US 2015 0037860A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0037860 A1 B0tes et al. (43) Pub. Date: Feb. 5, 2015
(54) METHODS FOR BOSYNTHESIS OF Publication Classification ISOPRENE (51) Int. C. (71) Applicant: INVISTA North America S.ár.l., CI2P 5/00 (2006.01) Wilmington, DE (US) CI2N 15/70 (2006.01) (52) U.S. C. (72) Inventors: Adriana Leonora Botes, Rosedale East CPC ...... CI2P5/007 (2013.01); C12N 15/70 (GB); Alex Van Eck Conradie, (2013.01) Eaglescliffe (GB) USPC ...... 435/167; 435/252.33: 435/252.3: 435/252.32; 435/252.34; 435/252.31; (21) Appl. No.: 14/452,201 435/254.3; 435/254.21: 435/254.23; 435/254.2: 435/254.11: 435/254.22 (22) Filed: Aug. 5, 2014 (57) ABSTRACT This document describes biochemical pathways for produc ing isoprene by forming two vinyl groups in a central precur Related U.S. Application Data sor produced from isobutyryl-CoA, 3-methyl-2-oxopen (60) Provisional application No. 61/862,401, filed on Aug. tanoate, or 4-methyl-2-oxopentanoate as well as recombinant 5, 2013. hosts for producing isoprene. Patent Application Publication Feb. 5, 2015 Sheet 1 of 19 US 2015/0037860 A1
FIGURE 1.
O O CoA Acetyl-CoA --sca {YN 1 -sc.AO Acetoacetyl-CoA EC 2.3.1.9 Acetyl-CoA Acetyl-CoA t HO -->Cs.O OH Q CoA 3-Hydroxy 3-methylglutaryl-CoA gy 2.H a. CoA Co a 2NAD(P)* Jyu HO OH (R)-mevalonate t ATP c N N e d ADP O HO o–F–o OH (R)-5-phosphomevalonate t ATP c N s na No ADP JyuO HO O-P-O-P-OH OH OH (R)-5-Diphosphomevalonate t ATP c a
g ADP PP CO2 N-1 -- P-O-P-OH HEos3.32 -- . . . EC 42.327 ) Nr. OH, OH Dimethylallyl diphosphate isoprene Isopentenyl diphosphate
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FIGURE 8
O O CoA Acetyl-CA ulus YN-1 S-CoA { S-CoA Acetoacetyl-CoA EC 2.3.1.9 acetyl-CoA T Acetyl-CoA S HO go s s - O X-sc.OH O CoA 3-Hydroxy 3-methylglutaryl-CoA TT 2:NAD(P)H QQ 2.H ADP a a + CO s CoA -- ATP + Pi 2NAD(P) HO N-17 Yu N-Y HQ, EC 2.7.1- a- ) HO S H) 21 HO OH EC 4.2.1- EC 4.1.1.33 (R)-mevalonate 3-methyl-pent-4-enoate1N3-hydroxy- isoprene ATP ADP + CO + Pi Patent Appl icat ion Publica ion Feb. 5, 2015 Sheet 9 of 19 US 2015/0037860 A1
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METHODS FOR BOSYNTHESIS OF a theoretical maximum yield of 25.2% (w/w) for the meva ISOPRENE lonate pathway, isoprene has been produced biocatalytically at a volumetric productivity of 2 g/(Lh) with a yield of 11% CROSS-REFERENCE TO RELATED (w/w) from glucose (Whited et al., 2010, supra). Particularly, APPLICATIONS the phosphate activation of mevalonate to 5-diphosphomeva lonate is energy intensive metabolically, requiring two moles 0001. This application claims priority to U.S. Application of ATP per mole of isoprene synthesis (FIG. 1). Accordingly, Ser. No. 61/862,401, filed Aug. 5, 2013, the disclosure of reducing the ATP consumption can improve the efficiency of which is incorporated by reference in its entirety. the pathway. TECHNICAL FIELD SUMMARY 0002 This invention relates to methods for biosynthesiz ing isoprene using one or more isolated enzymes Such as one 0012. The inventors have determined that it is possible to or more of a dehydratase, a monooxygenase, a cytochrome construct a biochemical pathway to synthesize isoprene from P450, an acyl-acp dehydrogenase, a mevalonate diphos (R)-mevalonate, 3-methyl-2-oxopentanoate, 4-methyl-2- phate decarboxylase, an acyl-acp decarboxylating oXopentanoate or isobutyryl-CoA, by introducing two vinyl groups without the need for terminal alcohol phosphoryla thioesterase, and a mevalonate-3-kinase; or using recombi tion. Such pathways rely on a dehydratase, monooxygenase, nant host cells expressing one or more Such enzymes. cytochrome P450, or dehydrogenase enzyme to introduce the BACKGROUND first vinyl group; and a MDD, mevalonate-3-kinase, acyl acp decarboxylating thioesterase (e.g., CurMTE) or a lina 0003) Isoprene is an important monomer for the produc lool dehydratase to introduce the second vinyl group into the tion of specialty elastomers including motor mounts/fittings, precursors leading to isoprene synthesis. The methods Surgical gloves, rubber bands, golfballs and shoes. Styrene described herein can include introducing the first vinyl group, isoprene-styrene block copolymers form a key component of introducing the second vinyl group, or introducing both the hot-melt pressure-sensitive adhesive formulations and cis first and second vinyl groups. poly-isoprene is utilised in the manufacture oftires (Whitedet 0013 Prior to the present invention, it was not known that al., Industrial Biotechnology, 2010, 6(3), 152-163). enzymes capable of introducing two vinyl groups, without the 0004. Manufacturers of rubber goods depend on either need for terminal alcohol phosphorylation, could be used to imported natural rubber from the Brazilian rubber tree or generate non-phosphorylated intermediates for the synthesis petroleum-based synthetic rubber polymers (Whited et al., of isoprene. Thus the invention provides enzymes that can 2010, supra). convert the central precursors mevalonate, 3-methyl-2-oxo 0005 Given a reliance on petrochemical feedstocks and pentanoate, 4-methyl-2-oxopentanoate or isobutyryl-CoA the harvesting of trees, biotechnology offers an alternative into isoprene. approach viabiocatalysis. Biocatalysis is the use of biological 0014. In some embodiments, 3-methyl-pent-2-enoyl-CoA catalysts, such as enzymes, to perform biochemical transfor or 4-methyl-pent-2-enoyl-CoA is formed by a 2-hydroxya mations of organic compounds. cyl-CoA dehydratase classified, for example, under EC 4.2. 0006. Accordingly, against this background, it is clear that 1.-, such as the gene products of HadBC (SEQID NOs: 3 and there is a need for Sustainable methods for producing inter 4) and its initiator Had I (SEQID NO: 2), or the gene products mediates, in particular isoprene, wherein the methods are of HgdAB (SEQ ID NOs: 6 and 7) and its initiator HagC biocatalysis based. (SEQID NO. 5). In some embodiments, the 2-hydroxyacyl 0007 Both bioderived feedstocks and petrochemical feed CoA dehydratase is the result of enzyme engineering. The stocks are viable starting materials for the biocatalysis pro 2-hydroxyacyl-CoA dehydratase enzymes isolated from CCSSCS. anaerobic bacteria possess a common catalytic mechanism 0008. The introduction of vinyl groups into medium car employed in amino acid degradation pathways. For example, bon chain length enzyme Substrates is a key consideration in the gene products of HadBC/HadI from Clostridium difficile synthesising isoprene via biocatalysis processes. catalyse the conversion of (R)-2-hydroxyisocaproyl-CoA to 0009. There are known metabolic pathways leading to the isocaprenoyl-CoA. Similarly, the gene products of HgdAB/ synthesis ofisoprene in prokaryotes such as Bacillis Subtillis HdgC catalyse the conversion of 2-hydroxyglutaryl-CoA to and eukaryotes such as Populus alba (Whited et al., 2010, glutaconyl-CoA (Kim et al., FEMS Microbiol. Reviews, Supra). 2004, 28,455-468). See FIGS. 2-5. 0010) Isoprene may be synthesized via two routes leading 0015. In some embodiments, the first vinyl group is intro to the precursor dimethylvinyl-PP, such as the mevalonate duced into 3-methyl-pent-2-enoyl-ACP, derived from the and the non-mevalonate pathway (Kuzuyama, Biosci. Bio central metabolite 3-methyl-2-oxopentanoate, which may be technol. Biochem., 2002, 66(8), 1619-1627). The mevalonate enzymatically converted in one or more steps to 3-methyl-3- pathway incorporates a decarboxylase enzyme, mevalonate hydroxypent-4-enoate or 3-methyl-3-sulphoryl-pent-4- diphosphate decarboxylase (hereafter MDD), that introduces enoyl-ACP (as shown, for example, in FIG. 2). It has been the first vinyl-group into the precursors leading to isoprene. demonstrated that the gene product of tcs D (SEQID NO: 14) The second vinyl-group is introduced by isoprene synthase from Streptomyces kanamyceticus has dehydrogenase activ (hereafter ISPS) in the final step in synthesizing isoprene. ity for straight and branch chain. C5acyl-ACP substrates (Mo 0011. The mevalonate pathway (FIG. 1) has been et al., JACS, 2011, 133,976-985). exploited in the biocatalytic production of isoprene using E. 0016. In some embodiments, the first vinyl group is intro coli as host. E. coli engineered with the mevalonate pathway duced forming 4-methyl-pent-2-enoyl-ACP derived from the requires three moles of acetyl-CoA, three moles of ATP and central metabolite 4-methyl-2-oxopentanoate or isobutyryl two moles of NAD(P)H to produce a mole ofisoprene. Given CoA, which may be enzymatically converted in one or more US 2015/0037860 A1 Feb. 5, 2015
steps to 4-methyl-3-hydroxypent-4-enoate, 4-methyl-3-sul 0024. In some embodiments, the second vinyl group is phoryl-pent-4-enoyl-ACP, or 3-methyl-3-buten-2-ol (see, for introduced by a linalool dehydratase classified, for example, example, FIG. 3, FIG. 6, and FIG. 7). It has been demon under EC 4.2.1.127 (Brodkorb et al., J. Biol. Chem., 2010, strated that the gene product of tcs) (SEQID NO: 14) from 285(40), 30436-30442) or a dehydratase classified under EC Streptomyces kanamyceticus has dehydrogenase activity for 4.2.1.- (Such as one isolated from species Such as Aquincola 4-methyl-pent-2-enoyl-ACP (Mo et al., 2011, supra). tertiaricarbonis or Methylibium petroleiphilum PM1; Schä 0017. In some embodiments, the first vinyl group is intro fer et al., 2011, supra) (FIG.3 and FIG. 7). duced into 3-methyl-3-hydroxy-pentanoate, which may be 0025. In one aspect, this document features a method for enzymatically converted in one or more steps to 3-methyl-3- enzymatically synthesizing isoprene. The method includes hydroxypent-4-enoate (see, for example, FIG. 4). It has been enzymatically introducing a terminal vinyl group into 3-me demonstrated that the monooxygenase encoded by mdp thyl-pent-2-enoyl-acp. 4-methyl-pent-2-enoyl-acp.3-me (SEQID NO: 15) introduces a terminal double bond into allyl thyl-3-hydroxy-pentanoate, 4-methyl-3-hydroxypentanoate, groups bound to a secondary alcohol (Schäfer et al., Appl. or mevalonate, and converting the resulting product in one or Environ. Microbiol., 2012, 78(17), 6280-6284). more steps to isoprene. The first vinyl group can be intro 0018. In some embodiments, the first vinyl group is intro duced using a dehydratase classified under EC 4.2.1.- (e.g., a duced into 4-methyl-3-hydroxypentanoate, which may be dehydratase having at least 70% homology to the amino acid enzymatically converted in one or more steps to 4-methyl-3- sequence set forth in SEQ ID NO: 22), a monooxygenase hydroxypent-4-enoate (see, for example, FIG. 5). (e.g., a monooxygenase having at least 70% homology to the 0019. In some embodiments, the first vinyl group is intro amino acid sequence set forth in SEQ ID NO: 15), a cyto duced into mevalonate, which can be converted enzymati chrome P450 reductase, oran acyl-acp dehydrogenase (e.g., cally in one or more steps to 3-hydroxy-3-methyl-pent-4- an acyl-acp dehydrogenase having at least 70% homology enoate (as shown, for example, in FIG. 8). to the amino acid sequence set forth in SEQID NO:14). For example, a terminal vinyl group can be introduced into 3-me 0020. In some embodiments, the 3-hydroxy functional thyl-pent-2-enoyl-acp or 4-methyl-pent-2-enoyl-acp group is introduced into 3-methyl-pent-2-enoyl-CoA or using an acyl-acp dehydrogenase. For example, a terminal 4-methyl-pent-2-enoyl-CoA by a (R)-specific enoyl-CoA vinyl group can be introduced into mevalonate using a dehy hydratase enzyme classified, for example, under EC 4.2.1. dratase classified under EC 4.2.1.—. For example, a terminal 119 such as the gene product of phal (SEQID NO: 16, Fukui vinyl group can be introduced into 3-methyl-3-hydroxy-pen et al., J. Bacteriol., 1998, 180(3), 667-673) or MaoC (SEQID tanoate or 4-methyl-3-hydroxypentanoate using a monooxy NO: 17: Park and Lee, J. Bacteriol., 2003, 185(18), 5291 genase or a cytochrome P450 reductase. 3-methyl-pent-2- 5397) or a bacterial (S)-specific enoyl-CoA hydratase classi enoyl-acp. 4-methyl-pent-2-enoyl-acp. 3-methyl-3- fied, for example, under EC 4.2.1.17 such as the gene product hydroxy-pentanoate, or 4-methyl-3-hydroxypentanoate, can of YsiB (SEQID NO: 1). In some embodiments, the enoyl be enzymatically formed from 3-methyl-2-oxopentanoate, CoA hydratase enzyme is the result of enzyme engineering. A 4-methyl-2-oxopentanoate, or isobutyryl-CoA. single enzyme candidate for the introduction of a 3-hydroxy functional group into 3-methylbuten-2-enoyl-CoA has been 0026. This document also features a method for enzymati identified previously in the cell free extract of Galactomyces cally synthesizing isoprene that includes enzymatically intro reessi, containing an enoyl-CoA hydratase classified in EC ducing a second terminal vinyl group into 3-methyl-3-hy 4.2.1.17 that converts 3-methylbuten-2-enoyl-CoA to 3-hy droxy-pent-4-enoate, 4-methyl-3-hydroxypent-4-enoate, droxy-3-methylbutanoyl-CoA (Lee et al., Appl. Environ. 4-methyl-3-sulphorylpent-4-enoyl-acp, or 3-methyl-3- Microbiol., 1997. 63(11), 4191-4195). Equivalentenoyl-CoA buten-2-ol to produce isoprene. The second vinyl group can hydratase activity from bacterial origin has not been identi be introduced using a mevalonate diphosphate decarboxy fied. See FIG. 4 and FIG. 5. lase, a mevalonate 3-kinase, an acyl-acp decarboxylating thioesterase, or a linalool dehydratase. The mevalonate 0021. In some embodiments, 4-methyl-3-oxopentanoyl diphosphate decarboxylase can have at least 70% homology ACP is formed by condensing isobutyryl-CoA and malonyl to the mevalonate diphosphate decarboxylase of any one of ACP using a B-ketoacyl-ACP-synthase enzyme classified, for the amino acid sequences set forth in SEQID NOS:8-11. The example, under EC 2.3.1-(EC 2.3.1.41 EC 2.3.1.79, or EC mevalonate 3-kinase can have at least 70% homology to the 2.3.1.80) such as the gene product of AnlF (SEQID NO: 18). amino acid sequence of SEQ ID NO: 12. The acyl-acp It has been demonstrated that the gene product of anlF con decarboxylating thioesterase can have at least 70% homology denses isobutyryl-CoA and malonyl-ACP (Lechner et al., to the amino acid sequence of SEQID NO: 21. The linalool ACS Synth. Biol., 2013, 207), 379-83). dehydratase can have at least 70% homology to the amino 0022. In some embodiments, the second vinyl group is acid sequence of SEQID NO: 13. The mevalonate diphos introduced into a medium chain carbon alkenoate by a meva phate decarboxylase can have a histidine at the positionalign lonate diphosphate decarboxylase (Leflurgy et al., J. Biol. ing with residue 74 of SEQID NO:11 and/or a phenylalanine Chem., 2010, 285(27), 20654-20663) or a mevalonate 3-ki at the position aligning with residue 145 of SEQID NO:11. nase (Vinokur et al., Biochemistry, 2014, 53(25), 4161 - The mevalonate diphosphate decarboxylase can have the 4168), converting 3-methyl-3-hydroxypent-4-enoate or amino acid sequence set forth in SEQID NO:11, except that 4-methyl-3-hydroxypent-4-enoate to isoprene (FIGS. 2-5). a histidine is substituted at position 74 for arginine and/or a 0023. In some embodiments, the second vinyl group is phenylalanine is substituted at position 145 for isoleucine. introduced into a medium chain carbon alkenoate by a decar For example, the mevalonate diphosphate decarboxylase can boxylating thioesterase (CurM TE), converting 3-methyl-3- convert3-methyl-3-hydroxypent-4-enoate or 4-methyl-3-hy sulphoryl-pent-4-enoyl-ACP or 4-methyl-3-sulphoryl-pent droxypent-4-enoate to isoprene. For example, a mevalonate 4-enoyl-ACP to isoprene (see, FIG. 2, FIG. 3 and FIG. 6; 3-kinase can convert 3-methyl-3-hydroxypent-4-enoate or Gehret et al., J. Biol. Chem., 2011, 286(16), 14445-14454). 4-methyl-3-hydroxypent-4-enoate to isoprene. For example, US 2015/0037860 A1 Feb. 5, 2015 the acyl-acp decarboxylating thioesterase can convert dehydratase. The host can include at least one exogenous 3-methyl-3-sulphorylpent-4-enoyl-acp or 4-methyl-3-sul nucleic acid encoding (i) the B-ketoacyl-ACP-synthase, (ii) phorylpent-4-enoyl-acp to isoprene. For example, the lina the acyl-ACP dehydrogenase, and (iii) the mevalonate lool dehydratase can convert 3-methyl-3-buten-2-ol to iso diphosphate decarboxylase, mevalonate 3-kinase, acyl-ACP prene. decarboxylating thioesterase, or linalool dehydratase. 0027. Any of the methods described herein can be per 0033. This document also features a recombinant host that formed using isolated enzymes. includes at least one exogenous nucleic acid encoding (i) a 0028. Any of the methods described herein can be per dehydratase classified under EC 4.2.1.—and (ii) a meva formed using cell lysates comprising the enzymes. lonate diphosphate decarboxylase or a mevalonate 3-kinase, 0029. Any of the methods described herein can be per the host producing isoprene. formed in a recombinant host. For example, the host can be a 0034. In any of the recombinant hosts, the enzymes from prokaryote selected from the group consisting of the genus the mevalonate pathway leading to isoprenoid synthesis, Such Escherichia Such as Escherichia coli: from the genus as enzymes classified under EC 2.3.1.9, EC 2.3.3.10, EC Clostridia Such as Clostridium liungdahli, Clostridium auto 1.1.1.34 or EC 1.1.1.88, can be introduced or gene dosed into ethanogenium or Clostridium kluyveri; from the genus the host that utilizes the non-mevalonate or 2-C-methyl-D- Corynebacteria Such as Corynebacterium glutamicum; from erythritol 4-phosphate pathway for isoprenoid synthesis. the genus Cupriavidus Such as Cupriavidus necator or 0035. In the recombinant host, the enzymes from the non Cupriavidus metallidurans; from the genus Pseudomonas mevalonate or 2-C-methyl-D-erythritol 4-phosphate pathway Such as Pseudomonas fluorescens or Pseudomonas putida; can be introduced into a host microorganism that utilizes the from the genus Bacillus such as Bacillus subtilis; or from the mevalonate pathway for isoprenoid synthesis. genus Rhodococcus Such as Rhodococcus equi. The host can 0036. In any of the recombinant hosts described herein, be a eukaryote selected from the group consisting of the genus the host can include one or more of the following attenuated Aspergillus Such as Aspergillus niger, from the genus Sac enzymes: the enzyme classified under EC 2.7.1.36 accepting charomyces such as Saccharomyces cerevisiae; from the mevalonate as Substrate, a polymer synthase, an acetate genus Pichia Such as Pichia pastoris; from the genus Yar kinase, a lactate dehydrogenase, an enzyme degrading phos rowia Such as Yarrowia lipolytica, from the genus Issatchen phoenolpyruvate to Succinate, and an enzyme degrading kia Such as Issathenkia Orientalis; from the genus Debaryo acetyl-CoA to ethanol. myces such as Debaryomyces hansenii; from the genus 0037. In any of the recombinant hosts described herein, Arxtula Such as Arxtula adenoinivorans; or from the genus the host can overexpress one or more genes encoding: an Kluyveromyces such as Kluyveromyces lactis. enzyme for 3'-phosphoadenosine-5'-phosphosulfate synthe 0030 The host can be subjected to a fermentation strategy sis, a puridine nucleotide transhydrogenase, a glyceralde entailing anaerobic, micro-aerobic or aerobic cultivation. A hyde-3-phosphate-dehydrogenase, a malic enzyme, a glu cell retention strategy using a ceramic hollow fiber membrane cose-6-phosphate dehydrogenase, and a fructose 1.6- can be employed to achieve and maintain a high cell density diphosphatase. during fermentation. 0038. In any of the recombinant hosts described herein, 0031. The principal carbon source fed to the fermentation the host can include a feedback inhibition resistant mutant of can derive from a biological or a non-biological feedstock. an acetolactate synthase. The biological feedstock can be, or can derive from, 0039. In any of the recombinant hosts described herein, monosaccharides, disaccharides, hemicellulose Such as the host can include an acetolactate synthase under control of levulinic acid and furfural, cellulose, lignocellulose, lignin, a promoter not subject to genetic repression by a branched triglycerides Such as glycerol and fatty acids, agricultural chain amino acid. waste or municipal waste. The non-biological feedstock can 0040. The reactions of the pathways described herein can be, or can derive from, either natural gas, syngas, CO/H, be performed in one or more cell (e.g., host cell) Strains (a) methanol, ethanol, non-volatile residue (NVR), caustic wash naturally expressing one or more relevant enzymes, (b) from cyclohexane oxidation processes or other waste stream genetically engineered to express one or more relevant from either the chemical or petrochemical industries. enzymes, or (c) naturally expressing one or more relevant 0032. This document also features a recombinant host pro enzymes and genetically engineered to express one or more ducing isoprene. The host includes at least one exogenous relevant enzymes. Alternatively, relevant enzymes can be nucleic acid encoding (i) a 2-hydroxyacyl-CoA dehydratase extracted from any of the above types of host cells and used in or a f-ketoacyl-ACP-synthase; (ii) an acyl-ACP dehydroge a purified or semi-purified form. Extracted enzymes can nase, a monooxygenase, a cytochrome P450, or a dehydratase optionally be immobilized to a solid substrate such as the classified under EC 4.2.1.—and (iii) a mevalonate diphos floors and/or walls of appropriate reaction vessels. Moreover, phate decarboxylase, a mevalonate 3-kinase, an acyl-ACP Such extracts include lysates (e.g., cell lysates) that can be decarboxylating thioesterase, or a linalool dehydratase, the used as sources of relevant enzymes. In the methods provided host producing isoprene. The host can include at least one by the document, all the steps can be performed in cells (e.g., exogenous nucleic acid encoding (i) the 2-hydroxyacyl-CoA host cells), all the steps can be performed using extracted dehydratase, (ii) the acyl-ACP dehydrogenase, and (iii) the enzymes, or some of the steps can be performed in cells and mevalonate diphosphate decarboxylase, mevalonate 3-ki others can be performed using extracted enzymes. nase, acyl-ACP decarboxylating thioesterase, or linalool 0041 Unless otherwise defined, all technical and scien dehydratase. The host can include at least one exogenous tific terms used herein have the same meaning as commonly nucleic acid encoding (i) the 2-hydroxyacyl-CoA dehy understood by one of ordinary skill in the art to which this dratase, (ii) the monooxygenase or cytochrome P450, and invention pertains. Although methods and materials similar (iii) the mevalonate diphosphate decarboxylase, mevalonate or equivalent to those described herein can be used to practice 3-kinase, acyl-ACP decarboxylating thioesterase, or linalool the invention, suitable methods and materials are described US 2015/0037860 A1 Feb. 5, 2015
below. All publications, patent applications, patents, and boxylase (see Genbank Accession No. AHF01884.1, SEQID other references mentioned herein are incorporated by refer NO:9), a Streptococcus pneumoniae mevalonate diphosphate ence in their entirety. In case of conflict, the present specifi decarboxylase (see Genbank Accession No. CAR68209.1, cation, including definitions, will control. In addition, the SEQ ID NO: 10), a Saccharomyces cerevisiae mevalonate materials, methods, and examples are illustrative only and not diphosphate decarboxylase (see Genbank Accession No. intended to be limiting. CAA66158.1, SEQID NO: 11), a Thermoplasma acidophi 0042. The details of one or more embodiments of the lum mevalonate 3-kinase (see Genbank Accession No. invention are set forth in the accompanying drawings and the CAC 12426.1, SEQID NO: 12), a Castellaniella defragrans description below. Other features, objects, and advantages of linalool dehydratase (see Genbank Accession No. the invention will be apparent from the description and the CBW30776.1, SEQID NO: 13), a Streptomyces kanamyceti drawings, and from the claims. The word “comprising in the cus acyl-ACP dehydrogenase encoded by tcs) (see Genbank claims may be replaced by “consisting essentially of or with Accession No. ADU56239.1, SEQID NO: 14), an Aquincola “consisting of according to standard practice in patent law. tertiaricarbonis monooxygenase encoded by mdp.J (see Gen bank Accession No. AER12131.1, SEQID NO:15), an Aero DESCRIPTION OF DRAWINGS monas punctata enoyl-CoA hydratase encoded by phal (see 0043 FIG. 1 is a schematic of an exemplary biochemical Genbank Accession No. BAA21816.1, SEQID NO: 16), an pathway leading to isoprene using (R)-mevalonate as a cen Escherichia coli enoyl-CoA hydratase encoded by MaoC (see tral precursor via isopentenyl diphosphate and dimethylallyl Genbank Accession No. AFY98994.1, SEQ ID NO: 17), a diphosphate. Streptomyces sp. CNH189 B-ketoacyl-ACP-synthase 0044 FIG. 2 is a schematic of exemplary biochemical encoded by AnlF (see Genbank Accession No. AFY98994.1, pathways leading to isoprene using 3-methyl-2-oxopen SEQ ID NO: 18), a Synechococcus PCC 7002 sulfotrans tanoate as a central precursor and an acyl-ACP dehydroge ferase domain encoded by OLSST (see Genbank Accession aSC. No. ACA99172.1, SEQID NO: 19), a Moorea producens 19L 0045 FIG. 3 is a schematic of exemplary biochemical sulfotransferase domain encoded by CurMST (see Genbank pathways leading to isoprene using 4-methyl-2-oxopen Accession No. ACV42478.1, SEQ ID NO: 20), a Moorea tanoate as a central precursor and an acyl-ACP dehydroge producens 19L thioesterase domain encoded by CurM TE aSC. (see Genbank Accession No. ACV42478.1, SEQID NO: 21) 0046 FIG. 4 is a schematic of an exemplary biochemical and an Elizabethkiingia meningoseptica oleate hydratase eno pathway leading to isoprene using 3-methyl-2-oxopen ded by ohy A (see Genbank Accession No. ACT54545.1, SEQ tanoate as a central precursor and a monooxygenase. ID NO: 22). 0047 FIG. 5 is a schematic of an exemplary biochemical 0.052 FIG. 10 is a graph of the results from a spectropho pathway leading to isoprene using 4-methyl-2-oxopen tometric enzyme assay for enoyl-CoA hydratase (encoded by pha.J) activity in the forward direction, accepting crotonyl tanoate as a central precursor and a monooxygenase. CoA as substrate. 0048 FIG. 6 is a schematic of exemplary biochemical 0053 FIG. 11 is a table providing the details of an LC-MS pathways leading to isoprene using isobutyryl-CoA as a cen analysis of an enzyme assay for enoyl-CoA hydratase (en tral precursor and an acyl-ACP dehydrogenase to introduce coded by pha.J) activity in the reverse direction, accepting the first vinyl group and a GHMP Superfamily enzyme to racemic 3-hydroxybutanoyl-CoA as substrate. introduce the second vinyl group. 0054 FIG. 12 is a table providing details of an LC-MS 0049 FIG. 7 is a schematic of exemplary biochemical analysis of an enzyme assay for enoyl-CoA hydratase (en pathways leading to isoprene using isobutyryl-CoA as a cen coded by pha.J) activity in the reverse direction, accepting tral precursor and an acyl-ACP dehydrogenase to introduce 3-methyl-3-hydroxypentanoyl-CoA as substrate. the first vinyl group and a dehydratase, such as linalool dehy 0055 FIG. 13 is a table providing details of an LC-MS dratase, to introduce the second vinyl group. analysis of an enzyme assay for enoyl-CoA hydratase (en 0050 FIG. 8 is a schematic of an exemplary biochemical coded by pha.J) activity in the reverse direction, accepting pathway leading to isoprene using (R)-mevalonate as a cen 4-methyl-3-hydroxypentanoyl-CoA as Substrate. tral precursor via 3-hydroxy-3-methyl-pent-4-enoate. 0056 FIG. 14 is a bar graph of the logarithmic GC-MS 0051 FIG.9 contains the amino acid sequences of a Bacil species abundance for GHMP Superfamily enzymes lus subtilis enoyl-CoA hydratase encoded by YsiB (see Gen (AAK33797.1, SEQID NO: 8: AHFO1884.1, SEQID NO:9; bank Accession No. CAA99573.1, SEQ ID NO:1), a CAR68209.1, SEQID NO: 10; CAA66158.1, SEQID NO: Clostridium difficile 2-hydroxyacyl-CoA dehydratase activa 11; CAA66158.1 having a histidine at position 74 instead of tor encoded by Had I (See Genbank Accession No. arginine; CAA66158.1 having a histidine at position 74 AAV40818.1, SEQID NO: 2), a Clostridium difficile 2-hy instead of arginine and a phenylalanine at position 145 droxyacyl-CoA dehydratase encoded by HadBC (see Gen instead of isoleucine; CAC12426.1, SEQ ID NO: 12) con bankAccession Nos. AAV40819.1 and AAV40820.1, SEQID Verting 3-methyl-3-hydroxypent-4-enoate to isoprene rela NO:3 and SEQID NO:4, respectively), an Acidaminococcus fermentans 2-hydroxyacyl-CoA dehydratase activator tive to the empty vector control. encoded by HgdC (See Genbank Accession No. CAA42196. 0057 FIG. 15 is a bar graph of the GC-MS peak area for 1, SEQ ID NO. 5), an Acidaminococcus fermentans 2-hy linalool dehydratase (CBW30776.1, SEQ ID NO: 13) con droxyacyl-CoA dehydratase encoded by HagAB (see Gen verting 3-methyl-3-buten-2-ol to isoprene relative to the bank Accession Nos. CAA32465.1 and CAA32466.1, SEQ empty vector control. ID NO: 6 and SEQID NO:7, respectively), a Streptococcus pyogenes mevalonate diphosphate decarboxylase (see Gen DETAILED DESCRIPTION bank Accession No. AAK33797.1, SEQID NO: 8), a Thio 0058. In particular, the invention provides enzymes and alkalimicrobium aerophilum mevalonate diphosphate decar recombinant host microorganisms for isoprene synthesis in US 2015/0037860 A1 Feb. 5, 2015 which two vinyl groups are introduced into central precursors produces that nucleic acid, protein, or compound as does a Such as 3-methyl-pent-2-enoyl-acp. 4-methyl-pent-2- host of the same particular type as it is found in nature. enoyl-acp. 3-methyl-3-hydroxy-pentanoate, 4-methyl-3- 0062 For example, depending on the host and the com hydroxypentanoate, or mevalonate, to produce isoprene in pounds produced by the host, one or more of the following one or more enzymatic steps. 3-methyl-pent-2-enoyl-acp. enzymes may be expressed in the host: 2-hydroxyacyl-CoA 4-methyl-pent-2-enoyl-acp. 3-methyl-3-hydroxy-pen dehydratase, a 3-hydroxyacyl-acp dehydratase, a (R)-2-hy tanoate, 4-methyl-3-hydroxypentanoate can be enzymati droxyacyl dehydrogenase, an acyl-ACP dehydrogenase Such cally produced from 3-methyl-2-oxopentanoate, 4-methyl-2- as the gene product of tcs), a monooxgyenase such as the oXopentanoate, or isobutyryl-CoA, in one or more enzymatic gene product of mdp.J., a (R)-specific enoyl-CoA hydratase steps. As used herein, the term “central precursor is used to Such as the gene product of pha or MaoC, a (S)-specific denote any metabolite in any metabolic pathway shown enoyl-CoA hydratase such as the gene product of YsiB, a herein leading to the synthesis ofisoprene. The term “central B-ketoacyl-ACP synthase such as the gene product of AnlF, a metabolite' is used herein to denote a metabolite that is pro mevalonate diphosphate decarboxylase, a mevalonate 3-ki duced in all microorganisms to support growth. nase, a decarboxylating thioesterase, a dehydratase, a linalool 0059. As such, host microorganisms described herein can dehydratase, an oleate hydratase Such as the gene product of include pathways that can be manipulated Such that isoprene ohyA, a kevitone hydrase, a carotenoid 1.2-hydratase, a CoA can be produced. In an endogenous pathway, the host micro transferase, a CoA ligase, an acyl transferase, a thioesterase, organism naturally expresses all of the enzymes catalyzing an acylacp thioesterase, a 3-hydroxyacyl-acp: CoA the reactions within the pathway. A host microorganism con transacylase, a Sulfotransferase, an acetoacetate decarboxy taining an engineered pathway does not naturally express all lase, a secondary alcohol dehydrogenase, a hydroxymethyl of the enzymes catalyzing the reactions within the pathway glutaryl-CoA reductase, a hydroxymethylglutaryl-CoA syn but has been engineered such that all of the enzymes within thase, or a 3-oxoacyl-acp reductase. the pathway are expressed in the host. 0063. In some embodiments, a recombinant host includes at least one exogenous nucleic acid encoding (i) a 2-hy 0060. The term “exogenous” as used herein with reference droxyacyl-CoA dehydratase or a B-ketoacyl-ACP-synthase: to a nucleic acid (or a protein) and a host refers to a nucleic (ii) an acyl-ACP dehydrogenase, a monooxygenase, a cyto acid that does not occur in (and cannot be obtained from) a chrome P450, or a dehydratase classified under EC 4.2.1.— cell of that particular type as it is found in nature or a protein and (iii) a mevalonate diphosphate decarboxylase, a meva encoded by Such a nucleic acid. Thus, a non-naturally-occur lonate 3-kinase, an acyl-ACP decarboxylating thioesterase, ring nucleic acid is considered to be exogenous to a host once or a linalool dehydratase, and produces isoprene. For in the host. It is important to note that non-naturally-occurring example, a host can include at least one exogenous nucleic nucleic acids can contain nucleic acid Subsequences or frag acid encoding a 2-hydroxyacyl-CoA dehydratase, an acyl ments of nucleic acid sequences that are found in nature ACP dehydrogenase, and a mevalonate diphosphate decar provided the nucleic acid as a whole does not exist in nature. boxylase, a mevalonate 3-kinase, an acyl-ACP decarboxylat For example, a nucleic acid molecule containing a genomic ing thioesterase, or a linalool dehydratase. For example, a DNA sequence within an expression vector is non-naturally host can include at least one exogenous nucleic acid encoding occurring nucleic acid, and thus is exogenous to a host cell (i) a 2-hydroxyacyl-CoA dehydratase, (ii) a monooxygenase once introduced into the host, since that nucleic acid molecule or a cytochrome P450, and (iii) a mevalonate diphosphate as a whole (genomic DNA plus vector DNA) does not exist in decarboxylase, mevalonate 3-kinase, acyl-ACP decarboxy nature. Thus, any vector, autonomously replicating plasmid, lating thioesterase, or linalool dehydratase. For example, a or virus (e.g., retrovirus, adenovirus, or herpesvirus) that as a host can include at least one exogenous nucleic acid encoding whole does not exist in nature is considered to be non-natu (i) a -ketoacyl-ACP-synthase, (ii) an acyl-ACP dehydroge rally-occurring nucleic acid. It follows that genomic DNA nase, and (iii) a mevalonate diphosphate decarboxylase, fragments produced by PCR or restriction endonuclease mevalonate 3-kinase, acyl-ACP decarboxylating treatment as well as cDNAs are considered to be non-natu thioesterase, or linalool dehydratase. rally-occurring nucleic acid since they exist as separate mol 0064. In some embodiments, a recombinant host can ecules not found in nature. It also follows that any nucleic acid include at least one exogenous nucleic acid encoding a (R)- containing a promoter sequence and polypeptide-encoding 2-hydroxyacyl dehydrogenase, a 2-hydroxyacyl-CoA dehy sequence (e.g., cDNA or genomic DNA) in an arrangement dratase and 2-hydroxyacyl-CoA dehydratase initiator, and an not found in nature is non-naturally-occurring nucleic acid. A acyl-acp dehydrogenase and produce 3-methyl-pent-2,4- nucleic acid that is naturally-occurring can be exogenous to a dienoyl-acp or 4-methyl-pent-2,4-dienoyl-acp. Such a particular host microorganism. For example, an entire chro host also can include one or more of the following exogenous mosome isolated from a cell of yeast X is an exogenous enzymes: a CoA transferase or a CoA ligase, and/or an acyl nucleic acid with respect to a cell of yeast y once that chro transferase and a 4' phosphopantetheinyl transferase, and pro mosome is introduced into a cell of yeast y. duce 3-methyl-pent-2,4-dienoyl-acp or 4-methyl-pent-2,4- 0061. In contrast, the term “endogenous” as used herein dienoyl-acp. Either of such hosts further can include an with reference to a nucleic acid (e.g., a gene) (or a protein)and exogenous 3-hydroxyacyl-acp dehydratase and further pro a host refers to a nucleic acid (or protein) that does occur in duce 3-methyl-3-hydroxypent-4-enoyl-acp or 4-methyl-3- (and can be obtained from) that particular hostas it is found in hydroxypent-4-enoyl-acp. See, FIGS. 2 and 3. nature. Moreover, a cell "endogenously expressing a nucleic 0065. In some embodiments, a recombinant host can acid (or protein) expresses that nucleic acid (or protein) as include an exogenous nucleic acid encoding one or more of a does a host of the same particular type as it is found in nature. B-ketoacyl-ACP-synthase and produce 4-methyl-3-hydroxy Moreover, a host "endogenously producing or that "endog pent-4-enoyl-acp. Such a host also can include one or more enously produces a nucleic acid, protein, or other compound of the following exogenous enzymes: a 3-oxoacyl-acp US 2015/0037860 A1 Feb. 5, 2015 reductase, a 3-hydroxyacyl-acp dehydratase, and an acyl phate decarboxylase or a mevalonate 3-kinase and produce CoA dehydrogenase and produce 4-methyl-3-hydroxypent isoprene. Such a host further can include one or more of the 4-enoyl-acp. See, FIG. 6 and FIG. 7. following exogenous enzymes: an acetyl-CoA C-acetyltrans 0066. In some embodiments, a recombinant host produc ferase, a hydroxymethylglutaryl-CoA synthase, and/or a ing 3-methyl-3-hydroxypent-4-enoyl-acp or 4-methyl-3- hydroxymethylglutaryl-CoA reductase, See, FIG. 8. hydroxypent-4-enoyl-acp further can include an exogenous 0074. Within an engineered pathway, the enzymes can be Sulfotransferase and an exogenous decarboxylating from a single source, i.e., from one species, or can be from thioesterase and produce isoprene. See, FIG. 2, FIG. 3, and multiple sources, i.e., different species. Nucleic acids encod FIG. 6. ing the enzymes described herein have been identified from 0067. In some embodiments, a recombinant host produc various organisms and are readily available in publicly avail ing 3-methyl-3-hydroxypent-4-enoyl-acp or 4-methyl-3- able databases such as GenBank or EMBL. hydroxypent-4-enoyl-acp can include one or more of an 0075 Any of the enzymes described herein that can be exogenous (R)-3-hydroxyacyl-ACP:CoA transacylase, and a used for isoprene production can have at least 70% sequence mevalonate diphosphate decarboxylase or a mevalonate 3-ki identity (homology) (e.g., at least 75%, 80%, 85%, 90%, nase and produce isoprene. Such a host also can include a 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence thioesterase or a CoA transferase, and produce isoprene. See, of the corresponding wild-type enzyme. FIG. 2, FIG. 3, and FIG. 6. 0076 For example, an enoyl-CoA hydratase described 0068. In some embodiments, a recombinant host produc herein can have at least 70% sequence identity (homology) ing 3-methyl-3-hydroxypent-4-enoyl-acp or 4-methyl-3- (e.g., at least 75%, 80%, 85%, 90%. 95%,97%.98%, 99%, or hydroxypent-4-enoyl-acp further can include one or more 100%) to the amino acid sequence of a Bacillus subtilis (Gen of an exogenous thioesterase (e.g., an acyl acp thioesterase) Bank Accession No. CAA99573.1, SEQ ID NO:1), a Aero and a mevalonate diphosphate decarboxylase or a mevalonate monas punctata (Genbank Accession No. BAA21816.1, SEQ 3-kinase and produce isoprene. See, FIG. 2, FIG.3, and FIG. ID NO: 16), or an Escherichia coli (Genbank Accession No. 6. AFY98994.1, SEQ ID NO: 17) enoyl-CoA hydratase. See, 0069. In some embodiments, a recombinant host produc FIG. 9. ing 4-methyl-3-hydroxypent-4-enoyl-acp can include one 0077. For example, a 2-hydroxyacyl-CoA dehydratase or more of an exogenous (R)-3-hydroxyacyl-ACP: CoA described herein can have at least 70% sequence identity transacylase, an acetoacetate decarboxylase, a secondary (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, alcohol dehydrogenase, and a linalool dehydratase and pro 98%, 99%, or 100%) to the amino acid sequence of a duce isoprene. Such a host also can include one or more of the Clostridium difficile 2-hydroxyacyl-CoA dehydratase following exogenous enzymes: a dehydrogenase, a CoA encoded by HadBC (Genbank Accession Nos. AAV40819.1 transferase, and/or a thioesterase and produce isoprene. See, and AAV40820.1, SEQID NO:3 and SEQID NO:4, respec FIG.3 and FIG. 7. tively) or an Acidaminococcus fermentans 2-hydroxyacyl 0070. In some embodiments, a recombinant host produc CoA dehydratase encoded by HagAB (Genbank Accession ing 4-methyl-3-hydroxypent-4-enoyl-acp can include one Nos. CAA32465.1 and CAA32466.1, SEQ ID NO: 6 and or more of an exogenous a thioesterase (e.g., an acyl-acp SEQ ID NO:7, respectively). See, FIG. 9. thioesterase), an acetoacetate decarboxylase, a secondary 0078. A 2-hydroxyacyl-CoA dehydratase activator alcohol dehydrogenase, and a linalool dehydratase and pro described herein can have at least 70% sequence identity duce isoprene. Such a host also can include an exogenous (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 3-oxoacyl-acp reductase and produce isoprene. See, FIG. 7. 98%, 99%, or 100%) to the amino acid sequence of a 0071. In some embodiments, a recombinant host can Clostridium difficile (Genbank Accession No. AAV40818.1, include at least one exogenous nucleic acid encoding a (R)- SEQID NO: 2) or an Acidaminococcus fermentans (Genbank 2-hydroxyacyl dehydrogenase, a 2-hydroxyacyl-CoA dehy Accession No. CAA42196.1, SEQID NO:5)2-hydroxyacyl dratase and 2-hydroxyacyl-CoA dehydratase initiator, and an CoA dehydratase activator. See, FIG. 9. (R)-specific or (S)-specific enoyl-CoA hydratase and produce 007.9 For example, a mevalonate diphosphate decarboxy 3-methyl-3-hydroxy-pentanoyl-CoA or 4-methyl-3-hy lase (MDD) described herein can have at least 70% sequence droxy-pentanoyl-CoA. Such a host also can include one or identity (homology) (e.g., at least 75%, 80%, 85%, 90%, more of the following exogenous enzymes: a CoA transferase 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence or a CoA ligase, and/or an acyl transferase and a 4' phospho of a Streptococcus pyogenes (Genbank Accession No. pantetheinyl transferase, and produce 3-methyl-3-hydroxy AAK33797.1, SEQ ID NO: 8), Thioalkalimicrobium aero pentanoyl-CoA or 4-methyl-3-hydroxy-pentanoyl-CoA. philum (Genbank Accession No. AHF01884.1, SEQID NO: Such hosts further can include an exogenous thioesterase or a 9), Saccharomyces cerevisiae (GenBank Accession No. CoA transferase and further produce 3-methyl-3-hydroxy CAA66158.1, SEQID NO:11), or Streptococcus pneumonia pentanoate or 4-methyl-3-hydroxypentanoate. Any of Such (GenBank Accession No. CAR68209.1, SEQ ID NO:10). hosts further can include a monooxygenase and produce See, FIG. 9. 3-methyl-3-hydroxy-pent-4-enoate or 4-methyl-3-hydroxy 0080 For example, a mevalonate 3-kinase described pent-4-enoate. See, FIG. 4 and FIG. 5. herein can have at least 70% sequence identity (homology) 0072 A recombinant host producing 3-methyl-3-hy (e.g., at least 75%, 80%, 85%, 90%. 95%,97%.98%, 99%, or droxy-pent-4-enoate or 4-methyl-3-hydroxypent-4-enoate 100%) to the amino acid sequence of a Thermoplasma aci can include a mevalonate diphosphate decarboxylase or a dophilum (Genbank Accession No. CAC 12426.1, SEQ ID mevalonate 3-kinase and produce isoprene. See, FIG. 4 and NO: 12) mevalonate 3-kinase. See, FIG.9. FIG.S. I0081 For example, a dehydratase described herein can 0073. In some embodiments, a recombinant host can have at least 70% sequence identity (homology) (e.g., at least include an exogneous dehydratase, and a mevalonate diphos 75%, 80%,85%, 90%, 95%,97%.98%,99%, or 100%) to the US 2015/0037860 A1 Feb. 5, 2015
amino acid sequence of the linalool dehydratase from Cas I0088. Once aligned, the number of matches is determined tellaniella defragrans (GenBank Accession No. CBW30776. by counting the number of positions where an identical amino 1, SEQID NO:13). See FIG.9. acid residue is presented in both sequences. The percent iden 0082 For example, an acyl-ACP dehydrogenase tity (homology) is determined by dividing the number of described herein can have at least 70% sequence identity matches by the length of the full-length polypeptide amino (homology) (e.g., at least 75%, 80%, 85%, 90%. 95%, 97%, acid sequence followed by multiplying the resulting value by 98%, 99%, or 100%) to the amino acid sequence of the 100. It is noted that the percent identity (homology) value is acyl-CoA dehydrogenase from Streptomyces kanamyceticus rounded to the nearest tenth. For example, 78.11, 78.12. (encoded by the tcs) gene) (GenBank Accession No. 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, ADU56239.1, SEQID NO: 14). See, FIG.9. 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is noted 0083. For example, a monooxygenase described herein that the length value will always be an integer. can have at least 70% sequence identity (homology) (e.g., at I0089. It will be appreciated that a number of nucleic acids least 75%, 80%, 85%, 90%, 95%,97%.98%, 99%, or 100%) can encode a polypeptide having a particular amino acid to the amino acid sequence of an Aquincola tertiaricarbonis sequence. The degeneracy of the genetic code is well known monooxygenase encoded by mdpi (Genbank Accession No. to the art; i.e., for many amino acids, there is more than one AER12131.1, SEQID NO: 15). See, FIG.9. nucleotide triplet that serves as the codon for the amino acid. 0084. For example, a f-ketoacyl-ACP synthase described For example, codons in the coding sequence for a given herein can have at least 70% sequence identity (homology) enzyme can be modified Such that optimal expression in a (e.g., at least 75%, 80%, 85%, 90%. 95%,97%.98%, 99%, or particular species (e.g., bacteria or fungus) is obtained, using 100%) to the amino acid sequence of a Streptomyces sp. appropriate codon bias tables for that species. CNH189 (Genbank Accession No. AFY98994.1, SEQ ID 0090 Functional fragments of any of the enzymes NO: 18) B-ketoacyl-ACP-synthase. See, FIG.9. described herein can also be used in the methods of the 0085 For example, a sulfotransferase described herein document. The term “functional fragment” as used herein can have at least 70% sequence identity (homology) (e.g., at refers to a peptide fragment of a protein that has at least 25% least 75%, 80%, 85%, 90%, 95%,97%.98%, 99%, or 100%) (e.g., at least:30%; 40%; 50%: 60%; 70%; 75%: 80%; 85%; to the amino acid sequence of a Synechococcus PCC 7002 90%; 95%: 98%: 99%; 100%; or even greater than 100%) of (Genbank Accession No. ACA99172.1, SEQ ID NO: 19) or the activity of the corresponding mature, full-length, wild Moorea producens 19L (Genbank Accession No. ACV42478. type protein. The functional fragment can generally, but not 1, SEQID NO: 20) sulfotransferase. See, FIG.9. always, be comprised of a continuous region of the protein, I0086 For example, a decarboxylating thioesterase wherein the region has functional activity. described herein can have at least 70% sequence identity 0091. This document also provides (i) functional variants (homology) (e.g., at least 75%, 80%, 85%, 90%. 95%, 97%, of the enzymes used in the methods of the document and (ii) 98%, 99%, or 100%) to the amino acid sequence of a Moorea functional variants of the functional fragments described producens 19L (Genbank Accession No. ACV42478.1, SEQ above. Functional variants of the enzymes and functional ID NO: 21) decarboxylating thioesterase. See, FIG.9. fragments can contain additions, deletions, or Substitutions 0087. The percent identity (homology) between two relative to the corresponding wild-type sequences. Enzymes amino acid sequences can be determined as follows. First, the with substitutions will generally have not more than 50 (e.g., amino acid sequences are aligned using the BLAST 2 not more than one, two, three, four, five, six, seven, eight, Sequences (B12seq) program from the stand-alone version of nine, ten, 12, 15.20,25.30.35.40, or 50) amino acid substitu BLASTZ containing BLASTP version 2.0.14. This stand tions (e.g., conservative substitutions). This applies to any of alone version of BLASTZ can be obtained from Fish & Rich the enzymes described herein and functional fragments. A ardson's web site (e.g., www.fr.com/blast?) or the U.S. gov conservative Substitution is a Substitution of one amino acid ernment's National Center for Biotechnology Information for another with similar characteristics. Conservative substi web site (www.ncbi.nlm.nih.gov). Instructions explaining tutions include Substitutions within the following groups: how to use the B12seq program can be found in the readme Valine, alanine and glycine; leucine, Valine, and isoleucine; file accompanying BLASTZ. B12seq. performs a comparison aspartic acid and glutamic acid; asparagine and glutamine: between two amino acid sequences using the BLASTP algo serine, cysteine, and threonine; lysine and arginine; and phe rithm. To compare two amino acid sequences, the options of nylalanine and tyrosine. The nonpolar hydrophobic amino B12seqare set as follows: —i is set to a file containing the first acids include alanine, leucine, isoleucine, Valine, proline, amino acid sequence to be compared (e.g., C:\Seq 1.txt); - is phenylalanine, tryptophan and methionine. The polar neutral set to a file containing the second amino acid sequence to be amino acids include glycine, serine, threonine, cysteine, compared (e.g., C:\Seq2.txt); - p is set to blastp; —o is set to tyrosine, asparagine and glutamine. The positively charged any desired file name (e.g., C:\output.txt); and all other (basic)amino acids include arginine, lysine and histidine. The options are left at their default setting. For example, the fol negatively charged (acidic) amino acids include aspartic acid lowing command can be used to generate an output file con and glutamic acid. Any Substitution of one member of the taining a comparison between two amino acid sequences: above-mentioned polar, basic or acidic groups by another C:\B12seq —i c:\Seq1.txt - c:\Seq2.txt —p blastp —o member of the same group can be deemed a conservative c:\output.txt. If the two compared sequences share homology Substitution. By contrast, a nonconservative Substitution is a (identity), then the designated output file will present those substitution of one amino acid for another with dissimilar regions of homology as aligned sequences. If the two com characteristics. pared sequences do not share homology (identity), then the 0092 Deletion variants can lack one, two, three, four, five, designated output file will not present aligned sequences. six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19. Similar procedures can be following for nucleic acid or 20 amino acid segments (of two or more amino acids) or sequences except that blastn is used. non-contiguous singleamino acids. Additions (addition vari US 2015/0037860 A1 Feb. 5, 2015
ants) include fusion proteins containing: (a) any of the 0097. In some embodiments, a 3-hydroxy functional enzymes described herein or a fragment thereof; and (b) group is introduced into 3-methyl-pent-2-enoyl-CoA or internal or terminal (C or N) irrelevant or heterologous amino 4-methyl-pent-2-enoyl-CoA by a (R)-specific enoyl-CoA acid sequences. In the context of Such fusion proteins, the hydratase enzyme classified, for example, under EC 4.2.1. term "heterologous amino acid sequences' refers to an amino 119 such as the gene product of phal (SEQID NO: 16, Fukui acid sequence other than (a). A heterologous sequence can be, et al., J. Bacteriol., 1998, 180(3), 667-673) or MaoC (SEQID for example a sequence used for purification of the recombi NO: 17: Park and Lee, J. Bacteriol., 2003, 185(18), 5291 nant protein (e.g., FLAG, polyhistidine (e.g., hexahistidine), 5397) or a bacterial (S)-specific enoyl-CoA hydratase classi hemagluttanin (HA), glutathione-S-transferase (GST), or fied, for example, under EC 4.2.1.17 such as the gene product maltosebinding protein (MBP)). Heterologous sequences of YsiB (SEQID NO: 1). See, for example, FIGS. 4 and 5. also can be proteins useful as detectable markers, for 0098. In some embodiments, the enoyl-CoA hydratase example, luciferase, green fluorescent protein (GFP), or enzyme is the result of enzyme engineering. A single enzyme chloramphenicol acetyl transferase (CAT). In some embodi candidate for the introduction of a 3-hydroxy functional ments, the fusion protein contains a signal sequence from group into 3-methylbuten-2-enoyl-CoA has been identified another protein. In certain host cells (e.g., yeast host cells), previously in the cell free extract of Galactomyces reessii, expression and/or secretion of the target protein can be containing an enoyl-CoA hydratase, classified in EC 4.2.1.17. increased through use of a heterologous signal sequence. In that converts 3-methylbuten-2-enoyl-CoA to 3-hydroxy-3- Some embodiments, the fusion protein can contain a carrier methylbutanoyl-CoA (Lee et al., Appl. Environ. Microbiol., (e.g., KLH) useful, e.g., in eliciting an immune response for 1997, 63(11), 4191-4195). Equivalent enoyl-CoA hydratase antibody generation) or ER or Golgi apparatus retention sig activity from bacterial origin has not been identified. See FIG. nals. Heterologous sequences can be of varying length and in 4 and FIG. 5. Some cases can be a longer sequences than the full-length 0099. In some embodiments, 4-methyl-3-oxopentanoyl target proteins to which the heterologous sequences are ACP is formed by condensing isobutyryl-CoA and malonyl attached. ACP using a B-ketoacyl-ACP-synthase enzyme classified, for 0093. Recombinant hosts can naturally express none or example, under EC 2.3.1.- (e.g., EC 2.3.1.41, EC 2.3.1.79, or Some (e.g., one or more, two or more, three or more, four or EC 2.3.1.80) such as the gene product of AnlF (SEQID NO: more, five or more, or six or more) of the enzymes of the 18). It has been demonstrated that the gene product of anlF pathways described herein. Endogenous genes of the recom condenses isobutyryl-CoA and malonyl-ACP (Lechner et al., binant hosts also can be disrupted to prevent the formation of ACS Synth. Biol., 2013, 207), 379-83). See, FIG. 6. undesirable metabolites or prevent the loss of intermediates in 0100. In some embodiments (FIG. 8), the central precur the pathway through other enzymes acting on Such interme Sor to 3-methyl-3-hydroxypent-4-enoate, acetyl-CoA, is con diates. Recombinant hosts can be referred to as recombinant verted to acetoacetyl-CoA by an acetyl-CoA C-acetyltrans host cells, engineered cells, or engineered hosts. Thus, as ferase classified, for example, under EC 2.3.1.9, followed by described herein, recombinant hosts can include nucleic acids conversion to 3-hydroxy-3-methylglutaryl-CoA by a encoding one or more of a decarboxylase, a kinase, a dehy hydroxymethylglutaryl-CoA synthase classified, for drogenase, a monooxygenase, an acyl acyl carrier protein example, under EC 2.3.3.10; followed by conversion to (R)- (acp) dehydrogenase, a dehydratase, a thioesterase, or a mevalonate by a hydroxymethylglutaryl-CoA reductase clas decarboxyating thioesterase as described in more detail sified under EC 1.1.1.88 or EC 1.1.1.34; followed by conver below. sion to 3-methyl-3-hydroxypent-4-enoate by an enzyme 0094. In addition, the production of isoprene can be per classified, for example, under EC 4.2.1.—such as an oleate formed in vitro using the isolated enzymes described herein, hydratase, (e.g., a the gene product of ohy A (SEQID NO: 22) using a lysate (e.g., a cell lysate) from a host microorganism or a dehydratase classified under EC 4.2.1.—(such as one as a source of the enzymes, or using a plurality of lysates from isolated from species such as Aquincola tertiaricarbonis or different host microorganisms as the source of the enzymes. Methylibium petroleiphilum PM1). 0101. In some embodiments, the dehydratase enzyme Production of Branched C5 Central Metabolite Backbones converting mevalonate to 3-methyl-3-hydroxypent-4-enoate 0095. In some embodiments, 3-methyl-pent-2-enoyl-CoA is the result of enzyme engineering to improve activity or or 4-methyl-pent-2-enoyl-CoA is formed by a 2-hydroxya specificity using the structure and wild-type residue diversity cyl-CoA dehydratase classified, for example, under EC 4.2. of for example, an oleate hydratase (SEQID NO: 22). 1.—, such as the gene product of HadBC (SEQID NO:3 and Enzymes Generating First Terminal Vinyl Group SEQ ID NO:4) and its initiator Had I (SEQID NO: 2), or by the gene product of HgdAB (SEQ ID NO: 6 and SEQ ID 0102. In some embodiments, a first terminal vinyl group is NO:7) and its initiator HagC (SEQID NO. 5). See, FIG. 2-5. introduced into 3-methyl-pent-2-enoyl-ACP forming 3-me 0096. In some embodiments, the 2-hydroxyacyl-CoA thyl-pent-2,4-dienoyl-acp and then enzymatically con dehydratase is the result of enzyme engineering. The 2-hy verted in one or more steps to 3-methyl-3-hydroxypent-4- droxyacyl-CoA dehydratase enzymes isolated from anaero enoate or 3-methyl-3-sulphoryl-pent-4-enoyl-ACP (as bic bacteria possess a common catalytic mechanism shown, for example, in FIG. 2). It has been demonstrated that employed in amino acid degradation pathways. For example, the gene product of tcs D (SEQID NO: 14) from Streptomyces the gene products of HadBC/HadI from Clostridium difficile kanamyceticus has dehydrogenase activity for straight and catalyse the conversion of (R)-2-hydroxyisocaproyl-CoA to branch chain C5 acyl-ACP substrates (Mo et al., JACS, 2011, isocaprenoyl-CoA. Similarly, the gene products of HgdAB/ 133,976-985). 3-methyl-pent-2-enoyl-ACP can be derived HdgC catalyse the conversion of 2-hydroxyglutaryl-CoA to from the central metabolite 3-methyl-2-oxopentanoate. glutaconyl-CoA (Kim et al., FEMS Microbiol. Reviews, 0103) In some embodiments, a first terminal vinyl group is 2004, 28,455-468). See FIGS. 2-5. introduced into 4-methyl-pent-2-enoyl-acp, forming 4-me US 2015/0037860 A1 Feb. 5, 2015 thyl-pent-2,4-dienoyl-acp, which may be enzymatically 3-methyl-3-buten-2-ol by a linalool dehydratase classified, converted in one or more steps to 4-methyl-3-hydroxypent for example, under EC 4.2.1.—such as EC 4.2.1.127 (SEQID 4-enoate (see, for example FIG. 3 and FIG. 6), 4-methyl-3- NO: 13, GenBank Accession No. CBW30776.1, Brodkorb et sulphoryl-pent-4-enoyl-ACP (see, for example, FIG. 3 and al., J. Biol. Chem., 2010, 285(40), 30436-30442) or a dehy FIG. 6), or 3-methyl-3-buten-2-ol (see, for example, FIG. 3 dratase classified under EC 4.2.1.—such as one isolated from and FIG. 7). It has been demonstrated that the gene product of a species such as Aquincola tertiaricarbonis or Methylibium tcsD (SEQID NO: 14) from Streptomyces kanamyceticus has petroleiphilum PM1; Schäfer et al., Appl. Environ. Micro dehydrogenase activity for 4-methyl-pent-2-enoyl-ACP (Mo biol., 2011, 77(17), 5981 - 5987). See, FIG.3 and FIG. 7. et al., 2011, Supra). 4-methyl-pent-2-enoyl-acp can be Pathways to 3-Methyl-3-Hydroxypent-4-Enoyl-acp derived from the central metabolite 4-methyl-2-oxopen 0110. In some embodiments (FIG. 2), the central precur tanoate or isobutyryl-CoA. sor to 3-methyl-3-hydroxypent-4-enoyl-acp. 3-methyl-2- 0104. In some embodiments, the first vinyl group is intro oXo-pentanoate, is converted to 3-methyl-2-hydroxypen duced into 3-methyl-3-hydroxy-pentanoate by a monooxgy tanoate by a (R)-2-hydroxyacyl dehydrogenase classified, for enase, forming 3-methyl-3-hydroxy-pent-4-enoate (see, for example, under EC 1.1.1.272 such as the gene product of example, FIG. 4). It has been demonstrated that the monooxy ldh A, followed by conversion to 3-methyl-2-hydroxy-pen genase encoded by mdpy (SEQ ID NO: 15) introduces a tanoyl-CoA by a CoA-transferase Such as the gene product of terminal double bond into allyl groups bound to a secondary HadA or GctAB (e.g., a glutaconate CoA transferase classi alcohol (Schäfer et al., Appl. Environ. Microbiol., 2012, fied, for example, under EC 2.8.3.12) or a CoA-ligase classi 78(17), 6280-6284). fied, for example, under EC 6.2.1.—(e.g., EC 6.2.1.2); fol 0105. In some embodiments, the first vinyl group is intro lowed by conversion to 3-methyl-pent-2-enoyl-CoA by a (R)- duced into 4-methyl-3-hydroxypentanoate, forming 4-me 2-Hydroxyacyl-CoA dehydratase such as the gene products thyl-3-hydroxypent-4-enoate (see, for example, FIG. 5). of HadBC (SEQIDNOs: 3 and 4) and the initiator Had I (SEQ 0106. In some embodiments, the first vinyl group is intro ID NO: 2) or the gene products of HgdAB (SEQID NOs: 6 duced into mevalonate, forming 3-hydroxy-3-methyl-pent-4- and 7) and the initiator HgdC (SEQID NO: 5); followed by enoate (as shown, for example, in FIG. 8). conversion to 3-methyl-pent-2-enoyl-acp by an acyl trans ferase Such as encoded by tcSA (see Genbank Accession No. Enzymes Generating Second Terminal Vinyl Group and ADU56236.1) or a 4' phosphopantetheinyl transferase such Producing Isoprene as encoded by s?p (see Genbank Accession No. CAA44858. 0107. In some embodiments, the second vinyl group is 1) or SVp (see Genbank Accession No. AAG43513.1); fol introduced into a medium chain carbon alkenoate such as lowed by conversion to 3-methyl-pent-2,4-dienoyl-ACP by 3-methyl-3-hydroxypent-4-enoate or 4-methyl-3-hydroxy an acyl-acp dehydrogenase Such as the gene product of pent-4-enoate by a GHMP Superfamily enzyme such as a Tcs.D (SEQID NO:14); followed by conversion to 3-methyl mevalonate diphosphate decarboxylase classified, for 3-hydroxypent-4-enoyl-ACP by a 3-hydroxyacyl-acp example, under EC 4.1.1.33 (e.g., SEQ ID NOs: 8-11) dehydratase classified, for example, under EC 4.2.1.59. (Lefurgy et al., J. Biol. Chem., 2010, 285(27), 20654-20663)) 0111. In some embodiments (FIG. 2), 3-methyl-3-hy or a mevalonate 3-kinase classified, for example, under EC droxypent-4-enoyl-acp, a central precursor to 3-methyl-3- 2.7.1.—(e.g., SEQID NO: 12) (Vinokur et al., Biochemistry, hydroxypent-4-enoate, is converted to 3-methyl-3-hydroxy 2014, 53 (25), 4161-4168), producing isoprene (FIGS. 2-6 pent-4-enoyl-CoA by a (R)-3-hydroxyacyl-ACP:CoA and 8). In some embodiments, a MDD has an amino acid transacylase such as the gene product of PhaG; followed by Substitution at one or both positions corresponding to amino conversion to 3-methyl-3-hydroxypent-4-enoate by a CoA acids 74 and/or 145 of the amino acid sequence set forth in tran?ferase such as the gene product of HadA or GctAB or by SEQ ID NO:11. For example, a histidine residue can be a thioesterase classified, for example, under EC 3.1.2.—Such Substituted for arginine at a position aligning with residue 74 as the gene product of tesB (e.g., GenBank Accession No. of SEQ ID NO:11 and/or a phenylalanine residue can be AAA24665.1) or YciA (See Genbank Accession No. Substituted for an isoleucine at a position aligning with resi BAA14785.1). due 145 of SEQ ID NO:11. In some embodiments, a MDD 0112. In some embodiments (FIG. 2), 3-methyl-3-hy has the amino acid sequence set forth in SEQ ID NO:11, droxypent-4-enoyl-ACP, a central precursor to 3-methyl-3- except that a histidine is Substituted at position 74 for arginine hydroxypent-4-enoate, is converted to 3-methyl-3-hydroxy and/or a phenylalanine is Substituted at position 145 for iso pent-4-enoate by a thioesterase such as an acyl acp leucine. thioesterase (e.g., the gene product encoded by GenBank 0108. In some embodiments, the second vinyl group is Accession No. AAO77182 or CCC78182.1). introduced into medium chain carbon alkenoate Such as 0113. In some embodiments (FIG. 2), 3-methyl-3-hy 3-methyl-3-sulphoryl-pent-4-enoyl-ACP or 4-methyl-3-sul droxypent-4-enoyl-acp, the central precursor to 3-methyl phoryl-pent-4-enoyl-ACP by a decarboxylating thioesterase 3-sulphoryl-pent-4-enoyl-acp, is converted to 3-methyl-3- (e.g., from Lyngbya majuscula (CurM TE), Moorea pro Sulphoryl-pent-4-enoyl-acp by a Sulfotransferase classified ducens (SEQ ID NO: 21), Pseudomonas entomophila, H. under EC 2.8.2.—such as the gene product of CurMST or ochraceum, Synechococcus PCC 7002, Cyanothece PCC OLS ST. 7424 or Cyanothece PCC 7822) (see Gehret et al., J. Biol. Pathways to 4-Methyl-3-Hydroxypent-4-Enoyl-acp Chem., 2011, 286(16), 14445-14454), converting 3-methyl 0114. In some embodiments (FIG. 3), the central precur 3-sulphoryl-pent-4-enoyl-ACP or 4-methyl-3-sulphoryl Sor to 4-methyl-3-hydroxypent-4-enoyl-acp. 4-methyl-2- pent-4-enoyl-ACP to isoprene (see, FIG. 2, FIG.3 and FIG. oXo-pentanoate, is converted to 4-methyl-2-hydroxypen 6). tanoate by a (R)-2-hydroxyacyl dehydrogenase classified, for 0109. In some embodiments, the second vinyl group is example, under EC 1.1.1.272 such as the gene product of introduced into a medium chain carbon alkenoate such as ldh A, followed by conversion to 4-methyl-2-hydroxy-pen US 2015/0037860 A1 Feb. 5, 2015 tanoyl-CoA by a CoA-transferase Such as the gene product of by a (R)-2-hydroxyacyl dehydrogenase classified, for HadA or GctAB or a CoA-ligase such as classified under EC example, under EC 1.1.1.272 such as the gene product of 6.2.1.—(2); followed by conversion to 4-methyl-pent-2- ldh A, followed by conversion to 3-methyl-2-hydroxy-pen enoyl-CoA by a (R)-2-Hydroxyacyl-CoA dehydratase such tanoyl-CoA by a CoA-transferase Such as the gene product of as the gene products HadBC and the initiator Had I; followed HadA or GctAB or a CoA-ligase such as classified under EC by conversion to 4-methyl-pent-2-enoyl-acp by an acyl 6.2.1.—(e.g., EC 6.2.1.2); followed by conversion to 3-me transferase such as the reaction with the gene product from thyl-pent-2-enoyl-CoA by a (R)-2-Hydroxyacyl-CoA dehy Tcs.A & Sfp/svp; followed by conversion to 4-methyl-pent-2, dratase such as the gene products of HadBC (SEQID NOs: 3 4-dienoyl-acp by an acyl-ACP dehydrogenase Such as the and 4) and the initiator HadI (SEQ ID NO:2) or the gene gene product of TcsD; followed by conversion to 4-methyl product HgdAB (SEQ ID NOs: 6 and 7) and the initiator 3-hydroxypent-4-enoyl-acp by a 3-hydroxyacyl-acp HgdC (SEQID NO:5); followed by conversion to 3-methyl dehydratase classified, for example, under EC 4.2.1.59 such 3-hydroxypentanoyl-CoA by an enoyl-CoA hydratase Such as the gene product of fabZ. as the gene product of phal (SEQID NO:16), MaoC (SEQID 0115. In some embodiments (FIG. 6), the central precur NO: 17) or YsiB (SEQID NO: 1); followed by conversion to Sor to 4-methyl-3-hydroxypent-4-enoyl-acp, isobutyryl 3-methyl-3-hydroxypentanoate by a CoA-transferase Such as CoA, is converted to 4-methyl-3-oxo-pentanoyl-acp by a the gene product of HadA or GctAB or a thioesterase such as B-ketoacyl-acp-synthase such as the gene product of AnIF: the gene product of tesB (e.g., GenBank Accession No. followed by conversion to 4-methyl-3-hydroxy-pentanoyl AAA24665.1) or YciA (see Genbank Accession No. acp by a 3-oxoacyl-acp reductase (EC 1.1.1.100) such as BAA 14785.1); followed by conversion to 3-methyl-3-hy the gene product of fabG or Anlo; followed by conversion to droxypent-4-enoate by a monooxygenase Such as the gene 4-methyl-pent-2-enoyl-acp by a 3-hydroxyacyl-acp dehy product of Mdp) (SEQ ID NO: 15) or a cytochrome P450 dratase (EC 4.2.1.59) such as the gene product of fabZ; fol such as the gene product of the CYP4 family. lowed by conversion to 4-methyl-pent-2,4-dienoyl-acp by I0120 In some embodiments, the enzymes shown in FIG.4 an acyl-acp dehydrogenase such as the gene product of are the result of enzyme engineering to improve activity or tcsD; followed by conversion to 4-methyl-3-hydroxypent-4- specificity using the enzyme structure and wild-type residue enoyl-acp by a 3-hydroxyacyl-acp hydratase such as EC diversity to inform the rational enzyme design. 4.2.1.59 such as the gene product of fabZ. 0116. In some embodiments (FIG. 7), the central precur Pathways to 4-Methyl-3-Hydroxypent-4-Enoate Sor to 4-methyl-3-hydroxypent-4-enoyl-acp, isobutyryl I0121. In some embodiments (FIG.3 and FIG. 6), the cen CoA, is converted to 4-methyl-3-oxo-pentanoyl-acp by a tral precursor to 4-methyl-3-hydroxypent-4-enoate, 4-me B-ketoacyl-acp-synthase such as the gene product of AnlF: thyl-3-hydroxypent-4-enoyl-acp, is converted to 4-methyl followed by conversion to 4-methyl-3-hydroxy-pentanoyl 3-hydroxypent-4-enoyl-CoA by (R)-3-hydroxyacl-acp: acp by a 3-oxoacyl-acp reductase (EC 1.1.1.100) such as the gene product of fabG or Anlo; followed by conversion to CoA transacylase such as the gene product of PhaG; followed 4-methyl-pent-2-enoyl-acp by a 3-hydroxyacyl-acp dehy by conversion to 4-methyl-3-hydroxypent-4-enoate by a dratase (EC 4.2.1.59) such as the gene product of fabZ; fol CoA-tran?ferase such as the gene product of HadA or GctAB lowed by conversion to 4-methyl-pent-2,4-dienoyl-acp by or by a thioesterase Such as the gene product of tesB (e.g., an acyl-acp dehydrogenase such as the gene product of GenBank Accession No. AAA24665.1) or YciA (see Gen tcsD; followed by conversion to 4-methyl-3-hydroxypent-4- bank Accession No. BAA 14785.1). I0122. In some embodiments (FIG.3 and FIG. 6), the cen enoyl-acp by a 3-hydroxyacyl-ACP hydratase such as EC tral precursor to 4-methyl-3-hydroxypent-4-enoate, 4-me 4.2.1.59 such as the gene product of fabZ. thyl-3-hydroxypent-4-enoyl-acp, is converted to 4-methyl Pathways to 3-Methyl-3-Hydroxypent-4-Enoate 3-hydroxypent-4-enoate by a thioesterase classified, for example, under EC 3.2.1.—Such as the Bacteroides thetaio 0117. In some embodiments (FIG. 2), the central precur taOmicron acyl-acp thioesterase (GenBank Accession No. sor to 3-methyl-3-hydroxypent-4-enoate, 3-methyl-3-hy AAO77182) or Lactobacillus plantarum thioesterase (Gen droxypent-4-enoyl-acp, is converted to 3-methyl-3-hy Bank Accession No. CCC78182.1). droxypent-4-enoyl-CoA by a (R)-3-hydroxyacyl-acp: CoA I0123. In some embodiments (FIG. 5), the central precur transacylase such as the gene product of PhaG; followed by Sor to 4-methyl-3-hydroxypent-4-enoate, 4-methyl-2-oxo conversion to 3-methyl-3-hydroxypent-4-enoate by a CoA pentanoate, is converted to 4-methyl-2-hydroxypentanoate transferase such as the gene product of HadA or GctAB or by by a (R)-2-hydroxyacyl dehydrogenase classified, for a thioesterase Such as the gene product oftesE (e.g., GenBank example, under EC 1.1.1.272 such as the gene product of Accession No. AAA24665.1) or YciA (See Genbank Acces ldh A, followed by conversion to 4-methyl-2-hydroxy-pen sion No. BAA14785.1). tanoyl-CoA by a CoA-transferase Such as the gene product of 0118. In some embodiments (FIG. 2), the central precur HadA or GctAB or a CoA-ligase classified, for example, sor to 3-methyl-3-hydroxypent-4-enoate, 3-methyl-3-hy under EC 6.2.1.—(e.g., EC 6.2.1.2); followed by conversion droxypent-4-enoyl-acp, is converted to 3-methyl-3-hy to 4-methyl-pent-2-enoyl-CoA by a (R)-2-Hydroxyacyl droxypent-4-enoate by a thioesterase classified, for example, CoA dehydratase such as the gene products of HadBC (SEQ under EC 3.2.1.—such as the Bacteroides thetaiotaOmicron ID NOs: 3 and 4) and the initiator Had I (SEQID NO:2) or the acyl-acp thioesterase (GenBank Accession No. AAO77182) gene products of HgdAB (SEQ ID NO: 6 and 7) and the or Lactobacillus plantarum thioesterase (GenBank Acces initiator HgdC (SEQ ID NO:5); followed by conversion to sion No. CCC78182.1). 4-methyl-3-hydroxypentanoyl-CoA by an enoyl-CoA 0119. In some embodiments (FIG. 4), the central precur hydratase such as the gene product of phal (SEQID NO: 16), sor to 3-methyl-3-hydroxypent-4-enoate, 3-methyl-2-oxo MaoC (SEQID NO: 17) or YsiB (SEQID NO: 1); followed pentanoate, is converted to 3-methyl-2-hydroxypentanoate by conversion to 4-methyl-3-hydroxypentanoate by a CoA US 2015/0037860 A1 Feb. 5, 2015
transferase such as the gene product of HadA or GctAB or a I0131 For example, the prokaryote can be a bacterium thioesterase such as the gene product of tesB (e.g., GenBank from the genus Escherichia such as Escherichia coli: from the Accession No. AAA24665.1) or YciA (e.g., Genbank Acces genus Clostridia Such as Clostridium ljungdahli, sion No. BAA 14785.1); followed by conversion to 4-methyl Clostridium autoethanogenium or Clostridium kluyveri; from 3-hydroxypent-4-enoate by a monooxygenase such as the the genus Corynebacteria Such as Corynebacterium gene product of Mdpy (SEQ ID NO: 15) or a cytochrome glutamicum; from the genus Cupriavidus Such as Cupriavi P450 such as the gene product of the CYP4 family. dus necator or Cupriavidus metallidurans; from the genus 0.124. In some embodiments, the enzymes shown in FIG.5 Pseudomonas such as Pseudomonas fluorescens, Pseudomo are the result of enzyme engineering to improve activity or nas putida or Pseudomonas Oleavorans; from the genus Delf specificity using the enzyme structure and wild-type residue tia Such as Delftia acidovorans; from the genus Bacillus Such diversity to inform the rational enzyme design. as Bacillus subtilis; from the genus Lactobacillus such as Pathway to 4-Methyl-3-Sulphoryl-Pent-4-Enoyl-acp Lactobacillus delbrueckii; or from the genus Lactococcus 0.125. In some embodiments, the central precursor to Such as Lactococcus lactis. Such prokaryotes also can be a 4-methyl-3-Sulphoryl-pent-4-enoyl-acp, 4-methyl-3-hy Source of genes to construct recombinant host cells described droxypent-4-enoyl-acp, is converted to 4-methyl-3-sulpho herein that are capable of producing isoprene or precursors ryl-pent-4-enoyl-acp by a Sulfotransferase Such as the gene thereof. product of CurMST or OLS ST. See, FIG.3 and FIG. 6. 0.132. In some embodiments, the host microorganism is a Pathways to 3-Methyl-3-Buten-2-ol eukaryote. For example, the eukaryote can be a filamentous fungus, e.g., one from the genus Aspergillus Such as Aspergil 0126. In some embodiments (e.g., FIG.3 and FIG. 7), the lus niger. Alternatively, the eukaryote can be a yeast, e.g., one central precursor to 3-methyl-3-buten-2-ol. 4-methyl-3-hy from the genus Saccharomyces such as Saccharomyces cer droxypent-4-enoyl-acp, can be converted to 4-methyl-3- evisiae; from the genus Pichia Such as Pichia pastoris; or hydroxypent-4-enoyl-acp by (R)-3-hydroxyacyl-acp: from the genus Yarrowia Such as Yarrowia lipolytica; from CoA transacylase such as the gene product of PhaG; followed the genus Issatchenkia Such as Issathenkia Orientalis; from by conversion to 4-methyl-3-oxopent-4-enoyl-CoA by a the genus Debaryomyces such as Debaryomyces hansenii; dehydrogenase classified, for example under EC 1.1.1.— from the genus Arxula Such as Arxula adenoinivorans; or such as EC 1.1.1.36 or EC 1.1.1.157); followed by conversion from the genus Kluyveromyces such as Kluyveromyces lactis. to 4-methyl-3-oxopent-4-enoate by a thioesterase classified Such eukaryotes also can be a source of genes to construct for example, under EC 3.2.1.11 or a CoA-transferase classi recombinant host cells described herein that are capable of fied under EC 2.8.3.—encoded by Ato AD or pcally; followed producing isoprene or precursors thereof. by conversion to 3-methyl-3-buten-2-one by an acetoacetate decarboxylase classified, for example, under EC 4.1.1.4; fol I0133. In some embodiments, isoprene is biosynthesized in lowed by conversion to 3-methyl-3-buten-2-olby a secondary a recombinant host using a fermentation strategy that can alcohol dehydrogenase, classified, for example, under EC include anaerobic, micro-aerobic or aerobic cultivation of the 1.1.1.B3, EC 1.1.1.B4 or EC 1.1.1.80. recombinant host. 0127. In some embodiments (e.g., FIG.3 and FIG. 7), the I0134. In some embodiments, isoprene is biosynthesized in central precursor to 3-methyl-3-buten-2-ol. 4-methyl-3-hy a recombinant host using a fermentation Strategy that uses an droxypent-4-enoyl-acp, can be converted to 4-methyl-3- alternate final electron acceptor to oxygen Such as nitrate. oXopent-4-enoyl-acp by a 3-oxoacyl-acp reductase Such I0135) In some embodiments, a cell retention strategy as an enzyme classified under EC 1.1.1.100 (e.g., the gene using, for example, ceramic hollow fiber membranes can be product of fabG or AnlG); following by conversion to 4-me employed to achieve and maintain a high cell density during thyl-3-oxopent-4-enoate by a thioesterase classified, for either fed batch or continuous fermentation in the synthesis of example, under EC 3.1.2.—Such as the Bacteroides thetaio isoprene. taOmicron acyl-acp thioesterase (GenBank Accession No. 0.136. In some embodiments, the biological feedstock can AAO77182) or Lactobacillus plantarum thioesterase (Gen be, can include, or can derive from, monosaccharides, disac Bank Accession No. CCC78182.1); followed by conversion charides, lignocellulose, hemicellulose, cellulose, lignin, to 3-methyl-3-buten-one by an acetoacetate decarboxylase levulinic acid & formic acid, triglycerides, glycerol, fatty classified, for example, under EC 4.1.1.4, followed by con acids, agricultural waste, condensed distillers’ solubles or version to 3-methyl-3-buten-2-ol by a secondary alcohol municipal waste. dehydrogenase, classified, for example, under EC 1.1.1.B3, EC 1.1.1.B4 or EC 1.1.18O. 0.137 The efficient catabolism of crude glycerol stemming 0128. In some embodiments, the enzymes shown in FIG.3 from the production of biodiesel has been demonstrated in and FIG. 7 are the result of enzyme engineering to improve several microorganisms such as Escherichia coli, Cupriavi activity or specificity using the enzyme structure and wild dus necator; Pseudomonas Oleavorans, Pseudomonas putida type residue diversity to inform the rational enzyme design. and Yarrowia lipolytica (Lee et al., Appl. Biochem. Biotech 0129. In some embodiments, the enzymes shown in FIG.3 mol., 2012, 166, 1801-1813; Yang et al., Biotechnology for and FIG. 7 are the result of enzyme engineering to improve Biofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Bio activity or specificity using the enzyme structure and wild technol., 2011, 90, 885-893). type residue diversity to inform the rational enzyme design. 0.138. The efficient catabolism of lignocellulosic-derived levulinic acid has been demonstrated in several organisms Cultivation Strategies Such as Cupriavidus necator and Pseudomonas putida in the 0130. In some embodiments, the nucleic acids encoding synthesis of 3-hydroxyvalerate via the precursor propanoyl the enzymes of the pathways described in FIGS. 2-8 are CoA (Jaremko and Yu, Journal of Biotechnology, 2011, 155, introduced into a host microorganism that is either a prokary 2011, 293-298; Martin and Prather, Journal of Biotechnology, ote or eukaryote. 2009, 139, 61-67). US 2015/0037860 A1 Feb. 5, 2015
0.139. The efficient catabolism of lignin-derived aromatic a growth, storage, or transport medium. Media can be liquid, compounds such benzoate analogues has been demonstrated semi-solid (e.g., gelatinous media), or frozen. The culture in several microorganisms such as Pseudomonas putida, includes the cells growing in the liquid or in?on the semi-solid Cupriavidus necator (Bugg et al., Current Opinion in Bio medium or being stored or transported in a storage or trans technology, 2011, 22, 394-400; Perez-Pantoja et al., FEMS port medium, including a frozen storage or transport medium. Microbiol. Rev., 2008, 32,736-794). The cultures are in a culture vessel or storage vessel or Sub 0140. The efficient utilization of agricultural waste, such strate (e.g., a culture dish, flask, or tube or a storage vial or as olive mill waste water has been demonstrated in several tube). microorganisms, including Yarrowia lipolytica (Papani kolaou et al., Bioresour: Technol., 2008,99(7), 2419 - 2428). Metabolic Engineering 0141. The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulo 0150. The present document provides methods involving sic, hemicellulosic, cane and beet molasses, cassava, corn and less than all the steps described for all the above pathways. other argricultural Sources has been demonstrated for several Such methods can involve, for example, one, two, three, four, microorganism such as Escherichia coli, Corynebacterium five, six, seven, eight, nine, ten, or more of such steps. Where glutamicum and Lactobacillus delbrueckii and Lactococcus less than all the steps are included in such a method, the first lactis (see, e.g., Hermann et al., Journal of Biotechnology, step can be any one of the steps listed. Furthermore, recom 2003, 104, 155-172: Wee et al., Food Technol. Biotechnol., binant hosts described herein can include any combination of 2006, 44(2), 163-172; Ohashi et al., Journal of Bioscience the above enzymes such that one or more of the steps, e.g., and Bioengineering, 1999, 87(5), 647-654). one, two, three, four, five, six, seven, eight, nine, ten, or more 0142. The efficient utilization of furfural, derived from a of such steps, can be performed within a recombinant host. variety of agricultural lignocellulosic sources, has been dem 0151. In addition, this document recognizes that where onstrated for Cupriavidus necator (Li et al., Biodegradation, enzymes have been described as accepting CoA-activated 2011, 22, 1215-1225). Substrates, analogous enzyme activities associated with 0143. In some embodiments, the non-biological feedstock acp-bound Substrates exist that are not necessarily in the can be or can derive from natural gas, syngas, CO/H, metha same enzyme class. nol, ethanol, benzoic acid, non-volatile residue (NVR) or a 0152 Also, this document recognizes that where enzymes caustic wash waste stream from cyclohexane oxidation pro have been described accepting (R)-enantiomers of Substrate, cesses, or terephthalic acid/isophthalic acid mixture waste analogous enzyme activities associated with (S)-enantiomer StreamS. Substrates exist that are not necessarily in the same enzyme 0144. The efficient catabolism of methanol has been dem class. onstrated for the methylotropic yeast Pichia pastoris. 0153. This document also recognizes that where an 0145 The efficient catabolism of ethanol has been dem enzyme is shown to accept a particular co-factor. Such as onstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. NADPH, or co-substrate, such as acetyl-CoA, many enzymes Acad. Sci. USA, 2008, 105(6) 2128-2133). are promiscuous in terms of accepting a number of different 0146 The efficient catabolism of CO, and H, which may co-factors or co-substrates in catalyzing a particular enzyme be derived from natural gas and other chemical and petro activity. Also, this document recognizes that where enzymes chemical sources, has been demonstrated for Cupriavidus have high specificity for e.g., a particular co-factor Such as necator (Prybylski et al., Energy, Sustainability and Society, NADH, an enzyme with similar or identical activity that has 2012, 2:11). high specificity for the co-factor NADPH may be in a differ 0147 The efficient catabolism of syngas has been demon ent enzyme class. strated for numerous microorganisms, such as Clostridium 0154) In some embodiments, the enzymes in the pathways liungdahlii and Clostridium autoethanogenium (Köpke et al., outlined herein can be the result of enzyme engineering via non-director rational enzyme designapproaches with aims of Applied and Environmental Microbiology, 2011, 77(15), improving activity, improving specificity, reducing feedback 5467-5475). inhibition, reducing repression, improving enzyme solubility, 0148. The efficient catabolism of the non-volatile residue waste stream from cyclohexane processes has been demon changing stereo-specificity, or changing co-factor specificity. strated for numerous microorganisms, such as Delftia aci 0.155. In some embodiments, the enzymes in the pathways dovorans and Cupriavidus necator (Ramsay et al., Applied outlined herein can be gene dosed, i.e., overexpressed, into and Environmental Microbiology, 1986, 52(1), 152-156). the resulting genetically modified organism via episomal or 0149. In some embodiments, substantially pure cultures of chromosomal integration approaches. recombinant host microorganisms are provided. As used 0156. In some embodiments, genome-scale system biol herein, a “substantially pure culture' of a recombinant host ogy techniques such as Flux Balance Analysis can be utilized microorganism is a culture of that microorganism in which to devise genome scale attenuation or knockout strategies for less than about 40% (i.e., less than about 35%; 30%; 25%: directing carbon flux to isoprene. 20%; 15%:10%; 5%; 2%; 1%: 0.5%; 0.25%: 0.1%; 0.01%; 0157 Attenuation strategies include, but are not limited 0.001%; 0.0001%; or even less) of the total number of viable to, the use of transposons, homologous recombination cells in the culture are viable cells other than the recombinant (double cross-over approach), mutagenesis, enzyme inhibi microorganism, e.g., bacterial, fungal (including yeast), tors and RNAi interference. mycoplasmal, or protozoan cells. The term “about in this 0158. In some embodiments, fluxomic, metabolomic and context means that the relevant percentage can be 15% of the transcriptomal data can be utilized to inform or Support specified percentage above or below the specified percentage. genome-scale system biology techniques, thereby devising Thus, for example, about 20% can be 17% to 23%. Such a genome scale attenuation or knockout strategies in directing culture of recombinant microorganisms includes the cells and carbon flux to isoprene. US 2015/0037860 A1 Feb. 5, 2015
0159. In some embodiments using hosts that naturally ilvN that are resistant to feedback inhibition by lysine and accumulate polyhydroxyalkanoates, the polymer synthase leucine, can be overexpressed in the host. enzymes can be attenuated in the host strain. 0173. In some embodiments, acetolactate synthase can be 0160. In some embodiments, the enzymes from the meva expressed under a promoter not subject to genetic repression lonate pathway, for example, EC 2.3.1.9, EC 2.3.3.10, EC by branch-chain amino acids (e.g., valine, leucine, or isoleu 1.1.1.34 or EC 1.1.1.88, are introduced or gene dosed into a cine). host microorganism that utilizes the non-mevalonate or 2-C- 0.174. In some embodiments, the efflux of isoprene across methyl-D-erythritol 4-phosphate pathway for isoprenoid the cell membrane to the extracellular media can be enhanced synthesis. or amplified by genetically engineering structural modifica 0161 In some embodiments, the enzymes from the non tions to the cell membrane or increasing any associated trans mevalonate or 2-C-methyl-D-erythritol 4-phosphate pathway porter activity for isoprene. leading to isoprenoid synthesis are introduced into a host microorganism that utilizes the mevalonate pathway for iso Producing Isoprene Using a Recombinant Host prenoid synthesis and EC 2.7.1.36 is attenuated. 0.175 Typically, isoprene is produced by providing a host 0162. In some embodiments, the enzymes responsible for microorganism and culturing the provided microorganism 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) synthesis with a culture medium containing a Suitable carbon Source as classified under EC 2.7.7.4 & EC 2.7.1.25 are constitutively described above. In general, the culture media and/or culture expressed in the host organisms. conditions can be such that the microorganisms grow to an 0163. In some embodiments requiring the intracellular adequate density and produce isoprene efficiently. For large availability of pyruvate for isoprene synthesis, a gene in an scale production processes, any method can be used Such as acetate synthesis pathway encoding an acetate kinase, such as those described elsewhere (Manual of Industrial Microbiol ack, can be attenuated (Shen et al., Appl. Environ. Microbiol., ogy and Biotechnology, 2" Edition, Editors: A. L. Demain 2011, 77(9), 2905-2915). and J. E. Davies, ASM Press; and Principles of Fermentation 0164. In some embodiments requiring the intracellular Technology, P. F. Stanbury and A. Whitaker, Pergamon). availability of pyruvate for isoprene synthesis, a gene encod Briefly, a large tank (e.g., a 100 gallon, 200 gallon, 500 gallon, ing the degradation of pyruvate to lactate Such as laha can be or more tank) containing an appropriate culture medium is attenuated (Shen et al., Appl. Environ. Microbiol., 2011, inoculated with a particular microorganism. After inocula 77(9), 2905-2915). tion, the microorganism is incubated to allow biomass to be 0.165. In some embodiments requiring the intracellular produced. Once a desired biomass is reached, the broth con availability of pyruvate for isoprene synthesis, a gene encod taining the microorganisms can be transferred to a second ing the degradation of phosphoenolpyruvate to Succinate Such tank. This second tank can be any size. For example, the as fraBC can be attenuated (see, e.g., Shen et al., 2011, Supra). second tank can be larger, Smaller, or the same size as the first 0166 In some embodiments requiring the intracellular tank. Typically, the second tank is larger than the first Such availability of pyruvate for isoprene synthesis, a gene encod that additional culture medium can be added to the broth from ing the degradation of acetyl-CoA to ethanol Such as adhE can the first tank. In addition, the culture medium within this be attenuated (Shen et al., 2011, supra). second tank can be the same as, or different from, that used in the first tank. 0167. In some embodiments, where pathways require 0176 Once transferred, the microorganisms can be incu excess NADPH co-factor in the synthesis ofisoprene, a puri bated to allow for the production ofisoprene. Once produced, dine nucleotide transhydrogenase gene Such as Udha can be any method can be used to isolate isoprene. overexpressed in the host organism (Brigham et al., Advanced 0177. Once produced, any method can be used to isolate Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090). isoprene. For example, isoprene can be recovered from the 0.168. In some embodiments, where pathways require fermenter off-gas stream as a volatile product as the boiling excess NADPH co-factor in the synthesis of isoprene, a glyc point of isoprene is 34.1° C. At a typical fermentation tem eraldehyde-3P-dehydrogenase gene such as GapN can be perature of approximately 30°C., isoprene has a high vapour overexpressed in the host organism (Brigham et al., 2012, pressure and can be stripped by the gas flow rate through the Supra). broth for recovery from the off-gas. Isoprene can be selec 0169. In some embodiments, where pathways require tively adsorbed onto, for example, an adsorbent and separated excess NADPH co-factor in the synthesis ofisoprene, a malic from the other off-gas components. Membrane separation enzyme gene Such as macA or maeB can be overexpressed in technology may also be employed to separate isoprene from the host organism (Brigham et al., 2012, Supra). the other off-gas compounds. Isoprene may desorbed from 0170 In some embodiments, where pathways require the adsorbent using, for example, nitrogen and condensed at excess NADPH co-factor in the synthesis of isoprene, a glu low temperature and high pressure. cose-6-phosphate dehydrogenase gene Such as Zwf can be overexpressed in the host organism (Lim et al., Journal of EXAMPLES Bioscience and Bioengineering, 2002, 93 (6), 543-549). 0171 In some embodiments, where pathways require Example 1 excess NADPH co-factor in the synthesis ofisoprene, a fruc Enzyme Activity of R-Specific Enoyl-CoA tose 1.6 diphosphatase gene Such as fbp can be overexpressed Hydratase Accepting in the host (Becker et al., Journal of Biotechnology, 2007, 3-Methyl-3-Hydroxypentanoyl-CoA and 132, 99-109). 4-Methyl-3-Hydroxypentanoyl-CoA as Substrate 0172. In some embodiments, a feedback inhibition resis tant mutant of an acetolactate synthase classified, for 0.178 The C-terminal his-tagged pha.J. gene (SEQID NO: example, under EC 2.2.1.6, such as mutants of ilvB and/or 16) from Aeromonas punctata was cloned into a pE23a US 2015/0037860 A1 Feb. 5, 2015 expression vector under the T7 promoter. The expression tanoyl-CoA and 4-methyl-3-hydroxypentanoyl-CoA as Sub vector was transformed into a BL21DE3 E. coli host. strate in the dehydration direction. Given the reversibility of 0179 The resulting recombinant E. coli strain was culti the enzyme reaction and the favoured hydration direction, the vated in a 1 L shake flask culture containing 100 mL. Luria enoyl-CoA hydratase encoded by pha.J from Aeromonas Broth media at 30°C., shaking at 200 rpm. The culture was punctata accepts 3-methyl-pent-2-enoyl-CoA and 4-methyl induced using 1 mM IPTG for 2 h. pent-2-enoyl-CoA as Substrate. 0180. The pellet from each of the induced shake flask cultures was harvested by centrifugation. The pellet was Example 2 resuspended in 20 mM HEPES (pH=7.2), 1 mM PMSF and 29 U benzonase. The resuspended pellet was lysed via soni Enzyme Activity of GHMP Superfamily Enzymes, cation. The cell debris was separated from the supernatant via Mevalonate Diphosphate Decarboxylase and centrifugation and filtered using a 0.2 um filter. Mevalonate-3-Kinase. Accepting 0181. The pha.J enzyme was purified from the supernatant 3-Methyl-3-Hydroxypent-4-Enoate as Substrate, using Ni-affinity chromatography and concentrated to 1.25 Forming Isoprene mg/mL. 0187. Each of the sequences encoding a C-terminal His 0182. The native enzyme activity assay in the forward tagged gene encoding the mevalonate diphosphate decar (hydration) direction was undertaken in a buffer composed of boxylase of SEQID NOs: 8, 9, 10, and 11 respectively (see 10 mM ammonium acetate (pH-8) and 1 mM of crotonyl FIG.9) and the mevalonate 3-kinase of SEQID NO: 12 (see CoA from Sigma-Aldrich at 30°C. The enzyme activity assay FIG.9) was cloned into a PD681-CH expression vector under reaction was initiated by adding 0.4 uM of purified enoyl control of the rhapBAD promoter, such that a C-terminal HIS CoA hydratase to the assay buffer containing the Substrate. tagged GHMP Superfamily enzyme could be produced. Each The enzyme encoded by phal accepted crotonyl-CoA as Sub expression vector was transformed into a BL21 DE3 E. coli strate as confirmed via spectrophotometry at 263 nm at 30°C. host. The resulting recombinant E. coli strains were cultivated The Substrate only control showed minimal spontaneous at 37° C. in a 5 L shake flask culture containing 1 L. Luria hydration of crotonyl-CoA as determined by spectrophotom Broth (LB) media and kanamycin antibiotic selection pres etry at 263 nm. See FIG. 10. sure, with shaking at 90 rpm. At an ODoo between 0.6 to 0.8, 0183 The native enzyme activity assay in the reverse (de the culture was induced with L-rhamnose to a final concen hydration) direction was undertaken in a buffer composed of tration of 2 g/L. The cultures was induced for 6 hat 37°C. The 10 mM ammonium acetate (pH-8) and 1 mM of racemic pellets from the induced shake flask cultures were harvested 3-hydroxybutanoyl-CoA. The enzyme activity assay reaction via centrifugation and stored at -20°C. was initiated by adding 5uMofpurified enoyl-CoA hydratase 0188 Each frozen pellet was thawed, resuspended and to the assay buffer containing the Substrate and incubated at lysed in a lysis buffer containing 50 mM Tris.HCl (pH=8.0), 30°C. for 1 h. The enzyme encoded by pha.J. accepted 3-hy 50 mMNaCl, 1 mM MgCl, 1% (w/v) TritonX-100, 1 mg/mL droxybutanoyl-CoA as substrate as confirmed via LC-MS. lysozyme and 10 U/mL benzonase for 1 h at 30°C. The cell The Substrate only control showed negligible spontaneous debris was removed via centrifugation. The GHMP Super dehydration of 3-hydroxybutanoyl-CoA. As demonstrated family enzymes were purified from the resulting Supernatant previously (Lan and Liao, PNAS, 2012, 109(16), 6018 using Ni-affinity chromatography and the eluate was buffer 6023), the enoyl-CoA hydratase encoded by pha) is revers exchanged and concentrated via ultrafiltration (10 kDa ible, though favors the forward (hydration) direction. See MWCO) into 100 mM HEPES (pH=7.0), 100 mM KCl to a FIG 11. final enzyme concentration of 200 uM. 0184 The non-native enzyme activity assay in the reverse 0189 Each enzyme activity assay was performed in an (dehydration) direction was undertaken in a buffer composed assay buffer composed of 100 mM HEPES (pH=7.0), 100 of 10 mMammonium acetate (pH-8) and 1 mM of 3-methyl mMKC1, 30 mMMgCl, 30 mMATP, 2 mMDTT and 10 mM 3-hydroxypentanoyl-CoA. The enzyme activity assay reac of 3-methyl-3-hydroxypent-4-enoate as substrate. Each tion was initiated by adding 5 uM of purified enoyl-CoA enzyme activity assay reaction was initiated by adding 0.5 mL hydratase to the assay buffer containing the Substrate and of the enzyme stock of SEQID NOs: 8, 9, 10, 11 or 12 to 0.5 incubated at 30° C. for 1 h. The enzyme encoded by phal mL of assay buffer in a 10 mL crimped glass vial and incu accepted 3-methyl-3-hydroxypentanoyl-CoA as Substrate as bating at 30°C. for 24 h. The headspace of each glass vial was confirmed via LC-MS. The substrate only control showed no analysed by GC-MS for isoprene and compared to the empty spontaneous dehydration of 3-methyl-3-hydroxypentanoyl vector control. The gene product of SEQID NOs: 8,9, 10, 11 CoA. See FIG. 12. and 12 accepted 3-methyl-3-hydroxypent-4-enoate as Sub 0185. The non-native enzyme activity assay in the reverse strate as confirmed via GC-MS (see FIG. 14) and synthesized (dehydration) direction was undertaken in a buffer composed isoprene as reaction product. of 10 mMammonium acetate (pH-8) and 1 mM of 4-methyl 3-hydroxypentanoyl-CoA. The enzyme activity assay reac Example 3 tion was initiated by adding 5 uM of purified enoyl-CoA hydratase to the assay buffer containing the Substrate and Enzyme Activity of Linalool Dehydratase Accepting incubated at 30° C. for 1 h. The enzyme encoded by phal 3-Methyl-3-Buten-2-ol as Substrate. Forming accepted 4-methyl-3-hydroxpentanoyl-CoA as Substrate as Isoprene confirmed via LC-MS. The substrate only control showed no 0190. A sequence encoding a C-terminal His-tag encoding spontaneous dehydration of 4-methyl-3-hydroxypentanoyl the linalool dehydratase of SEQID NO: 13 (see FIG.9) was CoA. See FIG. 13. cloned into apRT15 expression vector under control of the T7 0186 The enoyl-CoA hydratase encoded by pha.J from promoter Such that a C-terminal HIS tagged enzyme could be Aeromonas punctata accepted 3-methyl-3-hydroxypen produced. The expression vector was transformed into a US 2015/0037860 A1 Feb. 5, 2015 15
BL21 DE3 E. coli host. The resulting recombinant E. coli to the empty vector control (undertaken in triplicate). The strain was cultivated at 30° C. in a 1 L shake flask culture gene product of SEQID NO: 13 accepted 3-methyl-3-buten containing 100 mL Auto-Induction media and antibiotic 2-ol as substrate as confirmed via GC-MS (see FIG. 15) and selection pressure, shaking at 220 rpm overnight. The pellet synthesized isoprene as reaction product. from the induced shake flask culture was harvested via cen trifugation and used immediately in a whole cell assay. Other Embodiments 0191 The pellet was washed and resuspended in M9 mini 0.192 It is to be understood that while the invention has mal media to 160 mg/mL (wet weight) and dispensed into 10 been described in conjunction with the detailed description mL crimped glass vials in triplicate. The Substrate 3-methyl thereof, the foregoing description is intended to illustrate and 3-buten-2-ol was added to a final concentration of 20 mMand not limit the scope of the invention, which is defined by the incubated at 30°C. at 220 rpm for 48 h. The headspace of each Scope of the appended claims. Other aspects, advantages, and glass vial was analysed by GC-MS for isoprene and compared modifications are within the scope of the following claims.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 22
<21 Os SEQ ID NO 1 &211s LENGTH: 258 212s. TYPE: PRT <213> ORGANISM; Bacillus subtilis
<4 OOs SEQUENCE: 1
Met Asn Ala Ile Ser Luell Ala Wall Asp Glin Phe Wall Ala all Lieu. Thir 1. 5 15
Ile His Asn Pro Pro Ala ASn Ala Lieu. Ser Ser Arg Ile el Glu Glu 25 3 O
Luell Ser Ser Luell Asp Glin Cys Glu Thir Asp Ala Gly al Arg Ser 35 4 O 45
Ile Ile Ile His Gly Glu Gly Arg Phe Phe Ser Ala Gly A. la Asp Ile SO 55 60
Lys Glu Phe Thr Ser Luell Lys Gly ASn Glu Asp Ser Ser ell Lell Ala 65 70 7s 8O
Glu Arg Gly Glin Glin Luell Met Glu Arg Ile Glu Ser Phe P ro Llys Pro 85 90 95
Ile Ile Ala Ala Ile His Gly Ala Lieu. Gly Gly Gly L el Glu Lell 1.
Ala Met Ala His Ile Arg Ile Ala Glu Asp Ala Lys Lieu. Gly 115 12O 125
Luell Pro Glu Luell Asn. Luell Ile Pro Gly Phe Ala ly Thir Gin 13 O 14 O
Arg Lieu Pro Arg Wall Thir Ala Lieu. Glu eu. Ile Gly 145 15 O 155 16 O
Ser Gly Glu Pro Ile Ser Ala Lieu. Asp Lieu. Lieu Wall 1.65 17 O 17s
Ser Ile Gly Ala Asp Ala Wall Ile Glu Ala 18O
Luell Ala Ala Phe Ala Lys Ser Pro Glin. Thir Lieu. Ser Luell 195 2 OO
Luell Glu Lieu. Lieu. Tyr Ser Wall Ser Tyr Glu Ser Luell 210 22 O
Llys Lieu Glu Ala Arg Phe Gly Glu Ala Phe Glu Ser Asp Ala 225 23 O 235 24 O
Glu Gly Ile Glin Ala Phe Lieu. Glu Lys Arg Pro Phe 245 25 O 255 Gly Glu US 2015/0037860 A1 Feb. 5, 2015 16
- Continued
<210s, SEQ ID NO 2 &211s LENGTH: 266 212. TYPE: PRT <213> ORGANISM: Clostridium difficile
<4 OOs, SEQUENCE: 2 Met Tyr Thr Met Gly Lieu. Asp Ile Gly Ser Thr Ala Ser Lys Gly Val 1. 5 1O 15 Ile Lieu Lys Asn Gly Glu Asp Ile Val Ala Ser Glu Thir Ile Ser Ser 2O 25 3O Gly Thr Gly Thr Thr Gly Pro Ser Arg Val Lieu. Glu Lys Lieu. Tyr Gly 35 4 O 45 Llys Thr Gly Lieu Ala Arg Glu Asp Ile Llys Llys Val Val Val Thr Gly SO 55 6 O Tyr Gly Arg Met Asn Tyr Ser Asp Ala Asp Llys Glin Ile Ser Glu Lieu. 65 70 7s 8O Ser Cys His Ala Arg Gly Val Asin Phe Ile Ile Pro Glu Thr Arg Thr 85 90 95 Ile Ile Asp Ile Gly Gly Glin Asp Ala Lys Val Lieu Lys Lieu. Asp Asn 1OO 105 11 O Asn Gly Arg Lieu. Lieu. Asn. Phe Lieu Met Asn Asp Llys Cys Ala Ala Gly 115 12 O 125 Thr Gly Arg Phe Lieu. Asp Wal Met Ala Lys Ile Ile Glu Val Asp Wall 13 O 135 14 O Ser Glu Lieu. Gly Ser Ile Ser Met Asn Ser Glin Asn Glu Val Ser Ile 145 150 155 160 Ser Ser Thr Cys Thr Val Phe Ala Glu Ser Glu Val Ile Ser His Leu 1.65 17O 17s Ser Glu Asn Ala Lys Ile Glu Asp Ile Val Ala Gly Ile His Thir Ser 18O 185 19 O Val Ala Lys Arg Val Ser Ser Lieu Val Lys Arg Ile Gly Val Glin Arg 195 2OO 2O5 Asn Val Val Met Val Gly Gly Val Ala Arg Asn. Ser Gly Ile Val Arg 21 O 215 22O Ala Met Ala Arg Glu Ile Asn Thr Glu Ile Ile Val Pro Asp Ile Pro 225 23 O 235 24 O Glin Lieu. Thr Gly Ala Lieu. Gly Ala Ala Lieu. Tyr Ala Phe Asp Glu Ala 245 250 255 Lys Glu Ser Glin Lys Glu Val Lys Asn. Ile 26 O 265
<210s, SEQ ID NO 3 &211s LENGTH: 408 212. TYPE: PRT <213> ORGANISM: Clostridium difficile
<4 OOs, SEQUENCE: 3 Met Ser Glu Lys Lys Glu Ala Arg Val Val Ile Asn Asp Lieu. Lieu Ala 1. 5 1O 15
Glu Glin Tyr Ala Asn Ala Phe Lys Ala Lys Glu Glu Gly Arg Pro Val 2O 25 3O
Gly Trp Ser Thr Ser Val Phe Pro Glin Glu Lieu Ala Glu Val Phe Asp 35 4 O 45 US 2015/0037860 A1 Feb. 5, 2015 17
- Continued
Lieu. Asn Val Lieu. Tyr Pro Glu Asin Glin Ala Ala Gly Val Ala Ala Lys SO 55 6 O Lys Gly Ser Lieu. Glu Lieu. Cys Glu Ile Ala Glu Ser Lys Gly Tyr Ser 65 70 7s 8O Ile Asp Lieu. Cys Ala Tyr Ala Arg Thr Asn. Phe Gly Lieu. Lieu. Glu Asn 85 90 95 Gly Gly Cys Glu Ala Lieu. Asp Met Pro Ala Pro Asp Phe Lieu. Lieu. Cys 1OO 105 11 O Cys Asn. Asn. Ile Cys Asn Glin Val Ile Llys Trp Tyr Glu Asn. Ile Ser 115 12 O 125 Arg Glu Lieu. Asp Ile Pro Lieu. Ile Met Ile Asp Thir Thr Phe Asn. Asn 13 O 135 14 O Glu Asp Glu Val Thr Glin Ser Arg Ile Asp Tyr Ile Lys Ala Glin Phe 145 150 155 160 Glu Glu Ala Ile Lys Glin Lieu. Glu Ile Ile Ser Gly Llys Llys Phe Asp 1.65 17O 17s Pro Llys Llys Phe Glu Glu Val Met Lys Ile Ser Ala Glu Asn Gly Arg 18O 185 19 O Lieu. Trp Llys Tyr Ser Met Ser Leu Pro Ala Asp Ser Ser Pro Ser Pro 195 2OO 2O5 Met Asin Gly Phe Asp Leu Phe Thr Tyr Met Ala Val Ile Val Cys Ala 21 O 215 22O Arg Gly Llys Lys Glu Thir Thr Glu Ala Phe Llys Lieu Lieu. Ile Glu Glu 225 23 O 235 24 O Lieu. 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 26 O 265 27 O Tyr Lys Met Lys Thr Lieu Ala Lys Phe Gly Val Asn Met Thr Gly Ser 27s 28O 285 Val Tyr Pro His Ala Trp Ala Lieu. Glin Tyr Glu Val Asn Asp Lieu. Asp 29 O 295 3 OO Gly Met Ala Val Ala Tyr Ser Thr Met Phe Asn Asn Val Asn Lieu. Asp 3. OS 310 315 32O Arg Met Thir Lys Tyr Arg Val Asp Ser Lieu Val Glu Gly Lys Cys Asp 3.25 330 335 Gly Ala Phe Tyr His Met Asn Arg Ser Cys Llys Lieu Met Ser Lieu. Ile 34 O 345 35. O Glin Tyr Glu Met Glin Arg Arg Ala Ala Glu Glu Thr Gly Lieu Pro Tyr 355 360 365 Ala Gly Phe Asp Gly Asp Glin Ala Asp Pro Arg Ala Phe Thr Asn Ala 37 O 375 38O
Glin Phe Glu Thr Arg Ile Glin Gly Lieu Val Glu Val Met Glu Glu Arg 385 390 395 4 OO
Llys Llys Lieu. Asn Arg Gly Glu Ile 4 OS
<210s, SEQ ID NO 4 &211s LENGTH: 375 212. TYPE: PRT <213> ORGANISM: Clostridium difficile
<4 OOs, SEQUENCE: 4 US 2015/0037860 A1 Feb. 5, 2015 18
- Continued
Met Glu Ala Ile Lell Ser Met Glu Wall Wall Glu Asn Pro Asn 15
Ala Ala Wall Lys Lys Ser Glu Thir Gly Ala Ile Gly 25 3O
Phe Pro Wall Tyr Pro Glu Glu Ile Ile His Ala Ala Gly Met 35 4 O 45
Lell Pro Wall Gly Ile Trp Gly Gly Glin Thir Glu Lell Asp Luell Ala SO 55 6 O
Glin Tyr Phe Pro Ala Phe Ala Ser Ile Met Glin Ser Luell Glu 65 70 8O
Gly Luell Gly Ala Glu Luell Ser Gly Wall Ile Ile Pro 85 90 95
Gly Met Asp Thir Lell Ile Luell Gly Glin Asn Trp Lys Ser Ala 105 11 O
Wall Pro His Ile Ile Ser Luell Wall His Pro Glin Asn Arg 115 12 O 125
Lell Glu Ala Gly Wall Tyr Luell Ile Ser Glu Tyr Gly Wall Lys 13 O 135 14 O
Arg Glu Luell Glu Glu Ile Gly Tyr Glu Ile Glu Glu Ala Ile 145 150 155 160
His Glu Ser Ile Glu Wall Asn Glu His Arg Thir Met Arg Asp 1.65
Phe Wall Glu Wall Ala His Ser Asn Thir Ile Pro Ser Ile 18O 185 19 O
Arg Ser Luell Wall Ile Ser Gly Phe Phe Met Arg Lys Glu Glu His 195
Thir Glu Luell Wall Lys Asp Lell Ile Ala Luell Asn Ala Met Pro Glu 21 O 215 22O
Glu Wall Ser Gly Lys Wall Luell Luell Thir Gly Ile Luell Ala Asp 225 23 O 235 24 O
Ser Asp Ile Lell Asp Ile Luell Glu Asp ASn Asn Ile Ser Wall Wall 245 250 255
Ala Asp Asp Luell Ala Glin Glu Thir Arg Glin Phe Arg Thir Asp Wall Pro 26 O 265 27 O
Ala Gly Asp Asp Ala Lell Glu Arg Luell Ala Arg Glin Trp Ser Asn Ile 285
Glu Gly Cys Ser Lell Ala Tyr Asp Pro Lys Arg Gly Ser Luell 29 O 295 3 OO
Ile Wall Asp Glu Wall Lys Asp Ile Asp Gly Wall Ile Phe Cys 3. OS 310 315
Met Met Phe Cys Asp Pro Glu Glu Tyr Asp Pro Luell Wall Arg 3.25 330 335
Asp Ile Glu Asp Ser Gly Ile Pro Thir Luell Tyr Wall Glu Ile Asp 34 O 345 35. O
Glin Glin Thir Glin Asn Asn Glu Glin Ala Arg Thir Arg Ile Glin Thir Phe 355 360 365
Ala Glu Met Met Ser Lell Ala 37 O 375
<210s, SEQ ID NO 5 &211s LENGTH: 26 O US 2015/0037860 A1 Feb. 5, 2015 19
- Continued
212. TYPE: PRT <213> ORGANISM: Acidaminococcus fermentans
<4 OOs, SEQUENCE: 5 Met Ser Ile Tyr Thr Lieu. Gly Ile Asp Val Gly Ser Thr Ala Ser Lys 1. 5 1O 15 Cys Ile Ile Lieu Lys Asp Gly Lys Glu Ile Val Ala Lys Ser Lieu Val 2O 25 3O Ala Val Gly Thr Gly Thr Ser Gly Pro Ala Arg Ser Ile Ser Glu Val 35 4 O 45 Lieu. Glu Asn Ala His Met Lys Lys Glu Asp Met Ala Phe Thir Lieu Ala SO 55 6 O Thr Gly Tyr Gly Arg Asn. Ser Lieu. Glu Gly Ile Ala Asp Llys Glin Met 65 70 7s 8O Ser Glu Lieu 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 Glin Asp Wall Lys Val Ile His 1OO 105 11 O Val Glu Asn Gly Thr Met Thr Asn Phe Gln Met Asn Asp Llys Cys Ala 115 12 O 125 Ala Gly Thr Gly Arg Phe Lieu. Asp Wal Met Ala Asn. Ile Lieu. Glu Val 13 O 135 14 O Llys Val Ser Asp Lieu Ala Glu Lieu. Gly Ala Lys Ser Thr Lys Arg Val 145 150 155 16 O Ala Ile Ser Ser Thr Cys Thr Val Phe Ala Glu Ser Glu Val Ile Ser 1.65 17O 17s Glin Lieu. Ser Lys Gly Thr Asp Llys Ile Asp Ile Ile Ala Gly Ile His 18O 185 19 O Arg Ser Val Ala Ser Arg Val Ile Gly Lieu Ala Asn Arg Val Gly Ile 195 2OO 2O5 Val Lys Asp Val Val Met Thr Gly Gly Val Ala Glin Asn Tyr Gly Val 21 O 215 22O Arg Gly Ala Lieu. Glu Glu Gly Lieu. Gly Val Glu Ile Llys Thir Ser Pro 225 23 O 235 24 O Lieu Ala Glin Tyr Asn Gly Ala Lieu. Gly Ala Ala Lieu. Tyr Ala Tyr Lys 245 250 255 Lys Ala Ala Lys 26 O
<210s, SEQ ID NO 6 &211s LENGTH: 477 212. TYPE: PRT <213> ORGANISM: Acidaminococcus fermentans
<4 OOs, SEQUENCE: 6 Met Pro Llys Thr Val Ser Pro Gly Val Glin Ala Lieu. Arg Asp Val Val 1. 5 1O 15
Glu Lys Val Tyr Arg Glu Lieu. Arg Glu Ala Lys Glu Arg Gly Glu Lys 2O 25 3O
Val Gly Trp Ser Ser Ser Lys Phe Pro Cys Glu Lieu Ala Glu Ser Phe 35 4 O 45 Gly Lieu. His Val Gly Tyr Pro Glu Asn Glin Ala Ala Gly Ile Ala Ala SO 55 6 O US 2015/0037860 A1 Feb. 5, 2015 20
- Continued Asn Arg Asp Gly Glu Val Met Cys Glin Ala Ala Glu Asp Ile Gly Tyr 65 70 7s 8O Asp Asn Asp Ile Cys Gly Tyr Ala Arg Ile Ser Lieu Ala Tyr Ala Ala 85 90 95 Gly Phe Arg Gly Ala Asn Llys Met Asp Lys Asp Gly Asn Tyr Val Ile 1OO 105 11 O Asn Pro His Ser Gly Lys Glin Met Lys Asp Ala Asn Gly Lys Llys Val 115 12 O 125 Phe Asp Ala Asp Gly Llys Pro Val Ile Asp Pro Llys Thr Lieu Lys Pro 13 O 135 14 O Phe Ala Thir Thir Asp Asn. Ile Tyr Glu Ile Ala Ala Lieu Pro Glu Gly 145 150 155 160 Glu Glu Lys Thr Arg Arg Glin Asn Ala Lieu. His Llys Tyr Arg Gln Met 1.65 17O 17s Thr Met Pro Met Pro Asp Phe Val Lieu. Cys Cys Asn Asn Ile Cys Asn 18O 185 19 O Cys Met Thir Lys Trp Tyr Glu Asp Ile Ala Arg Arg His Asn. Ile Pro 195 2OO 2O5 Lieu. Ile Met Ile Asp Val Pro Tyr Asn. Glu Phe Asp His Val Asn. Glu 21 O 215 22O Ala Asn. Wall Lys Tyr Ile Arg Ser Glin Lieu. Asp Thr Ala Ile Arg Glin 225 23 O 235 24 O Met Glu Glu Ile Thr Gly Llys Llys Phe Asp Glu Asp Llys Phe Glu Gln 245 250 255 Cys Cys Glin Asn Ala Asn Arg Thr Ala Lys Ala Trp Lieu Lys Val Cys 26 O 265 27 O Asp Tyr Lieu. Glin Tyr Lys Pro Ala Pro Phe Asn Gly Phe Asp Leu Phe 27s 28O 285 Asn His Met Ala Asp Val Val Thir Ala Arg Gly Arg Val Glu Ala Ala 29 O 295 3 OO Glu Ala Phe Glu Lieu. Lieu Ala Lys Glu Lieu. Glu Gln His Wall Lys Glu 3. OS 310 315 32O Gly. Thir Thr Thr Ala Pro Phe Lys Glu Gln His Arg Ile Met Phe Glu 3.25 330 335 Gly Ile Pro Cys Trp Pro Llys Lieu Pro Asn Lieu. Phe Llys Pro Lieu Lys 34 O 345 35. O Ala Asn Gly Lieu. Asn. Ile Thr Gly Val Val Tyr Ala Pro Ala Phe Gly 355 360 365 Phe Val Tyr Asn. Asn Lieu. Asp Glu Lieu Val Lys Ala Tyr Cys Lys Ala 37 O 375 38O Pro Asn. Ser Val Ser Ile Glu Glin Gly Val Ala Trp Arg Glu Gly Lieu. 385 390 395 4 OO Ile Arg Asp Asn Llys Val Asp Gly Val Lieu Val His Tyr Asn Arg Ser 4 OS 41O 415
Cys Llys Pro Trp Ser Gly Tyr Met Pro Glu Met Glin Arg Arg Phe Thr 42O 425 43 O
Lys Asp Met Gly Ile Pro Thr Ala Gly Phe Asp Gly Asp Glin Ala Asp 435 44 O 445
Pro Arg Asn. Phe Asn Ala Ala Glin Tyr Glu Thir Arg Val Glin Gly Lieu. 450 45.5 460
Val Glu Ala Met Glu Ala Asn Asp Glu Lys Lys Gly Lys US 2015/0037860 A1 Feb. 5, 2015 21
- Continued
465 470 47s
<210s, SEQ ID NO 7 &211s LENGTH: 379 212. TYPE: PRT <213> ORGANISM: Acidaminococcus fermentans
<4 OO > SEQUENCE: 7 Met Ala Ile Ser Ala Lieu. Ile Glu Glu Phe Glin Llys Val Ser Ala Ser 1. 5 1O 15 Pro Llys Thr Met Lieu Ala Lys Tyr Lys Ala Glin Gly Llys Lys Ala Ile 2O 25 3O Gly Cys Lieu Pro Tyr Tyr Val Pro Glu Glu Lieu Val Tyr Ala Ala Gly 35 4 O 45 Met Val Pro Met Gly Val Trp Gly Cys Asn Gly Lys Glin Glu Val Arg SO 55 6 O Ser Lys Glu Tyr Cys Ala Ser Phe Tyr Cys Thr Ile Ala Glin Glin Ser 65 70 7s 8O Lieu. Glu Met Lieu. Lieu. Asp Gly. Thir Lieu. Asp Gly Lieu. Asp Gly Ile Ile 85 90 95 Thr Pro Val Lieu. Cys Asp Thr Lieu. Arg Pro Met Ser Glin Asin Phe Lys 1OO 105 11 O Val Ala Met Lys Asp Llys Met Pro Val Ile Phe Leu Ala His Pro Glin 115 12 O 125 Val Arg Glin Asn Ala Ala Gly Lys Glin Phe Thr Tyr Asp Ala Tyr Ser 13 O 135 14 O Glu Val Lys Gly His Lieu. Glu Glu Ile Cys Gly His Glu Ile Thr Asn 145 150 155 160 Asp Ala Ile Lieu. Asp Ala Ile Llys Val Tyr Asn Llys Ser Arg Ala Ala 1.65 17O 17s Arg Arg Glu Phe Cys Llys Lieu Ala Asn. Glu. His Pro Asp Lieu. Ile Pro 18O 185 19 O Ala Ser Val Arg Ala Thr Val Lieu. Arg Ala Ala Tyr Phe Met Lieu Lys 195 2OO 2O5 Asp Glu Tyr Thr Glu Lys Lieu. Glu Glu Lieu. Asn Lys Glu Lieu Ala Ala 21 O 215 22O Ala Pro Ala Gly Llys Phe Asp Gly. His Llys Val Val Val Ser Gly Ile 225 23 O 235 24 O Ile Tyr Asn Met Pro Gly Ile Lieu Lys Ala Met Asp Asp Asn Llys Lieu. 245 250 255 Ala Ile Ala Ala Asp Asp Cys Ala Tyr Glu Ser Arg Ser Phe Ala Val 26 O 265 27 O Asp Ala Pro Glu Asp Lieu. Asp Asn Gly Lieu. Glin Ala Lieu Ala Val Glin 27s 28O 285
Phe Ser Lys Gln Lys Asn Asp Val Lieu. Lieu. Tyr Asp Pro Glu Phe Ala 29 O 295 3 OO Lys Asn. Thir Arg Ser Glu. His Val Cys Asn Lieu Val Lys Glu Ser Gly 3. OS 310 315 32O
Ala Glu Gly Lieu. Ile Val Phe Met Met Glin Phe Cys Asp Pro Glu Glu 3.25 330 335 Met Glu Tyr Pro Asp Lieu Lys Lys Ala Lieu. Asp Ala His His Ile Pro 34 O 345 35. O US 2015/0037860 A1 Feb. 5, 2015 22
- Continued His Val Lys Ile Gly Val Asp Gln Met Thir Arg Asp Phe Gly Glin Ala 355 360 365
Gln Thr Ala Lieu. Glu Ala Phe Ala Glu Ser Lieu. 37 O 375
<210s, SEQ ID NO 8 &211s LENGTH: 314 212. TYPE: PRT <213> ORGANISM: Streptococcus pyogenes M1
<4 OOs, SEQUENCE: 8 Met Asp Pro Asn Val Ile Thr Val Thr Ser Tyr Ala Asn Ile Ala Ile 1. 5 1O 15 Ile Llys Tyr Trp Gly Lys Glu Asn Glin Ala Lys Met Ile Pro Ser Thr 2O 25 3O
Ser Ser Ile Ser Lieu. Thir Lieu. Glu Asn. Met Phe Thir Thir Thir Ser Wall 35 4 O 45 Ser Phe Leu Pro Asp Thr Ala Thr Ser Asp Glin Phe Tyr Ile Asin Gly SO 55 6 O Ile Lieu. Glin Asn Asp Glu Glu. His Thir Lys Ile Ser Ala Ile Ile Asp 65 70 7s 8O Glin Phe Arg Gln Pro Gly Glin Ala Phe Val Lys Met Glu Thr Glin Asn 85 90 95 Asn Met Pro Thr Ala Ala Gly Lieu Ser Ser Ser Ser Ser Gly Lieu. Ser 1OO 105 11 O Ala Lieu Val Lys Ala Cys Asp Gln Lieu. Phe Asp Thr Glin Lieu. Asp Glin 115 12 O 125 Lys Ala Lieu Ala Glin Lys Ala Lys Phe Ala Ser Gly Ser Ser Ser Arg 13 O 135 14 O Ser Phe Phe Gly Pro Val Ala Ala Trp Asp Lys Asp Ser Gly Ala Ile 145 150 155 160 Tyr Llys Val Glu Thir Asp Lieu Lys Met Ala Met Ile Met Lieu Val Lieu. 1.65 17O 17s Asn Ala Ala Lys Llys Pro Ile Ser Ser Arg Glu Gly Met Lys Lieu. Cys 18O 185 19 O Arg Asp Thir Ser Thr Thr Phe Asp Gln Trp Val Glu Gln Ser Ala Ile 195 2OO 2O5 Asp Tyr Gln His Met Lieu. Thir Tyr Lieu Lys Thr Asn Asn Phe Glu Lys 21 O 215 22O Val Gly Glin Lieu. Thr Glu Ala Asn Ala Lieu Ala Met His Ala Thir Thr 225 23 O 235 24 O Lys Thr Ala Asn Pro Pro Phe Ser Tyr Lieu. Thir Lys Glu Ser Tyr Glin 245 250 255
Ala Met Glu Ala Val Lys Glu Lieu. Arg Glin Glu Gly Phe Ala Cys Tyr 26 O 265 27 O
Phe Thr Met Asp Ala Gly Pro Asn Val Llys Val Lieu. Cys Lieu. Glu Lys 27s 28O 285
Asp Lieu Ala Glin Lieu Ala Glu Arg Lieu. Gly Lys Asn Tyr Arg Ile Ile 29 O 295 3 OO
Val Ser Lys Thir Lys Asp Lieu Pro Asp Wall 3. OS 310
<210s, SEQ ID NO 9 US 2015/0037860 A1 Feb. 5, 2015 23
- Continued
&211s LENGTH: 356 212. TYPE: PRT <213> ORGANISM: Thioalkalimicrobium aerophilum AL3 <4 OOs, SEQUENCE: 9 Met Pro Thr Pro Asn Pro Arg Glin Val Ala Phe Val Glin Ala Val Lieu. 1. 5 1O 15 Ala Thr Gly Lys Glin Ala Cys Ser Ser Ala Ser Thir Ile Llys Lieu. Glu 2O 25 3O Gly Lys Gly His Ala Pro Val Asn. Ile Ala Lieu. Ser Llys Tyr Trp Gly 35 4 O 45 Lys Arg Asp Thir Ile Lieu. Asn Lieu Pro Glin Asn Gly Ser Val Ser Ile SO 55 6 O Ser Lieu Pro Gly Lieu. Gly Thr Asp Thir Thr Lieu. Arg Pro Lieu Ala Ser 65 70 7s 8O Asp Ser Ser Glin Glin Val Thr Ala Glin Asp Arg Ile Ser Lieu. Asn Gly 85 90 95 Glin Glin Lieu. Asp Ala His Glin Pro Phe Ala His Arg Lieu. Ser Glin Phe 1OO 105 11 O Lieu. Asp Leu Phe Arg Thr Ala Glu Val Pro Phe Phe Glu Val Ile Thr 115 12 O 125 His Asn Thr Val Pro Thr Ala Ala Gly Lieu Ala Ser Ser Ala Ser Gly 13 O 135 14 O Tyr Ala Ala Lieu Val Lieu Ala Lieu. Asp Asp Lieu Phe ASn Trp Gln Lieu 145 150 155 160 Pro Ala Thr Glin Lieu. Ser Lieu. Lieu Ala Arg Lieu. Gly Ser Gly Ser Ala 1.65 17O 17s Ser Arg Ser Leu Phe Pro Gly Phe Ala Ile Trp His Ala Gly Glin Ser 18O 185 19 O Glu Glin Gly Lieu. Asp Ser Phe Ala Glu Ala Lieu. Asp Ala Pro Trp Pro 195 2OO 2O5 Asp Phe Cys Val Gly Lieu Val Glu Ile Asp Val Ala Glu, Llys Pro Val 21 O 215 22O Gly Ser Thr Ala Gly Met Glin Gln Thr Thr Ala Ala Cys Ala Leu Tyr 225 23 O 235 24 O Ser Ala Trp Pro Ala Glin Ala Glu Arg Asp Lys Ala Val Ile Ile Asn 245 250 255 Ala Ile Glin Glin Glin Asp Phe Ser Glin Lieu. Gly Ala Thr Ala Glu. His 26 O 265 27 O Asn Ala Leu Ser Met His Ala Thr Met Ile Ala Ser Trp Pro Pro Leu 27s 28O 285 Lieu. Tyr Trp Glin Ala Glu Ser Val Ile Ala Met Glin Llys Val Trp Ala 29 O 295 3 OO
Lieu. Arg Glin Glin Gly Val Glu Val Tyr Phe Thr Met Asp Ala Gly Pro 3. OS 310 315 32O
Asn Lieu Lys Lieu Lleu Phe Lieu Ala Ala Glin Llys Lys Ala Val Ser Ala 3.25 330 335
Ala Phe Ser Gly Lieu Lys Val Ile Glu Pro Phe Ala Lys Pro Asp Thr 34 O 345 35. O
Glin Ala Ala Ser 355 US 2015/0037860 A1 Feb. 5, 2015 24
- Continued
<210s, SEQ ID NO 10 &211s LENGTH: 317 212. TYPE: PRT <213> ORGANISM: Streptococcus pneumoniae
<4 OOs, SEQUENCE: 10 Met Asp Arg Glu Pro Val Thr Val Arg Ser Tyr Ala Asn. Ile Ala Ile 1. 5 1O 15 Ile Llys Tyr Trp Gly Llys Llys Lys Glu Lys Glu Met Val Pro Ala Thr 2O 25 3O Ser Ser Ile Ser Lieu. Thir Lieu. Glu Asn Met Tyr Thr Glu Thir Thr Lieu. 35 4 O 45 Ser Pro Leu Pro Ala Asn Val Thr Ala Asp Glu Phe Tyr Ile Asin Gly SO 55 6 O Glin Lieu. Glin Asn. Glu Val Glu. His Ala Lys Met Ser Lys Ile Ile Asp 65 70 7s 8O Arg Tyr Arg Pro Ala Gly Glu Gly Phe Val Arg Ile Asp Thr Glin Asn 85 90 95 Asn Met Pro Thr Ala Ala Gly Lieu Ser Ser Ser Ser Ser Gly Lieu. Ser 1OO 105 11 O Ala Lieu Val Lys Ala Cys Asn Ala Tyr Phe Llys Lieu. Gly Lieu. Asp Arg 115 12 O 125 Ser Glin Lieu Ala Glin Glu Ala Lys Phe Ala Ser Gly Ser Ser Ser Arg 13 O 135 14 O Ser Phe Tyr Gly Pro Lieu. Gly Ala Trp Asp Lys Asp Ser Gly Glu Ile 145 150 155 160 Tyr Pro Val Glu Thir Asp Lieu Lys Lieu Ala Met Ile Met Lieu Val Lieu. 1.65 17O 17s Glu Asp Llys Llys Llys Pro Ile Ser Ser Arg Asp Gly Met Lys Lieu. Cys 18O 185 19 O Val Glu Thir Ser Thr Thr Phe Asp Asp Trp Val Arg Glin Ser Glu Lys 195 2OO 2O5 Asp Tyr Glin Asp Met Lieu. Ile Tyr Lieu Lys Glu Asn Asp Phe Ala Lys 21 O 215 22O Ile Gly Glu Lieu. Thr Glu Lys Asn Ala Lieu Ala Met His Ala Thir Thr 225 23 O 235 24 O Lys Thr Ala Ser Pro Ala Phe Ser Tyr Lieu. Thr Asp Ala Ser Tyr Glu 245 250 255 Ala Met Asp Phe Val Arg Glin Lieu. Arg Glu Lys Gly Glu Ala Cys Tyr 26 O 265 27 O Phe Thr Met Asp Ala Gly Pro Asn Val Llys Val Phe Cys Glin Glu Lys 27s 28O 285 Asp Lieu. Glu. His Lieu. Ser Glu Ile Phe Gly His Arg Tyr Arg Lieu. Ile 29 O 295 3 OO
Val Ser Lys Thir Lys Asp Lieu. Ser Glin Asp Asp Cys Cys 3. OS 310 315
<210s, SEQ ID NO 11 &211s LENGTH: 396 212. TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae
<4 OOs, SEQUENCE: 11 Met Thr Val Tyr Thr Ala Ser Val Thr Ala Pro Val Asn Ile Ala Thr US 2015/0037860 A1 Feb. 5, 2015 25
- Continued
1. 5 1O 15 Lieu Lys Tyr Trp Gly Lys Arg Asp Thir Lys Lieu. Asn Lieu Pro Thr Asn 2O 25 3O Ser Ser Ile Ser Val Thir Lieu. Ser Glin Asp Asp Lieu. Arg Thir Lieu. Thir 35 4 O 45 Ser Ala Ala Thir Ala Pro Glu Phe Glu Arg Asp Thir Lieu. Trp Lieu. Asn SO 55 6 O Gly Glu Pro His Ser Ile Asp Asn. Glu Arg Thr Glin Asn. Cys Lieu. Arg 65 70 7s 8O Asp Lieu. Arg Glin Lieu. Arg Lys Glu Met Glu Ser Lys Asp Ala Ser Lieu 85 90 95 Pro Thr Lieu Ser Gln Trp Llys Lieu. His Ile Val Ser Glu Asn Asn Phe 1OO 105 11 O Pro Thr Ala Ala Gly Lieu Ala Ser Ser Ala Ala Gly Phe Ala Ala Lieu. 115 12 O 125 Val Ser Ala Ile Ala Lys Lieu. Tyr Gln Leu Pro Glin Ser Thr Ser Glu 13 O 135 14 O Ile Ser Arg Ile Ala Arg Lys Gly Ser Gly Ser Ala Cys Arg Ser Lieu. 145 150 155 160 Phe Gly Gly Tyr Val Ala Trp Glu Met Gly Lys Ala Glu Asp Gly His 1.65 17O 17s Asp Ser Met Ala Val Glin Ile Ala Asp Ser Ser Asp Trp Pro Gln Met 18O 185 19 O Lys Ala Cys Val Lieu Val Val Ser Asp Ile Llys Lys Asp Val Ser Ser 195 2OO 2O5 Thr Glin Gly Met Gln Lieu. Thr Val Ala Thr Ser Glu Lieu Phe Lys Glu 21 O 215 22O Arg Ile Glu. His Val Val Pro Lys Arg Phe Glu Wal Met Arg Lys Ala 225 23 O 235 24 O Ile Val Glu Lys Asp Phe Ala Thr Phe Ala Lys Glu Thr Met Met Asp 245 250 255 Ser Asn Ser Phe His Ala Thr Cys Lieu. Asp Ser Phe Pro Pro Ile Phe 26 O 265 27 O Tyr Met Asn Asp Thr Ser Lys Arg Ile Ile Ser Trp Cys His Thir Ile 27s 28O 285 Asn Glin Phe Tyr Gly Glu Thir Ile Val Ala Tyr Thr Phe Asp Ala Gly 29 O 295 3 OO Pro Asn Ala Val Lieu. Tyr Tyr Lieu Ala Glu Asn. Glu Ser Lys Lieu. Phe 3. OS 310 315 32O Ala Phe Ile Tyr Llys Lieu. Phe Gly Ser Val Pro Gly Trp Asp Llys Llys 3.25 330 335
Phe Thir Thr Glu Glin Lieu. Glu Ala Phe Asn His Glin Phe Glu Ser Ser 34 O 345 35. O
Asn Phe Thir Ala Arg Glu Lieu. Asp Lieu. Glu Lieu Gln Lys Asp Wall Ala 355 360 365
Arg Val Ile Lieu. Thr Glin Val Gly Ser Gly Pro Glin Glu Thir Asn Glu 37 O 375 38O
Ser Lieu. Ile Asp Ala Lys Thr Gly Lieu Pro Lys Glu 385 390 395
<210s, SEQ ID NO 12 US 2015/0037860 A1 Feb. 5, 2015 26
- Continued
&211s LENGTH: 318 212. TYPE: PRT <213> ORGANISM: Thermoplasma acidophilum
<4 OOs, SEQUENCE: 12 Met Thr Tyr Arg Ser Ile Gly Ser Thr Ala Tyr Pro Thr Ile Gly Val 1. 5 1O 15 Val Lieu. Leu Gly Gly Ile Ala Asn Pro Val Thr Arg Thr Pro Leu. His 2O 25 3O Thir Ser Ala Gly Ile Ala Tyr Ser Asp Ser Cys Gly Ser Ile Arg Ser 35 4 O 45 Glu Thr Arg Ile Tyr Ala Asp Glu Ala Thr His Ile Tyr Phe Asin Gly SO 55 6 O Thr Glu Ser Thir Asp Asp Asn Arg Ser Val Arg Arg Val Lieu. Asp Arg 65 70 7s 8O Tyr Ser Ser Val Phe Glu Glu Ala Phe Gly Thr Lys Thr Val Ser Tyr 85 90 95 Ser Ser Glin Asn. Phe Gly Ile Lieu. Ser Gly Ser Ser Asp Ala Gly Ala 1OO 105 11 O Ala Ser Ile Gly Ala Ala Ile Lieu. Gly Lieu Lys Pro Asp Lieu. Asp Pro 115 12 O 125 His Asp Val Glu Asn Asp Lieu. Arg Ala Val Ser Glu Ser Ala Gly Arg 13 O 135 14 O Ser Lieu Phe Gly Gly Lieu. Thir Ile Thir Trp Ser Asp Gly Phe His Ala 145 150 155 160 Tyr Thr Glu Lys Ile Lieu. Asp Pro Glu Ala Phe Ser Gly Tyr Ser Ile 1.65 17O 17s Val Ala Phe Ala Phe Asp Tyr Glin Arg Asin Pro Ser Asp Val Ile His 18O 185 19 O Glin Asn. Ile Val Arg Ser Asp Lieu. Tyr Pro Ala Arg Llys Llys His Ala 195 2OO 2O5 Asp Glu. His Ala His Met Ile Lys Glu Tyr Ala Lys Thr Asn Asp Ile 21 O 215 22O Lys Gly Ile Phe Asp Lieu Ala Glin Glu Asp Thr Glu Glu Tyr His Ser 225 23 O 235 24 O Ile Lieu. Arg Gly Val Gly Val Asin Val Ile Arg Glu Asn Met Glin Lys 245 250 255 Lieu. Ile Ser Tyr Lieu Lys Lieu. Ile Arg Lys Asp Tyr Trp Asn Ala Tyr 26 O 265 27 O Ile Val Thr Gly Gly Ser Asn Val Tyr Val Ala Val Glu Ser Glu Asn 27s 28O 285 Ala Asp Arg Lieu. Phe Ser Ile Glu Asn. Thir Phe Gly Ser Lys Llys Llys 29 O 295 3 OO Met Lieu. Arg Ile Val Gly Gly Ala Trp His Arg Arg Pro Glu 3. OS 310 315
<210s, SEQ ID NO 13 &211s LENGTH: 397 212. TYPE: PRT <213> ORGANISM: Castellaniella defragrans
<4 OOs, SEQUENCE: 13 Met Arg Phe Thr Lieu Lys Thir Thr Ala Ile Val Ser Ala Ala Ala Lieu. 1. 5 1O 15 US 2015/0037860 A1 Feb. 5, 2015 27
- Continued
Lieu Ala Gly Phe Gly Pro Pro Pro Arg Ala Ala Glu Lieu Pro Pro Gly 2O 25 3O Arg Lieu Ala Thir Thr Glu Asp Tyr Phe Ala Glin Glin Ala Lys Glin Ala 35 4 O 45 Val Thr Pro Asp Val Met Ala Gln Leu Ala Tyr Met Asn Tyr Ile Asp SO 55 6 O Phe Ile Ser Pro Phe Tyr Ser Arg Gly Cys Ser Phe Glu Ala Trp Glu 65 70 7s 8O Lieu Lys His Thr Pro Glin Arg Val Ile Llys Tyr Ser Ile Ala Phe Tyr 85 90 95 Ala Tyr Gly Lieu Ala Ser Val Ala Lieu. Ile Asp Pro Llys Lieu. Arg Ala 1OO 105 11 O Lieu Ala Gly. His Asp Lieu. Asp Ile Ala Val Ser Lys Met Lys Cys Llys 115 12 O 125 Arg Val Trp Gly Asp Trp Glu Glu Asp Gly Phe Gly. Thir Asp Pro Ile 13 O 135 14 O Glu Lys Glu Asn. Ile Met Tyr Lys Gly. His Lieu. Asn Lieu Met Tyr Gly 145 150 155 160 Lieu. Tyr Glin Lieu Val Thr Gly Ser Arg Arg Tyr Glu Ala Glu. His Ala 1.65 17O 17s His Lieu. Thir Arg Ile Ile His Asp Glu Ile Ala Ala Asn Pro Phe Ala 18O 185 19 O Gly Ile Val Cys Glu Pro Asp Asn Tyr Phe Val Glin Cys Asn Ser Val 195 2OO 2O5 Ala Tyr Lieu. Ser Lieu. Trp Val Tyr Asp Arg Lieu. His Gly Thr Asp Tyr 21 O 215 22O Arg Ala Ala Thr Arg Ala Trp Lieu. Asp Phe Ile Gln Lys Asp Lieu. Ile 225 23 O 235 24 O Asp Pro Glu Arg Gly Ala Phe Tyr Lieu Ser Tyr His Pro Glu Ser Gly 245 250 255 Ala Val Llys Pro Trp Ile Ser Ala Tyr Thr Thr Ala Trp Thr Lieu Ala 26 O 265 27 O Met Val His Gly Met Asp Pro Ala Phe Ser Glu Arg Tyr Tyr Pro Arg 27s 28O 285 Phe Lys Glin Thir Phe Val Glu Val Tyr Asp Glu Gly Arg Lys Ala Arg 29 O 295 3 OO Val Arg Glu Thir Ala Gly Thr Asp Asp Ala Asp Gly Gly Val Gly Lieu 3. OS 310 315 32O Ala Ser Ala Phe Thr Lieu Lleu Lieu Ala Arg Glu Met Gly Asp Glin Glin 3.25 330 335 Lieu. Phe Asp Gln Lieu. Lieu. Asn His Lieu. Glu Pro Pro Ala Lys Pro Ser 34 O 345 35. O Ile Val Ser Ala Ser Lieu. Arg Tyr Glu. His Pro Gly Ser Lieu. Leu Phe 355 360 365 Asp Glu Lieu. Lieu. Phe Lieu Ala Lys Val His Ala Gly Phe Gly Ala Lieu 37 O 375 38O
Lieu. Arg Met Pro Pro Pro Ala Ala Lys Lieu Ala Gly Lys 385 390 395
<210s, SEQ ID NO 14 &211s LENGTH: 386 US 2015/0037860 A1 Feb. 5, 2015 28
- Continued
212. TYPE: PRT <213> ORGANISM: Streptomyces kanamyceticus
<4 OOs, SEQUENCE: 14 Met Ser Glu Pro Glu. His Lieu. Asp Thr Val Arg Llys Phe Val Ala Glin 1. 5 1O 15 Glu Val Lieu. Gly Arg Glu Thir His Lieu. Asp Ser Lieu Ala Asp Ala Pro 2O 25 3O Lieu Ala Lieu. Tyr Glu Arg Phe Ala Glu Thr Gly Lieu Met Asn Trp Trp 35 4 O 45 Val Pro Glu Glu. His Gly Gly Lieu. Gly Lieu. Gly Lieu. Glu Asp Ser Val SO 55 6 O Arg Ile Val Ser Glu Lieu Ala Tyr Gly Asp Ala Gly Val Ala Phe Thr 65 70 7s 8O Lieu. Phe Leu Pro Ile Lieu. Thir Thr Ser Met Val Ser Trp Tyr Gly Ser 85 90 95 Ala Glu Lieu Lys Glu Lys Lieu. Lieu. Asp Pro Lieu Val Ala His Arg Gly 1OO 105 11 O Phe Cys Ala Thir Lieu. Gly Ser Glu. His Glu Ala Gly Ser Glu Lieu Ala 115 12 O 125 Lys Ile Ser Thr Val Val Arg Arg Asp Gly Glu Gly Lieu Val Lieu. Asp 13 O 135 14 O Gly Thr Lys Ala Phe Ser Thr Ser Thr Asp Phe Ala Glin Phe Leu Val 145 150 155 16 O Val Ile Ala Arg Ser Ala Glu Asn Pro Thr Arg Tyr Lieu Ala Val Ala 1.65 17O 17s Val Glu Arg Asp Ala Pro Gly Lieu. Arg Ile Asp Lys Arg Trp Asp Wall 18O 185 19 O Ile Gly Lieu. Arg Ala Ser Ala Thir Tyr Glin Val Ser Phe Ser Asp Cys 195 2OO 2O5 His Val Pro Ala Gly Asn Ala Lieu. Asp Gly His Gly Lieu. Arg Lieu. Lieu. 21 O 215 22O Glu Ile Gly Lieu. Asn Ala Ser Arg Ile Lieu. Ile Ala Ala Thr Ala Lieu. 225 23 O 235 24 O Gly Val Ala Arg Arg Ile Arg Asp Lieu. Cys Met Glu Tyr Ala Lys Thr 245 250 255 Llys Ser Lieu Lys Gly Ala Pro Lieu Val Asn Asp Ala Val Phe Ala Gly 26 O 265 27 O Arg Lieu. Gly Glin Phe Glu Met Glin Ile Glu Val Met Ala Asn Glin Cys 27s 28O 285 Lieu Ala Ala Ala Arg Thr Tyr Asp Ala Thir Ala Ala Arg Pro Asp Ala 29 O 295 3 OO
Ala Arg Thir Lieu. Lieu. Arg Glin Gly Ala Glin Llys Ser Ala Lieu. Thir Ala 3. OS 310 315 32O
Lys Met Phe Cys Gly Glin Thr Ala Trp Glin Ile Ala Ser Thr Ala Ser 3.25 330 335 Glu Met Phe Gly Gly Ile Gly Tyr Thr His Asp Val Pro Ile Gly Lys 34 O 345 35. O Lieu. Lieu. Arg Asp Val Arg His Ala Ser Ile Ile Glu Gly Gly Asp Asp 355 360 365
Val Lieu. Arg Asp Lieu Val Phe His Arg Phe Val Val Pro Thr Ala Lys 37 O 375 38O US 2015/0037860 A1 Feb. 5, 2015 29
- Continued
Arg Thr 385
<210s, SEQ ID NO 15 &211s LENGTH: 470 212. TYPE: PRT <213> ORGANISM: Aquincola tertiari carbonis <4 OOs, SEQUENCE: 15 Met Gly Asn Arg Glu Pro Lieu Ala Ala Ala Gly Glin Gly Thr Ala Tyr 1. 5 1O 15 Ser Gly Tyr Arg Lieu. Arg Asp Lieu. Glin Asn Val Ala Pro Thir Asn Lieu. 2O 25 3O Glu Ile Leu Arg Thr Gly Pro Gly Thr Pro Met Gly Glu Tyr Met Arg 35 4 O 45 Arg Tyr Trp Gln Pro Val Cys Lieu Ser Glin Glu Lieu. Thr Asp Val Pro SO 55 6 O Lys Ala Ile Arg Ile Lieu. His Glu Asp Lieu Val Ala Phe Arg Asp Arg 65 70 7s 8O Arg Gly Asn Val Gly Val Lieu. His Arg Lys Cys Ala His Arg Gly Ala 85 90 95 Ser Lieu. Glu Phe Gly Ile Val Glin Glu Arg Gly Ile Arg Cys Cys Tyr 1OO 105 11 O His Gly Trp His Phe Asp Val Asp Gly Ser Lieu Lieu. Glu Ala Pro Ala 115 12 O 125 Glu Pro Pro Asp Thr Lys Lieu Lys Glu Thr Val Cys Glin Gly Ala Tyr 13 O 135 14 O Pro Ala Phe Glu Arg Asn Gly Lieu Val Phe Ala Tyr Met Gly Pro Ala 145 150 155 160 Asp Arg Arg Pro Glu Phe Pro Val Phe Asp Gly Tyr Val Lieu Pro Llys 1.65 17O 17s Gly Thr Arg Lieu. Ile Pro Phe Ser Asn Val Phe Asp Cys Asn Trp Leu 18O 185 19 O Glin Val Tyr Glu Asn Glin Ile Asp His Tyr His Thr Ala Lieu. Lieu. His 195 2OO 2O5 Asn Asn Met Thr Val Ala Gly Val Asp Ala Lys Lieu Ala Asp Gly Ala 21 O 215 22O Thr Lieu. Glin Gly Gly Phe Gly Glu Met Pro Ile Ile Asp Trp His Pro 225 23 O 235 24 O Thir Asp Asp Asn. Asn Gly Met Ile Phe Thir Ala Gly Arg Arg Lieu. Ser 245 250 255 Asp Asp Glu Val Trp Ile Arg Ile Ser Glin Met Gly Lieu Pro Asn Trp 26 O 265 27 O
Met Glin Asn Ala Ala Ile Val Ala Ala Ala Pro Glin Arg His Ser Gly 27s 28O 285
Pro Ala Met Ser Arg Trp Glin Val Pro Val Asp Asp Glu. His Ser Ile 29 O 295 3 OO Ala Phe Gly Trp Arg His Phe Asn Asp Glu Val Asp Pro Glu. His Arg 3. OS 310 315 32O
Gly Arg Glu Glu Glu. Cys Gly Val Asp Llys Ile Asp Phe Lieu. Ile Gly 3.25 330 335 Gln Thr Arg His Arg Pro Tyr Glu Glu Thr Glin Arg Val Pro Gly Asp US 2015/0037860 A1 Feb. 5, 2015 30
- Continued
34 O 345 35. O Tyr Glu Ala Ile Val Ser Glin Gly Pro Ile Ala Lieu. His Gly Lieu. Glu 355 360 365 His Pro Gly Arg Ser Asp Val Gly Val Tyr Met Cys Arg Ser Lieu. Lieu. 37 O 375 38O Arg Asp Ala Val Ala Gly Lys Ala Pro Pro Asp Pro Val Arg Val Lys 385 390 395 4 OO Ala Gly Ser Thr Asp Gly Glin Thr Lieu Pro Arg Tyr Ala Ser Asp Ser 4 OS 41O 415 Arg Lieu. Arg Ile Arg Arg Arg Pro Ser Arg Glu Ala Asp Ser Asp Wall 42O 425 43 O Ile Arg Lys Ala Ala His Glin Val Phe Ala Ile Met Lys Glu. Cys Asp 435 44 O 445 Glu Lieu Pro Val Val Glin Arg Arg Pro His Val Lieu. Arg Arg Lieu. Asp 450 45.5 460
Glu Ile Glu Ala Ser Lieu 465 470
<210s, SEQ ID NO 16 &211s LENGTH: 134 212. TYPE: PRT <213> ORGANISM: Aeromonas punctata
<4 OOs, SEQUENCE: 16 Met Ser Ala Glin Ser Lieu. Glu Val Gly Glin Lys Ala Arg Lieu. Ser Lys 1. 5 1O 15 Arg Phe Gly Ala Ala Glu Val Ala Ala Phe Ala Ala Lieu. Ser Glu Asp 2O 25 3O Phe Asin Pro Leu. His Lieu. Asp Pro Ala Phe Ala Ala Thr Thr Ala Phe 35 4 O 45 Glu Arg Pro Ile Val His Gly Met Lieu. Lieu Ala Ser Lieu. Phe Ser Gly SO 55 6 O Lieu. Lieu. Gly Glin Glin Lieu Pro Gly Lys Gly Ser Ile Tyr Lieu. Gly Glin 65 70 7s 8O Ser Leu Ser Phe Lys Lieu Pro Val Phe Val Gly Asp Glu Val Thr Ala 85 90 95 Glu Val Glu Val Thr Ala Lieu. Arg Glu Asp Llys Pro Ile Ala Thr Lieu. 1OO 105 11 O Thir Thr Arg Ile Phe Thr Glin Gly Gly Ala Leu Ala Val Thr Gly Glu 115 12 O 125 Ala Val Val Lys Lieu Pro 13 O
<210s, SEQ ID NO 17 &211s LENGTH: 177 212. TYPE: PRT <213> ORGANISM: Escherichia coli
<4 OOs, SEQUENCE: 17 Met Lieu Ala Ala Ile Ser Lys Glin Trp Val Arg Gly Ala Lys Val Glu 1. 5 1O 15 Glu Asp Arg Ile His Pro Phe Arg Llys Tyr Phe Glu Glu Lieu. Glin Pro 2O 25 3O
Gly Asp Ser Lieu. Lieu. Thr Pro Arg Arg Thr Met Thr Glu Ala Asp Ile US 2015/0037860 A1 Feb. 5, 2015 31
- Continued
35 4 O 45 Val Asn. Phe Ala Cys Lieu. Ser Gly Asp His Phe Tyr Ala His Met Asp SO 55 6 O Lys Ile Ala Ala Ala Glu Ser Ile Phe Gly Glu Arg Val Val His Gly 65 70 7s 8O Tyr Phe Val Lieu. Ser Ala Ala Ala Gly Lieu. Phe Val Asp Ala Gly Val 85 90 95 Gly Pro Val Ile Ala Asn Tyr Gly Lieu. Glu Ser Lieu. Arg Phe Ile Glu 1OO 105 11 O Pro Val Llys Pro Gly Asp Thir Ile Glin Val Arg Lieu. Thir Cys Lys Arg 115 12 O 125 Llys Thr Lieu Lys Lys Glin Arg Ser Ala Glu Glu, Llys Pro Thr Gly Val 13 O 135 14 O Val Glu Trp Ala Val Glu Val Phe Asin Gln His Glin Thr Pro Val Ala 145 150 155 160 Lieu. Tyr Ser Ile Lieu. Thir Lieu Val Ala Arg Gln His Gly Asp Phe Val 1.65 17O 17s Asp
<210s, SEQ ID NO 18 &211s LENGTH: 345 212. TYPE: PRT <213> ORGANISM: Streptomyces sp. CNH189
<4 OOs, SEQUENCE: 18 Met Glu Met Ala Pro Gly Tyr Val Thr Ser Val Lieu. Gly Thr Gly Ser 1. 5 1O 15 Tyr Lieu Pro Glu Arg Val Val Thr Asn. Glu Glu Ile Glu Ala Arg Val 2O 25 3O Pro Gly Ala Ser Ala Glu Trp Ile Ala Val Arg Thr Ala Ile Val Glu 35 4 O 45 Arg Arg Tyr Ala Ala Pro Asp Glu Ala Ala Ser Asp Lieu Ala Wal His SO 55 6 O Ala Ala Arg Ala Ala Lieu. Asp Glin Ala Gly Lieu. Asp Ala Asp Gly Ile 65 70 7s 8O Asp Phe Ile Ile Val Ala Thir Thr Thr Gly Asp Ala Pro Ile Pro Ser 85 90 95 Thir Ala Ser Lieu Val Glin Lieu Ala Lieu. Gly Ala Arg Arg Ala Ala Cys 1OO 105 11 O Phe Asp Val Asn Ile Ala Cys Thr Gly Phe Val Thr Ala Leu Ser Ile 115 12 O 125 Ala Arg Ala Tyr Val Ala Lieu. Asp Pro Thir Thr Llys Val Lieu Val Ile 13 O 135 14 O
Gly. Thir Asp Val Trp Thr Arg Phe Ile Asp Phe Asp Asn Arg Ala Thr 145 150 155 160
Ser Val Lieu. Phe Gly Asp Gly Ala Gly Ala Ala Val Ile Gly Ser Val 1.65 17O 17s Pro His Ala Pro Gly Asp Pro Glu Arg Gly Lieu Lleu Lys Val Glu Lieu. 18O 185 19 O Val Ser Arg Gly Asp Ala His Glu Lieu. Ile Ser Met Pro Ala Gly Gly 195 2OO 2O5 Ser Arg Arg Pro Ala Ser Val Glu Thr Val Ala Asp Gly Gly His Lieu. US 2015/0037860 A1 Feb. 5, 2015 32
- Continued
21 O 215 22O Lieu. Ser Met Glin Gly Arg Gly Val Arg Asp Phe Val Lieu. Asp Asn. Wall 225 23 O 235 24 O Pro Gly Lieu. Ile Ala Gly Lieu. Lieu Lys Arg Ser Gly His Glu Pro Ala 245 250 255 Asp Val Glin His Phe Val Pro His Glin Ala Asn Gly Arg Lieu Val Glu 26 O 265 27 O Glu Lieu Ala Gly Ala Ser Gly Lieu Val Arg Ala Asp Thr His Lieu Pro 27s 28O 285 Lieu. Arg His Ser Gly Asn. Ile Gly Ser Ala Ser Val Pro Val Ala Lieu. 29 O 295 3 OO Asp Ala Ala Asn Arg Ser Gly Val Lieu. Arg Asp Gly Asp Lieu Val Lieu 3. OS 310 315 32O Lieu Ala Gly Phe Gly Ala Gly Met Ala Ala Gly Ala Ala Lieu. Lieu. Arg 3.25 330 335 Trp. Thir Ala Thr Glu Gly Gly Thr Arg 34 O 345
<210s, SEQ ID NO 19 &211s LENGTH: 313 212. TYPE: PRT <213> ORGANISM: Synechococcus PCC 7002
<4 OOs, SEQUENCE: 19 Ser Asn Ala Ser Glin Ser Lieu. Ser Val Lys Thr Llys Lys Glin Trp Glin 1. 5 1O 15 Llys Pro Asp His Lys Asn Pro Asn Pro Ile Ala Phe Ile Lieu. Ser Ser 2O 25 3O Pro Arg Ser Gly Ser Thr Lieu. Lieu. Arg Val Met Lieu Ala Gly His Pro 35 4 O 45 Gly Lieu. Tyr Ser Pro Pro Glu Lieu. His Lieu. Leu Pro Phe Glu. Thr Met SO 55 6 O Gly Asp Arg His Glin Glu Lieu. Gly Lieu. Ser His Lieu. Gly Glu Gly Lieu. 65 70 7s 8O Glin Arg Ala Lieu Met Asp Lieu. Glu Asn Lieu. Thr Pro Glu Ala Ser Glin 85 90 95 Ala Lys Val Asn Gln Trp Val Lys Ala Asn Thr Pro Ile Ala Asp Ile 1OO 105 11 O Tyr Ala Tyr Lieu. Glin Arg Glin Ala Glu Glin Arg Lieu. Lieu. Ile Asp Llys 115 12 O 125 Ser Pro Ser Tyr Gly Ser Asp Arg His Ile Lieu. Asp His Ser Glu Ile 13 O 135 14 O Lieu. Phe Asp Glin Ala Lys Tyr Ile His Lieu Val Arg His Pro Tyr Ala 145 150 155 160
Val Ile Glu Ser Phe Thr Arg Lieu. Arg Met Asp Llys Lieu. Lieu. Gly Ala 1.65 17O 17s
Glu Glin Glin Asn Pro Tyr Ala Lieu Ala Glu Ser Ile Trp Arg Thir Ser 18O 185 19 O
Asn Arg Asn. Ile Lieu. Asp Lieu. Gly Arg Thr Val Gly Ala Asp Arg Tyr 195 2OO 2O5
Lieu. Glin Val Ile Tyr Glu Asp Lieu Val Arg Asp Pro Arg Llys Val Lieu. 21 O 215 22O US 2015/0037860 A1 Feb. 5, 2015 33
- Continued Thir Asn. Ile Cys Asp Phe Lieu. Gly Val Asp Phe Asp Glu Ala Lieu. Lieu. 225 23 O 235 24 O Asn Pro Tyr Ser Gly Asp Arg Lieu. Thir Asp Gly Lieu. His Glin Glin Ser 245 250 255 Met Gly Val Gly Asp Pro Asn Phe Leu Gln His Lys Thr Ile Asp Pro 26 O 265 27 O Ala Lieu Ala Asp Llys Trp Arg Ser Ile Thr Lieu Pro Ala Ala Lieu. Glin 27s 28O 285 Lieu. Asp Thir Ile Glin Lieu Ala Glu Thir Phe Ala Tyr Asp Lieu Pro Glin 29 O 295 3 OO
Glu Pro Gln Lieu. Thir Pro Glin Thr Glin 3. OS 310
<210s, SEQ ID NO 2 O &211s LENGTH: 323 212. TYPE: PRT <213> ORGANISM: Moorea producens 19L
<4 OOs, SEQUENCE: 2O Ser Asn Ala Ser Pro Thir Ser Lieu. Glu Ile Phe Ala Thr Lys Ser Ser 1. 5 1O 15 Pro Ser Gly Asn. Ser Ala Arg Pro Ala Ser Val Ser Ser Arg Lieu Pro 2O 25 3O Gly Ile Ile Phe Ile Leu Ser Ser Pro Arg Ser Gly Ser Thr Lieu. Leu 35 4 O 45 Arg Val Met Leu Ala Gly His Ser Ser Leu Phe Ser Pro Pro Glu Lieu. SO 55 6 O His Lieu. Lieu Pro Phe Asn. Thir Met Lys Glu Arg Glin Glu Glin Lieu. Asn 65 70 7s 8O Lieu. Ser Tyr Lieu. Gly Glu Gly Lieu Gln Lys Thr Phe Met Glu Val Lys 85 90 95 Asn Lieu. Asp Ala Thr Ala Ser Glin Ala Lieu. Ile Lys Asp Lieu. Glu Ser 1OO 105 11 O Glin Asn Lieu. Ser Ile Glin Glin Val Tyr Gly Met Lieu. Glin Glu Asn. Ile 115 12 O 125 Ala Pro Arg Lieu. Lieu Val Asp Llys Ser Pro Thr Tyr Ala Met Glu Pro 13 O 135 14 O Thir Ile Lieu. Glu Arg Gly Glu Ala Lieu. Phe Ala Asn. Ser Lys Tyr Ile 145 150 155 160 Tyr Lieu Val Arg His Pro Tyr Ser Val Ile Glu Ser Phe Val Arg Met 1.65 17O 17s Arg Met Gln Lys Lieu Val Gly Lieu. Gly Glu Glu Asn Pro Tyr Arg Val 18O 185 19 O
Ala Glu Glin Val Trp Ala Lys Ser Asn Glin Asn. Ile Lieu. Asn. Phe Lieu 195 2OO 2O5
Ser Glin Lieu. Glu Pro Glu Arg Glin His Glin Ile Arg Tyr Glu Asp Lieu. 21 O 215 22O
Val Llys Llys Pro Glin Glin Val Lieu. Ser Glin Lieu. Cys Asp Phe Lieu. Asn 225 23 O 235 24 O
Val Pro Phe Glu Pro Glu Lieu. Leu Gln Pro Tyr Glin Gly Asp Arg Met 245 250 255
Thr Gly Gly Val His Ala Ala Ser Leu Ser Ile Ser Asp Pro Asn Phe 26 O 265 27 O US 2015/0037860 A1 Feb. 5, 2015 34
- Continued
Lieu Lys His Asn. Thir Ile Asp Glu Ser Lieu Ala Asp Llys Trp Llys Thr 27s 28O 285 Ile Gln Leu Pro Tyr Pro Leu Lys Ser Glu Thr Glin Arg Ile Ala Ser 29 O 295 3 OO Gln Leu Ser Tyr Glu Lieu Pro Asn Lieu Val Thir Thr Pro Thr Asn Glin 3. OS 310 315 32O
Glin Pro Glin
<210s, SEQ ID NO 21 &211s LENGTH: 286 212. TYPE: PRT <213> ORGANISM: Moorea producens 19L <4 OOs, SEQUENCE: 21 Ser Asn Ala Met Glu Glu Lys Phe Lieu. Glu Phe Gly Gly Asn Glin Ile 1. 5 1O 15 Cys Lieu. Cys Ser Trp Gly Ser Pro Glu. His Pro Val Val Lieu. Cys Ile 2O 25 3O His Gly Ile Lieu. Glu Glin Gly Lieu Ala Trp Glin Glu Val Ala Lieu Pro 35 4 O 45 Lieu Ala Ala Glin Gly Tyr Arg Val Val Ala Pro Asp Lieu. Phe Gly His SO 55 6 O Gly Arg Ser Ser His Lieu. Glu Met Val Thr Ser Tyr Ser Ser Lieu. Thr 65 70 75 8O Phe Lieu Ala Glin Ile Asp Arg Val Ile Glin Glu Lieu Pro Asp Glin Pro 85 90 95 Lieu. Lieu. Lieu Val Gly His Ser Met Gly Ala Met Lieu Ala Thir Ala Ile 1OO 105 11 O Ala Ser Val Arg Pro Llys Lys Ile Lys Glu Lieu. Ile Lieu Val Glu Lieu 115 12 O 125 Pro Lieu Pro Ala Glu Glu Ser Lys Lys Glu Ser Ala Val Asin Glin Lieu. 13 O 135 14 O Thir Thr Cys Lieu. Asp Tyr Lieu Ser Ser Thr Pro Gln His Pro Ile Phe 145 150 155 160 Pro Asp Wall Ala Thr Ala Ala Ser Arg Lieu. Arg Glin Ala Ile Pro Ser 1.65 17O 17s Lieu. Ser Glu Glu Phe Ser Tyr Ile Leu Ala Glin Arg Ile Thr Glin Pro 18O 185 19 O Asn Glin Gly Gly Val Arg Trp Ser Trp Asp Ala Ile Ile Arg Thr Arg 195 2OO 2O5 Ser Ile Lieu. Gly Lieu. Asn. Asn Lieu Pro Gly Gly Arg Ser Glin Tyr Lieu. 21 O 215 22O
Glu Met Leu Lys Ser Ile Glin Val Pro Thr Thr Lieu Val Tyr Gly Asp 225 23 O 235 24 O
Ser Ser Lys Lieu. Asn Arg Pro Glu Asp Lieu. Glin Glin Gln Lys Met Thr 245 250 255
Met Thr Glin Ala Lys Arg Val Phe Lieu. Ser Gly Gly His Asn Lieu. His 26 O 265 27 O
Ile Asp Ala Ala Ala Ala Lieu Ala Ser Lieu. Ile Lieu. Thir Ser 27s 28O 285
<210s, SEQ ID NO 22 US 2015/0037860 A1 Feb. 5, 2015 35
- Continued
&211s LENGTH: 646 212. TYPE: PRT <213> ORGANISM: Elizabethkiingia meningos eptica
<4 OOs, SEQUENCE: 22 Met Asn Pro Ile Thir Ser Llys Phe Asp Llys Val Lieu. Asn Ala Ser Ser 1. 5 1O 15 Glu Tyr Gly His Val Asn His Glu Pro Asp Ser Ser Lys Glu Glin Glin 2O 25 3O Arg Asn Thr Pro Gln Lys Ser Met Pro Phe Ser Asp Glin Ile Gly Asn 35 4 O 45 Tyr Glin Arg Asn Lys Gly Ile Pro Val Glin Ser Tyr Asp Asn. Ser Lys SO 55 6 O Ile Tyr Ile Ile Gly Ser Gly Ile Ala Gly Met Ser Ala Ala Tyr Tyr 65 70 7s 8O Phe Ile Arg Asp Gly His Val Pro Ala Lys Asn. Ile Thr Phe Lieu. Glu 85 90 95 Glin Lieu. His Ile Asp Gly Gly Ser Lieu. Asp Gly Ala Gly Asn Pro Thr 1OO 105 11 O Asp Gly Tyr Ile Ile Arg Gly Gly Arg Glu Met Asp Met Thr Tyr Glu 115 12 O 125 Asn Lieu. Trp Asp Met Phe Glin Asp Ile Pro Ala Lieu. Glu Met Pro Ala 13 O 135 14 O Pro Tyr Ser Val Lieu. Asp Glu Tyr Arg Lieu. Ile ASn Asp ASn Asp Ser 145 150 155 160 Asn Tyr Ser Lys Ala Arg Lieu. Ile Asn. Asn Lys Gly Glu Ile Lys Asp 1.65 17O 17s Phe Ser Llys Phe Gly Lieu. Asn Llys Met Asp Gln Lieu Ala Ile Ile Arg 18O 185 19 O Lieu. Lieu. Lieu Lys Asn Lys Glu Glu Lieu. Asp Asp Lieu. Thir Ile Glu Asp 195 2OO 2O5 Tyr Phe Ser Glu Ser Phe Leu Lys Ser Asin Phe Trp Thr Phe Trp Arg 21 O 215 22O Thir Met Phe Ala Phe Glu Asn Trp His Ser Lieu. Lieu. Glu Lieu Lys Lieu. 225 23 O 235 24 O Tyr Met His Arg Phe Lieu. His Ala Ile Asp Gly Lieu. Asn Asp Lieu. Ser 245 250 255 Ser Leu Val Phe Pro Llys Tyr Asn Glin Tyr Asp Thr Phe Val Thr Pro 26 O 265 27 O Lieu. Arg Llys Phe Lieu. Glin Glu Lys Gly Val Asn. Ile His Lieu. Asn Thr 27s 28O 285 Lieu Val Lys Asp Lieu. Asp Ile His Ile Asn Thr Glu Gly Llys Val Val 29 O 295 3 OO
Glu Gly Ile Ile Thr Glu Glin Asp Gly Lys Glu Val Lys Ile Pro Val 3. OS 310 315 32O
Gly Lys Asn Asp Tyr Val Ile Val Thir Thr Gly Ser Met Thr Glu Asp 3.25 330 335
Thr Phe Tyr Gly Asn Asn Lys Thr Ala Pro Ile Ile Gly Ile Asp Asn 34 O 345 35. O
Ser Thir Ser Gly Glin Ser Ala Gly Trp Llys Lieu. Trp Lys Asn Lieu Ala 355 360 365
Ala Lys Ser Glu Ile Phe Gly Llys Pro Glu Lys Phe Cys Ser Asn. Ile US 2015/0037860 A1 Feb. 5, 2015 36
- Continued
37 O 375 38O
Glu Llys Ser Ala Trp Glu Ser Ala Thr Luell Thir Pro Ser Ala 385 390 395 4 OO
Lell Ile Asp Lell Lys Glu Tyr Ser Wall ASn Asp Pro Tyr Ser Gly 4 OS 415
Thir Wall. Thir Gly Gly Ile Ile Thr Ile Thr Asp Ser Asn Trp Lieu. 425 43 O
Met Ser Phe Thir Asn Arg Glin Pro His Phe Pro Glu Glin Pro Asp 435 44 O 445
Asp Wall Luell Wall Lieu. Trp Wall Ala Luell Phe Met Asp Glu Gly 450 45.5 460
Asn Tyr Ile Lys Thir Met Luell Glu Thir Gly Asp Glu Ile Lieu. 465 470
Ala Glu Luell Tyr His Lieu. Gly Ile Glu Asp Gln Lieu. Glu Asn. Wall 485 490 495
Glin Asn Thir Ile Wall Arg Thir Ala Phe Met Pro Ile Thir Ser SOO 505
Met Phe Met Pro Arg Ala Gly Asp Arg Pro Arg Wall Wall Pro Glu 515 525
Gly Cys Lys Asn Lieu. Gly Lell Wall Gly Glin Phe Wall Glu Thir Asn. Asn 53 O 535 54 O
Asp Wall Wall Phe Thir Met Glu Ser Ser Wall Arg Thir Ala Arg Ile Ala 545 550 555 560
Wall Luell Lieu. Asn. Luell Asn Glin Wall Pro Asp Ile Asn. Pro 565 st O sts
Lell Glin Tyr Asp Ile Arg His Lieu. Luell Ala Ala Thir Luell Asn 585 59 O
Asp Asp Llys Pro Phe Wall Gly Glu Gly Lieu. Luell Arg Llys Wall Luell 595 605
Gly Thir Tyr Phe Glu His Wall Leul Pro Ala Gly Ala Ala Glu Glu Glu 610 615
Glu His Glu Ser Phe Ile Ala Glu His Wall Asn Phe Glu Trp 625 630 635 64 O Wall Gly Ile Arg Gly 645
1. A method for enzymatically synthesizing isoprene, said 6. The method of claim 2, where the dehydratase is classi method comprising enzymatically introducing a terminal fied under EC 4.2.1.—and has at least 70% homology to the vinyl group into 3-methyl-pent-2-enoyl-acp., 4-methyl amino acid sequence set forth in SEQID NO: 22. pent-2-enoyl-acp. 3-methyl-3-hydroxy-pentanoate, 4-me 7. The method of claim 1, wherein said terminal vinyl thyl-3-hydroxypentanoate, or mevalonate, and converting the group is introduced into 3-methyl-3-hydroxy-pentanoate or resulting product in one or more steps to isoprene. 4-methyl-3-hydroxypentanoate using a monooxygenase or a 2. The method of claim 1, wherein said first vinyl group is cytochrome P450 reductase. introduced using a dehydratase, a monooxygenase, a cyto 8. The method of claim 7, wherein said monooxygenase chrome P450 reductase, or an acyl-acp dehydrogenase. has at least 70% homology to the amino acid sequence set 3. The method of claim 1, wherein said terminal vinyl forth in SEQID NO: 15. group is introduced into 3-methyl-pent-2-enoyl-acp or 4-methyl-pent-2-enoyl-acp using an acyl-acp dehydroge 9. (canceled) aSC. 10. A method forenzymatically synthesizing isoprene, said method comprising enzymatically introducing a second ter 4. The method of claim 3, where said acyl-acp dehydro minal vinyl group into 3-methyl-3-hydroxy-pent-4-enoate, genase has at least 70% homology to the amino acid sequence 4-methyl-3-hydroxypent-4-enoate, 4-methyl-3-sulphoryl set forth in SEQID NO:14. pent-4-enoyl-acp, or 3-methyl-3-buten-2-ol to produce iso 5. (canceled) prene. US 2015/0037860 A1 Feb. 5, 2015 37
11. The method of claim 10, wherein said second vinyl myces hansenii; from the genus Arxula Such as Arxula group is introduced using a mevalonate diphosphate decar adenoinivorans; or from the genus Kluyveromyces such as boxylase, a mevalonate 3-kinase, an acyl-acp decarboxylat Kluyveromyces lactis. ing thioesterase, or a linalool dehydratase. 28. (canceled) 12. The method of claim 11, wherein said mevalonate 29. (canceled) diphosphate decarboxylase has at least 70% homology to the 30. The method of claim 24, wherein the principal carbon mevalonate diphosphate decarboxylase of any one of the source fed to the fermentation derives from a biological or a amino acid sequences set forth in SEQID NOS:8-11. non-biological feedstock. 13. The method of claim 11, wherein said mevalonate 31. The method of claim 30, wherein the biological feed diphosphate decarboxylase has a histidine at the position stock is, or derives from, monosaccharides, disaccharides, aligning with residue 74 of SEQID NO:11 and/or a pheny hemicellulose such as levulinic acid and furfural, cellulose, lalanine at the position aligning with residue 145 of SEQID lignocellulose, lignin, triglycerides such as glycerol and fatty NO:11. acids, agricultural waste or municipal waste, or wherein the 14. The method of claim 11, wherein said mevalonate non-biological feedstock is, or derives from, either natural diphosphate decarboxylase has the amino acid sequence set gas, syngas, CO2/H2, methanol, ethanol, non-volatile residue forth in SEQID NO:11, except that a histidine is substituted (NVR), caustic wash from cyclohexane oxidation processes at position 74 for arginine and/or a phenylalanine is Substi or other waste stream from either the chemical or petrochemi tuted at position 145 for isoleucine. cal industries. 15. The method of claim 11, wherein said mevalonate 32. (canceled) diphosphate decarboxylase converts 3-methyl-3-hydroxy 33. (canceled) pent-4-enoate or 4-methyl-3-hydroxypent-4-enoate to iso 34. (canceled) prene. 35. (canceled) 16. The method of claim 11, wherein said mevalonate 36. (canceled) 3-kinase converts 3-methyl-3-hydroxypent-4-enoate or 37. The method of claim 24, wherein said host comprises a 4-methyl-3-hydroxypent-4-enoate to isoprene. feedback inhibition resistant mutant of an acetolactate Syn 17. The method of claim 16, wherein said mevalonate thase. 3-kinase has at least 70% homology to the amino acid 38. The method of claim 24, wherein said host comprises sequence of SEQID NO: 12. an acetolactate synthase under control of a promoter not 18. The method of claim 10, wherein said acyl-acp decar subject to genetic repression by a branched chain amino acid. boxylating thioesterase converts 3-methyl-3-sulphorylpent 39. A recombinant host producing isoprene, said host com 4-enoyl-acp or 4-methyl-3-sulphorylpent-4-enoyl-acp to prising at least one exogenous nucleic acid encoding (i) a isoprene. 2-hydroxyacyl-CoA dehydratase or B-ketoacyl-ACP-syn 19. The method of claim 18, wherein said acyl-acp decar thase; (ii) an acyl-ACP dehydrogenase, a monooxygenase, a boxylating thioesterase has greater than 70% homology to the cytochrome P450, or a dehydratase classified under EC 4.2. amino acid sequence of SEQID NO: 21. 1.—and (iii) a mevalonate diphosphate decarboxylase, a 20. The method of claim 10, wherein said linalool dehy mevalonate 3-kinase, an acyl-ACP decarboxylating dratase converts 3-methyl-3-buten-2-ol to isoprene. thioesterase, or a linalool dehydratase, said host producing 21. The method of claim 20, wherein said linalool dehy isoprene. dratase has at least 70% homology to the amino acid sequence 40. The host of claim 39, wherein said host comprises at of SEQ ID NO: 13. least one exogenous nucleic acid encoding (i) said 2-hy 22. (canceled) droxyacyl-CoA dehydratase, (ii) said acyl-ACP dehydroge 23. (canceled) nase, and (iii) said mevalonate diphosphate decarboxylase, 24. The method of claim 1, wherein said method is per said mevalonate 3-kinase, said acyl-ACP decarboxylating formed in a recombinant host. thioesterase, or said linalool dehydratase. 25. (canceled) 41. The host of claim 39, wherein said host comprises at 26. The method of claim 24, where the recombinant host is least one exogenous nucleic acid encoding (i) said 2-hy a prokaryotic host selected from the genus Escherichia Such droxyacyl-CoA dehydratase, (ii) said monooxygenase or said as Escherichia coli; from the genus Clostridia Such as Clostridium ljungdahli, Clostridium autoethanogenium or cytochrome P450, and (iii) said mevalonate diphosphate Clostridium kluyveri; from the genus Corynebacteria Such as decarboxylase, said mevalonate 3-kinase, said acyl-ACP Corynebacterium glutamicum; from the genus Cupriavidus decarboxylating thioesterase, or said linalool dehydratase. Such as Cupriavidus necator or Cupriavidus metallidurans; 42. The host of claim 39, wherein said host comprises at from the genus Pseudomonas Such as Pseudomonas fluore least one exogenous nucleic acid encoding (i) said B-ketoa scens or Pseudomonas putida; from the genus Bacillus Such cyl-ACP-synthase, (ii) said acyl-ACP dehydrogenase, and as Bacillus subtillis; or from the genus Rhodococcus Such as (iii) said mevalonate diphosphate decarboxylase, said meva Rhodococcus equi. lonate 3-kinase, said acyl-ACP decarboxylating thioesterase, 27. The method of claim 24, where the recombinant host is or said linalool dehydratase. a eukaryotic host selected from the genus Aspergillus such as 43. A recombinant host producing isoprene, said host com Aspergillus niger; from the genus Saccharomyces such as prising at least one exogenous nucleic acid encoding (i) a Saccharomyces cerevisiae, from the genus Pichia Such as dehydratase classified under EC 4.2.1.—and (ii) a meva Pichia pastoris; from the genus Yarrowia Such as Yarrowia lonate diphosphate decarboxylase or a mevalonate 3-kinase, lipolytica, from the genus Issatchenkia Such as Issathenkia said host producing isoprene. Orientalis; from the genus Debaryomyces such as Debaryo k k k k k