of CO of at least 1.0 mmol CO/min/g biomass.

44. The selected bacterium of claim 43 wherein the bacterium has a specific uptake of CO of at least 2.0 mmol CO/min/g biomass.

45. The bacterium of claim 30 wherein the bacterium has a specific growth rate of at least 0.8 day.sup.-1.

46. The bacterium of claim 30 wherein the bacterium tolerates an alcohol concentration of up to 30 g/L of fermentation broth.

47. The bacterium of claim 46 wherein the bacterium tolerates an alcohol concentration of up to 70 g/L of fermentation broth.

Images