METHODS FOR BIOSYNTHESIS OF ISOBUTENE

Abstract

The document provides methods for biosynthesizing isobutene using one or more isolated enzymes such as one or more of an enoyl-CoA dehydratase, a 2-hydroxyacyl-CoA dehydratase, an isovaleryl-CoA/acyl-CoA dehydrogenase and a mevalonate diphosphate decarboxylase, or using recombinant host cells expressing one or more such enzymes.

Description

[0001] This application is a continuation of U.S. application Ser. No. 14/092,115 filed Nov. 27, 2013, which claims priority of U.S. Provisional Application Ser. No. 61/730,549, filed Nov. 28, 2012. The contents of the prior applications incorporated herein by reference in their entireties.

TECHNICAL FIELD

[0002] This document relates to methods for biosynthesizing isobutene using one or more isolated enzymes such as one or more of an enoyl-CoA dehydratase, a 2-hydroxyacyl-CoA dehydratase, an isovaleryl-CoA/acyl-CoA dehydrogenase, and a mevalonate diphosphate decarboxylase, or using recombinant host cells expressing one or more such enzymes.

BACKGROUND

[0003] Isobutene is an important monomer in the manufacture of fuel additives, butyl rubber polymer, and antioxidants (Bianca et al., Appl. Microbiol Biotechnol., 2012, 93, 1377-1387).

[0004] Manufacturers of goods using isobutene as feedstock depend on a number of petroleum-based sources, including (i) a C4 stream from a steam cracker separated from the butadiene, (ii) butene-butane fractions from a catalytic cracker and (iii) n-butane (from liquid petroleum gas) that is isomerized to isobutane and dehydrogenated to isobutene.

[0005] Given a reliance on petrochemical feedstocks and energy intensive processes, biotechnology offers an alternative approach via biocatalysis. Biocatalysis is the use of biological catalysts, such as enzymes or whole cells, to perform biochemical transformations of organic compounds.

[0006] Accordingly, against this background, it is clear that there is a need for sustainable methods for producing intermediates, in particular isobutene, wherein the methods are biocatalysis based. Both bioderived feedstocks and petrochemical feedstocks are viable starting materials for the biocatalysis processes.

[0007] The introduction of a double bond into a short branch chain aliphatic carbon substrate is a key consideration in synthesizing isobutene via a biocatalytic process. In this vain, a cytochrome P450 from Rhodotorula minuta var. texensis IFO 1102 forms isobutene via the decarboxylation of isovalerate. Also, it has been demonstrated that variants of oleate hydratase accept isobutanol and variants of mevalonate diphosphate decarboxylase accept 3-hydroxy-3-methylbutyrate as a substrate in the biosynthesis of isobutene. A number of enzymes have thus been identified as having catalytic activity in the synthesis of isobutene (Bianca et al., Appl. Microbiol Biotechnol., 2012, 93, 1377-1387).

[0008] However, the identified biochemical pathways leading to the precursors accepted for isobutene synthesis are carbon inefficient, reflected by low maximum theoretical yields on carbon. For example, the only pathways identified for exploiting the catalytic activity of mevalonate diphosphate decarboxylase have maximum theoretical yields of .about.0.2 [(g isobutene)/(g glucose)] (Bianca et al., 2012, supra). The economical production of isobutene using mevalonate diphosphate decarboxylase as final enzymatic step is thus challenged by carbon yield limitations.

SUMMARY

[0009] This document is based, at least in part, on constructing carbon efficient biochemical pathways for producing 3-methyl-3-hydroxy-butanoate, which can be converted to isobutene by a mevalonate diphosphate decarboxylase. Such pathways can rely on a 2-hydroxyacyl-CoA dehydratase or an isovaleryl-CoA/acyl-CoA dehydrogenase, and an enoyl-CoA hydratase (e.g., a (R)-specific enoyl-CoA hydratase) in synthesizing 3-methyl-3-hydroxy-butanoate. Prior to the present invention, it was not known that 2-hydroxyacyl-CoA dehydratase or an isovaleryl-CoA dehydrogenase combined with an enoyl-CoA hydratase could be utilized for the biological synthesis of 3-methyl-3-hydroxy-butanoate, leading to the synthesis of isobutene. Also, prior to the present invention, it was not known that an enoyl-CoA hydratase of bacterial origin could be utilized to synthesize 3-methyl-3-hydroxy-butanoate, leading to the synthesis of isobutene.

[0010] Thus, this document provides pathways and enzymes which can convert either of the central precursors 3-methyl-2-oxobutanoate or 4-methyl-2-oxopentanoate into isobutene via a common intermediate, 3-methyl-3-hydroxy-but-2-enoyl-CoA. As used herein, the term "central precursor" is used to denote any metabolite in any metabolic pathway shown herein leading to the synthesis of isobutene. The term "central metabolite" is used herein to denote a metabolite that is produced in all microorganisms to support growth.

[0011] In one aspect, this document features a method for synthesizing isobutene. The method includes (i) dehydrating 3-methyl-2-hydroxy-butanoyl-CoA and hydrating 3-methyl-but-2-enoyl-CoA, and converting the resulting product to isobutene. A 2-hydroxyacyl-CoA dehydratase can dehydrate 3-methyl-2-hydroxy-butanoyl-CoA. The 2-hydroxyacyl-CoA dehydratase can be encoded by the gene products HadI and HadBC or encoded by the gene products HgdC and HgdAB. The 2-hydroxyacyl-CoA dehydratase can have at least 70% sequence identity to the amino acid sequences set forth in SEQ ID NOs. 5 and 6; or can have at least 70% sequence identity to the amino acid sequences set forth in SEQ ID NOs. 8 and 9. A (R)-specific enoyl-CoA hydratase or an (S)-specific enoyl-CoA hydratase can hydrate 3-methyl-but-2-enoyl-CoA. The (R)-specific enoyl-CoA hydratase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:1-3. The (S)-specific enoyl-CoA hydratase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. This document also features a method for synthesizing isobutene. The method includes dehydrogenating 3-methyl-butanoyl-CoA and hydrating 3-methyl-but-2-enoyl-CoA, and converting the resulting product to isobutene. An isovaleryl-CoA or acyl-CoA dehydrogenase can dehydrogenate 3-methyl-butanoyl-CoA. The isovaleryl-CoA or acyl-CoA dehydrogenase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:12. A (R)-specific enoyl-CoA hydratase or an (S)-specific enoyl-CoA hydratase can hydrate 3-methyl-but-2-enoyl-CoA. The (R)-specific enoyl-CoA hydratase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:1-3. The (S)-specific enoyl-CoA hydratase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0012] The reactions of the pathways described herein can be performed in one or more cell (e.g., host cell) strains (a) naturally expressing one or more relevant enzymes, (b) genetically engineered to express one or more relevant enzymes, or (c) naturally expressing one or more relevant enzymes and genetically engineered to express one or more relevant enzymes. Alternatively, relevant enzymes can be extracted from any of the above types of host cells and used in a purified or semi-purified form. Extracted enzymes can optionally be immobilized to a solid substrate such as the floors and/or walls of appropriate reaction vessels. Moreover, such extracts include lysates (e.g., cell lysates) that can be used as sources of relevant enzymes. In the methods provided by the document, all the steps can be performed in cells (e.g., host cells), all the steps can be performed using extracted enzymes, or some of the steps can be performed in cells and others can be performed using extracted enzymes.

[0013] In any the methods described herein, the method can be performed in a recombinant host (e.g., a prokaryote or eukaryote). The prokaryotic host can be from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia acidovorans, from the genus Bacillus such as Bacillus subtilis; from the genes Lactobacillus such as Lactobacillus delbrueckii; from the genus Lactococcus such as Lactococcus lactis or from the genus Rhodococcus such as Rhodococcus equi. The eukaryotic host can be from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica or from the genus Issatchenkia such as Issatchenkia orientalis or from the genus Debaryomyces such as Debaryomyces hansenii or from the genus Arxula such as Arxula adenoinivorans or from the genus Kluyveromyces such as Kluyveromyces lactis.

[0014] In any the methods described herein, a fermentation strategy can be used that entails anaerobic, micro-aerobic or aerobic cultivation. A fermentation strategy can entail nutrient limitation such as nitrogen, phosphate or oxygen limitation. A cell retention strategy using a ceramic hollow fiber membrane can be employed to achieve and maintain a high cell density during fermentation. The principal carbon source fed to the fermentation can derive from a biological or non-biological feedstock. The biological feedstock can be, or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles or municipal waste. The non-biological feedstock can be, or can derive from, natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes.

[0015] In another aspect, this document features a recombinant host that includes an exogenous nucleic acid encoding a 2-hydroxyacyl-CoA dehydratase and an enoyl-CoA hydratase, the host producing isobutene. The host further can include a mevalonate diphosphate decarboxylase. The host further can include a (i) a 2-hydroxy-acyl dehydrogenase, (ii) a CoA transferase or a CoA-ligase, and (iii) a thioesterase.

[0016] In another aspect, this document features a recombinant host that includes an exogenous nucleic acid encoding an isovaleryl-CoA/acyl-CoA dehydrogenase and an enoyl-CoA hydratase, the host producing isobutene. The host further can include a mevalonate diphosphate decarboxylase. The host further can include a 4-methyl-2-oxo-pentanoate dehydrogenase complex and a thioesterase. The host further can include an indolepyruvate decarboxylase, a phenylacetaldehyde dehydrogenase, and a CoA-ligase.

[0017] In any of the recombinant hosts, the enoyl-CoA hydratase can be a (R)-specific enoyl-CoA hydratase having at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:1-3 or a (S)-specific enoyl-CoA hydratase having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0018] In any of the recombinant hosts, the host can naturally accumulate polyhydroxyalkanoates and comprise attenuated polymer synthase enzymes.

[0019] In any of the recombinant hosts, the host further can include one or more of the following attenuated enzymes: a phosphotransacetylase, an acetate kinase, an enzyme that degrades pyruvate to lactate, an enzyme that degrades phophoenolpyruvate to succinate, an enzyme degrading acetyl-CoA to ethanol, or the final branch chain amino acid transaminase.

[0020] In any of the recombinant hosts, the host further can overexpress a gene encoding one or more of the following: a puridine nucleotide transhydrogenase, a glyceraldehyde-3P-dehydrogenase, a malic enzyme, a glucose-6-phosphate dehydrogenase, a fructose 1,6 diphosphatase, or a feedback inhibition resistant mutant of an acetolactate synthase, wherein the acetolactate synthase is resistant to feedback inhibition by branch chain amino acids. The gene encoding the acetolactate synthase can be expressed using a promoter not subject to genetic repression by branch-chain amino acids.

[0021] In any of the recombinant hosts, the efflux of isobutene across the cell membrane can be enhanced or amplified by genetic engineering of the cell membrane or increases to an associated transporter activity for isobutene.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0023] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims. The word "comprising" in the claims may be replaced by "consisting essentially of" or with "consisting of," according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

[0024] FIG. 1 is a schematic of exemplary biochemical pathway leading to isobutene using 3-methyl-2-oxobutanoate as a central precursor.

[0025] FIG. 2 is a schematic of exemplary biochemical pathways leading to isobutene using 4-methyl-2-oxopentanoate as a central precursor.

[0026] FIG. 3 contains the amino acid sequence of an (R)-specific enoyl-CoA hydratase from Pseudomonas aeruginosa (encoded by the Phan gene) (GenBank Accession No. BAA92740, SEQ ID NO:1); Aeromonas punctata (GenBank Accession No. BAA21816.1, SEQ ID NO:2); and Pseudomonas putida (GenBank Accession No. NP_746661, SEQ ID NO:3), and a (S)-specific enoyl-CoA hydratase from Bacillus subtilis (GenBank Accesion No. CAA99573.1, SEQ ID NO:4).

[0027] FIG. 4 contains the amino acid sequence of a Clostridium difficile 2-hydroxyacyl-CoA dehydratase (encoded by a HadBC gene) (see GenBank Accession Nos. AAV40819.1 and AAV40820.1, SEQ ID NOs. 5 and 6, respectively) and a Clostridium difficile initiator (encoded by HadI) (GenBank Accession No. AAV40818.1, SEQ ID NO:7). FIG. 4 also contains the amino acid sequence of an Acidaminococcus fermentans 2-hydroxyacyl-CoA dehydratase encoded by the HgdAB gene (see GenBank Accession Nos. CAA32465.1 and CAA32466.1, SEQ ID NOs: 8 and 9, respectively) and an Acidaminococcus fermentans initiator encoded by HdgC (see GenBank Accession No. CAA42196.1, SEQ ID NO:10).

[0028] FIG. 5 contains the amino acid sequence of a Pseudomonas aeruginosa PAO1 isovaleryl-CoA dehydrogenase encoded by the liuA gene (GenBank Accession No. AAG05403.1, SEQ ID NO: 11) and the amino acid sequence of a Streptomyces avermitilis acyl-CoA dehydrogenase encoded by the acdH gene (GenBank Accession No. AAD44196.1, SEQ ID NO:12).

[0029] FIG. 6 is a graph of the absorbance units of crotonyl-CoA (substrate) over time in a spectrophotometric enzyme assay in the forward (hydrating) direction for an enoyl-CoA hydratase encoded by phaJ.

[0030] FIG. 7 is a table of results for the LC-MS analysis of an enzyme assay in the reverse (dehydrating) direction for an enoyl-CoA hydratase activity encoded by phaJ. The results indicate that the enoyl-CoA hydratase is reversible, favoring the forward hydration reaction.

[0031] FIG. 8 is a table of results for the LC-MS analysis of an enzyme assay in the reverse (dehydrating) direction for an enoyl-CoA hydratase encoded by phaJ. The results indicate that the enoyl-CoA hydratase accepted 3-methyl-3-hydroxybutanoyl-CoA as substrate. Given the reversibility of the enzyme reaction, the enoyl-CoA hydratase accepts 3-methyl-3-hydroxybut-2-enoyl-CoA as a substrate.

DETAILED DESCRIPTION

[0032] In particular, this document provides enzymes and recombinant host microorganisms for isobutene synthesis that can form a branch chain enoyl-CoA substrate, 3-methyl-but-2-enoyl-CoA, and hydrate the substrate to form 3-methyl-3-hydroxybutanoyl-CoA, which in turn can be converted after one or more enzymatic steps to isobutene by a mevalonate diphosphate decarboxylase. As such, host microorganisms described herein can include pathways that can be manipulated such that isobutene can be produced.

[0033] In an endogenous pathway, the host microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A host microorganism containing an engineered pathway does not naturally express all of the enzymes catalyzing the reactions within the pathway but has been engineered such that all of the enzymes within the pathway are expressed in the host. Within an engineered pathway, the enzymes can be from a single source, i.e., from one species, or can be from multiple sources, i.e., different species.

[0034] Nucleic acids encoding the enzymes described herein have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL. Any of the enzymes described herein that can be used for isobutene production can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of the corresponding wild-type enzyme. For example, an enoyl-CoA hydratase (e.g., a (R)-specific enoyl-CoA hydratase) described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of the Pseudomonas aeruginosa enoyl-CoA hydratase encoded by PhaJ1 gene (GenBank Accession No. BAA92740, SEQ ID NO:1), Aeromonas punctata enoyl-CoA hydratase (GenBank Accession No. BAA21816.1, SEQ ID NO:2), or Pseudomonas putida enoyl-CoA hydratase (GenBank Accession No. NP_746661, SEQ ID NO:3). See, FIG. 3. An enoyl-CoA hydratase also can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of the Bacillus subtilis (S)-specific enoyl CoA hydratase (GenBank Accession No. CAA99573.1, SEQ ID NO:4). See, FIG. 3.

[0035] For example, a 2-hydroxyacyl-CoA dehydratase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Clostridium difficile 2-hydroxyacyl-CoA dehydratase encoded by a HadBC gene (see GenBank Accession Nos. AAV40819.1 and AAV40820.1, SEQ ID NOs. 5 and 6, respectively) and its Clostridium difficile initiator encoded by HadI (GenBank Accession No. AAV40818.1, SEQ ID NO:7). The HadBC gene encodes the two subunits of the dehydratase. For example, a 2-hydroxyacyl-CoA dehydratase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Acidaminococcus fermentans 2-hydroxyacyl-CoA dehydratase encoded by the HgdAB gene (see GenBank Accession Nos. CAA32465.1 and CAA32466.1, SEQ ID NOs: 8 and 9, respectively) and its Acidaminococcus fermentans initiator encoded by HdgC (see GenBank Accession No. CAA42196.1, SEQ ID NO:10). The HgdAB gene encodes the two subunits of the dehydratase. See, FIG. 4.

[0036] For example, an isovaleryl-CoA dehydrogenase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Pseudomonas aeruginosa PAO1 isovaleryl-CoA dehydrogenase encoded by the liuA gene (GenBank Accession No. AAG05403.1, SEQ ID NO: 11).

[0037] For example, an acyl-CoA dehydrogenase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Streptomyces avermitilis acyl-CoA dehydrogenase encoded by the acdH gene (GenBank Accession No. AAD44196.1, SEQ ID NO: 12). See, FIG. 5.

[0038] The percent identity (homology) between two amino acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (e.g., www.fr.com/blast/) or the U.S. government's National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of Bl2seq are set as follows:--i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt);--j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt);--p is set to blastp;--o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq--i c:\seq1.txt--j c:\seq2.txt--p blastp--o c:\output.txt. If the two compared sequences share homology (identity), then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology (identity), then the designated output file will not present aligned sequences. Similar procedures can be following for nucleic acid sequences except that blastn is used.

[0039] Once aligned, the number of matches is determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity (homology) is determined by dividing the number of matches by the length of the full-length polypeptide amino acid sequence followed by multiplying the resulting value by 100. It is noted that the percent identity (homology) value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.

[0040] It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., bacteria or fungus) is obtained, using appropriate codon bias tables for that species.

[0041] Functional fragments of any of the enzymes described herein can also be used in the methods of the document. The term "functional fragment" as used herein refers to a peptide fragment of a protein that has at least 25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 100%; or even greater than 100%) of the activity of the corresponding mature, full-length, wild-type protein. The functional fragment can generally, but not always, be comprised of a continuous region of the protein, wherein the region has functional activity.

[0042] This document also provides (i) functional variants of the enzymes used in the methods of the document and (ii) functional variants of the functional fragments described above. Functional variants of the enzymes and functional fragments can contain additions, deletions, or substitutions relative to the corresponding wild-type sequences. Enzymes with substitutions will generally have not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservative substitutions). This applies to any of the enzymes described herein and functional fragments. A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a nonconservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

[0043] Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more amino acids) or non-contiguous single amino acids. Additions (addition variants) include fusion proteins containing: (a) any of the enzymes described herein or a fragment thereof; and (b) internal or terminal (C or N) irrelevant or heterologous amino acid sequences. In the context of such fusion proteins, the term "heterologous amino acid sequences" refers to an amino acid sequence other than (a). A heterologous sequence can be, for example a sequence used for purification of the recombinant protein (e.g., FLAG, polyhistidine (e.g., hexahistidine), hemaglutianin (HA), glutathione-S-transferase (GST), or maltosebinding protein (MBP)). Heterologous sequences also can be proteins useful as detectable markers, for example, luciferase, green fluorescent protein (GFP), or chloramphenicol acetyl transferase (CAT). In some embodiments, the fusion protein contains a signal sequence from another protein. In certain host cells (e.g., yeast host cells), expression and/or secretion of the target protein can be increased through use of a heterologous signal sequence. In some embodiments, the fusion protein can contain a carrier (e.g., KLH) useful, e.g., in eliciting an immune response for antibody generation) or ER or Golgi apparatus retention signals. Heterologous sequences can be of varying length and in some cases can be a longer sequences than the full-length target proteins to which the heterologous sequences are attached.

[0044] Recombinant hosts can naturally express none or some (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) of the enzymes of the pathways described herein. Endogenous genes of the recombinant hosts also can be disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Recombinant hosts can be referred to as recombinant host cells, engineered cells, or engineered hosts. Thus, as described herein, recombinant hosts can include nucleic acids encoding one or more of a decarboxylase, a dehydrogenase, a hydratase, a thioesterase, a Coenzyme A ligase, or a Coenzyme A transferase, as described in more detail below.

[0045] For example, recombinant hosts can include an exogenous nucleic acid encoding a 2-hydroxyacyl-CoA dehydratase and an enoyl-CoA hydratase, or an isovaleryl-CoA/acyl-CoA dehydrogenase and an enoyl-CoA hydratase. Such hosts can produce isobutene and include a nucleic acid encoding a mevalonate diphosphate decarboxylase. The hosts further can include (i) a 2-hydroxy-acyl dehydrogenase, (ii) a CoA transferase or a CoA-ligase, and/or (iii) a thioesterase or a CoA transferase. In some embodiments, the hosts further can include (i) a 4-methyl-2-oxo-pentanoate dehydrogenase, (ii) an indolepyruvate decarboxylase, a phenylacetaldehyde dehydrogenase and a CoA-ligase and/or (iii) a thioesterase.

[0046] The term "exogenous" as used herein with reference to a nucleic acid (or a protein) and a host refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.

[0047] In contrast, the term "endogenous" as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a host refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular host as it is found in nature. Moreover, a cell "endogenously expressing" a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature. Moreover, a host "endogenously producing" or that "endogenously produces" a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.

[0048] In addition, the production of isobutene can be performed in vitro using the isolated enzymes described herein, using a lysate (e.g., a cell lysate) from a host microorganism as a source of the enzymes, or using a plurality of lysates from different host microorganisms as the source of the enzymes.

[0049] In some embodiments, 3-methyl-but-2-enoyl-CoA is formed by an isovaleryl-CoA or acyl-CodA dehydrogenase enzyme classified, for example, under EC 1.3.8.4, such as the gene product of liuA or acdH. The liuA gene from Pseudomonas aeruginosa has dehydrogenase activity specific for isovaleryl-CoA (see, for example, Foster-Fromme and Jendrossek, FEMS Microbiol. Lett., 2008, 286, 78-84). The acdH gene from Streptomyces avermitilis also has dehydrogenase activity for isovaleryl-CoA (see, for example, Zhang et al., Microbiology, 1999, 145, 2323-2334).

[0050] In some embodiments, 3-methyl-but-2-enoyl-CoA is formed by a 2-hydroxyacyl-CoA dehydratase classified, for example, under EC 4.2.1.-, such as the product of the HadBC gene from Clostridium difficile and its initiator HadI (see FIG. 4), or the product of the HgdAB gene from Acidaminococcus fermentans and its initiator HdgC (see FIG. 4). In some embodiments, the 2-hydroxyacyl-CoA dehydratase is the result of enzyme engineering. The 2-hydroxyacyl-CoA dehydratase enzymes isolated from anaerobic bacteria possess a common catalytic mechanism employed in amino acid degradation pathways. For example, the gene products of HadBC/HadI from Clostridium difficile catalyse the conversion of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA. Similarly, the gene products of HgdAB/HdgC catalyse the conversion of 2-hydroxyglutaryl-CoA to glutaconyl-CoA (Kim et al., FEMS Microbiol. Reviews, 2004, 28, 455-468).

[0051] In some embodiments, the 3-hydroxy functional group is introduced into 3-methyl-but-2-enoyl-CoA by a R-specific enoyl-CoA hydratase enzyme classified, for example, under EC 4.2.119 such as the gene product of phaJ or MaoC or a bacterial (S)-specific enoyl-CoA hydratase classified, for example, under EC 4.2.1.17 such as the gene product of YsiB. In some embodiments, the enoyl-CoA hydratase enzyme is the result of enzyme engineering. A single enzyme candidate for the introduction of a 3-hydroxy functional group into 3-methylbuten-2-enoyl-CoA was identified in the cell free extract of Galactomyces reessii, containing an enoyl-CoA hydratase classified in EC 4.2.1.17 that converts 3-methylbuten-2-enoyl-CoA to 3-hydroxy-3-methylbutanoyl-CoA (Lee et al., Appl. Environ. Microbiol., 1997, 63(11), 4191-4195). Until now, an equivalent enoyl-CoA hydratase activity from bacterial origin had not been identified.

[0052] In some embodiments, the hydratase enzyme can be the result of enzyme engineering, using the enzyme structure of phaJ, EC 4.2.1.119 and EC 4.2.1.17 to inform rational enzyme design.

[0053] In some embodiments (FIG. 1), the central precursor to 3-methyl-but-2-enoyl-CoA, 3-methyl-2-oxo-butanoate, is converted to 3-methyl-2-hydroxy-butanoate by a 2-hydroxy-acyl dehydrogenase classified, for example, under EC 1.1.1.272, EC 1.1.1.345 or EC 1.1.1.28; followed by conversion to 3-methyl-2-hydroxybutanoyl-CoA by a CoA transferase classified, for example, under EC 2.8.3.- such as the gene product of GctAB or a CoA-ligase classified, for example, under EC 6.2.1.2; followed by conversion to 3-methyl-but-2-enoyl-CoA by a 2-hydroxy-acyl-CoA dehydratase such as the gene product of HadBC and the initiator HadI, or HgdAB and the initiator HgdC; followed by conversion to 3-methyl-3-hydroxy-butanoyl-CoA by an enoyl-CoA hydratase classified, for example, under EC 4.2.1.119 such as the gene product of phaJ or MaoC, or classified, for example, under EC 4.2.1.17 such as the gene product of YsiB; followed by conversion to 3-methyl-3-hydroxy-butanoate by a thioesterase classified, for example, under EC 3.1.2.- such as the gene product of tesA, tesB, or YciA, or a CoA-transferase classified, for example, under EC 2.8.3.- such as the gene product of GctAB; followed by conversion to isobutene by a mevalonate diphosphate decarboxylase classified, for example, under EC 4.1.1.33. The thioesterase encoded by YciA, has broad substrate specificity, hydrolysing a number of branch chain fatty acids such as isobutyryl-CoA, isovaleryl-CoA, as well as 3-hydroxy-3-methylglutaryl-CoA (Zhuang et al., Biochemistry, 2008, 47, 2789-2796).

[0054] In some embodiments, 3-methyl-2-oxobutanoate is synthesized from pyruvate, by conversion of pyruvate to 2-acetolactate by an acetolactate synthase classified, for example, under EC 2.2.1.6 such as the gene product of ilvB or ilvN; followed by conversion to 2,3-dihydroxyisovalerate by a dihydroxyisovalerate dehydrogenase classified, for example, under EC 1.1.1.86 such as the gene product of ilvC; followed by conversion to 3-methyl-2-oxo-butanoate by a 2,3-dihydroxyisovalerate dehydratase classified, for example, under EC 4.2.1.9 such as the gene product of ilvD.

[0055] In some embodiments (FIG. 2), the central precursor to 3-methyl-but-2-enoyl-CoA, 4-methyl-2-oxo-pentanoate, is converted to 3-methylbutanoate by a 4-methyl-2-oxo-pentanoate dehydrogenase complex classified, for example, under EC 1.2.1.- such as the gene products of pdhD, bfmBB, bfmBAA and bfmBAB, or classified under EC 1.2.7.7; followed by conversion to 3-methylbut-2-enoyl-CoA by an isovaleryl-CoA/acyl-CoA dehydrogenase classified, for example, under EC 1.3.8.4 such as the gene product of liuA or acdH; followed by conversion to 3-methyl-3-hydroxy-butanoyl-CoA by a (R)-specific enoyl-CoA hydratase classified, for example, under EC 4.2.1.119 such as the gene product of phaJ or MaoC, or classified, for example, under EC 4.2.1.17 such as the gene product of YsiB; followed by conversion to 3-methyl-3-hydroxy-butanoate by a thioesterase classified, for example, under EC 3.1.2.- such as the gene product of tesA, tesB, or YciA; followed by conversion to isobutene by a mevalonate diphosphate decarboxylase classified under EC 4.1.1.33.

[0056] In some embodiments (FIG. 2), the central precursor to 3-methyl-but-2-enoyl-CoA, 4-methyl-2-oxo-pentanoate, is converted to 3-methylbutanal by an indolepyruvate decarboxylase classified, for example, under EC 4.1.1.74 or EC 4.1.1.43; followed by conversion to 3-methyl-butanoate by a phenylacetaldehyde dehydrogenase classified, for example, under EC 1.2.1.5 or EC 1.2.1.39 such as the gene product of padA; followed by conversion to 3-methyl-butanoyl-CoA by a CoA-ligase classified, for example, under EC 6.2.1.2; followed by conversion to 3-methyl-but-2-enoyl-CoA by an isovaleryl-CoA/acyl-CoA dehydrogenase classified, for example, under EC 1.3.8.4 such as the gene product of liuA or acdH; followed by conversion to 3-methyl-3-hydroxy-butanoyl-CoA by a (R)-specific enoyl-CoA hydratase classified, for example, under EC 4.2.1.119 such as the gene product of phaJ or MaoC, or classified, for example, under EC 4.2.1.17 such as the gene product of YsiB; followed by conversion to 3-methyl-3-hydroxy-butanoate by a thioesterase classified, for example, under EC 3.1.2.- such as the gene product of tesA, tesB, or YciA; followed by conversion to isobutene by a mevalonate diphosphate decarboxylase classified under EC 4.1.1.33.

[0057] In some embodiments, the nucleic acids encoding the enzymes of the pathways described in FIG. 1 or 2 are introduced into a host microorganism that is either a prokaryote or eukaryote.

[0058] In some embodiments, the host microorganism is a prokaryote from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia acidovorans, from the genus Bacillus such as Bacillus subtilis; from the genes Lactobacillus such as Lactobacillus delbrueckii; from the genus Lactococcus such as Lactococcus lactis, or from the genus Rhodococcus such as Rhodococcus equi.

[0059] In some embodiments, the host microorganism is a eukaryote from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issatchenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis.

[0060] In some embodiments, isobutene is biosynthesized in a recombinant host using a fermentation strategy that can include anaerobic, micro-aerobic or aerobic cultivation of the recombinant host.

[0061] In some embodiments, isobutene is biosynthesized in a recombinant host under nutrient limiting conditions such as nitrogen, phosphate or oxygen limitation.

[0062] In some embodiments, isobutene is biosynthesized in a recombinant host using a fermentation strategy that uses an alternate final electron acceptor to oxygen such as nitrate.

[0063] In some embodiments, a cell retention strategy using, for example, ceramic hollow fiber membranes can be employed to achieve and maintain a high cell density during either fed batch or continuous fermentation in the synthesis of isobutene.

[0064] In some embodiments, the biological feedstock can be, can include, or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.

[0065] The efficient catabolism of crude glycerol stemming from the production of biodiesel has been demonstrated in several microorganisms such as Escherichia coli, Cupriavidus necator, Pseudomonas oleavorans, Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem. Biotechnol., 2012, 166, 1801-1813; Yang et al., Biotechnology for Biofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol., 2011, 90, 885-893).

[0066] The efficient catabolism of lignocellulosic-derived levulinic acid has been demonstrated in several organisms such as Cupriavidus necator and Pseudomonas putida in the synthesis of 3-hydroxyvalerate via the precursor propanoyl-CoA (Jaremko and Yu, Journal of Biotechnology, 2011, 155, 2011, 293-298; Martin and Prather, Journal of Biotechnology, 2009, 139, 61-67).

[0067] The efficient catabolism of lignin-derived aromatic compounds such benzoate analogues has been demonstrated in several microorganisms such as Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinion in Biotechnology, 2011, 22, 394-400; Perez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).

[0068] The efficient utilization of agricultural waste, such as olive mill waste water has been demonstrated in several microorganisms, including Yarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008, 99(7), 2419-2428).

[0069] The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other agricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcus lactis (see, e.g., Hermann et al, Journal of Biotechnology, 2003, 104, 155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2), 163-172; Ohashi et al., Journal of Bioscience and Bioengineering, 1999, 87(5), 647-654).

[0070] The efficient utilization of furfural, derived from a variety of agricultural lignocellulosic sources, has been demonstrated for Cupriavidus necator (Li et al., Biodegradation, 2011, 22, 1215-1225).

[0071] In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes.

[0072] The efficient catabolism of methanol has been demonstrated for the methylotropic yeast Pichia pastoris.

[0073] The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).

[0074] The efficient catabolism of CO.sub.2 and H.sub.2, which may be derived from natural gas and other chemical and petrochemical sources, has been demonstrated for Cupriavidus necator (Prybylski et al., Energy, Sustainability and Society, 2012, 2:11).

[0075] The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogenum (Kopke et al., Applied and Environmental Microbiology, 2011, 77(15), 5467-5475).

[0076] The efficient catabolism of the non-volatile residue waste stream from cyclohexane processes has been demonstrated for numerous microorganisms, such as Delftia acidovorans and Cupriavidus necator (Ramsay et al., Applied and Environmental Microbiology, 1986, 52(1), 152-156).

[0077] In some embodiments, substantially pure cultures of recombinant host microorganisms are provided. As used herein, a "substantially pure culture" of a recombinant host microorganism is a culture of that microorganism in which less than about 40% (i.e., less than about: 35%; 30%; 25%; 20%; 15%; 10%; 5%; 2%; 1%; 0.5%; 0.25%; 0.1%; 0.01%; 0.001%; 0.0001%; or even less) of the total number of viable cells in the culture are viable cells other than the recombinant microorganism, e.g., bacterial, fungal (including yeast), mycoplasmal, or protozoan cells. The term "about" in this context means that the relevant percentage can be 15% of the specified percentage above or below the specified percentage. Thus, for example, about 20% can be 17% to 23%. Such a culture of recombinant microorganisms includes the cells and a growth, storage, or transport medium. Media can be liquid, semi-solid (e.g., gelatinous media), or frozen. The culture includes the cells growing in the liquid or in/on the semi-solid medium or being stored or transported in a storage or transport medium, including a frozen storage or transport medium. The cultures are in a culture vessel or storage vessel or substrate (e.g., a culture dish, flask, or tube or a storage vial or tube).

Metabolic Engineering

[0078] The present document provides methods involving less than all the steps described for all the above pathways. Such methods can involve, for example, one, two, three, four, five, six, seven, eight, nine, ten, or more of such steps. Where less than all the steps are included in such a method, the first step can be any one of the steps listed.

[0079] Furthermore, recombinant hosts described herein can include any combination of the above enzymes such that one or more of the steps, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more of such steps, can be performed within a recombinant host.

[0080] In addition, this document recognizes that where enzymes have been described as accepting CoA-activated substrates, analogous enzyme activities associated with [acp]-bound substrates exist that are not necessarily in the same enzyme class.

[0081] Also, this document recognizes that where enzymes have been described accepting (R)-enantiomers of substrate, analogous enzyme activities associated with (S)-enantiomer substrates exist that are not necessarily in the same enzyme class.

[0082] This document also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or co-substrate, such as acetyl-CoA, many enzymes are promiscuous in terms of accepting a number of different co-factors or co-substrates in catalyzing a particular enzyme activity. Also, this document recognizes that where enzymes have high specificity for e.g., a particular co-factor such as NADH, an enzyme with similar or identical activity that has high specificity for the co-factor NADPH may be in a different enzyme class.

[0083] In some embodiments, the enzymes in the pathways outlined herein can be the result of enzyme engineering via non-direct or rational enzyme design approaches with aims of improving activity, improving specificity, reducing feedback inhibition, reducing repression, improving enzyme solubility, changing stereo-specificity, or changing co-factor specificity.

[0084] In some embodiments, the enzymes in the pathways outlined herein can be gene dosed, i.e., overexpressed, into the resulting genetically modified organism via episomal or chromosomal integration approaches.

[0085] In some embodiments, genome-scale system biology techniques such as Flux Balance Analysis can be utilized to devise genome scale attenuation or knockout strategies for directing carbon flux to isobutene.

[0086] Attenuation strategies include, but are not limited to, the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.

[0087] In some embodiments, fluxomic, metabolomic and transcriptomal data can be utilized to inform or support genome-scale system biology techniques, thereby devising genome scale attenuation or knockout strategies in directing carbon flux to isobutene.

[0088] In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the polymer synthase enzymes can be attenuated in the host strain.

[0089] In some embodiments requiring the intracellular availability of acetyl-CoA for isobutene synthesis, a host that is deficient (e.g., attenuated level of activity) in one or more enzymes in the acetate synthesis pathway can be used. For example, a host that is deficient in a phosphotransacetylase (encoded by the pta gene) can be used (Shen et al., Appl. Environ. Microbio., 2011, 77(9), 2905-2915).

[0090] In some embodiments requiring the intracellular availability of acetyl-CoA or pyruvate for isobutene synthesis, an enzyme degrading pyruvate to lactate can be attenuated (such as the gene product of ldhA) (Shen et al., Appl. Environ. Microbiol., 2011, 77(9), 2905-2915).

[0091] In some embodiments requiring the intracellular availability of pyruvate for isobutene synthesis, an enzyme that degrades phophoenolpyruvate to succinate, such as the gene product of frdBC, can be attenuated (see, e.g., Shen et al., 2011, supra).

[0092] In some embodiments requiring the intracellular availability of pyruvate for isobutene synthesis, a gene encoding an enzyme degrading acetyl-CoA to ethanol, such as the gene product of adhE, can be attenuated (Shen et al., 2011, supra).

[0093] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of isobutene, a gene encoding a puridine nucleotide transhydrogenase such as UdhA can be overexpressed in the host organism (Brigham et al., Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).

[0094] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of isobutene, a glyceraldehyde-3P-dehydrogenase gene such as GapN can be overexpressed in the host organism (Brigham et al., 2012, supra).

[0095] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of isobutene, a malic enzyme gene such as maeA or maeB can be overexpressed in the host organism (Brigham et al., 2012, supra).

[0096] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of isobutene, a glucose-6-phosphate dehydrogenase gene such as zwf can be overexpressed in the host organism (Lim et al., Journal of Bioscience and Bioengineering, 2002, 93(6), 543-549).

[0097] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of isobutene, a gene encoding a fructose 1,6 diphosphatase such as fbp can be overexpressed in the host (Becker et al., Journal of Biotechnology, 2007, 132, 99-109).

[0098] In some embodiments, a feedback inhibition resistant mutant of an acetolactate synthase classified, for example, under EC 2.2.1.6, such as mutants of ilvB and/or ilvN that are resistant to feedback inhibition by branch chain amino acids, can be overexpressed in the host.

[0099] In some embodiments, acetolactate synthase can be expressed under a promoter not subject to genetic repression by branch-chain amino acids (e.g., valine, leucine, or isoleucine).

[0100] In some embodiments, the branch chain amino acid transaminase encoded by ilvE is attenuated.

[0101] In some embodiments, the efflux of isobutene across the cell membrane to the extracellular media can be enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for isobutene.

Producing Isobutene Using a Recombinant Host

[0102] Typically, isobutene is produced by providing a host microorganism and culturing the provided microorganism with a culture medium containing a suitable carbon source as described above. In general, the culture media and/or culture conditions can be such that the microorganisms grow to an adequate density and produce isobutene efficiently. For large-scale production processes, any method can be used such as those described elsewhere (Manual of Industrial Microbiology and Biotechnology, 2.sup.nd Edition, Editors: A. L. Demain and J. E. Davies, ASM Press; and Principles of Fermentation Technology, P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g., a 100 gallon, 200 gallon, 500 gallon, or more tank) containing an appropriate culture medium is inoculated with a particular microorganism. After inoculation, the microorganism is incubated to allow biomass to be produced. Once a desired biomass is reached, the broth containing the microorganisms can be transferred to a second tank. This second tank can be any size. For example, the second tank can be larger, smaller, or the same size as the first tank. Typically, the second tank is larger than the first such that additional culture medium can be added to the broth from the first tank. In addition, the culture medium within this second tank can be the same as, or different from, that used in the first tank.

[0103] Once transferred, the microorganisms can be incubated to allow for the production of isobutene. Once produced, any method can be used to isolate isobutene. For example, isobutene can be recovered from the fermenter off-gas stream as a volatile product as the boiling point of isobutene is -6.9.degree. C. At a typical fermentation temperature of approximately 30.degree. C., isobutene boils off from the broth, stripped by the gas flow rate through the broth for recovery from the off-gas. Isobutene can be selectively adsorbed onto, for example, an adsorbent and separated from the other off-gas components. Membrane separation technology may also be employed to separate isobutene from the other off-gas compounds. Isobutene may desorbed from the adsorbent using, for example, nitrogen and condensed at low temperature and high pressure.

Example

Enzyme Activity of R-Specific Enoyl-CoA Hydratase Accepting 3-Methyl-3-Hydroxybutanoyl-CoA as Substrate

[0104] A C-terminal His-tagged phaJ gene from Aeromonas punctata, which encodes a R-specific enoyl-CoA hydratase (SEQ ID NO:2, see FIG. 3) was cloned into a pE23a expression vector under the T7 promoter. The expression vector was transformed into a BL21[DE3] E. coli host. The resulting recombinant E. coli strain was cultivated at 30.degree. C. in a 1 L shake flask culture containing 100 mL Luria Broth media, with shaking at 200 rpm. The culture was induced using 1 mM IPTG for 2 hours.

[0105] The pellet from each of the induced shake flask cultures was harvested by centrifugation. Each pellet was resuspended in 20 mM HEPES (pH=7.2 [-]), 1 mM PMSF and 29 units benzonase, and lysed via sonication. The cell debris was separated from the supernatant via centrifugation and filtered using a 0.2 .mu.m filter. The phaJ enzyme was purified from the supernatant using Ni-affinity chromatography and concentrated to 1.25 mg/mL.

[0106] The native enzyme activity assay in the forward (hydration) direction was undertaken in a buffer composed of 10 mM ammonium acetate (pH=8) and 1 mM of crotonyl-CoA (also known as 2-butenoyl-CoA) (Sigma-Aldrich) at 30.degree. C. The enzyme activity assay reaction was initiated by adding 0.4 .mu.M of purified enoyl-CoA hydratase to the assay buffer containing the substrate. The enoyl-CoA hydratase accepted crotonyl-CoA as substrate as confirmed via spectrophotometry at 263 nm at 30.degree. C. The substrate only control showed minimal spontaneous hydration of crotonyl-CoA as determined by spectrophotometry at 263 nm. See FIG. 6.

[0107] The native enzyme activity assay in the reverse (dehydration) direction was undertaken in a buffer composed of 10 mM ammonium acetate (pH=8) and 1 mM of racemic 3-hydroxybutanoyl-CoA. The enzyme activity assay reaction was initiated by adding 5 .mu.M of purified enoyl-CoA hydratase to the assay buffer containing the substrate and incubated at 30.degree. C. for 1 hour. The enoyl-CoA hydratase accepted 3-hydroxybutanoyl-CoA as substrate as confirmed via LC-MS. The substrate only control showed negligible spontaneous dehydration of 3-hydroxybutanoyl-CoA. As demonstrated previously (Lan and Liao, Proc. Natl. Acad. Sci. USA, 2012, 109(16), 6018-6023), the enoyl-CoA hydratase encoded by phaJ is reversible, though favors the forward (hydration) direction. See FIG. 7.

[0108] The non-native enzyme activity assay in the reverse (dehydration) direction was undertaken in a buffer composed of 10 mM ammonium acetate (pH=8) and 1 mM of 3-methyl-3-hydroxybutanoyl-CoA. The enzyme activity assay reaction was initiated by adding 5 .mu.M of purified enoyl-CoA hydratase to the assay buffer containing the substrate and incubated at 30.degree. C. for 1 hour. The enzyme encoded by phaJ accepted 3-methyl-3-hydroxybutanoyl-CoA as substrate as confirmed via LC-MS. The substrate only control showed no spontaneous dehydration of 3-methyl-3-hydroxybutanoyl-CoA. See FIG. 8.

[0109] The enoyl-CoA hydratase encoded by phaJ from Aeromonas punctata accepted 3-methyl-3-hydroxybutanoyl-CoA as substrate in the dehydration direction. Given the reversibility of the enzyme reaction and the favored hydration direction, the enoyl-CoA hydratase encoded by phaJ from Aeromonas punctata accepts 3-methyl-but-2-enoyl-CoA as substrate.

Other Embodiments

[0110] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

121156PRTPseudomonas aeruginosa 1Met Ser Gln Val Gln Asn Ile Pro Tyr Ala Glu Leu Glu Val Gly Gln 1 5 10 15 Lys Ala Glu Tyr Thr Ser Ser Ile Ala Glu Arg Asp Leu Gln Leu Phe 20 25 30 Ala Ala Val Ser Gly Asp Arg Asn Pro Val His Leu Asp Ala Ala Tyr 35 40 45 Ala Ala Thr Thr Gln Phe Lys Glu Arg Ile Ala His Gly Met Leu Ser 50 55 60 Gly Ala Leu Ile Ser Ala Ala Ile Ala Thr Val Leu Pro Gly Pro Gly65 70 75 80 Thr Ile Tyr Leu Gly Gln Thr Leu Arg Phe Thr Arg Pro Val Lys Leu 85 90 95 Gly Asp Asp Leu Lys Val Glu Leu Glu Val Leu Glu Lys Leu Pro Lys 100 105 110 Asn Arg Val Arg Met Ala Thr Arg Val Phe Asn Gln Ala Gly Lys Gln 115 120 125 Val Val Asp Gly Glu Ala Glu Ile Met Ala Pro Glu Glu Lys Leu Ser 130 135 140 Val Glu Leu Ala Glu Leu Pro Pro Ile Ser Ile Gly145 150 155 2134PRTAeromonas punctata 2Met Ser Ala Gln Ser Leu Glu Val Gly Gln Lys Ala Arg Leu Ser Lys 1 5 10 15 Arg Phe Gly Ala Ala Glu Val Ala Ala Phe Ala Ala Leu Ser Glu Asp 20 25 30 Phe Asn Pro Leu His Leu Asp Pro Ala Phe Ala Ala Thr Thr Ala Phe 35 40 45 Glu Arg Pro Ile Val His Gly Met Leu Leu Ala Ser Leu Phe Ser Gly 50 55 60 Leu Leu Gly Gln Gln Leu Pro Gly Lys Gly Ser Ile Tyr Leu Gly Gln65 70 75 80 Ser Leu Ser Phe Lys Leu Pro Val Phe Val Gly Asp Glu Val Thr Ala 85 90 95 Glu Val Glu Val Thr Ala Leu Arg Glu Asp Lys Pro Ile Ala Thr Leu 100 105 110 Thr Thr Arg Ile Phe Thr Gln Gly Gly Ala Leu Ala Val Thr Gly Glu 115 120 125 Ala Val Val Lys Leu Pro 130 3156PRTPseudomonas putida 3Met Ser Gln Val Thr Asn Thr Pro Tyr Glu Ala Leu Glu Val Gly Gln 1 5 10 15 Lys Ala Glu Tyr Lys Lys Ser Val Glu Glu Arg Asp Ile Gln Leu Phe 20 25 30 Ala Ala Met Ser Gly Asp His Asn Pro Val His Leu Asp Ala Glu Phe 35 40 45 Ala Ala Lys Ser Met Phe Arg Glu Arg Ile Ala His Gly Met Phe Ser 50 55 60 Gly Ala Leu Ile Ser Ala Ala Val Ala Cys Thr Leu Pro Gly Pro Gly65 70 75 80 Thr Ile Tyr Leu Gly Gln Gln Met Ser Phe Gln Lys Pro Val Lys Ile 85 90 95 Gly Asp Thr Leu Thr Val Arg Leu Glu Ile Leu Glu Lys Leu Pro Lys 100 105 110 Phe Lys Val Arg Ile Ala Thr Asn Val Tyr Asn Gln Asn Asp Glu Leu 115 120 125 Val Val Ala Gly Glu Ala Glu Ile Leu Ala Pro Arg Lys Gln Gln Thr 130 135 140 Val Glu Leu Val Ser Pro Pro Asn Phe Val Ala Ser145 150 155 4258PRTBacillus subtilis 4Met Asn Ala Ile Ser Leu Ala Val Asp Gln Phe Val Ala Val Leu Thr 1 5 10 15 Ile His Asn Pro Pro Ala Asn Ala Leu Ser Ser Arg Ile Leu Glu Glu 20 25 30 Leu Ser Ser Cys Leu Asp Gln Cys Glu Thr Asp Ala Gly Val Arg Ser 35 40 45 Ile Ile Ile His Gly Glu Gly Arg Phe Phe Ser Ala Gly Ala Asp Ile 50 55 60 Lys Glu Phe Thr Ser Leu Lys Gly Asn Glu Asp Ser Ser Leu Leu Ala65 70 75 80 Glu Arg Gly Gln Gln Leu Met Glu Arg Ile Glu Ser Phe Pro Lys Pro 85 90 95 Ile Ile Ala Ala Ile His Gly Ala Ala Leu Gly Gly Gly Leu Glu Leu 100 105 110 Ala Met Ala Cys His Ile Arg Ile Ala Ala Glu Asp Ala Lys Leu Gly 115 120 125 Leu Pro Glu Leu Asn Leu Gly Ile Ile Pro Gly Phe Ala Gly Thr Gln 130 135 140 Arg Leu Pro Arg Tyr Val Gly Thr Ala Lys Ala Leu Glu Leu Ile Gly145 150 155 160 Ser Gly Glu Pro Ile Ser Gly Lys Glu Ala Leu Asp Leu Gly Leu Val 165 170 175 Ser Ile Gly Ala Lys Asp Glu Ala Glu Val Ile Glu Lys Ala Lys Ala 180 185 190 Leu Ala Ala Lys Phe Ala Glu Lys Ser Pro Gln Thr Leu Ala Ser Leu 195 200 205 Leu Glu Leu Leu Tyr Ser Asn Lys Val Tyr Ser Tyr Glu Gly Ser Leu 210 215 220 Lys Leu Glu Ala Lys Arg Phe Gly Glu Ala Phe Glu Ser Glu Asp Ala225 230 235 240 Lys Glu Gly Ile Gln Ala Phe Leu Glu Lys Arg Lys Pro Gln Phe Lys 245 250 255 Gly Glu5408PRTClostridium difficile 5Met Ser Glu Lys Lys Glu Ala Arg Val Val Ile Asn Asp Leu Leu Ala 1 5 10 15 Glu Gln Tyr Ala Asn Ala Phe Lys Ala Lys Glu Glu Gly Arg Pro Val 20 25 30 Gly Trp Ser Thr Ser Val Phe Pro Gln Glu Leu Ala Glu Val Phe Asp 35 40 45 Leu Asn Val Leu Tyr Pro Glu Asn Gln Ala Ala Gly Val Ala Ala Lys 50 55 60 Lys Gly Ser Leu Glu Leu Cys Glu Ile Ala Glu Ser Lys Gly Tyr Ser65 70 75 80 Ile Asp Leu Cys Ala Tyr Ala Arg Thr Asn Phe Gly Leu Leu Glu Asn 85 90 95 Gly Gly Cys Glu Ala Leu Asp Met Pro Ala Pro Asp Phe Leu Leu Cys 100 105 110 Cys Asn Asn Ile Cys Asn Gln Val Ile Lys Trp Tyr Glu Asn Ile Ser 115 120 125 Arg Glu Leu Asp Ile Pro Leu Ile Met Ile Asp Thr Thr Phe Asn Asn 130 135 140 Glu Asp Glu Val Thr Gln Ser Arg Ile Asp Tyr Ile Lys Ala Gln Phe145 150 155 160 Glu Glu Ala Ile Lys Gln Leu Glu Ile Ile Ser Gly Lys Lys Phe Asp 165 170 175 Pro Lys Lys Phe Glu Glu Val Met Lys Ile Ser Ala Glu Asn Gly Arg 180 185 190 Leu Trp Lys Tyr Ser Met Ser Leu Pro Ala Asp Ser Ser Pro Ser Pro 195 200 205 Met Asn Gly Phe Asp Leu Phe Thr Tyr Met Ala Val Ile Val Cys Ala 210 215 220 Arg Gly Lys Lys Glu Thr Thr Glu Ala Phe Lys Leu Leu Ile Glu Glu225 230 235 240 Leu Glu Asp Asn Met Lys Thr Gly Lys Ser Ser Phe Arg Gly Glu Glu 245 250 255 Lys Tyr Arg Ile Met Met Glu Gly Ile Pro Cys Trp Pro Tyr Ile Gly 260 265 270 Tyr Lys Met Lys Thr Leu Ala Lys Phe Gly Val Asn Met Thr Gly Ser 275 280 285 Val Tyr Pro His Ala Trp Ala Leu Gln Tyr Glu Val Asn Asp Leu Asp 290 295 300 Gly Met Ala Val Ala Tyr Ser Thr Met Phe Asn Asn Val Asn Leu Asp305 310 315 320 Arg Met Thr Lys Tyr Arg Val Asp Ser Leu Val Glu Gly Lys Cys Asp 325 330 335 Gly Ala Phe Tyr His Met Asn Arg Ser Cys Lys Leu Met Ser Leu Ile 340 345 350 Gln Tyr Glu Met Gln Arg Arg Ala Ala Glu Glu Thr Gly Leu Pro Tyr 355 360 365 Ala Gly Phe Asp Gly Asp Gln Ala Asp Pro Arg Ala Phe Thr Asn Ala 370 375 380 Gln Phe Glu Thr Arg Ile Gln Gly Leu Val Glu Val Met Glu Glu Arg385 390 395 400 Lys Lys Leu Asn Arg Gly Glu Ile 405 6375PRTClostridium difficile 6Met Glu Ala Ile Leu Ser Lys Met Lys Glu Val Val Glu Asn Pro Asn 1 5 10 15 Ala Ala Val Lys Lys Tyr Lys Ser Glu Thr Gly Lys Lys Ala Ile Gly 20 25 30 Cys Phe Pro Val Tyr Cys Pro Glu Glu Ile Ile His Ala Ala Gly Met 35 40 45 Leu Pro Val Gly Ile Trp Gly Gly Gln Thr Glu Leu Asp Leu Ala Lys 50 55 60 Gln Tyr Phe Pro Ala Phe Ala Cys Ser Ile Met Gln Ser Cys Leu Glu65 70 75 80 Tyr Gly Leu Lys Gly Ala Tyr Asp Glu Leu Ser Gly Val Ile Ile Pro 85 90 95 Gly Met Cys Asp Thr Leu Ile Cys Leu Gly Gln Asn Trp Lys Ser Ala 100 105 110 Val Pro His Ile Lys Tyr Ile Ser Leu Val His Pro Gln Asn Arg Lys 115 120 125 Leu Glu Ala Gly Val Lys Tyr Leu Ile Ser Glu Tyr Lys Gly Val Lys 130 135 140 Arg Glu Leu Glu Glu Ile Cys Gly Tyr Glu Ile Glu Glu Ala Lys Ile145 150 155 160 His Glu Ser Ile Glu Val Tyr Asn Glu His Arg Lys Thr Met Arg Asp 165 170 175 Phe Val Glu Val Ala Tyr Lys His Ser Asn Thr Ile Lys Pro Ser Ile 180 185 190 Arg Ser Leu Val Ile Lys Ser Gly Phe Phe Met Arg Lys Glu Glu His 195 200 205 Thr Glu Leu Val Lys Asp Leu Ile Ala Lys Leu Asn Ala Met Pro Glu 210 215 220 Glu Val Cys Ser Gly Lys Lys Val Leu Leu Thr Gly Ile Leu Ala Asp225 230 235 240 Ser Lys Asp Ile Leu Asp Ile Leu Glu Asp Asn Asn Ile Ser Val Val 245 250 255 Ala Asp Asp Leu Ala Gln Glu Thr Arg Gln Phe Arg Thr Asp Val Pro 260 265 270 Ala Gly Asp Asp Ala Leu Glu Arg Leu Ala Arg Gln Trp Ser Asn Ile 275 280 285 Glu Gly Cys Ser Leu Ala Tyr Asp Pro Lys Lys Lys Arg Gly Ser Leu 290 295 300 Ile Val Asp Glu Val Lys Lys Lys Asp Ile Asp Gly Val Ile Phe Cys305 310 315 320 Met Met Lys Phe Cys Asp Pro Glu Glu Tyr Asp Tyr Pro Leu Val Arg 325 330 335 Lys Asp Ile Glu Asp Ser Gly Ile Pro Thr Leu Tyr Val Glu Ile Asp 340 345 350 Gln Gln Thr Gln Asn Asn Glu Gln Ala Arg Thr Arg Ile Gln Thr Phe 355 360 365 Ala Glu Met Met Ser Leu Ala 370 375 7266PRTClostridium difficile 7Met Tyr Thr Met Gly Leu Asp Ile Gly Ser Thr Ala Ser Lys Gly Val 1 5 10 15 Ile Leu Lys Asn Gly Glu Asp Ile Val Ala Ser Glu Thr Ile Ser Ser 20 25 30 Gly Thr Gly Thr Thr Gly Pro Ser Arg Val Leu Glu Lys Leu Tyr Gly 35 40 45 Lys Thr Gly Leu Ala Arg Glu Asp Ile Lys Lys Val Val Val Thr Gly 50 55 60 Tyr Gly Arg Met Asn Tyr Ser Asp Ala Asp Lys Gln Ile Ser Glu Leu65 70 75 80 Ser Cys His Ala Arg Gly Val Asn Phe Ile Ile Pro Glu Thr Arg Thr 85 90 95 Ile Ile Asp Ile Gly Gly Gln Asp Ala Lys Val Leu Lys Leu Asp Asn 100 105 110 Asn Gly Arg Leu Leu Asn Phe Leu Met Asn Asp Lys Cys Ala Ala Gly 115 120 125 Thr Gly Arg Phe Leu Asp Val Met Ala Lys Ile Ile Glu Val Asp Val 130 135 140 Ser Glu Leu Gly Ser Ile Ser Met Asn Ser Gln Asn Glu Val Ser Ile145 150 155 160 Ser Ser Thr Cys Thr Val Phe Ala Glu Ser Glu Val Ile Ser His Leu 165 170 175 Ser Glu Asn Ala Lys Ile Glu Asp Ile Val Ala Gly Ile His Thr Ser 180 185 190 Val Ala Lys Arg Val Ser Ser Leu Val Lys Arg Ile Gly Val Gln Arg 195 200 205 Asn Val Val Met Val Gly Gly Val Ala Arg Asn Ser Gly Ile Val Arg 210 215 220 Ala Met Ala Arg Glu Ile Asn Thr Glu Ile Ile Val Pro Asp Ile Pro225 230 235 240 Gln Leu Thr Gly Ala Leu Gly Ala Ala Leu Tyr Ala Phe Asp Glu Ala 245 250 255 Lys Glu Ser Gln Lys Glu Val Lys Asn Ile 260 265 8477PRTAcidaminococcus fermentans 8Met Pro Lys Thr Val Ser Pro Gly Val Gln Ala Leu Arg Asp Val Val 1 5 10 15 Glu Lys Val Tyr Arg Glu Leu Arg Glu Ala Lys Glu Arg Gly Glu Lys 20 25 30 Val Gly Trp Ser Ser Ser Lys Phe Pro Cys Glu Leu Ala Glu Ser Phe 35 40 45 Gly Leu His Val Gly Tyr Pro Glu Asn Gln Ala Ala Gly Ile Ala Ala 50 55 60 Asn Arg Asp Gly Glu Val Met Cys Gln Ala Ala Glu Asp Ile Gly Tyr65 70 75 80 Asp Asn Asp Ile Cys Gly Tyr Ala Arg Ile Ser Leu Ala Tyr Ala Ala 85 90 95 Gly Phe Arg Gly Ala Asn Lys Met Asp Lys Asp Gly Asn Tyr Val Ile 100 105 110 Asn Pro His Ser Gly Lys Gln Met Lys Asp Ala Asn Gly Lys Lys Val 115 120 125 Phe Asp Ala Asp Gly Lys Pro Val Ile Asp Pro Lys Thr Leu Lys Pro 130 135 140 Phe Ala Thr Thr Asp Asn Ile Tyr Glu Ile Ala Ala Leu Pro Glu Gly145 150 155 160 Glu Glu Lys Thr Arg Arg Gln Asn Ala Leu His Lys Tyr Arg Gln Met 165 170 175 Thr Met Pro Met Pro Asp Phe Val Leu Cys Cys Asn Asn Ile Cys Asn 180 185 190 Cys Met Thr Lys Trp Tyr Glu Asp Ile Ala Arg Arg His Asn Ile Pro 195 200 205 Leu Ile Met Ile Asp Val Pro Tyr Asn Glu Phe Asp His Val Asn Glu 210 215 220 Ala Asn Val Lys Tyr Ile Arg Ser Gln Leu Asp Thr Ala Ile Arg Gln225 230 235 240 Met Glu Glu Ile Thr Gly Lys Lys Phe Asp Glu Asp Lys Phe Glu Gln 245 250 255 Cys Cys Gln Asn Ala Asn Arg Thr Ala Lys Ala Trp Leu Lys Val Cys 260 265 270 Asp Tyr Leu Gln Tyr Lys Pro Ala Pro Phe Asn Gly Phe Asp Leu Phe 275 280 285 Asn His Met Ala Asp Val Val Thr Ala Arg Gly Arg Val Glu Ala Ala 290 295 300 Glu Ala Phe Glu Leu Leu Ala Lys Glu Leu Glu Gln His Val Lys Glu305 310 315 320 Gly Thr Thr Thr Ala Pro Phe Lys Glu Gln His Arg Ile Met Phe Glu 325 330 335 Gly Ile Pro Cys Trp Pro Lys Leu Pro Asn Leu Phe Lys Pro Leu Lys 340 345 350 Ala Asn Gly Leu Asn Ile Thr Gly Val Val Tyr Ala Pro Ala Phe Gly 355 360 365 Phe Val Tyr Asn Asn Leu Asp Glu Leu Val Lys Ala Tyr Cys Lys Ala 370 375 380 Pro Asn Ser Val Ser Ile Glu Gln Gly Val Ala Trp Arg Glu Gly Leu385 390 395 400 Ile Arg Asp Asn Lys Val Asp Gly Val Leu Val His Tyr Asn Arg Ser 405 410 415 Cys Lys Pro Trp Ser Gly Tyr Met Pro Glu Met Gln Arg Arg Phe Thr 420 425 430 Lys Asp Met Gly Ile Pro Thr Ala Gly Phe Asp Gly Asp Gln Ala Asp 435 440 445 Pro Arg Asn Phe Asn Ala Ala Gln Tyr Glu Thr Arg Val Gln Gly Leu 450 455 460 Val Glu Ala Met Glu Ala Asn Asp Glu Lys Lys Gly Lys465 470 475 9379PRTAcidaminococcus fermentans 9Met Ala Ile Ser Ala Leu Ile Glu Glu Phe Gln Lys Val Ser Ala Ser 1 5 10 15 Pro Lys Thr Met Leu Ala Lys Tyr Lys Ala Gln Gly Lys Lys Ala Ile 20 25 30 Gly Cys Leu Pro Tyr Tyr Val Pro Glu Glu Leu Val Tyr Ala Ala Gly 35 40 45

Met Val Pro Met Gly Val Trp Gly Cys Asn Gly Lys Gln Glu Val Arg 50 55 60 Ser Lys Glu Tyr Cys Ala Ser Phe Tyr Cys Thr Ile Ala Gln Gln Ser65 70 75 80 Leu Glu Met Leu Leu Asp Gly Thr Leu Asp Gly Leu Asp Gly Ile Ile 85 90 95 Thr Pro Val Leu Cys Asp Thr Leu Arg Pro Met Ser Gln Asn Phe Lys 100 105 110 Val Ala Met Lys Asp Lys Met Pro Val Ile Phe Leu Ala His Pro Gln 115 120 125 Val Arg Gln Asn Ala Ala Gly Lys Gln Phe Thr Tyr Asp Ala Tyr Ser 130 135 140 Glu Val Lys Gly His Leu Glu Glu Ile Cys Gly His Glu Ile Thr Asn145 150 155 160 Asp Ala Ile Leu Asp Ala Ile Lys Val Tyr Asn Lys Ser Arg Ala Ala 165 170 175 Arg Arg Glu Phe Cys Lys Leu Ala Asn Glu His Pro Asp Leu Ile Pro 180 185 190 Ala Ser Val Arg Ala Thr Val Leu Arg Ala Ala Tyr Phe Met Leu Lys 195 200 205 Asp Glu Tyr Thr Glu Lys Leu Glu Glu Leu Asn Lys Glu Leu Ala Ala 210 215 220 Ala Pro Ala Gly Lys Phe Asp Gly His Lys Val Val Val Ser Gly Ile225 230 235 240 Ile Tyr Asn Met Pro Gly Ile Leu Lys Ala Met Asp Asp Asn Lys Leu 245 250 255 Ala Ile Ala Ala Asp Asp Cys Ala Tyr Glu Ser Arg Ser Phe Ala Val 260 265 270 Asp Ala Pro Glu Asp Leu Asp Asn Gly Leu Gln Ala Leu Ala Val Gln 275 280 285 Phe Ser Lys Gln Lys Asn Asp Val Leu Leu Tyr Asp Pro Glu Phe Ala 290 295 300 Lys Asn Thr Arg Ser Glu His Val Cys Asn Leu Val Lys Glu Ser Gly305 310 315 320 Ala Glu Gly Leu Ile Val Phe Met Met Gln Phe Cys Asp Pro Glu Glu 325 330 335 Met Glu Tyr Pro Asp Leu Lys Lys Ala Leu Asp Ala His His Ile Pro 340 345 350 His Val Lys Ile Gly Val Asp Gln Met Thr Arg Asp Phe Gly Gln Ala 355 360 365 Gln Thr Ala Leu Glu Ala Phe Ala Glu Ser Leu 370 375 10260PRTAcidaminococcus fermentans 10Met Ser Ile Tyr Thr Leu Gly Ile Asp Val Gly Ser Thr Ala Ser Lys 1 5 10 15 Cys Ile Ile Leu Lys Asp Gly Lys Glu Ile Val Ala Lys Ser Leu Val 20 25 30 Ala Val Gly Thr Gly Thr Ser Gly Pro Ala Arg Ser Ile Ser Glu Val 35 40 45 Leu Glu Asn Ala His Met Lys Lys Glu Asp Met Ala Phe Thr Leu Ala 50 55 60 Thr Gly Tyr Gly Arg Asn Ser Leu Glu Gly Ile Ala Asp Lys Gln Met65 70 75 80 Ser Glu Leu Ser Cys His Ala Met Gly Ala Ser Phe Ile Trp Pro Asn 85 90 95 Val His Thr Val Ile Asp Ile Gly Gly Gln Asp Val Lys Val Ile His 100 105 110 Val Glu Asn Gly Thr Met Thr Asn Phe Gln Met Asn Asp Lys Cys Ala 115 120 125 Ala Gly Thr Gly Arg Phe Leu Asp Val Met Ala Asn Ile Leu Glu Val 130 135 140 Lys Val Ser Asp Leu Ala Glu Leu Gly Ala Lys Ser Thr Lys Arg Val145 150 155 160 Ala Ile Ser Ser Thr Cys Thr Val Phe Ala Glu Ser Glu Val Ile Ser 165 170 175 Gln Leu Ser Lys Gly Thr Asp Lys Ile Asp Ile Ile Ala Gly Ile His 180 185 190 Arg Ser Val Ala Ser Arg Val Ile Gly Leu Ala Asn Arg Val Gly Ile 195 200 205 Val Lys Asp Val Val Met Thr Gly Gly Val Ala Gln Asn Tyr Gly Val 210 215 220 Arg Gly Ala Leu Glu Glu Gly Leu Gly Val Glu Ile Lys Thr Ser Pro225 230 235 240 Leu Ala Gln Tyr Asn Gly Ala Leu Gly Ala Ala Leu Tyr Ala Tyr Lys 245 250 255 Lys Ala Ala Lys 260 11387PRTPseudomonas aeruginosa 11Met Thr Tyr Pro Ser Leu Asn Phe Ala Leu Gly Glu Thr Ile Asp Met 1 5 10 15 Leu Arg Asp Gln Val Arg Gly Phe Val Ala Ala Glu Leu Gln Pro Arg 20 25 30 Ala Ala Gln Ile Asp Gln Asp Asn Gln Phe Pro Met Asp Met Trp Arg 35 40 45 Lys Phe Gly Glu Met Gly Leu Leu Gly Ile Thr Val Asp Glu Glu Tyr 50 55 60 Gly Gly Ser Ala Leu Gly Tyr Leu Ala His Ala Val Val Met Glu Glu65 70 75 80 Ile Ser Arg Ala Ser Ala Ser Val Ala Leu Ser Tyr Gly Ala His Ser 85 90 95 Asn Leu Cys Val Asn Gln Ile Lys Arg Asn Gly Asn Ala Glu Gln Lys 100 105 110 Ala Arg Tyr Leu Pro Ala Leu Val Ser Gly Glu His Ile Gly Ala Leu 115 120 125 Ala Met Ser Glu Pro Asn Ala Gly Ser Asp Val Val Ser Met Lys Leu 130 135 140 Arg Ala Asp Arg Val Gly Asp Arg Phe Val Leu Asn Gly Ser Lys Met145 150 155 160 Trp Ile Thr Asn Gly Pro Asp Ala His Thr Tyr Val Ile Tyr Ala Lys 165 170 175 Thr Asp Ala Asp Lys Gly Ala His Gly Ile Thr Ala Phe Ile Val Glu 180 185 190 Arg Asp Trp Lys Gly Phe Ser Arg Gly Pro Lys Leu Asp Lys Leu Gly 195 200 205 Met Arg Gly Ser Asn Thr Cys Glu Leu Ile Phe Gln Asp Val Glu Val 210 215 220 Pro Glu Glu Asn Val Leu Gly Ala Val Asn Gly Gly Val Lys Val Leu225 230 235 240 Met Ser Gly Leu Asp Tyr Glu Arg Val Val Leu Ser Gly Gly Pro Val 245 250 255 Gly Ile Met Gln Ala Cys Met Asp Val Val Val Pro Tyr Ile His Asp 260 265 270 Arg Arg Gln Phe Gly Gln Ser Ile Gly Glu Phe Gln Leu Val Gln Gly 275 280 285 Lys Val Ala Asp Met Tyr Thr Ala Leu Asn Ala Ser Arg Ala Tyr Leu 290 295 300 Tyr Ala Val Ala Ala Ala Cys Asp Arg Gly Glu Thr Thr Arg Lys Asp305 310 315 320 Ala Ala Gly Val Ile Leu Tyr Ser Ala Glu Arg Ala Thr Gln Met Ala 325 330 335 Leu Asp Ala Ile Gln Ile Leu Gly Gly Asn Gly Tyr Ile Asn Glu Phe 340 345 350 Pro Thr Gly Arg Leu Leu Arg Asp Ala Lys Leu Tyr Glu Ile Gly Ala 355 360 365 Gly Thr Ser Glu Ile Arg Arg Met Leu Ile Gly Arg Glu Leu Phe Asn 370 375 380 Glu Thr Arg385 12386PRTStreptomyces avermitilis 12Met Asp His Arg Leu Thr Pro Glu Leu Glu Glu Leu Arg Arg Thr Val 1 5 10 15 Glu Glu Phe Ala His Asp Val Val Ala Pro Lys Ile Gly Asp Phe Tyr 20 25 30 Glu Arg His Glu Phe Pro Tyr Glu Ile Val Arg Glu Met Gly Arg Met 35 40 45 Gly Leu Phe Gly Leu Pro Phe Pro Glu Glu Tyr Gly Gly Met Gly Gly 50 55 60 Asp Tyr Leu Ala Leu Gly Ile Ala Leu Glu Glu Leu Ala Arg Val Asp65 70 75 80 Ser Ser Val Ala Ile Thr Leu Glu Ala Gly Val Ser Leu Gly Ala Met 85 90 95 Pro Ile His Leu Phe Gly Thr Asp Ala Gln Lys Ala Glu Trp Leu Pro 100 105 110 Arg Leu Cys Ser Gly Glu Ile Leu Gly Ala Phe Gly Leu Thr Glu Pro 115 120 125 Asp Gly Gly Ser Asp Ala Gly Ala Thr Arg Thr Thr Ala Arg Leu Asp 130 135 140 Glu Ser Thr Asn Glu Trp Val Ile Asn Gly Thr Lys Cys Phe Ile Thr145 150 155 160 Asn Ser Gly Thr Asp Ile Thr Gly Leu Val Thr Val Thr Ala Val Thr 165 170 175 Gly Arg Lys Pro Asp Gly Lys Pro Leu Ile Ser Ser Ile Ile Val Pro 180 185 190 Ser Gly Thr Pro Gly Phe Thr Val Ala Ala Pro Tyr Ser Lys Val Gly 195 200 205 Trp Asn Ala Ser Asp Thr Arg Glu Leu Ser Phe Ala Asp Val Arg Val 210 215 220 Pro Ala Ala Asn Leu Leu Gly Glu Gln Gly Arg Gly Tyr Ala Gln Phe225 230 235 240 Leu Arg Ile Leu Asp Glu Gly Arg Ile Ala Ile Ser Ala Leu Ala Thr 245 250 255 Gly Leu Ala Gln Gly Cys Val Asp Glu Ser Val Lys Tyr Ala Gly Glu 260 265 270 Arg His Ala Phe Gly Arg Asn Ile Gly Ala Tyr Gln Ala Ile Gln Phe 275 280 285 Lys Ile Ala Asp Met Glu Met Lys Ala His Met Ala Arg Val Gly Trp 290 295 300 Arg Asp Ala Ala Ser Arg Leu Val Ala Gly Glu Pro Phe Lys Lys Glu305 310 315 320 Ala Ala Ile Ala Lys Leu Tyr Ser Ser Thr Val Ala Val Asp Asn Ala 325 330 335 Arg Glu Ala Thr Gln Ile His Gly Gly Tyr Gly Phe Met Asn Glu Tyr 340 345 350 Pro Val Ala Arg Met Trp Arg Asp Ser Lys Ile Leu Glu Ile Gly Glu 355 360 365 Gly Thr Ser Glu Val Gln Arg Met Leu Ile Ala Arg Glu Leu Gly Leu 370 375 380 Val Gly385

Claims

1-25. (canceled)

26: A recombinant host or microorganism comprising at least one exogenous nucleic acid encoding a 2-hydroxyacyl-CoA dehydratase or isovaleryl-CoA/acyl-CoA dehydrogenase and an enoyl-CoA hydratase, said host or microorganism producing isobutene.

27: The host or microorganism of claim 26 further comprising a mevalonate diphosphate decarboxylase.

28: The host or microorganism of claim 26 further comprising a (i) a 2-hydroxy-acyl dehydrogenase, (ii) a CoA transferase or a CoA-ligase, and (iii) a thioesterase.

29-30. (canceled)

31: The host or microorganism of claim 26 further comprising a 4-methyl-2-oxo-pentanoate dehydrogenase complex and a thioesterase.

32: The host or microorganism of claim 26 further comprising an indolepyruvate decarboxylase, a phenylacetaldehyde dehydrogenase, and a CoA-ligase.

33: The host or microorganism of claim 26, wherein said enoyl-CoA hydratase is a (R)-specific enoyl-CoA hydratase having at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:1-3.

34: The host or microorganism of claim 26, wherein said enoyl-CoA hydratase is a (S)-specific enoyl-CoA hydratase having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

35: The host or microorganism of claim 26, wherein said host naturally accumulates polyhydroxyalkanoates and comprises attenuated polymer synthase enzymes.

36: The host or microorganism of claim 26 further comprising one or more of the following attenuated enzymes: a phosphotransacetylase, an acetate kinase, an enzyme that degrades pyruvate to lactate, an enzyme that degrades phophoenolpyruvate to succinate, an enzyme degrading acetyl-CoA to ethanol, or the final branch chain amino acid transaminase.

37: The host or microorganism of claim 26, wherein said host or microorganism expresses one or more of the following genes encoding: a puridine nucleotide transhydrogenase, a glyceraldehyde-3P-dehydrogenase, a malic enzyme, a glucose-6-phosphate dehydrogenase, a fructose 1,6 diphosphatase, or a feedback inhibition resistant mutant of an acetolactate synthase, wherein the acetolactate synthase is resistant to feedback inhibition by branch chain amino acids.

38: The host or microorganism of claim 37, wherein the gene encoding the acetolactate synthase is expressed using a promoter not subject to genetic repression by branch-chain amino acids.

39: The host or microorganism of claim 26, wherein the efflux of isobutene across the cell membrane is enhanced or amplified by genetic engineering of the cell membrane or increases to an associated transporter activity for isobutene.

40: The recombinant host or microorganism of claim 26 which is a prokaryotic host or microorganism from the genus Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia acidovorans, Bacillus, Lactobacillus, Lactococcus or Rhodococcus.

41: The recombinant host or microorganism of claim 26 which is a eukaryotic host or microorganism from the genus Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula or Kluyveromyces.