US009 169496B2

(12) United States Patent (10) Patent No.: US 9,169.496 B2 Marliere (45) Date of Patent: Oct. 27, 2015

(54) METHOD FOR THE ENZYMATIC (2013.01); C12N 9/1235 (2013.01); C12N 9/88 PRODUCTION OF BUTADIENE (2013.01); CI2P 7/04 (2013.01); C12P 7/24 (2013.01) (71) Applicant: Scientist of Fortune, S.A., Luxembourg (58) Field of Classification Search (LU) None See application file for complete search history. (72) Inventor: Philippe Marliere, Mouscron (BE) (56) References Cited (73) Assignee: Scientist of Fortune, S.A., Luxembourg (LU) U.S. PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this 5,855,881. A * 1/1999 Loike et al...... 424.942 patent is extended or adjusted under 35 2011/0300597 A1* 12/2011 Burk et al...... 435/167 U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.: 14/352,825 WO 2009111513 A1 9, 2009 WO 2011 14.0171 A 11, 2011 (22) PCT Filed: Oct. 18, 2012 (Continued) (86). PCT No.: PCT/EP2012/07O661 OTHER PUBLICATIONS S371 (c)(1), EC 1.1.1.34 (last viewed on Mar. 30, 2015).* (2) Date: Apr. 18, 2014 (Continued) (87) PCT Pub. No.: WO2013/057.194 Primary Examiner — Alexander Kim PCT Pub. Date: Apr. 25, 2013 (74) Attorney, Agent, or Firm — Michael M. Wales; InHouse Patent Counsel, LLC (65) Prior Publication Data (57) ABSTRACT US 2014/0256009 A1 Sep. 11, 2014 Described is a method for the enzymatic production of buta diene which allows to produce butadiene from crotyl alcohol. Related U.S. Application Data Also described are enzyme combinations and compositions (60) Provisional application No. 61/549,149, filed on Oct. containing Such enzyme combinations which allow the enzy 19, 2011. matic conversion of crotyl alcohol into butadiene. Further more, the invention relates to microorganisms which have (30) Foreign Application Priority Data been genetically modified so as to be able to produce butadi ene from crotyl alcohol. Oct. 19, 2011 (EP) ...... 11185854 Moreover, the invention relates to a method for the enzymatic production of crotyl alcohol from crotonyl-Coenzyme A. The (51) Int. Cl. obtained crotyl alcohol can be further converted into butadi CI2P 5/02 (2006.01) ene as described herein. Also described are enzyme combi CI2P 7/04 (2006.01) nations which allow to convert crotonyl-Coenzyme A into (Continued) crotyl alcohol as well as (micro)organisms which express (52) U.S. Cl. Such enzyme combinations. CPC ...... CI2P5/026 (2013.01); C12N 9/1205 32 Claims, 10 Drawing Sheets

N-1s US 9,169.496 B2 Page 2

(51) Int. Cl. Osterman et al., Characterization of mutation-induced changes in the CI2P 7/24 (2006.01) maize (Zea may’s L.) ADH1-1S1108 alcoholdehydrogenase, Journal CI2N 9/12 (2006.01) Biochem. Genet. (1993), vol. 31 (11-12), pp. 497-506. Copy pro CI2N 9/88 (2006.01) vided with the Abstract only.* XP 0.08660506.1 (last viewed on Mar. 31, 2015).* (56) References Cited International Preliminary Examination Report (IPER) for PCT/ EP2012/070661 mailed on May 1, 2014. FOREIGN PATENT DOCUMENTS Database CAPLUS Online Chemical Abstracts Service; 1958, Gorin, Y.A. et al.: “Diene hydrocarbons from unsaturated alcohols. I. WO 2012081723 A1 6, 2012 Catalytic dehydration of crotyl alcohol to butadiene'. XP002673746, WO 2012106516 A1 8, 2012 Database accession No. 1958:72071. WO 2012177710 A1 12/2012 Database WPI Week 198927 Thomson Scientific, London, GB: AN OTHER PUBLICATIONS 1989-195596 XP002673747, & JP 1 132391 A (Showa Denko KK) May 24, 1989. EC 1.1.1.1. (last viewed on Mar. 30, 2015).* Database WPI Week 201244 Thomson Scientific, London, GB: AN EC 1.2.1n2 (last viewed on Mar. 30, 2015).* 2012-HO2951 XP002694246, & WO 2012/081723 A1 (Mitsubishi Q08891-FACR2 ARATH (last viewed on Mar. 30, 2015).* Chem Corp) Jun. 21, 2012. Q96533-ADHX ARATH (last viewed on Mar. 30, 2015).* Havel, C. AL. Isopentenoid synthesis in isolated embryonic Q9SAH9-CCR2 ARATH (last viewed on Mar. 30, 2015).* Q60352-IPK METJA (last viewed on Mar. 30, 2015).* Drosophila cells. Possible regulation of 3-hydroxy-3-methylglutaryl Q58270-IDSA METJA (last viewed on Mar. 30, 2015).* coenzyme A reductase activity by shunted mevalonate carbon, Jour Q60337-THIL METJA (last viewed on Mar. 30, 2015).* nal of Biological Chemistry, Aug. 5, 1986, pp. 10150-10156, vol. Lin et al., Characterization of the monoterpene synthase genetps26, 261, No. 22, XPO08160854. the ortholog of a gene induced by insect herbivory in maize. Plant Physiol. (2008), vol. 146(3), pp. 940-951.* * cited by examiner U.S. Patent Oct. 27, 2015 Sheet 1 of 10 US 9,169.496 B2

1S-1Noh

Figure 1

O O O I 1s-1No-Noh -1S-1N1 Yo1">oh O H OH OH

Figure 2

-1S-1SO

Figure 3

N-1s Figure 4 U.S. Patent Oct. 27, 2015 Sheet 2 of 10 US 9,169.496 B2

L-Lactate NAD" Lactate dehydrogenase 340 nm. Crotyl alcohol ATP Pyruvate NADH Studied enzyme C Pyruvate kinase Crotyl phosphate ADP Phosphoenolpyruvate

Figure 5

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Figure 6 U.S. Patent Oct. 27, 2015 Sheet 3 of 10 US 9,169.496 B2

tes, As. 5-0.7 rain i{27-33)

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Figure 7

O O P P OH 2 * ...Ho1 -N-N Yo1 | SOH OH OH OH OH Dimethylallyl diphosphate isoprene Diphosphate

P P

4\-1 Ho1 OHNo1 OHNoh Croty diphosphate 1,3-Butadiene Diphosphate

Figure 8 U.S. Patent Oct. 27, 2015 Sheet 4 of 10 US 9,169.496 B2

NNH O H OH A Q l Qan (...)A e

-s-s-s-N-N- -N-frogSofo, : N Ö o O O k

OH Og-Ps O --

Figure 9

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Crotyl monophosphate Crotyl diphosphate 2OO

53.3 1513 ---235) 1CO 79.193.1 120.3

10

Figure 10a U.S. Patent Oct. 27, 2015 Sheet 5 of 10 US 9,169.496 B2

liters. Crotyl monophosphate 27 No croty diphosphate AO 154

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Figure 11 U.S. Patent Oct. 27, 2015 Sheet 6 of 10 US 9,169.496 B2

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i US 9, 169,496 B2 1. 2 METHOD FOR THE ENZYMATIC metabolic intermediate acetyl-Coenzyme A (in the following PRODUCTION OF BUTADIENE acetyl-CoA) as described herein. Thus, in a first aspect, the present invention relates to a This Application is a 371 National Phase filing of EP process for the production ofbutadiene in which butadiene is 2012070661 filed Oct. 18, 2012, which is a continuation of 5 produced by the enzymatic conversion of crotyl alcohol. Cro EP 11 858 544 which was filed on Oct. 19, 2011 and a tyl alcohol, also referred to as crotonyl alcohol or crotonol, is nonprovisional of U.S. Ser. No. 61/545,149 filed Oct. 19, an unsaturated alcohol of formula CHO (see FIG. 1). 2011, which are all incorporated by reference in their entirety. Another name for crotyl alcohol is But-2-en-1-ol. It can be The present invention relates to a method for the enzymatic produced by reduction of crotonaldehyde (see FIG. 3). 10 According to the present invention crotyl alcohol can be production of butadiene which allows to produce butadiene converted into butadiene by enzymatic reactions involving as from crotyl alcohol. The present invention also relates to intermediates crotyl phosphate and/or crotyl diphosphate. microorganisms which have been genetically modified so as Thus, the principle underlying the present invention is that to produce butadiene. crotyl alcohol is first enzymatically activated by the conver The present invention also relates to a method for the 15 sion into crotyl phosphate or crotyl diphosphate and is then enzymatic production of crotyl alcohol from crotonyl-Coen further converted into butadiene by the use of appropriate Zyme A. The obtained crotyl alcohol can be further converted enzymes as described below. into butadiene as described herein. The present invention Thus, the present invention relates, in a first aspect, to a furthermore relates to enzyme combinations which allow to method for the production ofbutadiene comprising the enzy convert crotonyl-Coenzyme A into crotyl alcohol as well as to matic conversion of crotyl alcohol into butadiene via crotyl (micro)organisms which express Such enzyme combinations. phosphate or crotyl diphosphate. Butadiene (1,3-butadiene) is a conjugated diene with the The enzymatic conversion of crotyl alcohol into butadiene formula CH (see FIG. 4). It is an important industrial can occur via different alternative routes. In a first aspect (A), chemical used as a monomer in the production of synthetic the present invention relates to a method for the production of rubber. There exist different possibilities to produce butadi 25 butadiene comprising the enzymatic conversion of crotyl ene. Butadiene is, for example, produced as a by product of alcohol into butadiene via crotyl phosphate wherein said the steam cracking process used to produce ethylene and method comprises the steps of other olefins. In this process butadiene occurs in the C4 (i) enzymatically converting crotyl alcohol into crotyl phos stream and is normally isolated from other byproducts by phate; and extraction into a polar aprotic solvent, such as acetonitrile, 30 (ii) enzymatically converting crotyl phosphate into butadi from which it is then stripped. Butadiene can also be pro CC. duced by the catalytic dehydrogenation of normal butane or it This alternative is in the following referred to as Alterna can be produced from ethanol. In the latter case, two different tive A and the different steps are referred to as A(i) and A(ii). processes are in use. In a single-step process, ethanol is con As regards step A(i), the enzymatic conversion of crotyl verted to butadiene, hydrogen and water at 400-450° C. over 35 alcohol into crotyl phosphate is a phosphorylation step and a metal oxide catalyst (Kirshenbaum, I. (1978), Butadiene. In can be achieved by enzymes which catalyze the transfer of a M. Grayson (Ed.), Encyclopedia of Chemical Technology, 3rd phospho group onto a molecule. Such as kinases. For ed., vol. 4, pp. 313-337. New York: John Wiley & Sons). In a example, enzymes which can be employed in this reaction are two-step process, ethanol is oxidized to acetaldehyde which enzymes which are classified as E.C.2.7.1, i.e. phosphotrans reacts with additional ethanol over a tantalum-promoted 40 ferases with an alcohol group as acceptor, preferably porous silica catalyst at 325-350° C. to yield butadiene (Kir enzymes which are classified as 2.7.1.50 (hydroxyethylthiaz shenbaum, I. (1978), loccit.). Butadiene can also be produced ole kinase) or which are classified as E.C. 2.7.1.89 (thiamine by catalytic dehydrogenation of normal butenes. kinase). Preferably, ATP is the donor of the phospho group in For the past two decades, genetic engineering technologies Such a reaction. Thus, in one embodiment the enzymatic have made possible the modification of the metabolism of 45 conversion of crotyl alcohol into crotyl phosphate can, e.g., micro-organisms, and hence their use to produce key Sub be achieved by the use of a hydroxyethylthiazole kinase (EC stances which they would otherwise produce at a low yield. 2.7.1.50). Hydroxyethylthiazole kinase is an enzyme which By enhancing naturally occurring metabolic pathways, these catalyzes the following reaction technologies open up new ways to bio-produce numerous ATP+4-methyl-5-(2-hydroxyethyl)thiazole." ADP+ compounds of industrial relevance. Several industrial com 50 4-methyl-5-(2-phosphoethyl)thiazole pounds such as amino-acids for animal feed, biodegradable The occurrence of this enzyme has been described for plastics or textile fibres are now routinely produced using several organisms, e.g. for E. coli, Bacillus subtilis, Rhizo genetically modified organisms. There are however no bio bium leguminosarum, Pyrococcus horikoshii OT3, Saccha processes using micro-organisms in place for the production romyces cerevisiae. of the major petrochemically derived molecules, in particular 55 In another embodiment the enzymatic conversion of crotyl butadiene, since no micro-organisms are known as natural alcohol into crotyl phosphate can, e.g., beachieved by the use producers of butadiene even in Small quantities. Given the of a thiamine kinase (EC 2.7.1.89). Thiamine kinase is an large amounts of rubber produced worldwide and the increas enzyme which catalyzes the following reaction ing environmental concerns and the limited resources for ATP+thiaminer ADP+thiamine phosphate producing butadiene using chemical processes, there is a need 60 to provide alternative, environmentally-friendly and Sustain The occurrence of this enzyme has been described for able processes for the production of butadiene. several organisms, e.g. for E. coli and Salmonella enterica. The present invention addresses this need and provides for Hydroxyethylthiazole is a moiety of thiamine and shares the first time a process by which butadiene can be produced with crotyl alcohol the following common structural motif enzymatically starting from crotyl alcohol. Crotyl alcohol 65 CH C CH CH, OH. itself can be provided by the enzymatic conversion of croto Thus, the inventor considers that a hydroxyethylthiazole nyl CoA which, in turn, can be provided starting from the kinase or a thiamine kinase could also act on other Substrates US 9, 169,496 B2 3 4 which contain this motif and found that, indeed, different water for forming terpenoid alcohols (Degenhardt et al., Phy tested hydroxyethylthiazole kinases and thiamine kinases tochemistry 70 (2009), 1621-1637). were capable of using crotyl alcohol as a Substrate and con The different terpene synthases share various structural Verting it into crotyl phosphate features. These include a highly conserved C-terminal In principle, any known hydroxyethylthiazole kinase can 5 domain, which contains their catalytic site and an aspartate be employed in the method according to the invention. In one rich DDXXD (SEQ ID NO: 19)motif essential for the diva aspect of the present invention, a hydroxyethylthiazole kinase lent metal ion (typically Mg2+ or Mn2+) assisted substrate of bacterial origin is used, such as a hydroxyethylthiazole binding in these enzymes (Green et al. Journal of biological kinase from a bacterium belonging to the genus Escherichia, chemistry, 284, 13, 8661-8669). In principle, any known Bacillus or Rhizobium, preferably of E. coli, Bacillus subtilis 10 enzyme which can be classified as belonging to the EC 4.2.3 or of R. leguminosarum. Amino acid and nucleotide enzyme Superfamily can be employed for the conversion of sequences for these enzymes are available. Examples for crotyl phosphate into butadiene. corresponding amino acid sequences are provided in SEQID Even more preferably the method according to the inven NOs: 1 to 3. In a particularly preferred embodiment any tion makes use of an isoprene synthase (EC 4.2.3.27), a protein showing an amino acid sequence as shown in any one 15 myrcene/ocimene synthase (EC 4.2.3.15), a farnesene Syn of SEQ ID NOs: 1 to 3 or showing an amino acid sequence thase (EC 4.2.3.46 or EC 4.2.3.47) or a pinene synthase (EC which is at least 80% homologous to any of SEQID NOs: 1 4.2.3.14). Also enzymes which are generally classified as to 3 and having the activity of a hydroxyethylthiazole kinase monoterpene synthases can be used. can be employed in a method according to the present inven In a particularly preferred embodiment, the dephosphory tion. 2O lation of crotyl phosphate to butadiene is achieved by an Moreover, in principle, any known thiamine kinase can be isoprene synthase (EC 4.2.3.27). Isoprene synthase is an employed in the method according to the invention. In one enzyme which catalyzes the following reaction: aspect of the present invention, a thiamine kinase of bacterial origin is used. Such as a thiamine kinase from a bacterium Dimethylallyl diphosphatev isoprene-diphosphate belonging to the genus Escherichia or Salmonella, preferably 25 This enzyme occurs in a number of organisms, in particular of E. coli or of Salmonella enterica. Amino acid and nucle in plants and some bacteria. The occurrence of this enzyme otide sequences for these enzymes are available. has, e.g., been described for Arabidopsis thaliana, a number In one embodiment of this method, step A(ii) consists of a of Populus species like P alba (UniProt accession numbers single enzymatic reaction in which crotyl phosphate is Q50L36, A9Q7C9, D8UY75 and D8UY76), P. nigra (Uni directly converted into butadiene. This option is in the fol- 30 Prot accession number AOPFK2), P canescence (UniProt lowing referred to as option A(ii.1). In this conversion of crotyl accession number Q9AR86; see also Köksal et al., J. Mol. phosphate into butadiene the phospho group is removed from Biol. 402 (2010), P tremuloides, P trichocarpa, Plobata, in crotyl phosphate with the simultaneous production of buta Quercus petraea, Quercus robur, Salix discolour, Pueraria diene. An enzyme which can catalyze this reaction is referred montana (UniProt accession number Q6EJ97), Mucuna pru to as a crotyl phosphate phosphate-lyase (butadiene forming). 35 riens, Vitis vinifera, Embryophyta and Bacillus subtilis. In Examples of enzymes which can catalyze the dephosphory principle, any known isoprene synthase can be employed in lation of crotyl phosphate into butadiene are enzymes which the method according to the invention. In a preferred embodi can be classified as belonging to the terpene synthase family. ment, the isoprene synthase employed in a method according Preferably such an enzyme belongs to the family of plant to the present invention is an isoprene synthase from a plant of terpene synthases. The terpene synthases constitute an 40 the genus Populus, more preferably from Populus tri enzyme family which comprises enzymes catalyzing the for chocarpa or Populus alba. In another preferred embodiment mation of numerous natural products always composed of the isoprene synthase employed in a method according to the carbon and hydrogen (terpenes) and sometimes also of oxy present invention is an isoprene synthase from Pueraria mon gen or other elements (terpenoids). Terpenoids are structur tana, preferably from Pueraria montana var. lobata (an ally diverse and widely distributed molecules corresponding 45 example for such a sequence is provided in SEQID NO: 7), or to well over 30000 defined natural compounds that have been from Vitis vinifera. Preferred isoprene synthases to be used in identified from all kingdoms of life. In plants, the members of the context of the present invention are the isoprene synthase the terpene synthase family are responsible for the synthesis of Populus alba (Sasaki et al.; FEBS Letters 579 (2005), of the various terpene molecules from two isomeric 5-carbon 2514-2518) or the isoprene synthases from Populus tri precursor “building blocks', isoprenyl diphosphate and pre- 50 chocarpa and Populus tremuloides which show very high nyl diphosphate, leading to 5-carbon isoprene, 10-carbon sequence homology to the isoprene synthase from Populus monoterpene, 15-carbon sesquiterpene and 20-carbon diter alba. A particularly preferred isoprene synthase is the iso penes” (Chen et al.: The Plant Journal 66 (2011), 212-229). prene synthase from Pueraria montana var. lobata (kudzu) The ability of terpene synthases to convert a prenyl diphos (Sharkey et al.: Plant Physiol. 137 (2005), 700-712). In a phate containing Substrate to diverse products during differ- 55 particularly preferred embodiment a protein showing an ent reaction cycles is one of the most unique traits of this amino acid sequence as shown in SEQID NOs: 7 or showing enzyme class. The common key step for the biosynthesis of an amino acid sequence which is at least 80% homologous to all terpenes is the reaction of terpene synthase on correspond SEQ ID NOs: 7 and having the activity of an isoprene syn ing diphosphate esters. The general mechanism of this thase can be employed in a method according to the present enzyme class induces the removal of the diphosphate group 60 invention. and the generation of an intermediate with carbocation as the The activity of an isoprene synthase can be measured first step. In the various terpene synthases, such intermediates according to methods known in the art, e.g. as described in further rearrange to generate the high number of terpene Silver and Fall (Plant Physiol (1991) 97, 1588-1591). In a skeletons observed in nature. In particular, the resulting cat typical assay, the enzyme is incubated with dimethylallyl ionic intermediate undergoes a series of cyclizations, hydride 65 diphosphate in the presence of the required co-factors, Mg+ shifts or other rearrangements until the reaction is terminated or Mn+ and K+ in sealed vials. At appropriate time volatiles by proton loss or the addition of a nucleophile, in particular compound in the headspace are collected with a gas-tight US 9, 169,496 B2 5 6 Syringe and analyzed for isoprene production by gas chroma C7E5V9), Zea mays (UniProt accession numbers Q2NM15, tography (GC). Crotyl monophosphate and crotyl diphos C7E5V8 and C7E5V7), Zea perennis (UniProt accession phate are structurally closely related to dimethylallyl diphos number C7E5WO) and Streptococcus coelicolor (Zhao et al., phate. In particular, the difference between crotyl J. Biol. Chem. 284 (2009), 36711-36719). In principle, any diphosphate and dimethylallyl diphosphate is just a methyl known beta-farnesene synthase can be employed in the group (see FIG. 8). The inventor considers that, therefore, an method according to the invention. In a preferred embodi isoprene synthase can also use crotyl diphosphate or crotyl ment, the beta-farnesene synthase employed in a method monophosphate as a Substrate. In principle, any known iso according to the present invention is a beta-farnesene Syn prene synthase can be employed in the method according to thase from Mentha piperita (Crock et al.; Proc. Natl. Acad. the invention. 10 Sci. USA 94 (1997), 12833-12838). In another particularly preferred embodiment, the enzyme Methods for the determination of farnesene synthase activ used for the conversion of crotyl phosphate into butadiene is ity are known in the art and are described, for example, in a myrcene/ocimene synthases (EC 4.2.3.15). Myrcene/ Greenetal. (Phytochemistry 68 (2007), 176-188). In a typical ocimene synthases (EC 4.2.3.15) are enzymes which natu assay farmesene synthase is added to an assay buffer contain rally catalyze the following reaction: 15 ing 50 mM BisTrisPropane (BTP) (pH 7.5), 10% (v/v) glyc Geranyl diphosphate (E)-beta-ocimene--diphos erol, 5 mM DTT. Tritiated farnesyl diphosphate and metal phate ions are added. Assays containing the protein are overlaid with 0.5 ml pentane and incubated for 1 hat 30°C. with gentle O shaking. Following addition of 20 mM EDTA (final concen tration) to stop enzymatic activity an aliquot of the pentane is Geranyl diphosphate Dy myrcene-diphosphate removed for scintillation analysis. The olefin products are These enzymes occur in a number of organisms, in particu also analyzed by GC-MS. lar in plants and animals, for example in Lotus japanicus, Pinene synthase (EC 4.2.3.14) is an enzyme which natu Phaseolus lunatus, Abies grandis, Arabidopsis thaliana (Uni rally catalyzes the following reaction: Prot accession number Q97.UH4), Actinidia chinensis, 25 Geranyl diphosphate alpha-pinene-diphosphate Perilla fructescens, Vitis vinifera, Ochtodes secundiramea and in Ips pini (UniProt accession number Q58GE8. In prin This enzyme occurs in a number of organisms, in particular ciple, any known myrcene/ocimene synthase can be in plants, for example in Abies grandis (UniProt accession employed in the method according to the invention. In a number O244475), Artemisia annua, Chamaecyparis for preferred embodiment, the myrcene/ocimene synthase 30 mosensis (UniProt accession number C3RSF5), Salvia offi employed in a method according to the present invention is a cinalis and Picea sitchensis (UniProt accession number beta-ocimene synthase from Lotus japanicus (Arimura et al.: Q6XDB5). Plant Physiol. 135 (2004), 1976-1983; an example for such an For the enzyme from Abies grandis a particular reaction enzyme is provided in SEQ ID NO: 9) or from Phaseolus was also observed (Schwab et al., Arch. Biochem. Biophys. lunatus (UniProt accession number B1P189; an example for 35 392 (2001), 123-136), namely the following: such an enzyme is provided in SEQID NO: 10). In a particu 6,7-dihydrogeranyl diphosphate' 6,7-dihy larly preferred embodiment the myrcene/ocimene synthase is dromyrcene-diphosphate an (E)-beta-ocimene synthase from Vitis vinifera (an example for such an enzyme is provided in SEQ ID NO: 12). In a In principle, any known pinene synthase can be employed particularly preferred embodiment any protein showing an 40 in the method according to the invention. In a preferred amino acid sequence as shown in any one of SEQID NOs: 9. embodiment, the pinene synthase employed in a method 10 or 12 or showing an amino acid sequence which is at least according to the present invention is a pinene synthase from 80% homologous to any of SEQ ID NOs: 9, 10 or 12 and Abies grandis (UniProt accession number O244475; Schwab having the activity of a beta-ocimene synthase can be et al., Arch. Biochem. Biophys. 392 (2001), 123-136). employed in a method according to the present invention. 45 Methods for the determination of pinene synthase activity The activity of an ocimene/myrcene synthase can be mea are known in the art and are described, for example, in sured as described, for example, in Arimura et al. (Plant Schwab et al. (Archives of Biochemistry and Biophysics 392 Physiology 135 (2004), 1976-1983. In a typical assay for (2001), 123-136). In a typical assay, the assay mixture for determining the activity, the enzyme is placed in screwcapped pinene synthase consists of 2 ml assay buffer (50 mM Tris/ glass test tube containing divalent metal ions, e.g. Mg+ 50 HCl, pH 7.5, 500 mM KC1, 1 mM MnC12, 5 mM dithiothrei and/or Mn+, and substrate, i.e. geranyl diphosphate. The tol, 0.05% NaHSO3, and 10% glycerol) containing 1 mg of aqueous layer is overlaid with pentane to trap Volatile com the purified protein. The reaction is initiated in a Teflon pounds. After incubation, the assay mixture is extracted with sealed screw-capped vial by the addition of 300 mM sub pentane a second time, both pentane fractions are pooled, strate. Following incubation at 25° C. for variable periods concentrated and analyzed by gas chromatography to quan 55 (0.5-24 h), the mixture is extracted with 1 ml of diethyl ether. tify ocimene? myrcene production. The biphasic mixture is vigorously mixed and then centri Beta-farnesene synthases (EC 4.2.3.47) naturally catalyze fuged to separate the phases. The organic extract is dried the following reaction: (MgSO4) and subjected to GC-MS and MDGC analysis. As indicated above, it is also possible to employ a monot (2E,6E)-farnesyl diphosphate (E)-beta-farnesene-- 60 erpene synthases in a method according to the invention. diphosphate Particularly preferred are the monoterpene synthase from This enzyme occurs in a number of organisms, in particular Melaleuca alternifolia described in Shelton et al. (Plant in plants and in bacteria, for example in Artemisia annua Physiol. Biochem. 42 (2004), 875-882; an example for such (UniProt accession number Q4VM12), Citrus junos (UniProt an enzyme is provided in SEQID NO: 11) or the monoterpene accession number Q94JS8), Oryza sativa (UniProt accession 65 synthase from Eucalyptus globulus (UniProt accession num number Q0J7R9), Pinus Sylvestris (UniProt accession num ber QOPCI4; an example for such an enzyme is provided in ber D7PCH9), Zea diploperennis (UniProt accession number SEQID NO: 8). In a particularly preferred embodiment any US 9, 169,496 B2 7 8 protein showing an amino acid sequence as shown in any one Genes encoding an isopentenyl phosphate kinase are also of SEQID NO: 11 or 8 or showing an amino acid sequence known from Methanothermobacter thermautotrophicus which is at least 80% homologous to any of SEQID NO: 11 (MTH) and from Thermoplasma acidophilum (THA) (Chen or 8 and having the activity of a monoterpene synthase can be and Poulter, Biochemistry 49 (2010), 207-217). For both employed in a method according to the present invention. 5 these enzymes crystal structures have been determined (Ma The present inventors have shown that different types of banglo et al., ACS Chem. Biol. 5 (2010), 517-527). The terpene synthases, e.g. isoprene synthases, (E)-beta-ocimene sequence of the isopentenyl phosphate kinase from Metha and monoterpene synthase from different plant organisms are nothermobacter thermautotrophicus is shown in SEQID NO: able to convert crotyl phosphate into butadiene (see Examples 5. The sequence of the isopentenyl phosphate kinase from 12 and 13 and FIG. 11). 10 The reactions catalyzed by the various terpene synthases, Thermoplasma acidophilum is shown in SEQID NO: 4. In a in particular the terpene synthases mentioned above, show particularly preferred embodiment any protein showing an certain common features. For example, the reactions cata amino acid sequence as shown in any one of SEQID NOs: 4 lyzed by isoprene synthases, by myrcene/ocimene synthases, to 6 or showing an amino acid sequence which is at least 80% by farmesene synthases, by pinene synthase and by monoter 15 homologous to any of SEQ ID NOs: 4 to 6 and having the pene synthases, respectively, are all believed to proceed activity of an isopentenyl phosphate kinase can be employed through a common mechanism in which, in a first step a in a method according to the present invention. carbocation is created by elimination of the diphosphate As regards step A(i2b), the enzymatic conversion of crotyl (PP), which is then followed by direct deprotonation so as to diphosphate into butadiene involves the removal of a diphos form the corresponding diene. It could be shown by the phate group from crotyl diphosphate with the simultaneous present inventors that enzymes which belong to the family of production ofbutadiene. An enzyme which can catalyze this terpene synthases are able to convert crotyl phosphate into reaction is referred to as a crotyl diphosphate diphosphate butadiene. lyase (butadiene forming). Examples of enzymes which can In another embodiment of the method according to the catalyze the dephosphorylation of crotyl diphosphate into invention step A(ii) consists of two enzymatic reactions com 25 butadiene are enzymes which can be classified as belonging prising: to the terpene synthase family. Preferably such an enzyme (a) the enzymatic conversion of crotyl phosphate into crotyl belongs to the family of plant terpene synthases. These diphosphate, and enzymes have already been disclosed in detail hereinabove in (b) the enzymatic conversion of crotyl diphosphate into buta connection with the conversion of crotyl phosphate into buta diene. 30 diene and the same applies here. This option is in the following referred to as option A(ii2) Preferably the terpene synthase is an isoprene synthase and the different steps are referred to as steps A(i2a) and (EC 4.2.3.27), a myrcene/ocimene synthase (EC 4.2.3.15), a A(ii.2b). farmesene synthase (EC 4.2.3.46 or EC 4.2.3.47) or a pinene As regards step A(ii2a), the enzymatic conversion of crotyl synthase (EC 4.2.3.14). Also enzymes which are generally phosphate into crotyl diphosphate can be achieved by the use 35 classified as monoterpene synthases can be used. Particularly of an enzyme which can catalyze the transfer of a phospho preferred the terpene synthase is an isoprene synthase, an group onto a molecule, such as kinases. Preferably, ATP is the (E)-beta ocimene synthase or a monoterpene synthase. donor of the phospho group in Such a reaction. The conver In a particularly preferred embodiment the dephosphory sion can in particular, e.g., be achieved by the use of an lation of crotyl diphosphate into butadiene is achieved by the isopentenyl phosphate kinase. This enzyme has so far not yet 40 use of an isoprene synthase (EC 4.2.3.27). As regards the been classified and, therefore, no EC number is available. It is isoprene synthase to be employed in the method, the same predicted to be a member of the amino acid kinase Superfam applies as has been described herein above. In a particularly ily, in particular the aspartokinase Superfamily. The enzyme preferred embodiment the isoprene synthase employed is an isopentenyl phosphate kinase catalyzes the following reac isoprene synthase from P. montana var. lobata. As explained tion: 45 above, crotyl diphosphate is structurally closely related to dimethylallyl diphosphate. Therefore, it is considered that an Isopentenyl phosphate+ATP isopentenyl diphos isoprene synthase can also use crotyl diphosphate as a Sub phate--ADP strate and can convert it into butadiene. This enzyme participates in an alternative branch of the In another particularly preferred embodiment, the dephos mevalonate pathway which has been discovered in the 50 phorylation of crotyl diphosphate into butadiene is achieved archaeon Methanocaldococcus jannaschii. It is a small mol by the use of a myrcene/ocimene synthase (EC 4.2.3.15). As ecule kinase. The primary amino acid sequence and the crys regards the myrcene/ocimene synthase to be employed in the tal structure of the isopentenyl phosphate kinase of Methano method, the same applies as has been described herein above. caldococcus jannaschii has already been disclosed as well as In a particularly preferred embodiment the myrcene/ocimene mutants which are able to use oligoprenyl monophosphates as 55 synthase employed is an (E)-beta ocimene synthase, most substrate (Dellas and Noel, ACS Chem. Biol. 5 (2010), 589 preferably an (E)-beta Ocimene synthase from Vitis vinifera or 601). The active site has been characterized and the amino from L. japonicus or from P. lunatus. acid residues crucial for binding and catalysis of the reaction In another particularly preferred embodiment, the dephos have been identified. Because of the high structural similarity phorylation of crotyl diphosphate into butadiene is achieved of isopentenyl phosphate and crotyl phosphate and the fact 60 by the use of a monoterpene synthase. As regards the monot that mutants of the isopentenyl phosphate kinase of Metha erpene synthase to be employed in the method, the same nocaldococcus jannaschii have already been shown to be able applies as has been described herein above. In a particularly to use other oligoprenyl monophosphates as Substrates, it can preferred embodiment the monoterpene synthase employed be expected that this enzyme or mutants thereof will also be is a monoterpene synthase from Eucalyptus globulus. able to convert crotyl phosphate into crotyl diphosphate. The 65 The present inventors have shown that different types of sequence of the isopentenyl phosphate kinase from Metha terpene synthases, e.g. isoprene synthase, (E)-beta-ocimene nocaldococcus jannaschii is shown in SEQID NO: 6. and monoterpene synthase from different plant organisms are US 9, 169,496 B2 10 able to convert crotyl diphosphate into butadiene (see can either be supplied externally or it can itselfbe provided by Examples 14 and 15 and FIG. 13). the reduction of crotonaldehyde (but-2-enal). This reduction In another aspect (B), the method according to the inven can, e.g., be achieved by chemical reactions as known to the tion comprises the two enzymatic steps of person skilled in the art. However, according to the present (I) enzymatically converting crotyl alcohol into crotyl invention it is preferable that the provision of crotyl alcohol is diphosphate, and achieved by the enzymatic conversion of crotonyl-Coenzyme (II) enzymatically converting crotyl diphosphate into butadi A (in the following crotonyl-CoA: see FIG.9) or of crotonal CC. dehyde into crotyl alcohol. Thus, in another embodiment the This alternative is in the following referred to as Alterna method according to the invention further comprises the step tive Band the different steps are referred to as B(I) and B(II). 10 of providing crotyl alcohol by the enzymatic conversion of In this embodiment the enzymatic conversion of crotyl crotonaldehyde into crotyl alcohol as described herein below. alcohol into crotyl diphosphate according to step B(I) con Crotonaldehyde (CH-CH=CHCHO; (2E)-but-2-enal) sists of a single enzymatic reaction in which crotyl alcohol is occurs naturally, e.g. in Soybean oils, and can be synthesized directly converted into crotyl diphosphate. chemically by the aldol condensation of acetaldehyde. Alter The direct enzymatic conversion of crotyl alcohol into 15 natively, the method according to the invention further com crotyl diphosphate in one step can, e.g., beachieved by the use prises the step of providing crotyl alcohol by the enzymatic of an enzyme which is able to catalyze the transfer of a conversion of crotonyl-CoA into crotyl alcohol as described diphosphate group, such as a diphosphotransferase, for below. Crotonyl-coenzyme A is a thioester between crotonic example enzymes which are classified as EC 2.7.6 (diphos acid and Coenzyme A. It is an intermediate in the fermenta photransferases). Examples are 2-amino-4-hydroxy-6-hy tion of butyric acid, and in the metabolism of lysine and droxymethyldihydropteridine diphosphokinase (EC 2.7.6.3) tryptophan. During degradation of these amino acids, C.-ke and thiamine diphosphokinase (EC 2.7.6.2). Preferably, ATP toadipate is produced which is converted into glutaryl-CoA is the donor of the diphosphate group in Such a reaction. by oxidative decarboxylation. Glutaryl-CoA is then con Thus, in one embodiment the direct enzymatic conversion Verted by glutaryl-CoA dehydrogenase into crotonyl-CoA, of crotyl alcohol into crotyl diphosphate in one step can be 25 which can be converted in two further steps into two mol achieved by the use of a 2-amino-4-hydroxy-6-hydroxymeth ecules of acetyl-CoA. Crotonyl-CoA is also a metabolite in yldihydropteridine diphosphokinase (EC 2.7.6.3). This the fermentation of glucose by Some obligatory anaerobe enzyme catalyzes the following reaction: bacteria in which butyric acid is produced, such as Clostridium acetobutyllicum. Moreover, crotonyl-CoA had 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropte 30 been isolated in Some microorganisms which assimilate ridine+ATP 2-amino-7,8-dihydro-4-hydroxy acetate via the so-called ethyl-malonyl-CoA pathway. It also 6-(diphosphooxymethyl)pteridine--AMP occurs as an intermediate in some metabolic pathways lead The occurrence of this enzyme has been described for ing to the assimilation of carbon dioxide, e.g. in the 3-hydrox several organisms, e.g. for E. coli, Plasmodium falciparum, yproprionate/4-hydroxybutyrate cycle or the dicarboxylate/ Plasmodium chabaudi, Streptococcus pneumoniae, Toxo 35 4-hydroxybutyrate cycle. plasma gondii, Yersinia pestis, Pneumocystis carinii, Haemo The present inventor also developed a method for enzy philus influenzae, S. cerevisiae, Arabidopsis thaliana and matically producing crotyl alcohol enzymatically starting Pisum sativum. from crotonaldehyde or from crotonyl-CoA. In principle, any known 2-amino-4-hydroxy-6-hydroxym Thus, in a second aspect, the present invention relates to a ethyldihydropteridine diphosphokinase can be employed in 40 method for producing crotyl alcohol. Such a method com the method according to the invention. prises the enzymatic conversion of crotonyl-CoA into cro In another embodiment the direct enzymatic conversion of tonaldehyde and the Subsequent enzymatic conversion of cro crotyl alcohol into crotyl diphosphate in one step can be tonaldehyde into crotyl alcohol. The first reaction may occur achieved by the use of a thiamine diphosphokinase (EC according to the following schemes: 2.7.6.2). This enzyme catalyzes the following reaction: 45 Crotonyl-CoA+NADH--H+ a crotonaldehyde--CoA+ ATP+thiamine AMP--thiamine diphosphate NAD The occurrence of this enzyme has been described for O several organisms, e.g. for Salmonella enterica, Plasmodium falciparum, Saccharomyces cerevisiae, Schizosaccharomy 50 Crotonyl-CoA+NADPH-i-H+ crotonaldehyde-- ces pombe, Candida albicans, Arabidopsis thaliana, Cae CoA-NADP norhabditis elegans, Rattus norvegicus, Mus musculus and This reaction is a reduction and can be catalyzed by various Homo sapiens. In principle, any known thiamine diphospho enzymes. In one aspect, it is possible to use for the above kinase can be employed in the method according to the inven indicated conversion of crotonyl-CoA to crotonaldehyde a tion. 55 hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34). This In step B(II) the obtained crotyl diphosphate is then further enzyme normally catalyzes the following reaction converted enzymatically into butadiene. This enzymatic con version of crotyl diphosphate into butadiene involves the (S)-3-hydroxy-methylglutaryl-CoA+2NADPH-i- removal of a diphosphate group and can, e.g., beachieved by His (R)-mevalonate+CoA+2NADP the use of terpene synthase as described herein above. As 60 Enzymes belonging to this class and catalyzing the above regards the preferred embodiments, the same applies as set shown conversion occur in organisms of all kingdoms, i.e. forth herein above. plants, animals, fungi, bacteria etc. and have extensively been In a particularly, preferred embodiment an isoprene syn described in the literature. Nucleotide and/or amino acid thase (EC 4.2.3.27) is employed for the conversion of crotyl sequences for Such enzymes have been determined for diphosphate into butadiene. 65 numerous organisms, in particular bacterial organisms. In The crotyl alcohol which is used as a substrate for the principle, any hydroxymethylglutaryl-CoA reductase (EC enzymatic production ofbutadiene according to the invention 1.1.1.34) can be used in the context of the present invention. US 9, 169,496 B2 11 12 Alternatively or in addition, the above described conver rium Pseudomonas putida MB-1 (Malone et al., Appl. Envi sion of crotonyl-CoA into crotonaldehyde can also be ronm. Microbiol. 65 (1999), 2622-2630) which uses NADH/ achieved by using an enzyme referred to as acetaldehyde NAD+ as a cofactor. dehydrogenase (EC 1.2.1.10). This enzyme normally cata In another embodiment it is also possible to use for the lyzes the following reaction above indicated conversion of crotonaldehyde to crotyl alco Acetyl-CoA+NADH+H+. acetaldehyde--CoA+ hol a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34). NAD This enzyme has already been described above and that what had been said above holds also true for this reaction. Enzymes belonging to this class and catalyzing the above Alternatively or in addition, the above described conver shown conversion occur in several types of bacteria, like e. 10 sion of crotonaldehyde into crotyl alcohol can also be coli, Acinetobacter sp., Leuconostoc mesenteroides, Pseudomonas sp. Clostridium beijerinckii, Clostridium achieved by using an alcohol dehydrogenase (EC 1.1.1.1). Kluyveri, Giardia intestinalis, Propionibacterium feudenre Such an enzyme normally catalyzes the following reaction ichii and Thermoanaerobacter ethanolicus. In principle, any Primary alcohol--NAD+ vir aldehyde--NADH--H acetaldehyde dehydrogenase (EC 1.2.1.10) can be used in the 15 Enzymes belonging to this class and catalyzing the above context of the present invention. shown conversion occur in organisms of all kingdoms, i.e. Alternatively or in addition, the above described conver plants, animals, fungi, bacteria etc. and have extensively been sion of crotonyl-CoA into crotonaldehyde can also be described in the literature. Nucleotide and/or amino acid achieved by using an enzyme referred to as aldehyde-alcohol sequences for Such enzymes have been determined for dehydrogenase, such as the aldehyde-alcohol dehydrogenase numerous organisms, in particular bacterial organisms. In as encoded by an adhE gene. Such an enzyme is bifunctional principle, any alcohol dehydrogenase (EC 1.1.1.1) can be in that it shows at least the enzymatic activities of an alcohol used in the context of the present invention. dehydrogenase and an aldehyde dehydrogenase, such as an Moreover, the conversion of crotonaldehyde to crotyl alco acetaldehyde dehydrogenase. An example for Such an hol can also be achieved by the use of an enzyme referred to enzyme is the aldehyde-alcohol dehydrogenase of E. coli 25 as an aldehyde reductase. Examples for Such enzymes are (adhE: UniProtKB/Swiss-Prot Accession number P0A9Q7: alcohol dehydrogenase (NADP+; EC 1.1.1.2), allyl-alcohol Jul. 27, 2011; Version 58). Corresponding enzymes are also dehydrogenase (EC 1.1.1.54), retinol dehydrogenase (EC known from other organisms, such as, e.g. Leuconostoc 1.1.1.105), sulcatone dehydrogenase (EC 1.1.1.260) and mesenteroides (Koo et al., Biotechnology Letters 27, 505 3-methylbutanal reductase (EC 1.1.1.265) 510), Polytomella sp. and Chlamydomonas reinhardtii (At 30 The enzymatic conversion of crotonyl-CoA into crotyl teia et al., Plant Mal. Biol. 53 (2003), 175-188). Genes encod alcohol may also occur according to the following schemes: ing such enzymes have been found in the genomes of several Gram-positive bacteria belonging to the categories bacilliand Crotonyl-CoA+2NADH+2H+y crotyl alcohol clostridia, in several gamma-proteobacteria, in actinobacte CoA-2NAD ria, in cyanobacteria and some amitochondriate protists (see Atteia et al., loc. cit.). 35 O Alternatively or in addition, the above described conver Crotonyl-CoA+2NADPH+2H+* crotyl alcohol sion of crotonyl-CoA into crotonaldehyde can also be CoA-2NADP achieved by using enzymes referred to as acyl-CoA reduc Similar to the above described conversions, this reaction tases. Examples for Such enzymes are cinnamoyl-CoA reduc 40 goes via crotonaldehyde as an intermediate. However, in this tase (EC 1.2.1.44), long-chain-fatty-acyl-CoA reductase (EC embodiment of the invention, the conversion of crotonyl 1.2.1.50) and malonyl-CoA reductase (malonate semialde CoA into crotyl alcohol is catalyzed by one enzyme which hyde-forming; EC 1.2.1.75). catalyses both reduction/hydrogenation steps. An enzyme According to the present invention the produced crotonal which may be employed in this conversion is an aldehyde dehyde is further converted into crotyl alcohol. The enzy 45 alcohol dehydrogenase, Such as the aldehyde-alcohol dehy matic conversion of crotonaldehyde into crotyl alcohol is a drogenase as encoded by an adhE gene which had already reduction/hydrogenation and may occur according to the fol been described above. lowing schemes: Another enzyme which may be used in this conversion is a Crotonaldehyde--NADH+H+ crotyl alcohol--NAD" hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) which 50 has already been described above. In a further preferred O embodiment the conversion of crotonyl-CoA into crotyl alco hol is achieved by the use of a short-chain dehydrogenase/ Crotonaldehyde--NADPH+H+ crotyl alcohol fatty acyl-CoA reductase. NADP The term “short-chain dehydrogenase/fatty acyl-CoA This reaction can be catalyzed by various enzymes. In one 55 reductase' or 'short-chain dehydrogenases/reductases aspect, it is possible to use for the above indicated conversion (SDR) in the context of the present invention refers to of crotonaldehyde to crotyl alcohol an enzyme which is enzymes which are characterized by the following features: known to be able to catalyze this reaction. One example is the 1. They catalyze a two-step reaction in which fatty acy-CoA aldo-keto reductase (AKR) encoded by the sakR1 gene. This is reduced to fatty alcohol. enzyme had been identified in Synechococcus sp. PCC 7002 60 2. They show a Substrate specificity for acyl-CoA containing and has been described in Dongyi et al. (Microbiol. 152 an aliphatic chain from 8 to 20 carbon atoms. (2006), 2013-2021). It uses NADPH/NADP+ as a cofactor. Preferably such enzymes are furthermore characterized by Another example is the aldo-keto reductase (AKR) encoded the feature that they show a specific motif in their primary by the At2g37770 gene, which had been identified in Arabi structure, i.e. amino acid sequence, namely they show two dopsis (Yamauchii et al. (J. Biol. Chem. 286 (2011) 6999 65 specific glycine motifs for NADP(H) binding. 7009). It uses NADPH/NADP+ as a cofactor. A further The short-chain dehydrogenase/fatty acyl-CoA reductase example is the 321-MB dehydrogenase from the soil bacte or short-chain dehydrogenases/reductases (SDR) enzymes US 9, 169,496 B2 13 14 constitute a family of enzymes, most of which are known to SEQID NO: 17. In a particularly preferred embodiment any be NAD- or NADP-dependent oxidoreductase (Jornwall H. et protein showing an amino acid sequence as shown in any one al., Biochemistry 34 (1995), 6003-6013). Recently, a novel of SEQID NOs: 13 to 17 or showing an amino acid sequence bacterial NADP-dependent reductase from Marinobacter which is at least 80% homologous to any of SEQID NOs: 13 aquaeolei VT8 was characterized (Willis et al., Biochemistry to 17 and having the activity of a short-chain dehydrogenase/ 50 (2011), 10550-10558). This enzyme catalyzes the four fatty acyl-CoA reductase can be employed in a method electron reduction of fatty acyl-CoA substrates to the corre according to the present invention. sponding fatty alcohols. The enzymatic conversion of fatty The methods according to the first and second aspect of the acyl-CoA into fatty alcohol occurs through an aldehyde inter present invention as described above may also be combined, mediate according to the following scheme: 10 i.e. it is possible that a method according to the first aspect of the invention for the production of butadiene from crotyl alcohol further comprises the steps of a method according to NADPH NADP the second aspect of the invention for the provision of crotyl O 15 alcohol by enzymatic reactions as described above. In another embodiment the methods according to the first --- and/or second aspect of the invention may also include the CoA further step of enzymatically providing crotonyl-CoA. This O NADPH NADP may be achieved by the enzymatic conversion of 3-hydroxy ul N-4 R-CH2OH butyryl-Coenzyme A into crotonyl-Coenzyme A. This reac R H tion may occur according to the following scheme: 3-hydroxybutyryl-Coenzyme A sa crotonyl-Coen The enzyme displays activity on fatty acyl-CoA Substrates zyme A+H2O ranging from 8 to 20 carbons in length (both Saturated and 25 This reaction corresponds to a Michael elimination and unsaturated) as well as on fatty aldehyde Substrates. Charac can, for example, be catalyzed by an enzyme called 3-hy teristically, proteins of this family possess two NAD(P)(H)- droxybutyryl-CoA dehydratase which is classified as EC binding motifs, which have the conserved sequence GXGX 4.2.1.55. This enzyme belongs to the family of lyases, spe (1-2x)G (SEQ ID NO: 20) (Willis et al., Biochemistry 50 cifically the hydro-lyases, which cleave carbon-oxygen (2011), 10550-10558; Jornwall H. et al., Biochemistry 34 30 bonds. The systematic name of this enzyme class is (3R)-3- (1995), 6003-6013). The first pattern, GTGFIG (SEQID NO: hydroxybutanoyl-CoA hydro-lyase (crotonoyl-CoA-form 18), is identified near the N-terminus and the second signature ing). Other names in common use include D-3-hydroxybu sequence, GXXXGXG (SEQID NO: 21), is located between tyryl coenzyme A dehydratase, D-3-hydroxybutyryl-CoA residues 384-390. dehydratase, enoyl coenzyme A hydrase (D), and (3R)-3- In principle any “short-chain dehydrogenase/fatty acyl 35 hydroxybutanoyl-CoA hydro-lyase. This enzyme partici CoA reductase' or “short-chain dehydrogenases/reductases pates in butanoate metabolism. Enzymes belonging to this (SDR) can be applied in the method according to the inven class and catalyzing the above shown conversion of 3-hy tion. droxybutyryl-Coenzyme A into crotonyl-Coenzyme A have Preferably, the short-chain dehydrogenase/fatty acyl-CoA been described to occur, e.g. in rat (Rattus norvegicus) and in reductase is a short-chain dehydrogenase/fatty acyl-CoA 40 Rhodospirillum rubrum. Nucleotide and/or amino acid reductase from a marine bacterium, preferably from the genus sequences for Such enzymes have been determined, e.g. for Marinobacter or Hahella, even more preferably from the Aeropyrum permix. In principle, any 3-hydroxybutyryl-CoA species Marinobacter aquaeolei, more preferably Marino dehydratase (EC 4.2.1.55) can be used in the context of the bacter aquaeolei VT8, Marinobacter manganoxydans, present invention. Marinobacter algicola, Marinobacter sp. ELB17 or Hahelly 45 Alternatively or in addition, the above described conver cheiuensis. Examples of Such enzymes are the short-chain sion of 3-hydroxybutyryl-Coenzyme A into crotonyl-Coen dehydrogenase/fatty acyl-CoA reductase from Marinobacter Zyme A can also be achieved by using an enzyme referred to aquaeolei VT8 (Uniprot accession number A1U3L3: Willis as enoyl-CoA hydratase (EC 4.2.1.17). Enoyl-CoA hydratase et al., Biochemistry 50 (2011), 10550-10558), the short-chain is an enzyme that normally hydrates the double bond between dehydrogenase from Marinobacter manganoxydans (Uni 50 the second and third carbons on acyl-CoA. However, it can prot accession number G6YQS9), the short-chain dehydro also be employed to catalyze the reaction in the reverse direc genase from Marinobacter algicola (Uniprotaccession num tion. This enzyme, also known as crotonase, is naturally ber A6EUH6), the short-chain dehydrogenase from involved in metabolizing fatty acids to produce both acetyl Marinobacter sp. ELB17 (Uniprot accession number CoA and energy. Enzymes belonging to this class have been A3JCC5) and the short-chain dehydrogenase from Hahella 55 described to occur, e.g. in rat (Rattus norvegicus), humans cheiuensis (Uniprot accession number Q2SCEO). (Homo sapiens), mouse (Mus musculus), wild boar (Sus The sequence of the short-chain dehydrogenase/fatty acyl scrofa), Bos taurus, E. coli, Clostridium acetobutylicum and CoA reductase from Marinobacter aquaeolei VT8 is shown Clostridium aminobutyricum. Nucleotide and/or amino acid in SEQID NO: 13. The sequence of the short-chain dehydro sequences for Such enzymes have been determined, e.g. for genase/fatty acyl-CoA reductase from Marinobacter manga 60 rat, humans and Bacillus subtilits. In principle, any enoyl noxydans is shown in SEQID NO: 14. The sequence of the CoA hydratase (EC 4.2.1.17) can be used in the context of the short-chain dehydrogenase/fatty acyl-CoA reductase from present invention. Marinobacter sp. ELB17 is shown in SEQ ID NO: 15. The In another embodiment it is also possible to use for the sequence of the short-chain dehydrogenase/fatty acyl-CoA above described conversion of 3-hydroxybutyryl-Coenzyme reductase from Marinobacter algicola is shown in SEQ ID 65 A into crotonyl-Coenzyme A an enoyl-CoA hydratase 2 (EC NO: 16. The sequence of the short-chain dehydrogenase/fatty 4.2.1.119) or a crotonyl-acyl-carrier-protein hydratase (EC acyl-CoA reductase from Hahella cheiuensis is shown in 4.2.1.58). US 9, 169,496 B2 15 16 In another embodiment the methods according to the first acellular reaction. Thus, in vitro preferably means in a cell and/or second aspect of the invention may also include the free system. The term “in vitro” in one embodiment means in further step of enzymatically providing 3-hydroxybutyryl the presence of isolated enzymes (or enzyme systems option Coenzyme A. This can be achieved by the enzymatic conver ally comprising possibly required cofactors). In one embodi sion of acetoacetyl-CoA into 3-hydroxybutyryl-Coenzyme ment, the enzymes employed in the method are used in puri A. This reaction may occur according to the following fied form. For carrying out the process in vitro the substrates scheme: for the reaction and the enzymes are incubated under condi acetoacetyl-CoA+NADH--H' 3 -hydroxybutyryl tions (buffer, temperature, coSubstrates, cofactors etc.) allow Coenzyme A+NAD" ing the enzymes to be active and the enzymatic conversion to 10 occur. The reaction is allowed to proceed for a time sufficient O to produce butadiene. The production of butadiene can be measured by methods known in the art, such as gas chroma acetoacetyl-CoA+NADPH-i-H' vie 3 -hydroxybutyryl tography possibly linked to mass spectrometry detection. Coenzyme A+NADP' The enzymes may be in any Suitable form allowing the This reaction is a reduction and can, e.g., be catalyzed by an 15 enzymatic reaction to take place. They may be purified or enzyme called acetoacetyl-CoA reductase which is classified partially purified or in the form of crude cellular extracts or as EC 1.1.1.36. Enzymes belonging to this class and cata partially purified extracts. It is also possible that the enzymes lyzing the above shown conversion of acetoactyl-CoA into are immobilized on a suitable carrier. 3-hydroxybutyryl-Coenzyme A occur in organisms of all In one embodiment of the method according to the inven kingdoms, i.e. plants, animals, fungi, bacteria etc. and have tion the substrate which is used in such an in vitro method is extensively been described in the literature. Nucleotide and/ crotyl alcohol which is converted by the use of the above or amino acid sequences for Such enzymes have been deter mentioned enzymes to butadiene. In another embodiment, the mined for numerous organisms, in particular bacterial organ Substrate used in Such an in vitro method is crotonaldehyde isms. In principle, any acetoacetyl-CoA reductase (EC which is first converted into crotyl alcohol as described above 1.1.1.36) can be used in the context of the present invention. 25 which is then in turn converted into butadiene as described In one embodiment the enzyme employed in the method above. according to the invention originates from E. coli. The in vitro method according to the invention may be In yet a further embodiment the methods according to the carried out in a one-pot-reaction, i.e. the Substrate is com first and/or second aspect of the invention may also include bined in one reaction mixture with the above described the further step of enzymatically providing acetoacetyl-CoA. 30 enzymes necessary for the conversion into butadiene and the This can be achieved by the enzymatic conversion of two reaction is allowed to proceed for a time sufficient to produce molecules acetyl-CoA into one molecule of acetoacetyl butadiene. Alternatively, the method may also be carried out CoA. Acetyl-CoA is a metabolic intermediate which occurs by effecting one or more enzymatic steps in a consecutive in all living organisms and plays a central role in metabolism. manner, i.e. by first mixing the Substrate with one or more Thus, according to the present invention, acetyl-CoA can, for 35 enzymes and allowing the reaction to proceed to an interme example, be converted into acetoacetyl-CoA by the following diate and then adding one or more further enzymes to convert reaction: the intermediate further either into an intermediate or into butadiene. 2 acetyl-CoA acetoacetyl-CoA CoA The in vitro method according to the invention furthermore This reaction is catalyzed by enzymes called acetyl-CoA 40 may comprise the step of collecting gaseous products, in C-acetyltransferases which are classified as EC 2.3.1.9. particular butadiene, degassing out of the reaction, i.e. recov Enzymes belonging to this class and catalyzing the above ering the products which degas, e.g., out of the culture. Thus, shown conversion of two molecules of acetyl-CoA into in one embodiment, the method is carried out in the presence acetoacetyl-CoA and CoA occur in organisms of all king of a system for collecting butadiene under gaseous form doms, i.e. plants, animals, fungi, bacteria etc. and have exten 45 during the reaction. sively been described in the literature. Nucleotide and/or As a matter of fact, butadiene adopts the gaseous state at amino acid sequences for Such enzymes have been deter room temperature and atmospheric pressure. The method mined for a variety of organisms, like Homo sapiens, Arabi according to the invention therefore does not require extrac dopsis thaliana, E. coli, Bacillus subtilis and Candida, to tion of the product from the reaction mixture, a step which is name just some examples. In principle, any acetyl-CoA 50 always very costly when performed at industrial scale. The C-acetyltransferase (EC 2.3.1.9) can be used in the context of evacuation and storage of butadiene and its possible Subse the present invention. quent physical separation from other gaseous Substances as Alternatively, the provision of acetoacetyl-CoA may also well as its chemical conversion can be performed according beachieved by the enzymatic conversion of acetyl-CoA and to any method known to one of skill in the art. For example, malonyl-CoA into acetoacetyl-CoA. This reaction is cata 55 butadiene can be separated from CO by the condensation of lyzed by an enzyme called acetoacetyl-CoA synthase. The CO, at low temperatures. CO can also be removed by polar gene encoding this enzyme was identified in the mevalonate Solvents, e.g. ethanolamine. Moreover, it can be isolated by pathway gene cluster for terpenoid production in a soil-iso adsorption on a hydrophobic membrane. lated Gram-positive Streptomyces sp. Strain CL190 (Oka In another embodiment the method according to the inven mura et al., PNAS USA 107 (2010) 11265-11270, 2010). 60 tion is carried out in culture, in the presence of an organism, Moreover a biosynthetic pathway using this enzyme for preferably a microorganism, producing at least the enzymes acetoacetyl-CoA production was recently developed in E. described above which are necessary to produce butadiene coli (Matsumoto K et al., Biosci. Biotechnol. Biochem, 75 according to a method of the invention according to the first (2), 364-366, 2011, enclosed) aspect by using one of the alternative routes A or B described The methods according to the present invention may be 65 above and starting either from crotyl alcohol or from cro carried out in vitro or in vivo. An in vitro reaction is under tonaldehyde. Thus, in such an embodiment of the invention, stood to be a reaction in which no cells are employed, i.e. an an organism, preferably a microorganism, that produces the US 9, 169,496 B2 17 18 enzymes specified in the description of alternatives A or B. expression of a different coding sequence, i.e., it is derived above, is used. It is possible to use a (micro)organism which from another gene, or is a synthetic promoter or a chimeric naturally produces one or more of the required enzymes and promoter. Preferably, the promoter is a promoter heterolo to genetically modify such a (micro)organism so that it gous to the organism/microorganism, i.e. a promoter which expresses also those enzymes which it does not naturally does naturally not occur in the respective organism/microor express. Preferably a (micro)organism is used which has been ganism. Even more preferably, the promoter is an inducible genetically modified as described hereinabove in connection promoter. Promoters for driving expression in different types with the second aspect of the invention so as to be able to of organisms, in particular in microorganisms, are well produce crotyl alcohol. known to the person skilled in the art. In alternative A1 it is for example possible to use Bacillus 10 subtilis which possesses a gene encoding the enzyme In a further embodiment the nucleic acid molecule is for hydroxyethylthiazole kinase and a gene encoding, a terpene eign to the organism/microorganism in that the encoded synthase, e.g. an isoprene synthase. Such a bacterium may be enzyme is not endogenous to the organism/microorganism, further genetically modified as described herein above so as i.e. is naturally not expressed by the organism/microorganism to be able to produce crotyl alcohol. 15 when it is not genetically modified. In other words, the In alternative B it is, e.g., possible to use E. coli or S. encoded enzyme is heterologous with respect to the organ cerevisiae, which both possess a gene encoding 2-amino-4- ism/microorganism. The foreign nucleic acid molecule may hydroxy-6-hydroxymethyldihydropteridine diphosphoki be present in the organism/microorganism in extrachromo nase, and to introduce into Such a microorganism a gene, for Somal form, e.g. as a plasmid, or stably integrated in the example from Bacillus subtilis encoding a terpene synthase, chromosome. A stable integration is preferred. Thus, the e.g. an isoprene synthase. Similarly, it is possible to use in genetic modification can consist, e.g. in integrating the cor alternative B as a microorganism B. subtilis and to genetically responding gene(s) encoding the enzyme(s) into the chromo modify it with a gene encoding a 2-amino-4-hydroxy-6-hy Some, or in expressing the enzyme(s) from a plasmid contain droxymethyldihydropteridine diphosphokinase, e.g. from E. ing a promoter upstream of the enzyme-coding sequence, the coli or from S. cerevisiae. Again, such microorganisms may 25 promoter and coding sequence preferably originating from be further genetically modified as described herein above so different organisms, or any other method known to one of as to be able to produce crotyl alcohol. skill in the art. Ifa (micro)organism is used which naturally expresses one In a preferred embodiment the (micro)organism of the of the required enzyme activities, it is possible to modify Such present invention is also genetically modified so as to be able a (micro)organism so that this activity is overexpressed in the 30 to produce crotyl alcohol as described herein above. (mircro)organism. This can, e.g., be achieved by effecting The organisms used in the invention can be prokaryotes or mutations in the promoter region of the corresponding gene eukaryotes, preferably, they are microorganisms such as bac So as to lead to a promoter which ensures a higher expression teria, yeasts, fungi or molds, or plant cells or animal cells. In of the gene. Alternatively, it is also possible to mutate the gene a particular embodiment, the microorganisms are bacteria, as such so as to lead to an enzyme showing a higher activity. 35 preferably of the genus Escherichia or Bacillus and even By using (micro)organisms which express the enzymes more preferably of the species Escherichia coli or Bacillus which are necessary according to alternative A or B as subtilis. described above, it is possible to carry out the method accord In another embodiment, the microorganisms are recombi ing to the invention directly in the culture medium, without nant bacteria of the genus Escherichia or Bacillus, preferably the need to separate or purify the enzymes. 40 of the species Escherichia coli or Bacillus subtilis, having In one embodiment, a (micro)organism is used having the been modified so as to endogenously produce crotyl alcohol natural or artificial property of endogenously producing cro and to convert it into butadiene. tyl alcohol, and also expressing or overexpressing the It is also possible to employ an extremophilic bacterium enzymes as described in connection with alternatives A and such as Thermus thermophilus, oranaerobic bacteria from the B, above, so as to produce butadiene directly from a carbon 45 family Clostridiae. Source present in Solution. In another embodiment, the (mi In one embodiment the microorganism is a fungus, more cro)organism which is used has the natural or artificial prop preferably a fungus of the genus Saccharomyces, Schizosac erty of endogenously producing crotonaldehyde and to con charomyces, Aspergillus, Trichoderma, Pichia or Kluyvero vert it into crotyl alcohol which can then be further converted myces and even more preferably of the species Saccharomy into butadiene. 50 ces cerevisiae, Schizosaccharomyces pombe, Aspergillus In one embodiment the organism employed in the method niger; Trichoderma reesei, Pichia pastoris or of the species according to the invention is an organism, preferably a micro Kluyveromyces lactis. In a particularly preferred embodiment organism, which has been genetically modified to contain one the microorganism is a recombinant yeast capable of produc or more foreign nucleic acid molecules encoding one or more ing crotyl alcohol and converting it into butadiene due to the of the enzymes as described above in connection with alter 55 expression of the enzymes described in connection with alter natives A or B. The term “foreign' in this context means that natives A or B, above. the nucleic acid molecule does not naturally occur in said In another embodiment, the method according to the inven organism/microorganism. This means that it does not occur in tion makes use of a photosynthetic microorganism expressing the same structure or at the same location in the organism/ at least the enzymes as described in connection with alterna microorganism. In one preferred embodiment, the foreign 60 tives A or B, above. Preferably, the microorganism is a pho nucleic acid molecule is a recombinant molecule comprising tosynthetic bacterium, or a microalgae. In a further embodi a promoter and a coding sequence encoding the respective ment the microorganism is analgae, more preferably analgae enzyme in which the promoter driving expression of the cod belonging to the diatomeae. Even more preferably such a ing sequence is heterologous with respect to the coding microorganism has the natural or artificial property of endog sequence. Heterologous in this context means that the pro 65 enously producing crotyl alcohol. In this case the microor moter is not the promoter naturally driving the expression of ganism would be capable of producing butadiene directly said coding sequence but is a promoter naturally driving from CO present in Solution. US 9, 169,496 B2 19 20 In another embodiment, it is possible to use a microorgan genes at certain stages of the culture Such as induction of gene ism which belongs to the group of acetogenic bacteria which expression by chemical inducers or by a temperature shift. are capable of converting CO (or CO+H) to produce acetyl In another embodiment the organism employed in the CoA via the so-called Wood-Ljungdahl pathway (Köpke et method according to the invention is a plant. In principle any al.; PNAS 10 (2010), 13087-13092). A fermentation process possible plant can be used, i.e. a monocotyledonous plant or using such microorganisms is known as Syngas fermentation. a dicotyledonous plant. It is preferable to use a plant which Strictly mesophilic anaerobes Such as C. liungdahli, C. ace can be cultivated on an agriculturally meaningful scale and ticum, Acetobacterium woodii, C. autoethanogenium, and C. which allows to produce large amounts of biomass. Examples carboxy deviron, are frequently being used in Syngas fermen are grasses like Lolium, cereals like rye, wheat, barley, oat, tation (Munasingheet et al.; Bioresource Technology 101 10 millet, maize, other starch storing plants like potato or Sugar (2010), 5013-5022). It is also conceivable to use in the method according to the storing plants like Sugar cane or Sugar beet. Conceivable is invention a combination of (micro)organisms wherein differ also the use of tobacco or of vegetable plants such as tomato, ent (micro)organisms express different enzymes as described pepper, cucumber, eggplant etc. Another possibility is the use above. In a further embodiment at least one of the microor 15 of oil storing plants such as rape seed, olives etc. Also con ganisms is capable of producing crotyl alcohol or, in an alter ceivable is the use of trees, in particular fast growing trees native embodiment, a further microorganism is used in the Such as eucalyptus, poplar or rubber tree (Hevea brasiliensis). method which is capable of producing crotyl alcohol. Particularly preferred is the use of plants which naturally In another embodiment the method according to the inven produce crotonaldehyde, e.g. soybeans. Such plants are pref tion makes use of a multicellular organism expressing at least erably further modified so as to be able to convert crotonal the enzymes as described in connection with alternatives A or dehyde into crotyl alcohol. B. above. Examples for Such organisms are plants or animals. In another embodiment, the method according to the inven In a particular embodiment, the method according to the tion is characterized by the conversion of a carbon Source, invention involves culturing microorganisms in standard cul Such as glucose, into crotyl alcohol (preferably via crotonyl ture conditions (30–37° C. at 1 atm, in a fermenter allowing 25 CoA and crotonaldehyde) followed by the conversion of cro aerobic growth of the bacteria) or non-standard conditions tyl alcohol into butadiene. (higher temperature to correspond to the culture conditions of In another embodiment, the method according to the inven thermophilic organisms, for example). tion comprises the production ofbutadiene from atmospheric In a further embodiment the method of the invention is CO or from CO artificially added to the culture medium. In carried out under microaerophilic conditions. This means that 30 this case the method is implemented in an organism which is the quantity of injected air is limiting so as to minimize able to carry out photosynthesis. Such as for example microal residual oxygen concentrations in the gaseous effluents con gae. taining butadiene. As described above, it is possible to use in the method In another embodiment the method according to the inven according to the inventiona (micro)organism which is geneti tion furthermore comprises the step of collecting the gaseous 35 cally modified so as to contain a nucleic acid molecule encod butadiene degassing out of the reaction. Thus in a preferred ing at least one of the enzymes as described above in connec embodiment, the method is carried out in the presence of a tion with alternatives A or B. Such a nucleic acid molecule system for collecting butadiene under gaseous form during encoding an enzyme as described above can be used alone or the reaction. as part of a vector. The nucleic acid molecules can further As a matter of fact, butadiene adopts the gaseous state at 40 comprise expression control sequences operably linked to the room temperature and atmospheric pressure. The method polynucleotide comprised in the nucleic acid molecule. The according to the invention therefore does not require extrac term “operatively linked' or “operably linked’, as used tion of butadiene from the liquid culture medium, a step throughout the present description, refers to a linkage which is always very costly when performed at industrial between one or more expression control sequences and the scale. The evacuation and storage of butadiene and its pos 45 coding region in the polynucleotide to be expressed in Such a sible Subsequent physical separation and chemical conver way that expression is achieved under conditions compatible sion can be performed according to any method known to one with the expression control sequence. of skill in the art and as described above. Expression comprises transcription of the heterologous In a particular embodiment, the method also comprises DNA sequence, preferably into a translatable mRNA. Regu detecting butadiene which is present in the gaseous phase. 50 latory elements ensuring expression in fungi as well as in The presence ofbutadiene in an environment of air or another bacteria, are well known to those skilled in the art. They gas, even in Small amounts, can be detected by using various encompass promoters, enhancers, termination signals, target techniques and in particular by using gas chromatography ing signals and the like. Examples are given further below in systems with infrared or flame ionization detection, or by connection with explanations concerning vectors. coupling with mass spectrometry. 55 Promoters for use in connection with the nucleic acid mol When the process according to the invention is carried out ecule may be homologous or heterologous with regard to its in vivo by using an organism/microorganism providing the origin and/or with regard to the gene to be expressed. Suitable respective enzyme activities, the organism, preferably micro promoters are for instance promoters which lend themselves organism, is cultivated under Suitable culture conditions to constitutive expression. However, promoters which are allowing the occurrence of the enzymatic reaction. The spe 60 only activated at a point in time determined by external influ cific culture conditions depend on the specific organism/mi ences can also be used. Artificial and/or chemically inducible croorganism employed but are well known to the person promoters may be used in this context. skilled in the art. The culture conditions are generally chosen The vectors can further comprise expression control in Such a manner that they allow the expression of the genes sequences operably linked to said polynucleotides contained encoding the enzymes for the respective reactions. 65 in the vectors. These expression control sequences may be Various methods are known to the person skilled in the art Suited to ensure transcription and synthesis of a translatable in order to improve and fine-tune the expression of certain RNA in bacteria or fungi. US 9, 169,496 B2 21 22 In addition, it is possible to insert different mutations into ing these properties are described in detail in the literature. the polynucleotides by methods usual in molecular biology Regulatory sequences for the expression in microorganisms (see for instance Sambrook and Russell (2001), Molecular (for instance E. coli, S. cerevisiae) are sufficiently described Cloning: A Laboratory Manual, CSH Press, Cold Spring Har in the literature. Promoters permitting a particularly high bor, N.Y., USA), leading to the synthesis of polypeptides expression of a downstream sequence are for instance the T7 possibly having modified biological properties. The introduc promoter (Studier et al., Methods in Enzymology 185 (1990), tion of point mutations is conceivable at positions at which a 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez modification of the amino acid sequence for instance influ and Chamberlin (Eds), Promoters, Structure and Function; ences the biological activity or the regulation of the polypep Praeger, New York, (1982), 462-481; DeBoer et al., Proc. tide. 10 Natl. Acad. Sci. USA (1983), 21-25), Ip1, rac (Boros et al., Moreover, mutants possessing a modified Substrate or Gene 42 (1986), 97-100). Inducible promoters are preferably product specificity can be prepared. Preferably, Such mutants used for the synthesis of polypeptides. These promoters often show an increased activity. Furthermore, the introduction of lead to higher polypeptide yields than do constitutive promot mutations into the polynucleotides encoding an enzyme as ers. In order to obtain an optimum amount of polypeptide, a defined above allows the gene expression rate and/or the 15 two-stage process is often used. First, the host cells are cul activity of the enzymes encoded by said polynucleotides to be tured under optimum conditions up to a relatively high cell optimized. density. In the second step, transcription is induced depend For genetically modifying bacteria or fungi, the polynucle ing on the type of promoter used. In this regard, a tac promoter otides encoding an enzyme as defined above or parts of these is particularly suitable which can be induced by lactose or molecules can be introduced into plasmids which permit IPTG (isopropyl-B-D-thiogalactopyranoside) (deBoer et mutagenesis or sequence modification by recombination of al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termina DNA sequences. Standard methods (see Sambrook and Rus tion signals for transcription are also described in the litera sell (2001), Molecular Cloning: A Laboratory Manual, CSH ture. Press, Cold Spring Harbor, N.Y., USA) allow base exchanges The transformation of the host cell with a polynucleotide or to be performed or natural or synthetic sequences to be added. 25 vector according to the invention can be carried out by stan DNA fragments can be connected to each other by applying dard methods, as for instance described in Sambrook and adapters and linkers to the fragments. Moreover, engineering Russell (2001), Molecular Cloning: A Laboratory Manual, measures which provide suitable restriction sites or remove CSH Press, Cold Spring Harbor, N.Y., USA; Methods in Surplus DNA or restriction sites can be used. In those cases, in Yeast Genetics, A Laboratory Course Manual, Cold Spring which insertions, deletions or Substitutions are possible, in 30 Harbor Laboratory Press, 1990. The host cell is cultured in vitro mutagenesis, “primer repair, restriction or ligation can nutrient media meeting the requirements of the particular host be used. In general, a sequence analysis, restriction analysis cell used, in particular in respect of the pH value, temperature, and other methods of biochemistry and molecular biology are salt concentration, aeration, antibiotics, vitamins, trace ele carried out as analysis methods. mentS etc. The polynucleotide introduced into a (micro)organism is 35 The present invention also relates to an organism, prefer expressed so as to lead to the production of a polypeptide ably a microorganism, which is able to express the enzymes having any of the activities described above. An overview of required for the conversion of crotyl alcohol into butadiene different expression systems is for instance contained in according to alternative A or B of the method of the invention Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (according to the first aspect) as described above and which is (Methods in Enzymology 153 (1987), 516-544) and in Saw 40 able to convert crotyl alcohol into butadiene. ers et al. (Applied Microbiology and Biotechnology 46 Thus, the present invention also relates to a (micro)organ (1996), 1-9), Billman-Jacobe (Current Opinion in Biotech ism which expresses nology 7 (1996), 500-4), Hockney (Trends in Biotechnology A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1.50); or 12 (1994), 456-463), Griffiths et al., (Methods in Molecular (ii) a thiamine kinase (EC 2.7.1.89); and Biology 75 (1997), 427-440). An overview of yeast expres 45 (b)(i) a terpene synthase, e.g. an isoprene synthase (EC sion systems is for instance given by Hensing et al. (Antonie 4.2.3.27); or van Leuwenhoek 67 (1995), 261-279), Bussineau et al. (De (ii) an isopentenyl phosphate kinase and a terpene syn velopments in Biological Standardization 83 (1994), 13-19), thase, e.g. an isoprene synthase (EC 4.2.3.27); or Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93, B)(a)(i) a 2-amino-4-hydroxy-6-hydroxymethyldihydropte Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), 50 ridine diphosphokinase (EC 2.7.6.3); or Vedvick (Current Opinion in Biotechnology 2 (1991), 742 (ii) a thiamine diphosphokinase (EC 2.7.6.2); and 745) and Buckholz (Bio/Technology 9 (1991), 1067-1072). (b) a terpene synthase, e.g. an isoprene synthase (EC Expression vectors have been widely described in the lit 4.2.3.27), and which is capable of converting crotyl alcohol erature. As a rule, they contain not only a selection marker into butadiene. As regards preferred embodiments, the gene and a replication-origin ensuring replication in the host 55 same applies as has been set forth above in connection with selected, but also a bacterial or viral promoter, and in most the method according to the invention. cases a termination signal for transcription. Between the pro As regards in particular the terpene synthase and the pre moter and the termination signal there is in general at least ferred embodiments of terpene synthases to be expressed by one restriction site or a polylinker which enables the insertion the (micro)organism, the same applies as has been set forth of a coding DNA sequence. The DNA sequence naturally 60 above in connection with the method according to the inven controlling the transcription of the corresponding gene can be tion. used as the promoter sequence, if it is active in the selected Thus, in one preferred embodiment the terpene synthase is host organism. However, this sequence can also be exchanged (a) an isoprene synthase (EC 4.2.3.27); or for other promoter sequences. It is possible to use promoters (b) a myrcene/ocimene synthase (EC 4.2.3.15); or ensuring constitutive expression of the gene and inducible 65 (c) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or promoters which permit a deliberate control of the expression (d) a pinene synthase (EC 4.2.3.14); or of the gene. Bacterial and viral promoter sequences possess (e) a monoterpene synthase. US 9, 169,496 B2 23 24 The present invention also relates to an organism, prefer generally belong to the EC classification EC 1.3.1 and include ably a microorganism, which is able to express the enzymes acyl-CoA dehydrogenase (NADP+, EC 1.3.1.8), enoyl-acyl required for the conversion of crotonyl-CoA into crotonalde carrier-protein reductase (NADH: EC 1.3.1.9), enoyl-acyl hyde and/or crotylalcohol as described in connection with the carrier-protein reductase (NADPH: EC 1.3.1.10), cis-2- second aspect of the invention. Thus, the present invention 5 enoyl-CoA reductase (NADPH: EC 1.3.1.37) and trans-2- also relates to a (mirco)organism which expresses enoyl-CoA reductase (NADPH: EC 1.3.1.38). Thus, in one (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); embodiment the organism is genetically modified so as to and/or decrease the activity of enzymes which may lead to a reduc (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or tion of crotonyl-CoA to butyryl-CoA and thus to a diversion (iii) an alcohol dehydrogenase (EC 1.1.1.1); and/or 10 of crotonyl-CoA into other pathways. Such a reduction of (iv) an aldehydefalcohol dehydrogenase; and/or activity can be achieved by methods known to the person (v) an acyl-CoA reductase; and/or skilled in the art and include, for example, the decrease of the (vi) an aldo-keto reductase (AKR); and/or expression of the respective gene(s) coding for the respective (vii) an aldehyde reductase; and/or enzyme(s) by known methods such as antisense approaches, (viii) a short-chain dehydrogenase/fatty acyl-CoA reductase 15 siRNA approaches or the like. In case the respective enzyme (ix) and which is capable of converting crotonyl-CoA into activity is not necessary for Survival of the microorganism, it crotonaldehyde and/or crotyl alcohol. can also be knocked out completely, e.g. by disrupting the The present invention also relates to an organism, prefer gene or completely deleting the gene. ably a microorganism, which is further able to express the The present invention also relates to a composition com enzymes required for the conversion of 3-hydroxybutyryl prising CoA into crotonyl-CoA. Thus, the present invention also A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1.50); or relates to a (mirco)organism which expresses a 3-hydroxybu (ii) a thiamine kinase (EC 2.7.1.89); and tyryl-CoA dehydratase (EC 4.2.1.55) and/or an enoyl-CoA (b)(i) a terpene synthase, e.g. an isoprene synthase (EC hydratase (EC 4.2.1.17) and/or an enoyl-CoA hydratase 2 4.2.3.27); or (EC 4.2.1.119) and/or a crotonyl-acyl-carrier-protein 25 (ii) an isopentenyl phosphate kinase and a terpene syn hydratase (EC 4.2.1.58) and which is capable of converting thase, e.g. an isoprene synthase (EC 4.2.3.27); or 3-hydroxybutyryl-CoA into crotonyl-CoA. B)(a)(i) a 2-amino-4-hydroxy-6-hydroxymethyldihydropte The present invention also relates to an organism, prefer ridine diphosphokinase EC 2.7.6.3); or ably a microorganism, which is further able to express the (ii) a thiamine diphosphokinase (EC 2.7.6.2); and enzymes required for the conversion of acetoacetyl-CoA into 30 (b) a terpene synthase, e.g. an isoprene synthase (EC 3-hydroxybutyryl-CoA. Thus, the present invention also 4.2.3.27). relates to a (mirco)organism which further expresses an Such a composition may also comprise crotyl alcohol. As acetoacetyl-CoA reductase (EC 1.1.1.36) and which is regards preferred embodiments, the same applies as has been capable of converting acetoacetyl-CoA into 3-hydroxybu set forth above in connection with the method according to tyryl-CoA. 35 the invention. Finally, the present invention also relates to an organism, As regards in particular the terpene synthase and the pre preferably a microorganism, which is further able to express ferred embodiments of terpene synthases to be expressed by the enzymes required for the enzymatic production of the (micro)organism, the same applies as has been set forth acetoacetyl-CoA. This production may be achieved by the above in connection with the method according to the inven conversion of acetyl-CoA into acetoacetyl-CoA or by the 40 tion. conversion of acetyl-CoA and malonyl-CoA into acetoacetyl Thus, in one preferred embodiment the terpene synthase is CoA. Thus, the present invention also relates to a (mirco) (f) an isoprene synthase (EC 4.2.3.27); or organism which further expresses an acetyl-CoA C-acetyl (g) a myrcene/ocimene synthase (EC 4.2.3.15); or transferase (EC 2.3.1.9) and which is capable of converting (h) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or acetyl-CoA into acetoacetyl-CoA and/or which further 45 (i) a pinene synthase (EC 4.2.3.14); or expresses a acetoacetyl-CoA synthase and which is capable (i) a monoterpene synthase. of converting acetyl-CoA and malonyl-CoA into acetoacetyl The present invention also relates to a composition com CoA. prising In one embodiment an organism according to the present (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); invention is a recombinant organism in the sense that it is 50 and/or genetically modified due to the introduction of at least one (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or nucleic acid molecule encoding at least one of the above (iii) an alcohol dehydrogenase (EC 1.1.1.1); and/or mentioned enzymes. Preferably such a nucleic acid molecule (iv) an aldehydefalcohol dehydrogenase; and/or is heterologous with regard to the organism which means that (v) an acyl-CoA reductase; and/or it does not naturally occur in said organism. 55 (vi) an aldo-keto reductase (AKR); and/or The microorganism is preferably a bacterium, a yeast or a (vii) an aldehyde reductase; and/or fungus. In another preferred embodiment the organism is a (viii) a short-chain dehydrogenase/fatty acyl-CoA reductase. plant or non-human animal. As regards other preferred Moreover, the present invention also relates to Such a com embodiments, the same applies as has been set forth above in position which further comprises a 3-hydroxybutyryl-CoA connection with the method according to the invention. 60 dehydratase (EC 4.2.1.55) and/or an enoyl-CoA hydratase In an embodiment according to the present invention in (EC 4.2.1.17) and/or an enoyl-CoA hydratase 2 (EC which an organism, preferably a microorganism, is employed 4.2.1.119) and/or a crotonyl-acyl-carrier-protein hydratase which is capable of providing crotonyl-CoA. Such a (micro) (EC 4.2.1.58). The present invention also relates to a compo organism is advantageously further genetically modified so as sition which also comprises an acetoacetyl-CoA reductase to avoid diverting of the crotonyl-CoA into other pathways. It 65 (EC 1.1.1.3.6). Finally, the present invention also relates to a is known, for example, that crotonyl-CoA can be reduced by composition which also comprises an acetyl-CoA C-acetyl a variety of enzymes to lead to butyryl-CoA. These enzymes transferase (EC 2.3.1.9) or an acetoacetyl-CoA synthase. US 9, 169,496 B2 25 26 The present invention also relates to the use of a combina FIG. 5 shows a scheme of the ADP quantification assay, tion of enzymes comprising: monitoring NADH consumption by the decrease of absor A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1.50); or bance at 340 nm. (ii) a thiamine kinase (EC 2.7.1.89); and FIG. 6 shows a mass spectrum of an enzymatic assay with (b)(i) a terpene synthase, e.g. an isoprene synthase (EC 5 hydroxyethylthiazole kinase from E. coli. 4.2.3.27); or FIG. 7 shows a mass spectrum of a control assay without (ii) an isopentenyl phosphate kinase and a terpene Syn enzyme. thase, e.g. an isoprene synthase (EC 4.2.3.27); or FIG. 8 shows a comparison between dimethylallyl diphos B) (a)(i) a 2-amino-4-hydroxy-6-hydroxymethyldihydropte phate and crotyl diphosphate and their conversion into iso ridine diphosphokinase EC 2.7.6.3); or 10 prene (2-methyl-buta-1,3-diene) and butadiene, respectively. (ii) a thiamine diphosphokinase (EC 2.7.6.2); and FIG. 9 shows the formula of crotonyl-Coenzyme A (b) a terpene synthase, e.g. an isoprene synthase (EC FIG. 10 shows the MS spectrum of the trans crotyl mono 4.2.3.27); phosphate phosphorylation reaction catalyzed by isopentenyl for the production of butadiene from crotyl alcohol. As monophosphate kinase from M. jannaschii (a) and of a con regards preferred embodiments, the same applies as has been 15 trol assay without enzyme (b). set forth above in connection with the method according to FIG. 11 shows 1,3-butadiene production from trans crotyl the invention. monophosphate catalyzed by terpene synthases. As regards in particular the terpene synthase and the pre FIG. 12 shows the mass spectrum of commercial 1,3-buta ferred embodiments of terpene synthases to be expressed by diene (a) and 1,3-butadiene produced from transcrotyl mono the (micro)organism, the same applies as has been set forth 20 phosphate in an enzymatic reaction catalyzed by monoter above in connection with the method according to the inven pene synthase from E. globulus (b). tion. FIG. 13 shows 1,3-butadiene production from trans crotyl Thus, in one preferred embodiment the terpene synthase is diphosphate catalyzed by terpene synthases. (k) an isoprene synthase (EC 4.2.3.27); or FIG. 14 shows a time courses of NADPH oxidation in (1) a myrcene/ocimene synthase (EC 4.2.3.15); or 25 crotonyl-CoA reduction assay with reductase from Hahella (m) a farmesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or cheiuensis and varying concentrations of NADPH. (n) a pinene synthase (EC 4.2.3.14); or FIG.15 shows a chromatogram of the crotonyl-CoA reduc (o) a monoterpene synthase. tion reaction catalyzed by acyl-CoA reductase from H. The present invention also relates to the use of cheiuensis. (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34): 30 Other aspects and advantages of the invention will be and/or described in the following examples, which are given for (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or purposes of illustration and not by way of limitation. (iii) an alcohol dehydrogenase (EC 1.1.1.1); and/or (iv) an aldehydefalcohol dehydrogenase; and/or EXAMPLES (v) an acyl-CoA reductase; and/or 35 (vi) an aldo-keto reductase (AKR); and/or Example 1 (vii) an aldehyde reductase; and/or (viii) a short-chain dehydrogenase/fatty acyl-CoA reductase Cloning, Expression and Purification of Enzymes for the conversion of crotonyl-CoA into crotonaldehyde and/ or crotyl alcohol. 40 Cloning, Bacterial Cultures and Expression of Proteins Furthermore the present invention also relates to the use of The genes encoding studied enzymes were cloned in pET a combination of enzymes comprising 25b vector (Novagen). A stretch of 6 histidine codons was (a) an acetoacetyl-CoA reductase (EC 1.1.1.36); and inserted after the methionine initiation codon to provide an (b)(i) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55); affinity tag for purification. Competent E. coli BL21 (DE3) and or 45 cells (Novagen) were transformed with these vectors accord (ii) an enoyl-CoA hydratase (EC 4.2.1.17); and/or ing to the heat shock procedure. The transformed cells were (iii) an enoyl-CoA hydratase 2 (EC 4.2.1.119); and/or grown with shaking (160 rpm) on ZYM-5052 auto-induction (iv) a crotonyl-acyl-carrier-protein hydratase (EC medium (Studier F.W. Prot. Exp. Pur. 41 (2005), 207-234) for 4.2.1.58) 6 hat 37°C. and protein expression was continued at 28°C. for the production of crotonyl-CoA from acetoacetyl-CoA. 50 or 20° C. overnight (approximately 16 h). The cells were The present invention also relates to the use of a combina collected by centrifugation at 4°C., 10,000 rpm for 20 min tion of enzymes comprising and the pellets were frozen at -80° C. (a)(i) an acetyl-CoA C-acetyltransferase (EC 2.3.1.9) and/or Protein Purification and Concentration (ii) an acetoacetyl-CoA synthase; and The pellets from 200 ml of culture cells were thawed on ice (b) an acetoacetyl-CoA reductase (EC 1.1.1.36): 55 and resuspended in 5 ml of NaHPO pH 8 containing 300 (c) (i) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55); mM NaCl, 5 mM MgCl, and 1 mM DTT. Twenty microliters and/or of lysonase (Novagen) were added. Cells were incubated 10 (ii) an enoyl-CoA hydratase (EC 4.2.1.17); and/or minutes at room temperature and then returned to ice for 20 (iii) an enoyl-CoA hydratase 2 (EC 4.2.1.119); and/or minutes. Cell lysis was completed by sonication for 3x15 (iv) a crotonyl-acyl-carrier-protein hydratase (EC 60 seconds. The bacterial extracts were then clarified by cen 4.2.1.58) trifugation at 4° C., 10,000 rpm for 20 min. The clarified for the production of crotonyl-CoA from acetyl-CoA. bacterial lysates were loaded on PROTINO-1000 Ni-TED FIG. 1 shows the chemical structure of crotonyl alcohol. column (Macherey-Nagel) allowing adsorption of 6-His FIG. 2 shows the chemical structure of crotyl phosphate. tagged proteins. Columns were washed and the enzymes of and crotyl diphosphate 65 interest were eluted with 4 ml of 50 mM NaHPO pH 8 FIG.3 shows the chemical structure of crotonaldehyde. containing 300mMNaCl,5 mMMgCl, 1 mMDTT,250 mM FIG. 4 shows the chemical structure of butadiene. imidazole. Eluates were then concentrated and desalted on US 9, 169,496 B2 27 28 Amicon Ultra-4 10 kDa filter unit (Millipore) and resus supernatant was transferred into a clean vial. The MS spectra pended in 0.25 ml 50 mM Tris-HCl pH 7.4 containing 0.5 mM were obtained on an ion trap mass spectrometer (Esquire DTT and 5 mM MgCl2. Protein concentrations were quanti 3000, Bruker) in negative ion mode by direct injection of the fied according to the Bradford method. The purity of proteins sample using a syringe pump operated at a flow rate of 2 ml/h. The presence of crotyl monophosphate was evaluated. MS thus purified varied from 50% to 90%. 5 analysis showed an M-H-ion at m/z, 151, corresponding to Example 2 crotyl monophosphate, from the enzymatic sample but not from the controls (FIGS. 6 and 7). Screening for Crotyl Alcohol Phosphorylation Example 4 Activity 10 Screening for 1,3-Butadiene Production from Crotyl The release of ADP that is associated with crotyl alcohol Monophosphate Using Purified Isoprene Synthases phosphorylation was quantified using the pyruvate kinase/ lactate dehydrogenase coupled assay (FIG. 5). The purified Crotyl monophosphate is synthesized upon request by a 4-methyl-5-(2-hydroxyethyl)thiazole kinases from Escheri- 15 company specialized in custom synthesis (Syntheval, chia coli (SEQID NO:2), Bacillus subtilis (SEQ ID NO:1), France). Rhizobium leguminosarum (SEQ ID NO:3) were evaluated The enzymatic assays are carried out under the following for their ability to phosphorylate crotyl alcohol releasing conditions at 37° C.: ADP. The studied enzymatic reaction was carried out under 50 mM Tris-HC1 pH7.5 the following conditions at 37°C.: 2O 1 to 200 mM cis or trans crotyl monophosphate 50 mM Tris-HCl pH 7.5 1 mM DTT 10 mM MgCl, 1 to 20 mM MgCl, 100 mM KC1 1 to 5 mg/ml isoprene synthase 5 nM ATP The enzyme-free control reaction is carried out in parallel. O4 nM NADH 25 The enzymatic mixture is incubated at 37° C. for 72 h in a sealed vial (Interchim). 1 mM Phosphoenolpyruvate Volatile compounds in the headspace of the reaction mix 3 U/ml Lactate dehydrogenase ture are collected using a gas Syringe equipped with an anti 1.5 U/ml Pyruvate kinase backup mechanism and are directly injected into a GC-430 50 mM crotyl alcohol, mixture cis and trans gas chromatograph (Brucker) equipped with an FID detector The pH was adjusted to 7.5 30 and a GAS-PRO column (Agilent). The enzymatic reaction Each assay was started by addition of a particular enzyme product is identified by direct comparison with standard 1,3- at a concentration 0.05 mg/ml and the disappearance of butadiene (Sigma). NADH was monitored by following the absorbance at 340 The identity of the gas is further confirmed in GC/MS nM. analyses. Assays with hydroxyethylthiazole kinase from the E. coli 35 and Rh. leguminosarum gave rise to a reproducible and sig Example 5 nificant increase in ADP production in the presence of crotyl alcohol (Table 1). Mass spectrometry was then used to verify Screening for Crotyl Monophosphate the formation of crotyl monophosphate in the assay with the Phosphorylation Activity E. coli enzyme. 40 Sequences ofisopentenyl monophosphate kinases inferred TABLE 1. from the genomes of several members of the Archaea, in particular Methanothermobacter (SEQID NO:5), Methano 4-methyl-5-(2-hydroxyethyl) Activity, micromole? caldococcus (SEQ ID NO:6) and Thermoplasma (SEQ ID thiazole kinase min mg protein 45 NO:4) genus, are generated by oligonucleotide concatenation E. coi O.220 to fit the codon usage of E. coli. A stretch of 6 histidine codons Rh. legitiminosartin O.O87 is inserted after the methionine initiation codon to provide an B. subtiis O.O14 affinity tag for purification. The genes thus synthesized are cloned in a pBT25b expression vector and the proteins are 50 produced according to the protocol described in Example 1. Example 3 The enzymes are then assayed using the method described in Example 2 with crotyl monophosphate concentrations vary Mass Spectrometry Analysis of the Crotyl Alcohol ing from 0 to 50 mM. The release of ADP that is associated Phosphorylation Reaction with crotyl monophosphate phosphorylation is quantified 55 using the pyruvate kinase/lactate dehydrogenase coupled The desired enzymatic reactions were carried out under the assay. Each assay is started by addition of particular enzyme following conditions: (at a final concentration from 0.05 mg/ml to 1 mg/ml) and the 50 mM Tris-HC1 pH7.5 disappearance of NADH is monitored by following the absor 10 mM MgCl2 bance at 340 nM. 50 mM cis or trans crotyl alcohol 60 20 mM ATP Example 6 0.1 mg/ml purified hydroxythiazolekinase from E. coli (SEQ ID NO:2) Mass Spectrometry Analysis of the Crotyl The control reactions without enzyme, without substrate Monophosphate Phosphorylation Reaction and without ATP were run in parallel. The assays were incu- 65 bated overnight without shaking at 37° C. Typically, an ali Enzymatic assays are run in 50 mM Tris-HCl pH 7.5, quot of 2001 reaction was removed, centrifuged and the contained 5 mM MgCl2, 20 mM ATP 2 mM f-mercaptoet US 9, 169,496 B2 29 30 hanol and crotyl monophosphate varying in the range from 0 Example 2. Kinetic parameters obtained for purified 4-me to 50 mM in a final volume of 0.25 ml. The reactions are thyl-5-(2-hydroxyethyl)thiazole kinase from E. coli are pre initiated with the addition of purified isoprenol monophos sented in Table 2. phate kinase and incubated overnight at 37–55°C. The control reactions contain no enzyme. Following incubation samples 5 TABLE 2 are processed by mass spectrometry analysis. An aliquot of 200 ul reaction is removed, centrifuged and the Supernatant is Kinetic parameters transferred to a clean vial. The MS spectra are obtained onion Substrate K, mM kars' trapp mass spectrometer (Esquire 3000, Bruker) in negative ion mode by direct injection of sample using a syringe pump 10 Ciscrotyl alcohol 13.6 O.19 operated at a flow rate of 2 ml/h. Transcrotyl alcohol 30 O.11 Example 7 Example 10 Screening for 1,3-Butadiene Production from Crotyl 15 Diphosphate Using Purified Isoprene Synthases Mass Spectrometry Analysis of the Crotyl Monophosphate Phosphorylation Reaction Crotyl diphosphate is synthesized upon request by a com pany specialized in custom synthesis, Syntheval (France). The enzymatic reactions were carried out under the follow The enzymatic assays are carried out under the following ing conditions: conditions at 37° C.: 50 mM Tris-HCl pH 7.5 50 mM Tris-HC1 pH7.5 10 mM MgCl, 1 to 200 mM cis or trans crotyl diphosphate 100 mM KC1 1 mM DTT 25 50 mM transcrotyl monophosphate 1 to 20 mM MgCl, 20 mM ATP 1 to 5 mg/ml isoprene synthase 0.1 mg/ml purified isopentenyl monophosphate kinase The enzyme-free control reaction is carried out in parallel. The enzymatic mixture is incubated at 37° C. for 72 h in a Control assays were performed in which either no enzyme sealed vial (Interchim). was added, or no Substrate was added. The assays were incu 30 bated overnight without shaking at 37° C. Typically, an ali Volatile compounds in the headspace of the reaction mix quot of 200 ul reaction was removed, centrifuged and the ture are collected using a gas Syringe equipped with an anti Supernatant was transferred into a clean vial. The MS spectra backup mechanism and are directly injected into a GC-430 were obtained on ion trap mass spectrometer (Esquire 3000, gas chromatograph (Brucker) equipped with an FID detector Bruker) in negative ion mode by direct injection of sample and a GAS-PRO column (Agilent). The enzymatic reaction using a syringe pump operated at a flow rate of 2 ml/h. The product is identified by direct comparison with standard 1,3- 35 presence of crotyl diphosphate was evaluated. MS analysis butadiene (Sigma). showed an M-H ion at m/Z 231.5, corresponding to crotyl The identity of the gas is further confirmed in GC/MS diphosphate, from the enzymatic samples but not from the analyses. controls. Examples of mass spectrums of enzymatic assay Example 8 40 with isopentenyl monophosphate kinase from M. jannaschii and control assay without enzyme are shown in FIGS. 10a Screening of Hydroxymethylglutaryl-CoA and 10b. (Hmg-CoA) Reductases Using Crotonyl-CoA as a Substrate Example 11 45 Sequences of hydroxymethylglutaryl-CoA reductases Kinetic Parameters of the Crotyl Monophosphate inferred from the genomes of prokaryotic and eukaryotic Phosphorylation Reaction organisms are generated to fit the codon usage of E. coli. A Ciscrotyl monophosphate and trans crotyl monophosphate stretch of 6 histidine codons is inserted after the methionine were synthesized upon request by a company specialized in initiation codon to provide an affinity tag for purification. The 50 genes thus synthesized are cloned in a pET25b expression custom synthesis (Syntheval, France). Kinetic parameters for vector and the proteins are produced according to the protocol the phosphorylation of these substrates were determined described in Example 1. The reductase activity of the purified using the spectrophotometric assay described in Example 2. enzymes using crotonyl-CoA as a Substrate is then deter Kinetic parameters obtained with purified isopentenyl mono mined by measuring the initial decrease in absorbance at 340 55 phosphate kinases from different members of the Archaea nm due to the NADPH oxidation. The standard assay is per kingdom are presented in Table 3 (cis crotyl monophosphate formed at pH 7.5, 50 mM phosphate buffer, containing 10 as a Substrate) and Table 4 (trans crotyl monophosphate as a mM dithiothreitol, 0.1 mM NADPH and crotonyl-CoA at substrate). concentration varying from 0 to 10 mM. 60 TABLE 3 Example 9 Isopentenyl monophosphate Kinetic parameters Kinetic Parameters of Crotyl Alcohol Phosphorylation kinase K, mM kars' 65 Methanocaidococcus O.20 3.4 Kinetic parameters of crotyl alcohol phosphorylation were iannaschii determined using the spectrophotometric assay described in US 9, 169,496 B2 32 TABLE 3-continued 50 mM Tris-HCl pH 7.5 20 mM MgCl, Isopentenyl 20 mMKC1 monophosphate Kinetic parameters 2 mMDTT kinase K, mM kees' 5 0-25 mM trans crotyl monophosphate Meihanothermobacter O.94 5.7 The reaction was initiated by addition of 0.25 mg of puri thermautotrophicus fied monoterpene synthase from Eucalyptus globulus to 0.5 Thermoplasma O.61 1.8 ml of reaction mixture. An enzyme-free control reaction was acidophilum carried out in parallel. Assays were incubated at 37° C. for 10 0.5-4h in a sealed vial of 1.5 ml (Interchim) with shaking. 1,3-butadiene production was analyzed using the GC/FID TABLE 4 procedure described in Example 12. Monoterpene synthase Kinetic parameters from E. globulus was found to have a K value of 6 mM and 15 akes of at least 0.2x10 sec'. Enzyme K, mM kars' Example 14 Meihanocaidococcus O49 3.0 iannaschii Meihanothermobacter O45 8.7 Enzyme Catalyzed Production of 1,3-Butadiene from thermautotrophicus 2O Trans Crotyl Diphosphate with Purified Terpene Thermoplasma 1 2.2 acidophilum Synthases The enzymatic assays were carried out under the following conditions at 37° C.: Example 12 25 50 mM Tris-HCl pH 7.5 Enzyme Catalyzed Production of 1,3-Butadiene from 25 mM trans crotyl diphosphate Trans Crotyl Monophosphate with Purified Terpene 2 mMDTT Synthases 50 mM MgCl, 50 mMKC1 The enzymatic assays were carried out under the following 30 2 mg of the purified terpene synthase was added to 0.5 ml of conditions at 37° C.: reaction mixture. An enzyme-free control reaction was 50 mM Tris-HCl pH 7.5 carried out in parallel. Assays were incubated at 37°C. for 25 mM trans crotyl monophosphate 24h in a sealed vial of 1.5 ml (Interchim) with shaking. 2 mMDTT One ml of the gaseous phase was then collected and 50 mM MgCl, 35 directly injected into a Varian GC-430 gas chromatograph 50 mM KC1 equipped with a flame ionization detector (FID). Nitrogen 2 mg of the purified terpene synthase was added to 0.5 ml of was used as carrier gas with a flow rate of 1.5 ml/min. Volatile reaction mixture. An enzyme-free control reaction was compounds were chromatographically separated on RT-Alu carried out in parallel. Assays were incubated at 37°C. for mina Bond/Na2SO4 column (Restek) using an isothermal 24h in a sealed vial of 1.5 ml (Interchim) with shaking. 40 One ml of the gaseous phase was then collected and mode at 130°C. The enzymatic reaction product was identi directly injected into a Varian GC-430 gas chromatograph fied by direct comparison with 1,3-butadiene standard equipped with a flame ionization detector (FID). Nitrogen (Sigma). Several terpene synthases were shown to catalyze was used as carrier gas with a flow rate of 1.5 ml/min. Volatile butadiene production from trans crotyl diphosphate (FIG. compounds were chromatographically separated on RT-Alu 13). mina Bond/Na2SO4 column (Restek) using an isothermal mode at 130°C. The enzymatic reaction product was identi Example 15 fied by direct comparison with 1,3-butadiene standard (Sigma). Several terpene synthases were shown to catalyze Kinetic Parameters of Enzyme Catalyzed Production butadiene production from transcrotyl monophosphate (FIG. of 1,3-Butadiene from Trans Crotyl Diphosphate 11). 50 Gas chromatography-mass spectrometry was then used to The kinetic parameters of enzyme catalyzed production of confirm the identity of the product detected by GC/FID. 1,3-butadiene from trans crotyl monophosphate were mea Assay with E. globulus enzyme (SEQ ID NO: 8) was ana Sured under the following conditions: lyzed on a Varian 3400CX gas chromatograph equipped with 50 mM Tris-HCl pH 7.5 Varian Saturn 3 mass selective detector. A mass spectrum of 55 20 mM MgCl, 1,3-butadiene obtained by enzymatic conversion of trans cro 20 mMKC1 tyl monophosphate was similar to that of commercial 1.3- 2 mMDTT butadiene (FIGS. 12a and 12b). 0-25 mM trans crotyl diphosphate The reaction was initiated by addition of 0.25 mg of puri Example 13 60 fied monoterpene synthase from Eucalyptus globulus to 0.5 ml of reaction mixture. An enzyme-free control reaction was Kinetic Parameters of Enzyme Catalyzed Production carried out in parallel. Assays were incubated at 37° C. for of 1,3-Butadiene from Trans Crotyl Monophosphate 0.5-4h in a sealed vial of 1.5 ml (Interchim) with shaking. 1,3-butadiene production was analyzed using the GC/FID The kinetic parameters of enzyme catalyzed production of 65 procedure described in Example 12. Monoterpene synthase 1,3-butadiene from trans crotyl monophosphate were mea from E. globulus was found to have a K value of 7 mMand Sured under the following conditions: ak of at least 0.3x10" sec'. US 9, 169,496 B2 33 34 Example 16 Example 17 HPLC Studies of Enzymatic Reduction of Screening of Short-Chain Crotonyl-CoA Dehydrogenases/Reductases with Crotonyl-CoA as Substrate The enzymatic assays were carried out under the following conditions: 50 mM Potassium phosphate pH 7.5 Sequences of short-chain dehydrogenases/reductases 100 mM KC1 inferred from the genomes of prokaryotic organisms were 24 mM trans crotonyl-CoA generated by oligonucleotide concatenation to fit the codon 10 80 nM NADPH usage of E. coli. A stretch of 6 histidine codons is inserted The reactions were initiated by addition of 150 lug of puri after the methionine initiation codon to provide an affinity tag fied dehydrogenase/reductase to 150 ul of reaction mixture. for purification. The genes thus synthesized were cloned in a Assays were incubated at 37° C. for 0.5-6 h. The reactions pET25b expression vector and the proteins were produced were stopped by heating at 65° C. for 5 minutes, reaction according to the protocol described in Example 1. 15 mixtures were centrifuged and 120 ul of the clarified super natant were transferred into a clean vial. The reaction prod For the reductase assay, a reaction mixture containing 50 ucts were then extracted with an equal volume of ethyl mM potassium phosphate pH 7.5, 0.1-0.4 mM NADPH, 100 acetate. 100 ul of the upper ethyl acetate phase was trans mM NaCl, 5 mM trans crotonyl-CoA and 0.5-1 mg/ml ferred into a clean vial for HPLC analysis. Commercial cro enzyme in a total Volume of 120 ul was used and the reaction tonaldehyde and crotyl alcohol were used as reference. was carried out at 37° C. for 20 min. Control assays were HPLC-UV analysis was performed using a 1260 Inifinity LC performed in which either no enzyme was added, or no Sub System (Agilent). 10ul of samples were separated on Zorbax strate was added. Each sample was continuously monitored SB-Aq column (250x4.6 mm, 3.5 um particle size) with a for the decrease of NADPH at 340 nm on a SpectraMax mobile phase flow rate of 1.5 ml/min. The mobile phase 25 consisted of 95:5 (v/v) HO/Acetonitrile containing 8.4 mM Plus384 UV/Vis Microplate Reader (Molecular Device). sulfuric acid. Retention time for trans crotyl alcohol and Several enzymes demonstrated crotonyl-CoA reductase crotonaldehyde in these conditions were 4.3 and 5.4 min, activity with NADPH as co-substrate (FIG. 14 which shows a respectively. time courses of NADPH oxidation in crotonyl-CoA reduction The HPLC analysis showed that crotonaldehyde and crotyl assay with reductase from Hahella cheiuensis (SEQID NO: 30 alcohol were formed by enzyme catalyzed reduction of croto 17) and varying concentrations of NADPH, Table 5). nyl-CoA. A typical chromatogram obtained with short chain alcohol dehydrogenase-like protein from H. cheuensis is TABLE 5 shown on FIG. 15. These data indicate that the short-chain dehydrogenase/ Activity, reductase catalyzes the four-electron reduction of crotonyl Imol/min.img 35 Enzyme protein CoA to crotyl alcohol via the aldehyde intermediate. Short-chain dehydrogenase/reductase 1.4 Example 18 (Fatty alcohol forming acyl-CoA reductase) rom Marinobacter Kinetic Parameters of Crotyl Alcohol Production aquaeolei VT8 (SEQ ID NO: 13) 40 from Trans Crotonyl-CoA Short chain alcohol dehydrogenase- 7.5 ike protein from Marinobacter manganoxydans Kinetic parameters values towards NADPH were deter (SEQID NO: 14) mined at fixed concentration of crotonyl-CoA (5 mM) and Short chain alcohol dehydrogenase- 4 varying NADPH concentration from 0 to 0.8 mM. NADPH ike protein from oxidation was measured spectrophotometrically at 340 nm Marinobacter algicola 45 (SEQID NO: 16) according to the procedure described in Example 16. Short chain alcohol dehydrogenase- 4.9 The short chain alcohol dehydrogenase-like protein from ike protein from H. cheuensis was found to have a K of 1 mM and a k of Hahella cheuensis 6.0 sec' towards NADPH as substrate. Short chain alcohol dehydrogenase- 1.2 ike protein from 50 Kinetic parameters values towards crotonyl-CoA were Marinobacter determined at fixed concentration of NADPH (80 mM) and sp. ELB17 (SEQID NO: 15) varying crotonyl-CoA concentration from 0 to 32 mM. Kinetic parameters for the overall reaction were determined by crotyl alcohol quantification using HPLC procedure The products of enzymatic reduction of crotonyl-CoA 55 described in Example 17. The short chain alcohol dehydro were next analyzed by high-performance liquid chromatog genase-like protein from H. cheuensis was found to have a raphy (HPLC). K of 5 mMandak of at least 0.05 sec'.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 21

SEO ID NO 1 LENGTH: 272 TYPE PRT ORGANISM; Bacillus subtilis US 9, 169,496 B2 35 36 - Continued

<4 OOs, SEQUENCE: 1.

Met Asp Ala Glin Ser Ala Ala Luell Thir Ala Wall Arg Arg His 1. 15

Ser Pro Luell Wall His Ser Ile Thir Asn Asn Wall Wall Thir Asn Phe Thir 25

Ala Asn Gly Luell Lell Ala Lell Gly Ala Ser Pro Wall Met Ala Ala 35 4 O 45

Glu Glu Wall Ala Asp Met Ala Ile Ala Ala Luell Wall Luell SO 55

Asn Ile Gly Thir Lell Ser Glu Ser Wall Glu Met Ile Ile Ala 65 70

Gly Ser Ala Asn Glu His Gly Wall Pro Wall Lell Asp Pro Wall 85 90 95

Gly Ala Gly Ala Thir Pro Phe Arg Thir Ser Arg Asp Ile Ile 105 11 O

Arg Glu Wall Arg Lell Ala Ala Ile Arg ASn Ala Glu Ile Ala 115 12 O 125

His Thir Wall Gly Wall Thir Asp Trp Luell Wall Asp Ala Gly 13 O 135

Glu Gly Gly Gly Asp Ile Ile Arg Luell Glin Ala Ala Glin Lys 145 150 155 160

Lell Asn Thir Wall Ile Ala Ile Thir Gly Wall Wall Ile Ala Asp 1.65

Thr Ser His Wall Tyr Thr Lieu His Asn His Lieu Luell Thr Lys 18O 185 19 O

Wall Thir Gly Ala Gly Lell Luell Thir Ser Wall Wall Gly Ala Phe Cys 195

Ala Wall Glu Glu Asn Pro Lell Phe Ala Ala Ile Ala Ala Ile Ser Ser 21 O 215 22O

Tyr Gly Wall Ala Ala Glin Lell Ala Ala Glin Glin Thir Ala Asp Gly 225 23 O 235 24 O

Pro Gly Ser Phe Glin Ile Glu Luell Luell Asn Lys Lell Ser Thir Wall Thir 245 250 255

Glu Glin Asp Wall Glin Glu Trp Ala Thir Ile Glu Arg Wall Thir Wall Ser 26 O 265 27 O

<210s, SEQ ID NO 2 &211s LENGTH: 262 212. TYPE : PRT &213s ORGANISM: E. coli

<4 OOs, SEQUENCE: 2

Met Glin Val Asp Lell Lell Gly Ser Ala Glin Ser Ala His Ala Luell His 1. 1O 15

Lell Phe His Glin His Ser Pro Luell Wall His Cys Met Thir Asn Asp Wall 25

Wall Glin Thir Phe Thir Ala Asn Thir Luell Luell Ala Lell Gly Ala Ser Pro 35 4 O 45

Ala Met Wall Ile Glu Thir Glu Glu Ala Ser Glin Phe Ala Ala Ile Ala SO 55 6 O

Ser Ala Luell Luell Ile Asn Wall Gly Thir Luell Thir Glin Pro Arg Ala Glin 65 70 8O

Ala Met Arg Ala Ala Wall Glu Glin Ala Lys Ser Ser Glin Thir Pro Trp 85 90 95 US 9, 169,496 B2 37 38 - Continued

Thir Luell Asp Pro Wall Ala Wall Gly Ala Luell Asp Tyr Arg Arg His Phe 105 11 O

His Glu Luell Lell Ser Phe Lys Pro Ala Ala Ile Arg Gly Asn Ala 115 12 O 125

Ser Glu Ile Met Ala Lell Ala Gly Ile Ala ASn Gly Gly Arg Gly Wall 13 O 135 14 O

Asp Thir Thir Asp Ala Ala Ala Asn Ala Ile Pro Ala Ala Glin Thir Luell 145 150 155 160

Ala Arg Glu Thir Gly Ala Ile Wall Wall Wall Thir Gly Glu Met Asp Tyr 1.65 17O 17s

Wall Thir Asp Gly His Arg Ile Ile Gly Ile His Gly Gly Asp Pro Luell 18O 185 19 O

Met Thir Lys Wall Wall Gly Thir Gly Cys Ala Luell Ser Ala Wall Wall Ala 195

Ala Cys Ala Lell Pro Gly Asp Thir Luell Glu Asn Wall Ala Ser Ala 21 O 215 22O

Cys His Trp Met Lys Glin Ala Gly Glu Arg Ala Wall Ala Arg Ser Glu 225 23 O 235 24 O

Gly Pro Gly Ser Phe Wall Pro His Phe Luell Asp Ala Lell Trp Glin Luell 245 250 255

Thir Glin Glu Wall Glin Ala 26 O

SEQ ID NO 3 LENGTH: 267 TYPE : PRT ORGANISM: Rhizobium leguminosarum

< 4 OOs SEQUENCE: 3

Met Glin Thr Arg Thir Thir Pro Gly Ala Met Luell Ala Met Arg Glu 1. 5 1O 15

Lys Pro Pro Luell Wall Glin Ile Thir Asn Tyr Wall Ala Met Asn Ile 2O 25 3O

Ala Ala Asn Wall Lell Lell Ala Ala Gly Ala Ser Pro Ala Met Wall His 35 4 O 45

Ala Ala Glu Glu Ala Gly Glu Phe Ala Ala Ile Ala Ser Ala Luell Thir SO 55 6 O

Ile Asn Ile Gly Thir Lell Ser Thir Glin Trp Ile Asp Gly Met Glin Ala 65 70

Ala Ala Ala Ala Thir Ser Ala Gly Lys Pro Trp Wall Luell Asp Pro 85 90 95

Wall Ala His Tyr Ala Thir Ala Phe Arg Arg ASn Ala Wall Ala Glu Luell 105 11 O

Lell Ala Luell Pro Thir Ile Ile Arg Gly ASn Ala Ser Glu Ile Ile 115 12 O 125

Ala Luell Ala Gly Gly Glu Ser Arg Gly Glin Gly Wall Asp Ser Arg Asp 13 O 135 14 O

Pro Wall Glu Glin Ala Glu Gly Ser Ala Arg Trp Lell Ala Glu Arg Glin 145 150 155 160

Arg Ala Wall Wall Ala Wall Thir Gly Ala Wall Asp Phe Wall Thir Asp Gly 1.65 17O 17s

Glu Arg Ala Wall Arg Ile Glu Gly Gly Ser Ala Lell Met Pro Glin Wall 18O 185 19 O US 9, 169,496 B2 39 40 - Continued

Thir Ala Lieu. Gly Cys Ser Lieu. Thir Cys Lieu Val Gly Ala Phe Ala Ala 195 2OO 2O5 Thr Ala Pro Glu Asp Ile Phe Gly Ala Thr Val Ala Ala Leu Ser Thr 21 O 215 22O Phe Ala Ile Ala Gly Glu Glu Ala Ala Lieu. Gly Ala Ala Gly Pro Gly 225 23 O 235 24 O Ser Phe Ser Trp Arg Phe Lieu. Asp Ala Lieu Ala Ala Lieu. Asp Ala Glu 245 250 255 Thir Lieu. Asp Ala Arg Ala Arg Ile Ser Ala Ala 26 O 265

<210s, SEQ ID NO 4 &211s LENGTH: 245 212. TYPE: PRT <213> ORGANISM: Thermoplasma acidophilum

<4 OOs, SEQUENCE: 4 Met Met Ile Lieu Lys Ile Gly Gly Ser Val Ile Thr Asp Llys Ser Ala 1. 5 1O 15 Tyr Arg Thr Ala Arg Thr Tyr Ala Ile Arg Ser Ile Val Llys Val Lieu. 2O 25 3O Ser Gly Ile Glu Asp Leu Val Cys Val Val His Gly Gly Gly Ser Phe 35 4 O 45 Gly His Ile Lys Ala Met Glu Phe Gly Lieu Pro Gly Pro Lys Asn Pro SO 55 6 O Arg Ser Ser Ile Gly Tyr Ser Ile Val His Arg Asp Met Glu Asn Lieu 65 70 75 8O Asp Lieu Met Val Ile Asp Ala Met Ile Glu Met Gly Met Arg Pro Ile 85 90 95 Ser Val Pro Ile Ser Ala Lieu. Arg Tyr Asp Gly Arg Phe Asp Tyr Thr 1OO 105 11 O Pro Leu. Ile Arg Tyr Ile Asp Ala Gly Phe Val Pro Val Ser Tyr Gly 115 12 O 125 Asp Val Tyr Ile Lys Asp Glu. His Ser Tyr Gly Ile Tyr Ser Gly Asp 13 O 135 14 O Asp Ile Met Ala Asp Met Ala Glu Lieu. Lieu Lys Pro Asp Val Ala Val 145 150 155 160 Phe Lieu. Thir Asp Wall Asp Gly Ile Tyr Ser Lys Asp Pro Lys Arg Asn 1.65 17O 17s Pro Asp Ala Val Lieu. Lieu. Arg Asp Ile Asp Thr Asn. Ile Thir Phe Asp 18O 185 19 O Arg Val Glin Asn Asp Val Thr Gly Gly Ile Gly Llys Llys Phe Glu Ser 195 2OO 2O5 Met Val Lys Met Lys Ser Ser Val Lys Asn Gly Val Tyr Lieu. Ile Asn 21 O 215 22O Gly Asn His Pro Glu Arg Ile Gly Asp Ile Gly Lys Glu Ser Phe Ile 225 23 O 235 24 O

Gly Thr Val Ile Arg 245

<210s, SEQ ID NO 5 &211s LENGTH: 266 212. TYPE: PRT <213> ORGANISM: Methanothermobacter thermautotrophicus str. Delta H

<4 OOs, SEQUENCE: 5 US 9, 169,496 B2 41 42 - Continued

Met Ile Ile Luell Lell Gly Gly Ser Wall Ile Thir Arg Lys Asp Ser 15

Glu Glu Pro Ala Asp Arg Asp Asn Luell Glu Arg Ile Ala Ser Glu 25

Ile Gly Asn Ala Ser Pro Ser Ser Luell Met Ile Wall His Gly Ala Gly 35 4 O 45

Ser Phe Gly His Pro Phe Ala Gly Glu Tyr Arg Ile Gly Ser Glu Ile SO 55 6 O

Glu Asn Glu Glu Asp Lell Arg Arg Arg Arg Phe Gly Phe Ala Luell Thir 65 70

Glin Asn Trp Wall Lys Lell Asn Ser His Wall Asp Ala Luell Luell 85 90 95

Ala Glu Gly Ile Pro Ala Wall Ser Met Glin Pro Ser Ala Phe Ile Arg 105 11 O

Ala His Ala Gly Arg Ile Ser His Ala Asp Ile Ser Lell Ile Arg Ser 115 12 O 125

Luell Glu Glu Gly Met Wall Pro Wall Wall Gly Asp Wall Wall Luell 13 O 135 14 O

Asp Ser Asp Arg Arg Lell Phe Ser Wall Ile Ser Gly Asp Glin Luell 145 150 155 160

Ile Asn His Phe Ser Lell Luell Met Pro Glu Arg Wall Ile Luell Gly 1.65 17O 17s

Thir Asp Wall Asp Gly Wall Thir Arg Asn Pro His Pro Asp 18O 185 19 O

Ala Arg Lieu Lieu Asp Wall Ile Gly Ser Lieu Asp Asp Lieu Glu Ser Lieu 195 2O5

Asp Gly Thir Luell Asn Thir Asp Wall Thir Gly Gly Met Wall Gly Ile 21 O 215 22O

Arg Glu Luell Luell Lell Lell Ala Glu Gly Wall Glu Ser Glu Ile Ile 225 23 O 235 24 O

Asn Ala Ala Wall Pro Gly Asn Ile Glu Arg Ala Lell Lell Gly Glu Glu 245 250 255

Wall Arg Gly Thir Ile Thir Gly Lys His 26 O 265

<210s, SEQ ID NO 6 &211s LENGTH: 26 O 212. TYPE : PRT &213s ORGANISM: Methano caldococcus jannaschii

<4 OOs, SEQUENCE: 6

Met Lieu. Thir Ile Lell Lell Gly Gly Ser Ile Lell Ser Asp Lys Asn 1. 1O 15

Wall Pro Tyr Ser Ile Trp Asp Asn Luell Glu Arg Ile Ala Met Glu 2O 25 3O

Ile Asn Ala Lell Asp Tyr Asn Glin Asn Lys Glu Ile 35 4 O 45

Lell Luell Wall His Gly Gly Gly Ala Phe Gly His Pro Wall Ala 55 6 O

Lys Luell Ile Glu Asp Gly Ile Phe Ile Asn Met Glu 65 70 7s 8O

Gly Phe Trp Glu Ile Glin Arg Ala Met Arg Arg Phe Asn Asn Ile 85 90 95

Ile Ile Asp Thir Lell Glin Ser Asp Ile Pro Ala Wall Ser Ile Glin 1OO 105 11 O US 9, 169,496 B2 43 44 - Continued

Pro Ser Ser Phe Wall Wall Phe Gly Asp Luell Ile Phe Asp Thir Ser 115 12 O 125

Ala Glu Met Lell Lys Arg Asn Luell Wall Pro Wall Ile His Gly 135 14 O

Asp Wall Ile Asp Asp Asn Gly Tyr Arg Ile Ile Ser Gly Asp 145 150 155 160

Asp Wall Pro Tyr Lell Ala Asn Glu Luell Lys Ala Asp Luell Ile Luell 1.65 17O 17s

Thir Asp Wall Asp Gly Wall Luell Ile Asp Asn Pro Ile 18O 185 19 O

Arg Asp Asn Asn Ile Tyr Ile Luell Asn Tyr Luell Ser Gly 195 2O5

Ser Ser Ile Asp Wall Thir Gly Gly Met Tyr Ile Asp Met 215 22O

Ile Arg Asn Cys Arg Gly Phe Wall Phe Asn Gly Asn Ala 225 23 O 235 24 O

Asn Asn Ile Lys Ala Lell Luell Gly Glu Wall Glu Gly Thir Glu Ile 245 250 255

Asp Phe Ser Glu 26 O

<210s, SEQ ID NO 7 &211s LENGTH: 608 212. TYPE : PRT &213s ORGANISM: Pueraria lobata

<4 OOs, SEQUENCE:

Met Ala Thir Asn Lell Lell Luell Ser Asn Lys Lell Ser Ser Pro Thir 1. 15

Thir Pro Ser Thir Arg Phe Pro Glin Ser Asn Phe Ile Thir Glin 2O 25 3O

Thir Ser Luell Ala Asn Pro Lys Pro Trp Arg Wall Ile Ala Thir 35 4 O 45

Ser Ser Glin Phe Thir Glin Ile Thir Glu His ASn Ser Arg Arg Ser Ala SO 55 6 O

Asn Tyr Glin Pro Asn Lell Trp Asn Phe Glu Phe Lell Glin Ser Luell Glu 65 70 8O

Asn Asp Luell Wall Glu Luell Glu Glu Lys Ala Thir Luell Glu 85 90 95

Glu Glu Wall Arg Cys Met Ile Asn Arg Wall Asp Thir Glin Pro Luell Ser 1OO 105 11 O

Lell Luell Glu Luell Ile Asp Asp Wall Glin Arg Luell Gly Lell Thir 115 12 O 125

Phe Glu Asp Ile Ile Lys Ala Luell Glu ASn Ile Wall Luell Luell Asp 13 O 135 14 O

Glu Asn Asn Lys Ser Asp Luell His Ala Thir Ala Luell Ser Phe 145 150 155 160

Arg Luell Luell Arg Glin His Gly Phe Glu Wall Ser Glin Asp Wall Phe Glu 1.65 17O 17s

Arg Phe Asp Lys Glu Gly Gly Phe Ser Gly Glu Lell Lys Gly Asp 18O 185 19 O

Wall Glin Gly Luell Lell Ser Lell Tyr Glu Ala Ser Lell Gly Phe Glu 195 2OO 2O5 US 9, 169,496 B2 45 46 - Continued

Gly Glu Asn Luell Lell Glu Glu Ala Arg Thir Phe Ser Ile Thir His Luell 21 O 215 22O

Lys Asn Asn Luell Glu Gly Ile Asn Thir Lys Wall Ala Glu Glin Wall 225 23 O 235 24 O

Ser His Ala Luell Glu Lell Pro His Glin Arg Lell His Arg Luell Glu 245 250 255

Ala Arg Trp Phe Lell Asp Glu Pro Glu Pro His His Glin 26 O 265 27 O

Lell Luell Luell Glu Lell Ala Luell Asp Phe ASn Met Wall Glin Thir Luell 285

His Glin Glu Lell Glin Asp Luell Ser Arg Trp Trp Thir Glu Met Gly 29 O 295 3 OO

Lell Ala Ser Lell Asp Phe Wall Arg Asp Arg Lell Met Glu Wall Tyr 3. OS 310 315

Phe Trp Ala Luell Gly Met Ala Pro Asp Pro Glin Phe Gly Glu Cys Arg 3.25 330 335

Ala Wall Thir Met Phe Gly Luell Wall Thir Ile Ile Asp Asp Wall 34 O 345 35. O

Asp Wall Gly Thir Lell Asp Glu Luell Glin Lell Phe Thir Asp Ala 355 360 365

Wall Glu Arg Trp Asp Wall Asn Ala Ile Asn Thir Lell Pro Asp Met 37 O 375

Lys Luell Phe Lell Ala Lell Tyr Asn Thir Wall Asn Asp Thir Ser Tyr 385 390 395 4 OO

Ser Ile Lieu Lys Glu Lys Gly His Asn Asn Lieu. Ser Luell Thr Lys 4 OS 415

Ser Trp Arg Glu Lell Ala Phe Luell Glin Glu Ala Lys Trp Ser 425 43 O

Asn Asn Lys Ile Ile Pro Ala Phe Ser Lell Glu Asn Ala Ser 435 44 O 445

Wall Ser Ser Ser Gly Wall Ala Luell Luell Ala Pro Ser Phe Ser Wall 450 45.5 460

Cys Glin Glin Glu Asp Ile Ser Asp His Ala Lell Arg Ser Luell Thir 465 470

Asp Phe His Lell Wall Arg Ser Ser Cys Wall Ile Phe Arg Luell 485 490 495

Asn Asp Luell Thir Ser Ala Ala Glu Luell Glu Arg Gly Glu Thir Thir 505

Asn Ser Ile Ser Met His Glu Asn Asp Gly Thir Ser Glu Glu 515 525

Glin Ala Arg Glu Lell Arg Luell Ile Asp Ala Glu Trp 53 O 535 54 O

Met Asn Arg Arg Wall Ser Asp Ser Thir Luell Lell Pro Ala Phe 5.45 550 555 560

Met Glu Ile Wall Asn Met Ala Arg Wall Ser His Thir Tyr Glin 565 st O sts

Gly Asp Lell Gly Arg Pro Asp Ala Thir Glu Asn Arg Ile 585 59 O

Luell Luell Ile Asp Pro Phe Pro Ile ASn Glin Lell Met Wall 595 6OO 605

<210s, SEQ ID NO 8 &211s LENGTH: 582 212. TYPE : PRT US 9, 169,496 B2 47 48 - Continued <213> ORGANISM: Eucalyptus globulus

<4 OOs, SEQUENCE: 8 Met Ala Lieu. Arg Lieu Lleu Phe Thr Pro His Leu Pro Val Lieu. Ser Ser 1. 5 1O 15 Arg Arg Ala Asn Gly Arg Val Arg Cys Ser Ala Ser Thr Glin Ile Ser 2O 25 3O Asp Pro Glin Glu Gly Arg Arg Ser Ala Asn Tyr Glin Pro Ser Val Trip 35 4 O 45 Thr Tyr Asn Tyr Lieu. Glin Ser Ile Val Ala Gly Glu Gly Arg Glin Ser SO 55 6 O Arg Arg Glu Val Glu Glin Glin Lys Glu Lys Val Glin Ile Lieu. Glu Glu 65 70 7s 8O Glu Val Arg Gly Ala Lieu. Asn Asp Glu Lys Ala Glu Thir Phe Thir Ile 85 90 95 Phe Ala Thr Val Asp Asp Ile Glin Arg Lieu. Gly Lieu. Gly Asp His Phe 1OO 105 11 O Glu Glu Asp Ile Ser Asn Ala Lieu. Arg Arg Cys Val Ser Lys Gly Ala 115 12 O 125 Val Phe Met Ser Leu Gln Lys Ser Lieu. His Gly Thr Ala Leu Gly Phe 13 O 135 14 O Arg Lieu. Lieu. Arg Gln His Gly Tyr Glu Val Ser Glin Asp Val Phe Lys 145 150 155 160 Ile Phe Lieu. Asp Glu Ser Gly Ser Phe Val Lys Thr Lieu. Gly Gly Asp 1.65 17O 17s Val Glin Gly Val Lieu. Ser Lieu. Tyr Glu Ala Ser His Lieu Ala Phe Glu 18O 185 19 O Glu Glu Asp Ile Lieu. His Lys Ala Arg Ser Phe Ala Ile Llys His Lieu. 195 2OO 2O5 Glu Asn Lieu. Asn. Ser Asp Wall Asp Lys Asp Lieu. Glin Asp Glin Val Lys 21 O 215 22O His Glu Lieu. Glu Lieu Pro Lieu. His Arg Arg Met Pro Lieu. Lieu. Glu Ala 225 23 O 235 24 O Arg Arg Ser Ile Glu Ala Tyr Ser Arg Arg Glu Tyr Thr Asn Pro Glin 245 250 255 Ile Lieu. Glu Lieu Ala Lieu. Thir Asp Phe Asin Val Ser Glin Ser Thr Lieu. 26 O 265 27 O Glin Arg Asp Lieu. Glin Glu Met Lieu. Gly Trp Trp Asn. Asn Thr Gly Lieu. 27s 28O 285 Ala Lys Arg Lieu. Ser Phe Ala Arg Asp Arg Lieu. Ile Glu. Cys Phe Phe 29 O 295 3 OO Trp Ala Val Gly Ile Ala His Glu Pro Ser Lieu. Ser Ile Cys Arg Llys 3. OS 310 315 32O Ala Val Thir Lys Ala Phe Ala Lieu. Ile Lieu Val Lieu. Asp Asp Val Tyr 3.25 330 335

Asp Val Phe Gly Thr Lieu. Glu Glu Lieu. Glu Lieu. Phe Thr Glu Ala Val 34 O 345 35. O

Arg Arg Trp Asp Lieu. Asn Ala Val Glu Asp Lieu Pro Val Tyr Met Lys 355 360 365

Lieu. Cys Tyr Lieu Ala Lieu. Tyr Asn. Ser Val Asn. Glu Met Ala Tyr Glu 37 O 375 38O

Thir Lieu Lys Glu Lys Gly Glu Asn Val Ile Pro Tyr Lieu Ala Lys Ala 385 390 395 4 OO US 9, 169,496 B2 49 50 - Continued

Trp Asp Luell Cys Ala Phe Luell Glin Glu Ala Lys Trp Ser Asn 4 OS 415

Ser Arg Ile Ile Pro Gly Wall Glu Glu Tyr Luell Asn Asn Gly Trp Wall 425 43 O

Ser Ser Ser Gly Ser Wall Met Luell Ile His Ala Phe Luell Ala Ser 435 44 O 445

Pro Ser Ile Arg Glu Glu Luell Glu Ser Luell Glu His His Asp 450 45.5 460

Lell Luell Arg Luell Pro Ser Lell Ile Phe Arg Luell Thir Asn Asp Ile Ala 465 470

Ser Ser Ser Ala Glu Lell Glu Arg Gly Glu Thir Thir Asn Ser Ile Arg 485 490 495

Phe Met Glin Glu Gly Ile Ser Glu Luell Glu Ala Arg Glu SOO 505

Wall Glu Glu Ile Asp Thir Ala Trp Met Asn Met 515 525

Wall Asp Arg Ser Thir Phe Asn Glin Ser Phe Wall Arg Met Thir Asn 53 O 535 54 O

Lell Ala Arg Met Ala His Wall Glin Asp Gly Asp Ala Ile Gly 5.45 550 555 560

Ser Pro Asp Asp Lell Ser Trp Asn Arg Wall His Ser Lell Ile Ile Lys 565 st O sts

Pro Ile Ser Pro Ala Ala

SEO ID NO 9 LENGTH: 595 TYPE : PRT ORGANISM: Lotus japonicus

< 4 OOs SEQUENCE:

Met Ala Glin Ser Phe Ser Met Wall Luell Asn Ser Ser Phe Thir Ser His 1. 5 15

Pro Ile Phe Cys Lys Pro Glin Luell Ile Ile Arg Gly His Asn Luell 25

Lell Glin Gly His Arg Ile Asn Ser Pro Ile Pro Tyr Ala Ser Thir 35 4 O 45

Ser Ser Thir Ser Wall Ser Glin Arg Ser Ala Asn Glin Pro Asn SO 55 6 O

Ile Trp Asn Tyr Asp Tyr Lell Glin Ser Luell Lys Lell Gly Tyr Ala Asp 65 70

Ala His Glu Asp Met Ala Luell Glin Glu Glu Wall Arg Arg 85 90 95

Ile Ile Asp Asp Ala Glu Ile Trp Thir Thir Lell Glu Luell Ile 1OO 105 11 O

Asp Asp Wall Lell Gly Luell Gly Tyr His Phe Glu Glu Ile 115 12 O 125

Arg Glu Wall Luell Asn Phe Luell Ser Luell ASn Thir Wall His Arg 13 O 135 14 O

Ser Luell Asp Thir Ala Lell Phe Arg Luell Lell Arg Glu Tyr Gly 145 150 155 160

Ser Asp Wall Ser Ala Asp Ile Phe Glu Arg Phe Lell Asp Glin Asn Gly 1.65 17O 17s

Asn Phe Thir Ser Lell Wall Asn Asn Wall Lys Gly Met Luell Ser Luell 18O 185 19 O US 9, 169,496 B2 51 52 - Continued

Glu Ala Ser Phe Lell Ser Tyr Glu Gly Glu Glin Ile Luell Asp Lys 195 2O5

Ala Asn Ala Phe Thir Ser Phe His Luell Lys Ser Ile His Glu Glu Asp 21 O 215

Ile Asn Asn Ile Lell Lell Glu Glin Wall Asn His Ala Lell Glu Luell Pro 225 23 O 235 24 O

Lell His Arg Arg Ile His Arg Luell Glu Ala Arg Trp Thir Glu Ser 245 250 255

Ser Arg Arg Lys Asp Ala Asn Trp Wall Luell Lell Glu Ala Ala Lys 26 O 265 27 O

Lell Asp Phe Asn Met Wall Glin Ser Thir Luell Glin Asp Luell Glin Glu 27s 28O 285

Met Ser Arg Trp Trp Gly Met Gly Luell Ala Pro Luell Ser Phe 29 O 295 3 OO

Ser Arg Asp Arg Lell Met Glu Phe Phe Trp Thir Wall Gly Met Ala 3. OS 310 315

Phe Glu Pro Tyr Ser Asp Luell Arg Lys Gly Lell Thir Wall Thir 3.25 330 335

Ser Luell Ile Thir Thir Ile Asp Asp Ile Asp Wall His Gly Thir Luell 34 O 345 35. O

Glu Glu Luell Glu Lell Phe Thir Ala Ile Wall Glu Ser Trp Asp Ile Lys 355 360 365

Ala Met Glin Wall Lell Pro Glu Met Ile Ser Phe Luell Ala Luell 37 O 375

Tyr Asn Thir Wall Asn Glu Lell Ala Asp Ala Lell Arg Glu Glin Gly 385 390 395 4 OO

His Asp Ile Luell Pro Lell Thir Ala Trp Ser Asp Met Luell Lys 4 OS 415

Ala Phe Luell Glin Glu Ala Trp Cys Arg Glu His Luell Pro Lys 425 43 O

Phe Glu His Lell Asn Asn Ala Trp Wall Ser Wall Ser Gly Wall Wall 435 44 O 445

Ile Luell Thir His Ala Phe Luell Luell Asn His Asn Thir Thir Glu 450 45.5 460

Wall Luell Glu Ala Lell Glu Asn His Ala Luell Lell Arg Pro Ser 465 470

Ile Ile Phe Arg Lell Asn Asp Luell Gly Thir Ser Thir Ala Glu Luell 485 490 495

Glin Arg Gly Glu Wall Ala Asn Ser Ile Luell Ser Met His Glu Asn SOO 505

Asp Ile Gly Glu Glu Ser Ala His Glin His Ile His Ser Luell Luell Asn 515 525

Glu Thir Trp Lys Met Asn Arg Asp Arg Phe Ile His Ser Pro Phe 53 O 535 54 O

Pro Glu Pro Phe Wall Glu Ile Ala Thir Asn Luell Ala Arg Ile Ala Glin 5.45 550 555 560

Thir Glin Thir Gly Asp Gly His Gly Ala Pro Asp Ser Ile Ala 565 st O sts

Asn Arg Wall Lys Ser Lell Ile Ile Glu Pro Ile Wall Luell Asn Gly 58O 585 59 O

Asp Ile Tyr 595 US 9, 169,496 B2 53 - Continued

<210s, SEQ ID NO 10 &211s LENGTH: 593 212. TYPE : PRT &213s ORGANISM: Phaseolus lunatus

<4 OOs, SEQUENCE: 10

Met Lieu. Luell Asn Ser Ser Phe Ile Ser Arg Wall Thir Phe Ala Lys Pro 1. 5 1O 15

Lell Pro Wall Ala Pro Asn Luell Luell His Arg Arg Ile Ile Phe Pro 2O 25 3O

Arg Asn Gly Thir Thir Ile Asn Wall Asn Ala Ser Glu Arg Ser 35 4 O 45

Ala Asn Tyr Glin Pro Asn Lell Trp Thir Asp Phe Lell Glin Ser Luell SO 55 6 O

Lys His Ala Ala Asp Thir Arg Glu Asp Arg Ala Glin Luell 65 70

Glin Glu Glu Wall Arg Met Ile Asp Glu Asn Ser Asp Met Trp 85 90 95

Lell Luell Glu Lell Ile Asn Asp Wall Arg Lell Gly Luell Ser Tyr 105 11 O

His Asp Glu Ile Gly Glu Ala Luell Luell Arg Phe His Ser Ser 115 12 O 125

Ala Thir Phe Ser Gly Thir Ile Wall His Arg Ser Lell His Glu Thir Ala 13 O 135 14 O

Lell Phe Arg Lell Lell Arg Glu Gly Tyr Asp Wall Thir Ala Asp 145 150 155 16 O

Met Phe Glu Arg Phe Glu Arg Asn Gly His Phe Ala Ser Luell 1.65 17O 17s

Met Ser Asp Wall Lys Gly Met Luell Ser Luell Tyr Glin Ala Ser Phe Luell 18O 185 19 O

Gly Tyr Glu Gly Glu Glin Ile Luell Asp Asp Ala Ala Phe Ser Ser 195 2O5

Phe His Luell Ser Wall Lell Ser Glu Gly Arg Asn Asn Met Wall Luell 21 O 215 22O

Glu Glu Wall Asn His Ala Lell Glu Luell Pro Luell His His Arg Ile Glin 225 23 O 235 24 O

Arg Luell Glu Ala Arg Trp Ile Glu Tyr Tyr Ala Glin Arg Asp 245 250 255

Ser Asn Arg Wall Lell Lell Glu Ala Ala Luell Asp Phe Asn Ile Luell 26 O 265 27 O

Glin Ser Thir Luell Glin Asn Asp Luell Glin Glu Wall Ser Arg Trp Trp 27s 285

Gly Met Gly Luell Ala Ser Lys Luell Ser Phe Ser Arg Asp Arg Luell Met 29 O 295 3 OO

Glu Phe Phe Trp Ala Ala Gly Met Wall Phe Glu Pro Glin Phe Ser 3. OS 310 315

Asp Luell Arg Gly Lell Thir Wall Ala Ser Lell Ile Thir Thir Ile 3.25 330 335

Asp Asp Wall Tyr Asp Wall Gly Thir Luell Glu Glu Lell Glu Luell Phe 34 O 345 35. O

Thir Ala Ala Wall Glu Ser Trp Asp Wall Lys Ala Ile Glin Wall Luell Pro 355 360 365

Asp Tyr Met Lys Ile Phe Luell Ala Luell Tyr Asn Thir Wall Asn Glu 37 O 375 38O US 9, 169,496 B2 55 56 - Continued

Phe Ala Asp Ala Lell Glu Glin Gly Glin Asp Ile Luell Pro Tyr 385 390 395 4 OO

Lell Thir Ala Trp Ser Asp Luell Luell Lys Ala Phe Lell Glin Glu Ala 4 OS 415

Trp Ser Arg Asp Arg His Met Pro Arg Phe Asn Asp Tyr Luell Asn 425 43 O

Asn Ala Trp Wall Ser Wall Ser Gly Wall Wall Luell Lell Thir His Ala Tyr 435 44 O 445

Phe Luell Luell Asn His Ser Ile Thir Glu Glu Ala Lell Glu Ser Luell Asp 450 45.5 460

Ser His Ser Lell Lell Glin Asn Thir Ser Luell Wall Phe Arg Luell Cys 465 470

Asn Asp Luell Gly Thir Ser Ala Glu Luell Glu Arg Gly Glu Ala Ala 485 490 495

Ser Ser Ile Luell Cys Arg Arg Glu Ser Gly Ala Ser Glu Glu Gly SOO 505

Ala Lys His Ile Ser Luell Luell Asn Glu Thir Trp Met 515 525

Asn Glu Asp Arg Wall Ser Glin Ser Pro Phe Pro Lys Ala Phe Wall Glu 53 O 535 54 O

Thir Ala Met Asn Lell Ala Arg Ile Ser His Cys Thir Glin Gly 5.45 550 555 560

Asp Gly His Gly Ala Pro Asp Ser Thir Ala Asn Arg Ile Arg Ser 565 st O sts

Lell Ile Ile Glu Pro Ile Ala Luell Tyr Glu Thir Glu Ile Ser Thir Ser 58O 585 59 O Tyr

<210s, SEQ ID NO 11 &211s LENGTH: 583 212. TYPE : PRT <213> ORGANISM: Melaleuca alternifolia

<4 OOs, SEQUENCE: 11

Met Ala Lieu. Arg Lell Lell Ser Thir Pro His Luell Pro Glin Luell Cys Ser 1. 5 15

Arg Arg Wall Ser Gly Arg Wall His Cys Ser Ala Ser Thir Glin Wall Ser 2O 25

Asp Ala Glin Gly Gly Arg Arg Ser Ala Asn Glin Pro Ser Wall Trp 35 4 O 45

Thir Tyr Asn Lell Glin Ser Luell Wall Ala Asp Asp Ile Arg Arg Ser SO 55 6 O

Arg Arg Glu Wall Glu Glin Glu Arg Glu Lys Ala Glin Ile Luell Glu Glu 65 70

Asp Wall Arg Gly Ala Lell Asn Asp Gly Asn Ala Glu Pro Met Ala Ile 85 90 95

Phe Ala Luell Wall Asp Asp Ile Glin Arg Luell Gly Lell Gly Arg Phe 105 11 O

Glu Glu Asp Ile Ser Ala Luell Arg Arg Lell Ser Glin Ala 115 12 O 125

Wall Thir Gly Ser Lell Glin Lys Ser Luell His Gly Thir Ala Luell Ser Phe 13 O 135 14 O

Arg Wall Luell Arg Glin His Gly Phe Glu Wall Ser Glin Asp Wall Phe Lys 145 150 155 160 US 9, 169,496 B2 57 58 - Continued

Ile Phe Met Asp Glu Ser Gly Ser Phe Met Lys Thir Lell Gly Gly Asp 1.65 17s

Wall Glin Gly Met Lell Ser Lell Glu Ala Ser His Lell Ala Phe Glu 18O 185 19 O

Glu Glu Asp Ile Lell His Ala Thir Phe Ala Ile His Luell 195 2OO

Glu Asn Luell Asn His Asp Ile Asp Glin Asp Luell Glin Asp His Wall Asn 21 O 215

His Glu Luell Glu Lell Pro Lell His Arg Arg Met Pro Lell Luell Glu Ala 225 23 O 235 24 O

Arg Arg Phe Ile Glu Ala Ser Arg Arg Ser Asn Wall Asn Pro Arg 245 250 255

Ile Luell Glu Luell Ala Wall Met Phe Asn Ser Ser Glin Luell Thir Luell 26 O 265 27 O

Glin Arg Asp Luell Glin Asp Met Luell Gly Trp Trp Asn Asn Wall Gly Luell 285

Ala Lys Arg Luell Ser Phe Ala Arg Asp Arg Luell Met Glu Phe Phe 29 O 295 3 OO

Trp Ala Wall Gly Ile Ala Arg Glu Pro Ala Luell Ser Asn Arg Lys 3. OS 310 315

Gly Wall Thir Ala Phe Ser Luell Ile Luell Wall Lell Asp Asp Wall Tyr 3.25 330 335

Asp Wall Phe Gly Thir Lell Asp Glu Luell Glu Luell Phe Thir Asp Ala Wall 34 O 345 35. O

Arg Arg Trp His Glu Asp Ala Wall Glu Asn Luell Pro Gly Met Lys 355 360 365

Lell Cys Phe Luell Ala Lell Tyr Asn Ser Wall ASn Asp Met Ala Glu 37 O 375

Thir Luell Glu Thir Gly Glu Asn Wall Thir Pro Lell Thir Wall 385 390 395 4 OO

Trp Asp Luell Cys Ala Phe Luell Glin Glu Ala Trp Ser Tyr 4 OS 415

Asn Ile Thir Pro Gly Wall Glu Glu Tyr Luell Asn Asn Gly Trp Wall 425 43 O

Ser Ser Ser Gly Glin Wall Met Luell Thir His Ala Phe Luell Ser Ser 435 44 O 445

Pro Ser Luell Arg Glu Glu Luell Glu Ser Luell Glu His His Asp 450 45.5 460

Lell Luell Arg Luell Pro Ser Lell Ile Phe Arg Luell Thir Asn Asp Luell Ala 465 470

Thir Ser Ser Ala Glu Lell Gly Arg Gly Glu Thir Thir Asn Ser Ile Luell 485 490 495

Met Arg Glu Gly Phe Ser Glu Ser Glu Ala Arg Glin SOO 505

Wall Ile Glu Glin Ile Asp Thir Ala Trp Arg Glin Met Asn Met 515 525

Wall Asp His Ser Thir Phe Asn Arg Ser Phe Met Glin Met Thir Asn 53 O 535 54 O

Lell Ala Arg Met Ala His Wall Glin Asp Gly Asp Ala Ile Gly 5.45 550 555 560

Ala Pro Asp Asp Glin Ser Trp Asn Arg Wall His Ser Lell Ile Ile Lys 565 st O sts US 9, 169,496 B2 59 - Continued

Pro Val Ser Leu Ala Pro Cys 58O

<210s, SEQ ID NO 12 &211s LENGTH: 6O1 212. TYPE: PRT <213> ORGANISM: Witis vinifera

<4 OOs, SEQUENCE: 12 Met Ala Lieu. His Leu Phe Tyr Phe Pro Lys Gln Cys Phe Lieu. Thr His 1. 5 1O 15 Asn Lieu Pro Gly His Pro Met Lys Llys Pro Pro Arg Gly Thr Thr Ala 2O 25 3O Glin Ile Arg Cys Ser Ala Asn Glu Glin Ser Phe Ser Leu Met Thr Glu 35 4 O 45 Ser Arg Arg Ser Ala His Tyr Glin Pro Ala Phe Trp Ser Tyr Asp Phe SO 55 6 O Val Glu Ser Lieu Lys Lys Arg Glu Glu Ile Cys Asp Gly Ser Val Lys 65 70 7s 8O Glu Lieu. Glu Lys Met Tyr Glu Asp Arg Ala Arg Llys Lieu. Glu Asp Glu 85 90 95 Val Llys Trp Met Ile His Glu Lys Ser Ala Glu Pro Lieu. Thir Lieu. Lieu 1OO 105 11 O Glu Phe Ile Asp Asp Ile Glin Arg Lieu. Gly Lieu. Gly His Arg Phe Glu 115 12 O 125 Asn Asp Ile Lys Arg Ser Lieu. Asp Llys Ile Lieu Lleu Lieu. Glu Gly Ser 13 O 135 14 O Asn Ala Gly Lys Gly Glu Ser Lieu. His His Thr Ala Lieu. Arg Phe Arg 145 150 155 160 Ile Leu Lys Gln His Gly Tyr Llys Val Ser Glin Glu Val Phe Glu Gly 1.65 17O 17s Phe Thr Asp Glin Asn Gly His Phe Lys Ala Cys Lieu. Cys Lys Asp Wall 18O 185 19 O Lys Gly Met Lieu. Ser Lieu. Tyr Glu Ala Ser Tyr Lieu Ala Ser Glu Gly 195 2OO 2O5 Glu Thir Lieu. Lieu. His Glu Ala Met Ala Phe Lieu Lys Met His Lieu Lys 21 O 215 22O Asp Lieu. Glu Gly Thr Lieu. Asp Llys Ser Lieu. Glu Glu Lieu Val Asn His 225 23 O 235 24 O Ala Met Glu Lieu Pro Lieu. His Arg Arg Met Pro Arg Lieu. Glu Ala Arg 245 250 255 Trp Phe Ile Glu Ala Tyr Lys Arg Arg Glu Gly Ala Asp Asp Val Lieu. 26 O 265 27 O Lieu. Glu Lieu Ala Ile Lieu. Asp Phe Asn Met Val Glin Trp Thir Lieu. Glin 27s 28O 285 Asp Asp Lieu. Glin Asp Met Ser Arg Trp Trip Lys Asp Met Gly Lieu Ala 29 O 295 3 OO Ser Lys Lieu. His Phe Ala Arg Asp Arg Lieu Met Glu. Cys Phe Phe Trp 3. OS 310 315 32O

Thr Val Gly Met Ala Phe Glu Pro Glu Phe Ser Asn Cys Arg Lys Gly 3.25 330 335

Lieu. Thir Lys Val Thr Ser Phe Ile Thr Thr Ile Asp Asp Val Tyr Asp 34 O 345 35. O

Val Tyr Gly Ser Val Asp Glu Lieu. Glu Lieu. Phe Thr Asp Ala Val Ala 355 360 365 US 9, 169,496 B2 61 62 - Continued

Arg Trp Asp Ile Asn Met Wall Asn Asn Luell Pro Gly Tyr Met Lys Luell 37 O 375 38O

Cys Phe Luell Ala Lell Tyr Asn Thir Wall Asn Glu Met Ala Asp Thir 385 390 395 4 OO

Lell Glu Glin Gly His Asn Ile Luell Pro Lell Thir Ala Trp 4 OS 415

Ala Luell Cys Lys Wall Phe Luell Wall Glu Ala Trp Cys His 425 43 O

Glu Thir Pro Thir Phe Glu Glu Tyr Luell Glu Asn Gly Trp Arg Ser 435 44 O 445

Wall Ser Gly Ala Ala Ile Lell Ile His Ala Phe Lell Met Ser 450 45.5 460

Asn Ile Thir Glu Ala Lell Glu Luell Glu Asn Asp His Glu Luell 465 470 48O

Lell Arg Trp Pro Ser Thir Ile Phe Arg Luell Cys Asn Asp Luell Ala Thir 485 490 495

Ser Ala Glu Lell Glu Arg Gly Glu Ser Ala Asn Ser Ile Ser SOO 505

Met His Glin Thir Gly Wall Ser Glu Glu Asp Ala Arg Glu His Met 515 525

Ile Luell Ile Asp Glu Ser Trp Met Asn Wall Arg Glu 53 O 535 54 O

Met Asp Ser Asp Ser Pro Phe Ala Pro Phe Wall Glu Thir Ala Ile 5.45 550 555 560

Asn Luell Ala Arg Ile Ala Glin Thir Tyr Glin Tyr Gly Asp Ser His 565 st O sts

Gly Ala Pro Asp Ala Arg Ser Lys Arg Wall Lell Ser Luell Ile Wall 585 59 O

Glu Pro Ile Pro Met Asn Lell Lys 595 6OO

SEQ ID NO 13 LENGTH: 661 TYPE : PRT ORGANISM: Marinobacter aquaeolei VT8

< 4 OOs SEQUENCE: 13

Met Asn Tyr Phe Lell Thir Gly Gly Thir Gly Phe Ile Gly Arg Phe Luell 1. 5 15

Wall Glu Luell Lell Ala Arg Gly Gly Thir Wall Wall Luell Wall Arg 25

Glu Glin Glin Asp Lell Glu Arg Luell Arg Glu Arg Trp Gly Ala 4 O 45

Asp Asp Glin Wall Ala Wall Ile Gly Asp Lell Thir Ser Asn SO 55 6 O

Lell Gly Ile Asp Ala Lys Thir Luell Ser Luell Gly Asn Ile Asp 65 70

His Wall Phe His Lell Ala Ala Wall Asp Met Gly Ala Asp Glu Glu 85 90 95

Ala Glin Ala Ala Thir Asn Ile Glu Gly Thir Arg Ala Ala Wall Glin Ala 105 11 O

Ala Glu Ala Met Gly Ala His Phe His His Wall Ser Ser Ile Ala 115 12 O 125 US 9, 169,496 B2 63 64 - Continued

Ala Ala Gly Luell Phe Gly Ile Phe Arg Glu Asp Met Phe Glu Glu 13 O 135 14 O

Ala Glu Luell Asp His Pro Luell Arg Thir Lys His Glu Ser Glu 145 150 155 160

Wall Wall Arg Glu Glu Wall Pro Phe Arg Ile Arg Pro 1.65 17O

Gly Met Wall Ile Gly His Ser Glu Thir Gly Glu Met Asp Lys Wall Asp 18O 185 19 O

Gly Pro Tyr Phe Phe Met Ile Glin Lys Ile Arg His Ala Luell 195

Pro Glin Trp Wall Pro Thir Ile Gly Ile Glu Gly Gly Arg Luell Asn Ile 21 O 215 22O

Wall Pro Wall Asp Phe Wall Wall Asp Ala Luell Asp His Ile Ala His Luell 225 23 O 235 24 O

Glu Gly Glu Asp Gly Asn Phe His Luell Wall Asp Ser Asp Pro 245 250 255

Wall Gly Glu Ile Lell Asn Ile Phe Glu Ala Gly His Ala Pro 26 O 265 27 O

Arg Met Gly Met Arg Ile Asp Ser Arg Met Phe Gly Phe Ile Pro Pro 28O 285

Phe Ile Arg Glin Ser Ile Lys Asn Luell Pro Pro Wall Arg Ile Thir 29 O 295 3 OO

Gly Ala Luell Luell Asp Asp Met Gly Ile Pro Pro Ser Wall Met Ser Phe 3. OS 310 315

Ile Asn Tyr Pro Thr Arg Phe Asp Thr Arg Glu Lieu Glu Arg Wall Lieu 3.25 330 335

Gly Thir Asp Ile Glu Wall Pro Arg Luell Pro Ser Ala Pro Wall 34 O 345 35. O

Ile Trp Asp Trp Glu Arg Asn Luell Asp Pro Asp Lell Phe Asp 355 360 365

Arg Thir Luell Gly Thir Wall Glu Gly Wall Cys Wall Wall Thir Gly 37 O 375

Ala Thir Ser Gly Ile Gly Lell Ala Thir Ala Glu Lell Ala Glu Ala 385 390 395 4 OO

Gly Ala Ile Luell Wall Ile Gly Ala Arg Thir Glu Thir Luell Asp Glu 4 OS 415

Wall Ala Ala Ser Lell Glu Ala Gly Gly ASn Wall His Ala Glin 425 43 O

Asp Phe Ser Asp Met Asp Asp Asp Arg Phe Wall Thir Wall 435 44 O 445

Lell Asp Asn His Gly His Wall Asp Wall Luell Wall Asn Asn Ala Gly Arg 450 45.5 460

Ser Ile Arg Arg Ser Lell Ala Luell Ser Phe Asp Arg Phe His Asp Phe 465 470

Glu Arg Thir Met Glin Lell Asn Tyr Phe Gly Ser Wall Arg Luell Ile Met 485 490 495

Gly Phe Ala Pro Ala Met Lell Glu Arg Arg Arg Gly His Wall Wall Asn SOO 505

Ile Ser Ser Ile Gly Wall Lell Thir Asn Ala Pro Arg Phe Ser Ala Tyr 515 52O 525

Wall Ser Ser Ser Ala Lell Asp Ala Phe Ser Arg Ala Ala Ala 53 O 535 54 O US 9, 169,496 B2 65 66 - Continued

Glu Trp Ser Asp Arg Asn Wall Thir Phe Th Thir Ile Asn Met Pro Luell 5.45 550 555 560

Wall Thir Met Ile Ala Pro Thir Lys Ile Tyr Asp Ser Wall Pro 565 st O sts

Thir Luell Thir Asp Glu Ala Ala Glin Met Wall Ala Asp Ala Ile Wall 585 59 O

Arg Pro Ile Ala Thir Arg Lieu. Gly Wall Phe Ala Glin Wall 595 605

Lell His Ala Lel Ala Pro Lys Met Gly Glu Ile Ile Met Asn Thir Gly 610 615

Tyr Arg Met Phe Pro Asp Ser Pro Ala Ala Ala Gly Ser Ser Gly 625 630 635 64 O

Glu Pro Wall Ser Thir Glu Glin Wall Ala Phe Ala Ala Ile Met 645 650 655

Arg Gly Ile Tyr Trp 660

SEQ ID NO 14 LENGTH: 54 O TYPE : PRT ORGANISM: Marinobacter manganoxydans

< 4 OOs SEQUENCE: 14

Met Asn Tyr Phe Lell Thir Gly Gly Thir Gly Phe Ile Gly Arg Phe Luell 1. 5 1O 15

Wall Glu Lys Luell Lell Ala Arg Gly Gly Thir Wall His Wall Luell Wall Arg 25 30

Glu Glin Ser Glin Asp Lell Asp Lieu. Arg Glu Arg Trp Gly Ala 35 4 O 45

Asp Glu Thir Glin Wall Ala Wall Ile Gly Asp Lell Thir Ser Asn SO 55 6 O

Lell Gly Ile Asp Ala Lys Thir Met Ala Lieu. Gly Ile Asp 65 70 7s

His Phe Phe His Lell Ala Ala Wall Asp Met Gly Ala Asp Glu Glu 85 90 95

Ala Glin Glin Ala Thir Asn Ile Glu Gly Thr Arg Ala Ala Wall Asn Ala 105 11 O

Ala Glu Ala Met Gly Ala His Phe His His Wall Ser Ser Ile Ala 115 12 O 125

Ala Ala Gly Luell Phe Gly Ile Phe Arg Glu Asp Met Phe Glu Glu 13 O 135 14 O

Ala Glu Luell Asp His Pro Luell Arg Thr His Glu Ser Glu 145 150 155 160

Wall Wall Arg Glu Glu Wall Pro Phe Arg Ile Arg Pro 1.65 17O

Gly Met Wall Ile Gly His Thir Ala Thir Gly Glu Met Asp Lys Wall Asp 18O 185 19 O

Gly Pro Tyr Phe Phe Met Ile Gln Lys Ile Arg His Ala Luell 195 2O5

Pro Glin Trp Wall Pro Thir Ile Gly Wall Glu Gly Gly Arg Luell Asn Ile 21 O 215 22O

Wall Pro Wall Asp Phe Wall Wall Asn Ala Met Asp His Ile Ala His Luell 225 23 O 235 24 O

Glu Gly Glu Asp Gly Lys Phe His Lieu Wall Asp Thir Asp Pro 245 250 255 US 9, 169,496 B2 67 68 - Continued

Wall Gly Glu Ile Lell Asn Ile Phe Ser Glu Ala Gly His Ala Pro 26 O 265 27 O

Arg Met Gly Met Arg Ile Asp Ser Arg Met Phe Gly Phe Ile Pro Pro 28O 285

Phe Ile Arg Glin Ser Lell Lys Asn Luell Pro Pro Wall Arg Luell Thir 29 O 295 3 OO

Ser Ala Ile Lieu. Asp Asp Met Gly Ile Pro Pro Ser Wall Met Ser Phe 3. OS 310 315

Ile Asn Tyr Pro Thir Arg Phe Asp Ala Arg Glu Thir Glu Arg Wall Luell 3.25 330 335

Gly Thir Gly Ile Glu Wall Pro Arg Luell Pro Asp Ala Pro Wall 34 O 345 35. O

Ile Trp Asp Glu Arg Asn Luell Asp Pro Asp Lell Phe Asp 355 360 365

Arg Thir Luell Thir Wall Glu Gly Arg Wall Cys Wall Wall Thir Gly 37 O 375

Ala Thir Ser Gly Ile Gly Lell Ala Thir Ala Glin Lell Ala Asp Ala 385 390 395 4 OO

Gly Ala Ile Lieu Wall Ile Gly Ala Arg Lys Luell Glu Arg Luell Lys Glu 4 OS 415

Wall Ala Ala Glu Lieu. Glu Ser Arg Gly Ala Ser Wall His Ala Pro 42O 425 43 O

Asp Phe Ser Asp Met Asp Ala Asp Glu Phe Wall Thir Wall 435 44 O 445

Lell Asp Asn His Gly Glin Wall Asp Wall Luell Wall Asn Asn Ala Gly Arg 450 45.5 460

Ser Ile Arg Arg Ser Lell Asp Luell Ser Phe Asp Arg Phe His Asp Phe 465 470

Glu Arg Thir Met Glin Lell Asn Phe Gly Ser Wall Arg Luell Ile Met 485 490 495

Gly Phe Ala Pro Llys Met Lell Glu Asn Arg Arg Gly His Wall Wall Asn SOO 505 51O

Ile Ser Ser Ile Gly Wall Lell Thir Asn Ala Pro Arg Phe Ser Ala Tyr 515 52O 525

Wall Ala Ser Llys Ser Ala Lell Asp Ala Phe Ser Arg 53 O 535 54 O

<210s, SEQ ID NO 15 &211s LENGTH: 661 212. TYPE : PRT <213> ORGANISM: Marinobacter sp. ELB17

<4 OOs, SEQUENCE: 15

Met Asn Tyr Phe Val Thir Gly Gly Thir Gly Phe Ile Gly Arg Phe Luell 1. 5 15

Ile Ala Arg Lieu. Luell Ala Arg Gly Ala Ile Wall His Wall Luell Wall Arg 25

Glu Glin Ser Wall Glin Lys Lell Ala Asp Luell Arg Glu Lys Luell Gly Ala 35 4 O 45

Asp Glu Glin Ile Ala Wall Wall Gly Asp Lell Thir Ala Pro Gly SO 55 6 O

Lell Gly Luell Asp Llys Lys Thir Luell Glin Luell Ser Gly Ile Asp 65 70 7s 8O US 9, 169,496 B2 69 70 - Continued

His Phe Phe His Lell Ala Ala Ile Asp Met Ser Ala Ser Glu Glu 85 90 95

Ser Glin Glin Ala Ala Asn Ile Asp Gly Thir Arg Ala Ala Wall Ala Ala 105 11 O

Ala Glu Ala Luell Gly Ala Gly Ile Phe His His Wall Ser Ser Ile Ala 115 12 O 125

Wall Ala Gly Luell Phe Gly Thir Phe Arg Glu Asp Met Phe Ala Glu 13 O 135 14 O

Ala Gly Luell Asp His Pro Phe Ser Thir His Glu Ser Glu 145 150 155 160

Arg Wall Wall Arg Asp Glu Luell Pro Phe Arg Ile Arg Pro 1.65 17O

Gly Met Wall Ile Gly Asp Ser Ala Thir Gly Glu Met Asp Lys Wall Asp 18O 185 19 O

Gly Pro Tyr Phe Phe Met Ile Glin Lys Ile Arg Gly Ala Luell 195

Pro Glin Trp Wall Pro Thir Ile Gly Luell Glu Gly Gly Arg Luell Asn Ile 21 O 215 22O

Wall Pro Wall Asn Phe Wall Ala Asp Ala Luell Asp His Ile Ala His Luell 225 23 O 235 24 O

Pro Asp Glu Asp Gly Lys Phe His Luell Wall Asp Ser Asp Pro 245 250 255

Wall Gly Glu Ile Lell Asn Ile Phe Glu Ala Gly His Ala Pro 26 O 265 27 O

Met Gly Met Arg Ile Asp Ser Arg Met Phe Gly Phe Wall Pro Pro 28O 285

Phe Ile Arg Glin Ser Lell Lys Asn Luell Pro Pro Wall Arg Met Gly 29 O 295 3 OO

Arg Ala Luell Luell Asp Asp Lell Gly Ile Pro Ala Ser Wall Luell Ser Phe 3. OS 310 315

Ile Asn Tyr Pro Thir Arg Phe Asp Ala Arg Glu Thir Glu Arg Wall Luell 3.25 330 335

Glin Gly Thir Gly Ile Glu Wall Pro Arg Luell Pro Asp Ala Pro Wall 34 O 345 35. O

Ile Trp Asp Trp Glu Arg Asn Luell Asp Pro Asp Lell Phe Thir Asp 355 360 365

Arg Thir Luell Arg Gly Thir Wall Glu Gly Wall Cys Wall Wall Thir Gly 37 O 375

Ala Thir Ser Gly Ile Gly Lell Ala Thir Ala Glu Lell Ala Asp Ala 385 390 395 4 OO

Gly Ala Ile Luell Wall Ile Gly Ala Arg Thir Glin Glu Thir Luell Asp Glin 4 OS 415

Wall Ser Ala Glin Lell Asn Ala Arg Gly Ala Asp Wall His Ala Glin 425 43 O

Asp Phe Ala Asp Met Asp Ala Asp Arg Phe Ile Glin Thir Wall 435 44 O 445

Ser Glu Asn His Gly Ala Wall Asp Wall Luell Ile Asn Asn Ala Gly Arg 450 45.5 460

Ser Ile Arg Arg Ser Lell Asp Ser Phe Asp Arg Phe His Asp Phe 465 470

Glu Arg Thir Met Glin Lell Asn Phe Gly Ser Lell Arg Luell Ile Met 485 490 495 US 9, 169,496 B2 71 72 - Continued

Gly Phe Ala Pro Ala Met Lell Glu Arg Arg Arg Gly His Ile Ile Asn SOO 505

Ile Ser Ser Ile Gly Wall Lell Thir Asn Ala Pro Arg Phe Ser Ala Tyr 515 52O 525

Wall Ala Ser Ala Ala Lell Asp Ser Phe Ser Arg Ala Ala Ala 53 O 535 54 O

Glu Trp Ser Asp Arg His Wall Phe Thir Thir Ile Asn Met Pro Luell 5.45 550 555 560

Wall Thir Pro Met Ile Ala Pro Thir Lys Ile Asp Ser Wall Pro 565 st O sts

Thir Luell Ser Pro Glu Glu Ala Ala Asp Met Wall Wall Asn Ala Ile Wall 58O 585 59 O

Arg Pro Ile Ala Thir Arg Met Gly Wall Phe Ala Glin Wall 595 605

Lell Asn Ala Wall Ala Pro Lys Ala Ser Glu Ile Lell Met Asn Thir Gly 610 615

Tyr Met Phe Pro Asp Ser Met Pro Lys Gly Glu Wall Ser 625 630 635 64 O

Ala Glu Gly Ala Ser Thir Asp Glin Wall Ala Phe Ala Ala Ile Met 645 650 655

Arg Gly Ile His Trp 660

<210s, SEQ ID NO 16 &211s LENGTH: 661 212. TYPE PRT &213s ORGANISM: Marinobacter algicola DG893

<4 OOs, SEQUENCE: 16

Met Asn Tyr Phe Lell Thir Gly Gly Thir Gly Phe Ile Gly Arg Phe Luell 1. 5 15

Wall Glu Lys Luell Lell Ala Arg Gly Gly Thir Wall His Wall Luell Wall Arg 25

Glu Glin Ser Glin Glu Lell Asp Luell Arg Glu Arg Trp Gly Ala 35 4 O 45

Asp Glu Ser Arg Wall Ala Wall Ile Gly Asp Lell Thir Ser Pro Asn SO 55 6 O

Lell Gly Ile Asp Ala Lys Thir Met Ser Luell Gly Asn Ile Asp 65 70 8O

His Phe Phe His Lell Ala Ala Wall Asp Met Gly Ala Asp Glu 85 90 95

Ser Glin Glin Ala Thir Asn Ile Glu Gly Thir His Ser Ala Wall Asn Ala 105 11 O

Ala Ala Ala Met Glu Ala Gly Cys Phe His His Wall Ser Ser Ile Ala 115 12 O 125

Ala Ala Gly Luell Phe Gly Thir Phe Arg Glu Asp Met Phe Glu Glu 13 O 135 14 O

Ala Glu Luell Asp His Pro Luell Luell Thir His Glu Ser Glu 145 150 155 160

Wall Wall Arg Glu Ser Wall Pro Phe Arg Ile Arg Pro 1.65 17O

Gly Met Wall Wall Gly His Ser Thir Gly Glu Met Asp Lys Wall Asp 18O 185 19 O

Gly Pro Tyr Phe Phe Met Ile Glin Lys Ile Arg His Ala Luell 195 2OO 2O5 US 9, 169,496 B2 73 74 - Continued

Pro Glin Trp Wall Pro Thir Ile Gly Ile Glu Gly Gly Arg Luell Asn Ile 21 O 215 22O

Wall Pro Wall Asp Phe Wall Wall Asn Ala Met Asp His Ile Ala His Luell 225 23 O 235 24 O

Gly Glu Asp Gly Asn Phe His Luell Wall Asp Ser Asp Pro Tyr 245 250 255

Wall Gly Glu Ile Lell Asn Ile Phe Ser Glu Ala Gly His Ala Pro 26 O 265 27 O

Arg Met Ala Met Arg Ile Asp Ser Arg Met Phe Gly Phe Wall Pro Pro 28O 285

Phe Ile Arg Glin Ser Lell Lys Asn Luell Pro Pro Wall Arg Luell Thir 29 O 295 3 OO

Thir Ala Luell Luell Asp Asp Met Gly Ile Pro Pro Ser Wall Luell Ser Phe 3. OS 310 315

Ile Asn Tyr Pro Thir Arg Phe Asp Ala Arg Glu Thir Glu Arg Wall Luell 3.25 330 335

Asp Thir Gly Ile Wall Wall Pro Arg Luell Glu Ser Ala Ala Wall 34 O 345 35. O

Lell Trp Asp Phe Trp Glu Arg Asn Luell Asp Pro Asp Lell Phe Asp 355 360 365

Arg Thir Luell Arg Gly Thir Wall Glu Gly Wall Cys Wall Ile Thir Gly 37 O 375

Gly Thir Ser Gly Ile Gly Lell Ala Thir Ala Glin Lell Ala Asp Ala 385 390 395 4 OO

Gly Ala Ile Luell Wall Ile Gly Ala Arg Lys Glu Arg Luell Met Glu 4 OS 415

Wall Ala Ala Glu Lell Glu Ala Arg Gly Gly ASn Wall His Ala Glin 425 43 O

Asp Phe Ala Asp Met Asp Asp Asp Arg Phe Wall Thir Wall 435 44 O 445

Lell Asp Asn His Gly His Wall Asp Wall Luell Wall Asn Asn Ala Gly Arg 450 45.5 460

Ser Ile Arg Arg Ser Lell Ala Luell Ser Phe Asp Arg Phe His Asp Phe 465 470

Glu Arg Thir Met Glin Lell Asn Tyr Phe Gly Ser Wall Arg Luell Ile Met 485 490 495

Gly Phe Ala Pro Ala Met Lell Glu Arg Arg Arg Gly His Wall Wall Asn SOO 505

Ile Ser Ser Ile Gly Wall Lell Thir Asn Ala Pro Arg Phe Ser Ala Tyr 515 525

Wall Ala Ser Ser Ala Lell Asp Thir Phe Ser Arg Ala Ala Ala 53 O 535 54 O

Glu Trp Ser Asp Arg Asn Wall Thir Phe Thir Thir Ile Asn Met Pro Luell 5.45 550 555 560

Wall Thir Pro Met Ile Ala Pro Thir Lys Ile Asp Ser Wall Pro 565 st O sts

Thir Luell Thir Pro Asp Glu Ala Ala Glu Met Wall Ala Asp Ala Ile Wall 585 59 O

Arg Pro Ile Ala Thir Arg Luell Gly Ile Phe Ala Glin Wall 595 605

Met Glin Ala Luell Ala Pro Lys Met Gly Glu Ile Wall Met Asn Thir Gly 610 615 62O US 9, 169,496 B2 75 76 - Continued Tyr Arg Met Phe Pro Asp Ser Pro Ala Ala Ala Gly Ser Arg Ser Gly 625 630 635 64 O

Ala Llys Pro Llys Val Ser Ser Glu Glin Val Ala Phe Ala Ala Ile Met 645 650 655 Arg Gly Ile Tyr Trp 660

<210s, SEQ ID NO 17 &211s LENGTH: 661 212. TYPE : PRT <213s ORGANISM: Hahella cheuensis

<4 OOs, SEQUENCE: 17

Met Asn Tyr Phe Wall Thir Gly Gly Thir Gly Phe Ile Gly Arg Phe Luell 1. 5 15

Wall Pro Lys Luell Lell Arg Gly Gly Thir Wall Tyr Lell Luell Wall 25 3O

Glu Ala Ser Luell Pro Lell Asp Glu Luell Arg Glu Arg Trp Asn Ala 35 4 O 45

Ser Asp Glu Glin Wall Wall Gly Wall Wall Gly Asp Lell Ala Glin Pro Met SO 55 6 O

Lell Gly Wall Ser Glu Lys Asp Ala Ala Met Luell Arg Gly Wall Gly 65 70

His Phe Phe His Lell Ala Ala Ile Asp Met Glin Ala Ser Ala Glu 85 90 95

Ser Glin Glu Glin Ala Asn Ile Glu Gly Thir Arg Asn Ala Wall Luell 105 11 O

Ala Asp Ser Luell Ala Ala Cys Phe His His Wall Ser Ser Ile Ala 115 12 O 125

Ala Ala Gly Luell Tyr Arg Gly Ile Phe Arg Glu Asp Met Phe Glu Glu 13 O 135 14 O

Ala Glu Luell Asp Asn Pro Luell Arg Thir His Glu Ser Glu 145 150 155 160

Wall Wall Arg Glu Glu Glin Thir Pro Trp Arg Wall Arg Pro 1.65 17O

Gly Met Wall Wall Gly His Ser Thir Gly Glu Ile Asp Lys Ile Asp 18O 185 19 O

Gly Pro Tyr Phe Phe Luell Ile Glin Lys Lell Arg Ser Ala Luell 195

Pro Glin Trp Met Pro Thir Wall Gly Luell Glu Gly Gly Arg Ile Asn Ile 21 O 215 22O

Wall Pro Wall Asp Phe Wall Wall Asp Ala Met Asp His Ile Ala His Ala 225 23 O 235 24 O

Glu Gly Glu Asp Gly Lys Phe His Luell Thir Asp Pro Asp Pro Tyr 245 250 255

Wall Gly Glu Ile Lell Asn Ile Phe Ala Glu Ala Gly His Ala Pro 26 O 265 27 O

Met Ala Met Arg Ile Asp Ala Arg Met Phe Gly Phe Ile Pro Pro 27s 285

Met Ile Arg Glin Gly Ile Ala Arg Luell Pro Pro Wall Glin Arg Met Lys 29 O 295 3 OO

Asn Ala Wall Luell Asn Asp Lell Gly Ile Pro Asp Glu Wall Met Ser Phe 3. OS 310 315 32O

Ile Asn Pro Thir Arg Phe Asp Asn Arg Glu Thir Glu Arg Luell Luell 3.25 330 335 US 9, 169,496 B2 77 - Continued

Lys Gly Thr Ala Ile Ala Val Pro Arg Lieu. Glin Asp Tyr Ser Pro Ala 34 O 345 35. O Ile Trp Asp Tyr Trp Glu Arg His Lieu. Asp Pro Asp Lieu. His Lys Asp 355 360 365 Arg Thr Lieu. Arg Gly Ala Val Glu Gly Arg Val Cys Val Ile Thr Gly 37 O 375 38O Ala Thir Ser Gly Ile Gly Lieu. Ser Ala Ala Arg Llys Lieu Ala Glu Ala 385 390 395 4 OO Gly Ala Lys Val Val Ile Ala Ala Arg Thir Lieu. Glu Lys Lieu. Glin Glu 4 OS 41O 415 Val Llys Lys Glu Lieu. Glu Glu Lieu. Gly Gly Glu Val Tyr Glu Tyr Ser 42O 425 43 O Val Asp Lieu. Ser Asp Lieu. Glu Asp Cys Asp Arg Phe Val Ala Asn Val 435 44 O 445 Lieu Lys Asp Lieu. Gly His Val Asp Val Lieu Val Asn. Asn Ala Gly Arg 450 45.5 460 Ser Ile Arg Arg Ser Ile Gln His Ala Phe Asp Arg Phe His Asp Phe 465 470 47s 48O Glu Arg Thr Met Gln Lieu. Asn Tyr Phe Gly Ser Lieu. Arg Lieu. Ile Met 485 490 495 Gly Phe Ala Pro Ser Met Lieu. Glu Arg Arg Arg Gly His Ile Val Asn SOO 505 51O Ile Ser Ser Ile Gly Val Lieu. Thir Asn Ala Pro Arg Phe Ser Ala Tyr 515 52O 525 Val Ala Ser Lys Ala Ala Lieu. Asp Ala Phe Ser Arg Cys Ala Ala Ala 53 O 535 54 O Glu Phe Ser Asp Lys Asn Val Thr Phe Thr Thr Ile Asn Met Pro Leu 5.45 550 555 560 Val Arg Thr Pro Met Ile Ser Pro Thr Lys Ile Tyr Asp Ser Val Pro 565 st O sts Thir Lieu. Thr Pro Glu Glu Ala Ala Asp Lieu Val Ala Glu Ala Ile Ile 58O 585 59 O His Arg Pro Lys Arg Ile Ala Thr Arg Lieu. Gly Val Phe Ala Glin Val 595 6OO 605 Lieu. His Ser Met Ala Pro Llys Phe Ser Glu Ile Ile Met Asn Thr Gly 610 615 62O Phe Llys Met Phe Pro Asp Ser Ser Ala Ala Thr Gly Gly Lys Asp Gly 625 630 635 64 O Glu Lys Pro Llys Val Ser Thr Glu Glin Val Ala Phe Ala Ala Ile Met 645 650 655 Arg Gly Ile His Trp 660

<210s, SEQ ID NO 18 &211s LENGTH: 6 212. TYPE: PRT <213s ORGANISM: Unknown 22 Os. FEATURE: <223> OTHER INFORMATION: NAD(P) (H) -binding motif conserved region found in multiple species

<4 OOs, SEQUENCE: 18 Gly Thr Gly Phe Ile Gly 1. 5 US 9, 169,496 B2 79 80 - Continued

<210s, SEQ ID NO 19 &211s LENGTH: 5 212. TYPE: PRT <213s ORGANISM: Unknown 22 Os. FEATURE: <223> OTHER INFORMATION: Terpene synthase highly conserved C-terminal domain found in multiple species 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (4) <223> OTHER INFORMATION: Xaa can be any amino acid <4 OOs, SEQUENCE: 19 Asp Asp Xaa Xala Asp 1. 5

<210s, SEQ ID NO 2 O &211s LENGTH: 7 212. TYPE: PRT <213s ORGANISM: Unknown 22 Os. FEATURE: <223> OTHER INFORMATION: NAD(P) (H) -binding motif conserved region found in multiple species 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) <223> OTHER INFORMATION: Xaa can be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) . . (5) <223> OTHER INFORMATION: Xaa can be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) <223> OTHER INFORMATION: Xaa can be des-Xaa or any amino acid

<4 OOs, SEQUENCE: 2O Gly Xaa Gly Xaa Xaa Xaa Gly 1. 5

<210s, SEQ ID NO 21 &211s LENGTH: 7 212. TYPE: PRT <213s ORGANISM: Unknown 22 Os. FEATURE: <223> OTHER INFORMATION: NAD(P) (H) -binding motif conserved region found in multiple species 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (4) <223> OTHER INFORMATION: Xaa can be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) <223> OTHER INFORMATION: Xaa can be any amino acid

<4 OOs, SEQUENCE: 21 Gly Xaa Xala Xala Gly Xaa Gly 1. 5

The invention claimed is: using a terpene synthase selected from an isoprene Syn 1. A method for the production ofbutadiene comprising the thase (EC 4.2.3.27), a myrcene/ocimene synthase (EC enzymatic conversion of crotyl alcohol into butadiene 60 4.2.3.15), a farmesene synthase (EC 4.2.3.46 or EC wherein the method comprises: 4.2.3.47), a pinene synthase (EC 4.2.3.14) or a monot (i) enzymatically converting crotyl alcohol into crotyl erpene synthase. monophosphate using a hydroxyethylthiazole kinase 2. The method of claim 1 further comprising the enzymatic (EC 2.7.1.50) or a thiamine kinase (EC 2.7.1.89); and conversion of crotonyl-CoA into crotyl alcohol. (ii) enzymatically converting crotyl monophosphate into 65 3. The method of claim 2 wherein crotonyl-CoA is enzy butadiene, wherein the crotyl monophosphate is directly matically converted into crotonaldehyde and then crotonal converted into butadiene in a single enzymatic reaction dehyde is enzymatically converted into crotyl alcohol. US 9, 169,496 B2 81 82 4. The method of claim 3, wherein the enzymatic conver 15. The method of claim 13 wherein the enzymatic con sion of crotonyl-CoA into crotonaldehyde is achieved by the version of crotonaldehyde into crotyl alcohol is achieved by use of the use of an enzyme selected from: (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); and/or O (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or (ii) an alcohol dehydrogenase (EC 1.1.1.1); or (iii) an acyl-CoA reductase. (iii) an aldehyde reductase; or 5. The method of claim 3 wherein the enzymatic conver (iv) an aldo-keto reductase. sion of crotonaldehyde into crotyl alcohol is achieved by the 16. The method of claim 13 wherein the enzymatic con use of an enzyme selected from: 10 version of crotonyl-CoA into crotyl alcohol is achieved by the (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); use of an aldehyde/alcohol dehydrogenase or by the use of a (ii) an alcohol dehydrogenase (EC 1.1.1.1); hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) or by (iii) an aldehyde reductase; or the use of a short-chain dehydrogenase/fatty acyl-CoA reduc (iv) an aldo-keto reductase. tase. 6. The method of claim 3 wherein the enzymatic conver 15 sion of crotonyl-CoA into crotyl alcohol is achieved by the 17. A method of producing butadiene comprising enzy use of an aldehyde/alcohol dehydrogenase or by the use of a matically converting crotyl alcohol into butadiene compris hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) or by ing an in vitro cell-free system using a combination of the use of a short-chain dehydrogenase/fatty acyl-CoA reduc enzymes comprising: tase. (a) a hydroxyethylthiazole kinase (EC 2.7.1.50) or a thia 7. The method of claim 1 further comprising the step of mine kinase (EC 2.7.1.89); and providing crotyl alcohol by the enzymatic conversion of cro (b) an isopentenyl phosphate kinase; and tonaldehyde into crotyl alcohol. (c) a terpene synthase wherein said terpene synthase is 8. The method of claim 7 wherein the conversion of cro selected from an isoprene synthase (EC 4.2.3.27), a tonaldehyde into crotyl alcohol is achieved by the use of an 25 myrcene/ocimene synthase (EC 4.2.3.15); a farnesene enzyme selected from: synthase (EC 4.2.3.46 or EC 4.2.3.47); a pinene syn (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); thase (EC 4.2.3.14) or a monoterpene synthase. (ii) an alcohol dehydrogenase (EC 1.1.1.1); 18. A recombinant microorganism or plant cell compris (iii) an aldehyde reductase; or 1ng: (iv) an aldo-keto reductase. 30 (a) an overexpressed hydroxyethylthiazole kinase (EC 9. A method for the production ofbutadiene comprising the 2.7.1.50) or a thiamine kinase (EC 2.7.1.89); and enzymatic conversion of crotyl alcohol into butadiene (b) an overexpressed isopentenyl phosphate kinase; and wherein the method comprises: (c) an overexpressed terpene synthase, wherein said ter (a) enzymatically converting crotyl alcohol into crotyl pene synthase is selected from an isoprene synthase (EC monophosphate using a hydroxyethylthiazole kinase 35 4.2.3.27), a myrcene/ocimene synthase (EC 4.2.3.15); a (EC 2.7.1.50) or a thiamine kinase (EC 2.7.1.89); farmesene synthase (EC 4.2.3.46 or EC 4.2.3.47); a (b) enzymatically converting crotyl monophosphate into pinene synthase (EC 4.2.3.14) or a monoterpene Syn crotyl diphosphate using isopentenyl phosphate kinase; thase and which is capable of converting crotyl alcohol and into butadiene. (c) enzymatically converting crotyl diphosphate into buta 40 19. The recombinant microorganism or plant cell of claim diene using a terpene synthase selected from an isoprene 18, wherein the recombinant microorganism or plant cell synthase (EC 4.2.3.27), a myrcene/ocimene synthase further expresses an enzyme selected from: (EC 4.2.3.15); a farmesene synthase (EC 4.2.3.46 or EC (a) hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); 4.2.3.47); a pinene synthase (EC 4.2.3.14) or a monot (b) aldehyde/alcohol dehydrogenase; or erpene synthase. 45 (c) Short-chain dehydrogenase/fatty acyl-CoA reductase 10. The method of claim 9 further comprising the step of and which is capable of converting crotonyl-CoA into buta providing crotyl alcohol by the enzymatic conversion of cro diene. tonaldehyde into crotyl alcohol. 20. The recombinant microorganism or plant cell of claim 11. The method of claim 10 wherein the conversion of 18, wherein the recombinant microorganism or plant cell crotonaldehyde into crotyl alcohol is achieved by the use of 50 further expresses an enzyme selected from: (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); (a) acyl-CoA reductase or acetaldehyde dehydrogenase and/or (EC 1.2.1.10) or hydroxymethylglutaryl-CoA reductase (ii) an alcohol dehydrogenase (EC 1.1.1.1); and/or (EC 1.1.1.34) or aldehyde/alcohol dehydrogenase or (iii) an aldehyde reductase; and/or short-chain dehydrogenase/fatty acyl-CoA reductase (iv) an aldo-keto reductase. 55 and 12. The method of claim 9 further comprising the enzy (b) alcoholdehydrogenase (EC 1.1.1.1) or aldehyde reduc matic conversion of crotonyl-CoA into crotyl alcohol. tase or aldo-keto reductase 13. The method of claim 12 wherein crotonyl-CoA is enzy and which is capable of converting crotonyl-CoA into buta matically converted into crotonaldehyde and then crotonal diene. dehyde is enzymatically converted into crotyl alcohol. 60 21. The recombinant microorganism or plant cell of claim 14. The method of claim 13, wherein the enzymatic con 20, wherein the acyl-CoA reductase is selected from: version of crotonyl-CoA into crotonaldehyde is achieved by (a) cinnamoyl-CoA reductase (EC 1.2.1.44); the use of (b) long-chain-fatty-acyl-CoA reductase (EC 1.2.1.50); or (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); (c) malonyl-CoA reductase (EC 1.2.1.75). and/or 65 22. The recombinant microorganism or plant cell of claim (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or 18, wherein the recombinant microorganism or plant cell (iii) an acyl-CoA reductase. further expresses an enzyme selected from: US 9, 169,496 B2 83 84 (a) acyl-CoA reductase or acetaldehyde dehydrogenase and which is capable of converting crotonyl-CoA into crotyl (EC 1.2.1.10); and alcohol. (b) alcoholdehydrogenase (EC 1.1.1.1) or aldehyde reduc 28. The recombinant microorganism or plant cell of claim tase or aldo-keto reductase or hydroxymethylglutaryl CoA reductase (EC 1.1.1.34) or aldehyde/alcoholdehy 27, wherein the acyl-CoA reductase is selected from: drogenase or short-chain dehydrogenase/fatty acyl-CoA (a) cinnamoyl-CoA reductase (EC 1.2.1.44); reductase (b) long-chain-fatty-acyl-CoA reductase (EC 1.2.1.50); or and which is capable of converting crotonyl-CoA into buta (c) malonyl-CoA reductase (EC 1.2.1.75). diene. 29. A recombinant microorganism or plant cell compris 1ng: 23. The recombinant microorganism or plant cell of claim 10 22, wherein the acyl-CoA reductase is selected from: (a) an overexpressed heterologous acyl-CoA reductase or (a) cinnamoyl-CoA reductase (EC 1.2.1.44); acetaldehyde dehydrogenase (EC 1.2.1.10); and (b) long-chain-fatty-acyl-CoA reductase (EC 1.2.1.50); or (b) an overexpressed heterologous alcohol dehydrogenase (c) malonyl-CoA reductase (EC 1.2.1.75). (EC 1.1.1.1) or aldehyde reductase or aldo-keto reduc 24. An in vitro, cell-free system comprising: tase or hydroxymethylglutaryl-CoA reductase (EC (a) a hydroxyethylthiazole kinase (EC 2.7.1.50) or a thia 15 1.1.1.34) or aldehyde/alcohol dehydrogenase or short mine kinase (EC 2.7.1.89); and chain dehydrogenase/fatty acyl-CoA reductase (b) an isopentenyl phosphate kinase; and and which is capable of converting crotonyl-CoA into crotyl (c) a terpene synthase wherein said terpene synthase is alcohol. selected from an isoprene synthase (EC 4.2.3.27), a 30. The recombinant microorganism or plant cell of claim myrcene/ocimene synthase (EC 4.2.3.15); a farmesene 29, wherein the acyl-CoA reductase is selected from: synthase (EC 4.2.3.46 or EC 4.2.3.47); a pinene syn (a) cinnamoyl-CoA reductase (EC 1.2.1.44); thase (EC 4.2.3.14) or a monoterpene synthase. (b) long-chain-fatty-acyl-CoA reductase (EC 1.2.1.50); or 25. The in vitro cell-free system of claim 24 further com (c) malonyl-CoA reductase (EC 1.2.1.75). prising crotyl alcohol. 31. A recombinant microorganism or plant cell compris 26. A recombinant microorganism or plant cell which over 25 1ng: expresses a heterologous enzyme selected from: (a) an overexpressed hydroxyethylthiazole kinase (EC (a) hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34): 2.7.1.50) or a thiamine kinase (EC 2.7.1.89); and Or (b) an overexpressed terpene synthase selected from an (b) short-chain dehydrogenase/fatty acyl-CoA reductase, isoprene synthase (EC 4.2.3.27), a myrcene/ocimene and which is capable of converting crotonyl-CoA into crotyl 30 synthase (EC 4.2.3.15), a farnesene synthase (EC alcohol. 4.2.3.46 or EC 4.2.3.47), a pinene synthase (EC 27. A recombinant microorganism or plant cell compris 4.2.3.14) or a monoterpene synthase. 1ng: 32. An in vitro, cell-free system comprising: (a) an overexpressed heterologous acyl-CoA reductase or (a) a hydroxyethylthiazole kinase (EC 2.7.1.50) or a thia acetaldehyde dehydrogenase (EC 1.2.1.10) or 35 mine kinase (EC 2.7.1.89); and hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) or (b) a terpene synthase selected from an isoprene synthase aldehyde/alcohol dehydrogenase or short-chain dehy (EC 4.2.3.27), a myrcene/ocimene synthase (EC drogenase/fatty acyl-CoA reductase; and 4.2.3.15), a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47), a pinene synthase (EC 4.2.3.14) or a monot (b) an overexpressed heterologous alcohol dehydrogenase 40 (EC 1.1.1.1) or aldehyde reductase or aldo-keto reduc erpene synthase. tase