# Patent

## Title
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.

## Background
This application claims priority of U.S. Provisional Application Ser. No. 61/730,549, filed Nov. 28, 2012. The contents of the prior application is incorporated herein by reference in its entirety.


TECHNICAL FIELD
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
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).
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.
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.
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.
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).
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  ̃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
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.
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.
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.
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.
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 subtillis; 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 Issathenkia 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. 
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, CO2/H2, methanol, ethanol, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes.
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.
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.
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.
In any of the recombinant hosts, the host can naturally accumulate polyhydroxyalkanoates and comprise attenuated polymer synthase enzymes.
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.
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.
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.
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.
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
FIG. 1 is a schematic of exemplary biochemical pathway leading to isobutene using 3-methyl-2-oxobutanoate as a central precursor.
FIG. 2 is a schematic of exemplary biochemical pathways leading to isobutene using 4-methyl-2-oxopentanoate as a central precursor.
FIG. 3 contains the amino acid sequence of an (R)-specific enoyl-CoA hydratase from Pseudomonas aeruginosa (encoded by the PhaJ1 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 Accession No. CAA99573.1, SEQ ID NO:4).
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).
FIG. 5 contains the amino acid sequence of a Pseudomonas aeruginosa PAUL 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).
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.
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.
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
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.
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.
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 Pha II 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.
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.
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).
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.
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 (B12seq) 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 (worldwide web address fr.com/blast/) or the U.S. government's National Center for Biotechnology Information worldwide web address ncbi.nlm.nih.gov). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of B12seq 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:\B12seq-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.
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.
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.
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.
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.
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), hemagluttanin (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.
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.
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.
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.
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.
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.
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).
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).
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.
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.
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).
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.
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 phca 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.
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.
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.
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 subtillis; 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. 
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 Issathenkia 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. 
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.
In some embodiments, isobutene is biosynthesized in a recombinant host under nutrient limiting conditions such as nitrogen, phosphate or oxygen limitation.
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.
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.
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.
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).
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).
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; Pérez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).
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).
The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other argricultural 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).
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).
In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO2/H2, methanol, ethanol, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes.
The efficient catabolism of methanol has been demonstrated for the methylotropic yeast Pichia pastoris. 
The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).
The efficient catabolism of CO2 and H2, 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).
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).
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).
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
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.
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.
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.
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.
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.
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.
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.
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.
Attenuation strategies include, but are not limited to, the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.
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.
In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the polymer synthase enzymes can be attenuated in the host strain.
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).
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).
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).
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).
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).
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).
In some embodiments, where pathways require excess NADPH co-factor in the synthesis of isobutene, a malic enzyme gene such as macA or maeB can be overexpressed in the host organism (Brigham et al., 2012, supra).
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).
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).
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.
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).
In some embodiments, the branch chain amino acid transaminase encoded by ilvE is attenuated.
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
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, 2nd 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.
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° C. At a typical fermentation temperature of approximately 30° 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
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° 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.
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 μm filter. The phaJ enzyme was purified from the supernatant using Ni-affinity chromatography and concentrated to 1.25 mg/mL.
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° C. The enzyme activity assay reaction was initiated by adding 0.4 μM 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° C. The substrate only control showed minimal spontaneous hydration of crotonyl-CoA as determined by spectrophotometry at 263 nm. See FIG. 6.
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 μM of purified enoyl-CoA hydratase to the assay buffer containing the substrate and incubated at 30° 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.
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 μM of purified enoyl-CoA hydratase to the assay buffer containing the substrate and incubated at 30° 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.
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
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.

## Claims

1. A method for synthesizing isobutene, said method comprising:
(a) culturing a recombinant host cell genetically engineered to synthesize isobutene via said method in a culture medium in the presence of a fermentable carbon source, said recombinant host cell comprising exogenous nucleic acids encoding polypeptides having the activities of:
(i) a 2-hydroxyacyl-CoA dehydratase, wherein said polypeptide has 2-hydroxyacyl-CoA dehydratase activity and comprises the amino acid sequences selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9;
(ii) an (R)-specific enoyl-CoA hydratase, wherein said polypeptide has (R)-specific enoyl-CoA hydratase activity and comprises the amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;
(iii) a thioesterase classified under 3.1.2.-; and
(iv) a mevalonate diphosphate decarboxylase classified under 4.1.1.33;
(b) enzymatically dehydrating 3-methyl-2-hydroxy-butanoyl-CoA to form 3-methyl-but-2-enoyl-CoA using said polypeptide having the 2-hydroxyacyl-CoA dehydratase activity in said host cell;
(c) enzymatically hydrating the 3-methyl-but-2-enoyl-CoA to form 3-methyl-3-hydroxy butanoyl-CoA using said polypeptide having the (R)-specific enoyl-CoA hydratase activity in said host cell;
(d) enzymatically converting 3-methyl-3-hydroxy butanoyl-CoA to 3-methyl-3-hydroxybutanoate using said polypeptide having the thioesterase activity in said host cell; and
(e) enzymatically converting 3-methyl-3-hydroxybutanoate to isobutene using said polypeptide having the mevalonate diphosphate decarboxylase activity in said host cell.

2. The method of claim 1, wherein said 2-hydroxyacyl-CoA dehydratase is the gene products:
activator of 2-hydroxyisocaproyl-CoA dehydratase (HadI) and 2-hydroxyisocaproyl-CoA dehydratase (HadBC); or
activator of (R)-hydroxyglutaryl-CoA dehydratase B (HgdC) and 2-hydoxyglutaryl-CoA dehydratase B (hgdB) protein precursor (HgdAB).

3. The method of claim 1, where the host is a prokaryote.

4. The method according to claim 3, where the prokaryotic host is from the genus selected from the group consisting of Escherichia; Clostridia; Corynebacteria; Cupriavidus; Pseudomonas; Delftia acidovorans, Bacillus; Lactobacillus; Lactococcus and Rhodococcus.

5. The method according to claim 1, where the fermentable carbon source is a feedstock selected from cellulose, lignin, levulinic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles and municipal waste.

6. The method according to claim 1, where the fermentable carbon source is a feedstock selected from natural gas, syngas, CO2/H2, methanol and ethanol.

7. The method of claim 1, wherein the host is a eukaryote.

8. The method according to claim 7, where the eukaryotic host is from the genus selected from the group consisting of Aspergillus; Saccharomyces; Pichia; Yarrowia; Issatchenkia; Debaryomyces; Arxula and Kluyveromyces.

9. The method according to claim 1, where the fermentable carbon source is a feedstock selected from monosaccharides, disaccharides, lignocellulose, hemicellulose and formic acid.