US 20140256009 A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0256009 A1 Marliere (43) Pub. Date: Sep. 11, 2014
(54) METHOD FOR THE ENZYMATIC CI2N 9/88 (2006.01) PRODUCTION OF BUTADIENE CI2P 7/04 (2006.01) (52) U.S. Cl. (71) Applicant: SCIENTIST OF FORTUNE, S.A., CPC, C12P5/026 (2013.01); C12P 7/04 (2013.01); Luxembourg (LU) CI2N 9/1205 (2013.01); C12N 9/88 (2013.01); CI2N 9/1235 (2013.01) (72) Inventor: Philippe Marliere, Mouscron (BE) USPC 435/157:435/167; 435/252.33: 435/252.31; (73) Assignee: SCIENTIST OF FORTUNE, S.A., 435/252.3; 435/254.11: 435/254.21: 435/254.2: Luxembourg (LU) 435/254.3; 435/254.6:435/254.23; 435/257.2: 435/194 (21) Appl. No.: 14/352,825 (22) PCT Filed: Oct. 18, 2012 (57) ABSTRACT (86). PCT No.: PCT/EP2012/07O661 S371 (c)(1), Described is a method for the enzymatic production of buta (2), (4) Date: Apr. 18, 2014 diene which allows to produce butadiene from crotyl alcohol. Also described are enzyme combinations and compositions Related U.S. Application Data containing Such enzyme combinations which allow the enzy (60) Provisional application No. 61/549,149, filed on Oct. matic conversion of crotyl alcohol into butadiene. Further 19, 2011. more, the invention relates to microorganisms which have been genetically modified so as to be able to produce butadi (30) Foreign Application Priority Data ene from crotyl alcohol. Moreover, the invention relates to a method for the enzymatic Oct. 19, 2011 (EP) ...... 111858,544 production of crotyl alcohol from crotonyl-Coenzyme A. The Publication Classification obtained crotyl alcohol can be further converted into butadi ene as described herein. Also described are enzyme combi (51) Int. Cl. nations which allow to convert crotonyl-Coenzyme A into CI2P 5/02 (2006.01) crotyl alcohol as well as (micro)organisms which express CI2N 9/12 (2006.01) Such enzyme combinations. Patent Application Publication Sep. 11, 2014 Sheet 1 of 10 US 2014/0256009 A1
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METHOD FOR THE ENZYMATIC 0006 Thus, in a first aspect, the present invention relates PRODUCTION OF BUTADIENE to a process for the production of butadiene in which butadi ene is produced by the enzymatic conversion of crotyl alco 0001. The present invention relates to a method for the hol. Crotyl alcohol, also referred to as crotonyl alcohol or enzymatic production ofbutadiene which allows to produce crotonol, is an unsaturated alcohol of formula CHO (see butadiene from crotyl alcohol. The present invention also FIG. 1). Another name for crotyl alcohol is But-2-en-1-ol. It relates to microorganisms which have been genetically modi can be produced by reduction of crotonaldehyde (see FIG.3). fied so as to produce butadiene. According to the present invention crotyl alcohol can be 0002 The present invention also relates to a method for converted into butadiene by enzymatic reactions involving as the enzymatic production of crotyl alcohol from crotonyl intermediates crotyl phosphate and/or crotyl diphosphate. Coenzyme A. The obtained crotyl alcohol can be further Thus, the principle underlying the present invention is that converted into butadiene as described herein. The present crotyl alcohol is first enzymatically activated by the conver invention furthermore relates to enzyme combinations which sion into crotyl phosphate or crotyl diphosphate and is then allow to convert crotonyl-Coenzyme A into crotyl alcohol as further converted into butadiene by the use of appropriate well as to (micro)organisms which express such enzyme enzymes as described below. combinations. 0007 Thus, the present invention relates, in a first aspect, 0003. Butadiene (1,3-butadiene) is a conjugated diene to a method for the production ofbutadiene comprising the with the formula CH (see FIG. 4). It is an important indus enzymatic conversion of crotyl alcohol into butadiene via trial chemical used as a monomer in the production of syn crotyl phosphate or crotyl diphosphate. thetic rubber. There exist different possibilities to produce 0008. The enzymatic conversion of crotyl alcohol into butadiene. Butadiene is, for example, produced as a by prod butadiene can occur via different alternative routes. In a first uct of the steam cracking process used to produce ethylene aspect (A), the present invention relates to a method for the and other olefins. In this process butadiene occurs in the C4 production of butadiene comprising the enzymatic conver stream and is normally isolated from other byproducts by sion of crotyl alcohol into butadiene via crotyl phosphate extraction into a polar aprotic solvent, such as acetonitrile, wherein said method comprises the steps of from which it is then stripped. Butadiene can also be pro duced by the catalytic dehydrogenation of normal butane or it (i) enzymatically converting crotyl alcohol into crotyl phos can be produced from ethanol. In the latter case, two different phate; and processes are in use. In a single-step process, ethanol is con (ii) enzymatically converting crotyl phosphate into butadi verted to butadiene, hydrogen and water at 400-450° C. over CC. a metal oxide catalyst (Kirshenbaum, I. (1978), Butadiene. In 0009. This alternative is in the following referred to as M. Grayson (Ed.), Encyclopedia of Chemical Technology, 3rd Alternative A and the different steps are referred to as A(i) and ed., vol. 4, pp. 313-337. New York: John Wiley & Sons). In a A(ii). two-step process, ethanol is oxidized to acetaldehyde which 0010. As regards step A(i), the enzymatic conversion of reacts with additional ethanol over a tantalum-promoted crotyl alcohol into crotyl phosphate is a phosphorylation step porous silica catalyst at 325-350° C. to yield butadiene (Kir and can be achieved by enzymes which catalyze the transfer shenbaum, I. (1978), loccit.). Butadiene can also be produced of a phospho group onto a molecule. Such as kinases. For by catalytic dehydrogenation of normal butenes. example, enzymes which can be employed in this reaction are 0004 For the past two decades, genetic engineering tech enzymes which are classified as E.C.2.7.1, i.e. phosphotrans nologies have made possible the modification of the metabo ferases with an alcohol group as acceptor, preferably lism of micro-organisms, and hence their use to produce key enzymes which are classified as 2.7.1.50 (hydroxyethylthiaz substances which they would otherwise produce at a low ole kinase) or which are classified as E.C. 2.7.1.89 (thiamine yield. By enhancing naturally occurring metabolic pathways, kinase). Preferably, ATP is the donor of the phospho group in these technologies open up new ways to bio-produce numer Such a reaction. Thus, in one embodiment the enzymatic ous compounds of industrial relevance. Several industrial conversion of crotyl alcohol into crotyl phosphate can, e.g., compounds Such as amino-acids for animal feed, biodegrad be achieved by the use of a hydroxyethylthiazole kinase (EC able plastics or textile fibres are now routinely produced using 2.7.1.50). Hydroxyethylthiazole kinase is an enzyme which genetically modified organisms. There are however no bio catalyzes the following reaction processes using micro-organisms in place for the production ATP+4-methyl-5-(2-hydroxyethyl)thiazole ADP+ of the major petrochemically derived molecules, in particular 4-methyl-5-(2-phosphoethyl)thiazole butadiene, since no micro-organisms are known as natural producers of butadiene even in Small quantities. Given the 0011. The occurrence of this enzyme has been described large amounts of rubber produced worldwide and the increas for several organisms, e.g. for E. coli, Bacillus subtilis, Rhizo ing environmental concerns and the limited resources for bium leguminosarum, Pyrococcus horikoshii OT3, Saccha producing butadiene using chemical processes, there is a need romyces cerevisiae. to provide alternative, environmentally-friendly and Sustain 0012. In another embodiment the enzymatic conversion of able processes for the production of butadiene. crotyl alcohol into crotyl phosphate can, e.g., beachieved by 0005. The present invention addresses this need and pro the use of a thiamine kinase (EC 2.7.1.89). Thiamine kinase is vides for the first time a process by which butadiene can be an enzyme which catalyzes the following reaction produced enzymatically starting from crotyl alcohol. Crotyl ATP+thiamines ADP+thiamine phosphate alcohol itself can be provided by the enzymatic conversion of crotonyl CoA which, in turn, can be provided starting from 0013 The occurrence of this enzyme has been described the metabolic intermediate acetyl-Coenzyme A (in the fol for several organisms, e.g. for E. coli and Salmonella lowing acetyl-CoA) as described herein. enterica. US 2014/0256009 A1 Sep. 11, 2014
0014) Hydroxyethylthiazole is a moiety of thiamine and ing diphosphate esters. The general mechanism of this shares with crotyl alcohol the following common structural enzyme class induces the removal of the diphosphate group motif CH-C CH-CH OH. and the generation of an intermediate with carbocation as the 0015 Thus, the inventor considers that a hydroxyethylthi first step. In the various terpene synthases, such intermediates azole kinase or a thiamine kinase could also act on other further rearrange to generate the high number of terpene substrates which contain this motif and found that, indeed, skeletons observed in nature. In particular, the resulting cat different tested hydroxyethylthiazole kinases and thiamine ionic intermediate undergoes a series of cyclizations, hydride kinases were capable of using crotyl alcohol as a Substrate and shifts or other rearrangements until the reaction is terminated converting it into crotyl phosphate by proton loss or the addition of a nucleophile, in particular 0016. In principle, any known hydroxyethylthiazole water for forming terpenoid alcohols (Degenhardt et al., Phy kinase can be employed in the method according to the inven tochemistry 70 (2009), 1621-1637). tion. In one aspect of the present invention, a hydroxyethylthi 0020. The different terpene synthases share various struc azole kinase of bacterial origin is used. Such as a hydroxyeth tural features. These include a highly conserved C-terminal ylthiazole kinase from a bacterium belonging to the genus domain, which contains their catalytic site and an aspartate Escherichia, Bacillus or Rhizobium, preferably of E. coli, rich DDXXD motifessential for the divalent metalion (typi Bacillus subtilis or of R. leguminosarum. Amino acid and cally Mg2+ or Mn2+) assisted substrate binding in these nucleotide sequences for these enzymes are available. enzymes (Green et al. Journal of biological chemistry, 284. Examples for corresponding amino acid sequences are pro 13, 8661-8669). In principle, any known enzyme which can vided in SEQ ID NOs: 1 to 3. In a particularly preferred be classified as belonging to the EC 4.2.3 enzyme superfamily embodiment any protein showing an amino acid sequence as can be employed for the conversion of crotyl phosphate into shown in any one of SEQID NOs: 1 to 3 or showing an amino butadiene. acid sequence which is at least 80% homologous to any of 0021. Even more preferably the method according to the SEQID NOs: 1 to 3 and having the activity of a hydroxyeth invention makes use of an isoprene synthase (EC 4.2.3.27), a ylthiazole kinase can be employed in a method according to myrcene/ocimene synthase (EC 4.2.3.15), a farnesene Syn the present invention. thase (EC 4.2.3.46 or EC 4.2.3.47) or a pinene synthase (EC 0017 Moreover, in principle, any known thiamine kinase 4.2.3.14). Also enzymes which are generally classified as can be employed in the method according to the invention. In monoterpene synthases can be used. one aspect of the present invention, a thiamine kinase of 0022. In a particularly preferred embodiment, the dephos bacterial origin is used, such as a thiamine kinase from a phorylation of crotyl phosphate to butadiene is achieved by an bacterium belonging to the genus Escherichia or Salmonella, isoprene synthase (EC 4.2.3.27). Isoprene synthase is an preferably of E. coli or of Salmonella enterica. Amino acid enzyme which catalyzes the following reaction: and nucleotide sequences for these enzymes are available. Dimethylallyl diphosphatev isoprene-diphosphate 0018. In one embodiment of this method, step A(ii) con sists of a single enzymatic reaction in which crotyl phosphate 0023 This enzyme occurs in a number of organisms, in is directly converted into butadiene. This option is in the particular in plants and some bacteria. The occurrence of this following referred to as option A(ii.1). In this conversion of enzyme has, e.g., been described for Arabidopsis thaliana, a crotyl phosphate into butadiene the phospho group is number of Populus species like P alba (UniProt accession removed from crotyl phosphate with the simultaneous pro numbers Q50L36, A9Q7C9, D8UY75 and D8UY76), P duction of butadiene. An enzyme which can catalyze this nigra (UniProt accession number AOPFK2), P canescence reaction is referred to as a crotyl phosphate phosphate-lyase (UniProt accession number Q9AR86; see also KÖksal et al., J. (butadiene forming). Examples of enzymes which can cata Mol. Biol. 402 (2010), P tremuloides, P. trichocarpa, P. lyze the dephosphorylation of crotyl phosphate into butadi lobata, in Quercus petraea, Quercus robur, Salix discolour, ene are enzymes which can be classified as belonging to the Pueraria montana (UniProt accession number Q6EJ97), terpene synthase family. Preferably Such an enzyme belongs Mucuna pruriens, Vitis vinifera, Embryophyta and Bacillus to the family of plant terpene synthases. The terpene Syn subtilis. In principle, any known isoprene synthase can be thases constitute an enzyme family which comprises employed in the method according to the invention. In a enzymes catalyzing the formation of numerous natural prod preferred embodiment, the isoprene synthase employed in a ucts always composed of carbon and hydrogen (terpenes) and method according to the present invention is an isoprene Sometimes also of oxygen or other elements (terpenoids). synthase from a plant of the genus Populus, more preferably Terpenoids are structurally diverse and widely distributed from Populus trichocarpa or Populus alba. In another pre molecules corresponding to well over 30000 defined natural ferred embodiment the isoprene synthase employed in a compounds that have been identified from all kingdoms of method according to the present invention is an isoprene life. In plants, the members of the terpene synthase family are synthase from Pueraria montana, preferably from Pueraria responsible for the synthesis of the various terpene molecules montana var. lobata (an example for Such a sequence is pro from two isomeric 5-carbon precursor “building blocks, iso vided in SEQ ID NO: 7), or from Vitis vinifera. Preferred prenyl diphosphate and prenyl diphosphate, leading to 5-car isoprene synthases to be used in the context of the present bon isoprene, 10-carbon monoterpene, 15-carbon sesquiter invention are the isoprene synthase of Populus alba (Sasaki et pene and 20-carbon diterpenes' (Chen et al.; The Plant al.; FEBS Letters 579 (2005), 2514-2518) or the isoprene Journal 66 (2011), 212-229). synthases from Populus trichocarpa and Populus tremuloides 0019. The ability of terpene synthases to convert a prenyl which show very high sequence homology to the isoprene diphosphate containing Substrate to diverse products during synthase from Populus alba. Aparticularly preferred isoprene different reaction cycles is one of the most unique traits of this synthase is the isoprene synthase from Pueraria montana var. enzyme class. The common key step for the biosynthesis of lobata (kudzu) (Sharkey et al.: Plant Physiol. 137 (2005), all terpenes is the reaction of terpene synthase on correspond 700-712). In a particularly preferred embodiment a protein US 2014/0256009 A1 Sep. 11, 2014
showing an amino acid sequence as shown in SEQID NOs: 7 After incubation, the assay mixture is extracted with pentane or showing an amino acid sequence which is at least 80% a second time, both pentane fractions are pooled, concen homologous to SEQID NOs: 7 and having the activity of an trated and analyzed by gas chromatography to quantify isoprene synthase can be employed in a method according to ocimene/myrcene production. the present invention. 0028 Beta-farnesene synthases (EC 4.2.3.47) naturally 0024. The activity of an isoprene synthase can be mea catalyze the following reaction: Sured according to methods known in the art, e.g. as described (2E,6E)-farnesyl diphosphate (E)-beta-farnesene-- in Silver and Fall (Plant Physiol (1991) 97, 1588-1591). In a diphosphate typical assay, the enzyme is incubated with dimethylallyl 0029. This enzyme occurs in a number of organisms, in diphosphate in the presence of the required co-factors, Mg" particular in plants and in bacteria, for example in Artemisia or Mn" and K" in sealed vials. At appropriate time volatiles annua (UniProt accession number Q4VM12), Citrus junos compound in the headspace are collected with a gas-tight (UniProt accession number Q94JS8), Oryza sativa (UniProt Syringe and analyzed for isoprene production by gas chroma accession number Q0J7R9), Pinus Sylvestris (UniProt acces tography (GC). Crotyl monophosphate and crotyl diphos sion number D7PCH9), Zea diploperennis (UniProt acces phate are structurally closely related to dimethylallyl diphos sion number C7E5V9), Zea mays (UniProt accession num phate. In particular, the difference between crotyl bers Q2NM15, C7E5 V8 and C7E5V7), Zea perennis diphosphate and dimethylallyl diphosphate is just a methyl (UniProt accession number C7E5WO) and Streptococcus group (see FIG. 8). The inventor considers that, therefore, an coelicolor (Zhao et al., J. Biol. Chem. 284 (2009), 36711 isoprene synthase can also use crotyl diphosphate or crotyl 36719). In principle, any known beta-farnesene synthase can monophosphate as a Substrate. In principle, any known iso be employed in the method according to the invention. In a prene synthase can be employed in the method according to preferred embodiment, the beta-farnesene synthase the invention. employed in a method according to the present invention is a 0025. In another particularly preferred embodiment, the beta-farnesene synthase from Mentha piperita (Crock et al.: enzyme used for the conversion of crotyl phosphate into Proc. Natl. Acad. Sci. USA 94 (1997), 12833-12838). butadiene is a myrcene/ocimene synthases (EC 4.2.3.15). 0030 Methods for the determination of farnesene syn Myrcene/ocimene synthases (EC 4.2.3.15) are enzymes thase activity are known in the art and are described, for which naturally catalyze the following reaction: example, in Green et al. (Phytochemistry 68 (2007), 176 Geranyl diphosphate (E)-beta-ocimene--diphos 188). In a typical assay farnesene synthase is added to an phate assay buffer containing 50 mM BisTrispropane (BTP) (pH 7.5), 10% (v/v) glycerol, 5 mM DTT. Tritiated farnesyl O diphosphate and metal ions are added. Assays containing the Geranyl diphosphate -> myrcene-diphosphate protein are overlaid with 0.5 ml pentane and incubated for 1 h at 30°C. with gentle shaking. Following addition of 20 mM 0026. These enzymes occur in a number of organisms, in EDTA (final concentration) to stop enzymatic activity an particular in plants and animals, for example in Lotus japani aliquot of the pentane is removed for Scintillation analysis. cus, Phaseolus lunatus, Abies grandis, Arabidopsis thaliana The olefin products are also analyzed by GC-MS. (UniProt accession number Q97.UH4), Actinidia chinensis, 0031 Pinene synthase (EC 4.2.3.14) is an enzyme which Perilla fructescens, Vitis vinifera, Ochtodes secundiramea naturally catalyzes the following reaction: and in Ips pini (UniProt accession number Q58GE8. In prin Geranyl diphosphate alpha-pinene-diphosphate ciple, any known myrcene/ocimene synthase can be employed in the method according to the invention. In a 0032. This enzyme occurs in a number of organisms, in preferred embodiment, the myrcene/ocimene synthase particular in plants, for example in Abies grandis (UniProt employed in a method according to the present invention is a accession number O244475), Artemisia annua, Chamaecy beta-ocimene synthase from Lotus japanicus (Arimura et al.: paris formosensis (UniProt accession number C3RSF5), Plant Physiol. 135 (2004), 1976-1983; an example for such an Salvia officinalis and Picea sitchensis (UniProt accession enzyme is provided in SEQ ID NO: 9) or from Phaseolus number Q6XDB5). lunatus (UniProt accession number B1P189; an example for 0033 For the enzyme from Abies grandis a particular such an enzyme is provided in SEQID NO: 10). In a particu reaction was also observed (Schwab et al., Arch. Biochem. larly preferred embodiment the myrcene/ocimene synthase is Biophys. 392 (2001), 123-136), namely the following: an (E)-beta-ocimene synthase from Vitis vinifera (an example 6,7-dihydrogeranyl diphosphate' 6,7-dihy for such an enzyme is provided in SEQ ID NO: 12). In a dromyrcene-diphosphate particularly preferred embodiment any protein showing an 0034. In principle, any known pinene synthase can be amino acid sequence as shown in any one of SEQID NOs: 9. employed in the method according to the invention. In a 10 or 12 or showing an amino acid sequence which is at least preferred embodiment, the pinene synthase employed in a 80% homologous to any of SEQ ID NOs: 9, 10 or 12 and method according to the present invention is a pinene Syn having the activity of a beta-ocimene synthase can be thase from Abies grandis (UniProt accession number employed in a method according to the present invention. O244475; Schwab et al., Arch. Biochem. Biophys. 392 0027. The activity of an ocimene/myrcene synthase can be (2001), 123-136). measured as described, for example, in Arimura et al. (Plant 0035 Methods for the determination of pinene synthase Physiology 135 (2004), 1976-1983. In a typical assay for activity are known in the art and are described, for example, in determining the activity, the enzyme is placed in screwcapped Schwab et al. (Archives of Biochemistry and Biophysics 392 glass test tube containing divalent metal ions, e.g. Mg" and/ (2001), 123-136). In a typical assay, the assay mixture for or Mn", and substrate, i.e. geranyldiphosphate. The aqueous pinene synthase consists of 2 ml assay buffer (50 mM Tris/ layer is overlaid with pentane to trap Volatile compounds. HCl, pH 7.5, 500 mM KC1, 1 mM MnC12, 5 mM dithiothrei US 2014/0256009 A1 Sep. 11, 2014 tol, 0.05% NaHSO3, and 10% glycerol) containing 1 mg of 0042. This enzyme participates in an alternative branch of the purified protein. The reaction is initiated in a Teflon the mevalonate pathway which has been discovered in the sealed screw-capped vial by the addition of 300 mM sub archaeon Methanocaldococcus jannaschii. It is a small mol strate. Following incubation at 25° C. for variable periods ecule kinase. The primary amino acid sequence and the crys (0.5-24 h), the mixture is extracted with 1 ml of diethyl ether. tal structure of the isopentenyl phosphate kinase of Methano The biphasic mixture is vigorously mixed and then centri caldococcus jannaschii has already been disclosed as well as fuged to separate the phases. The organic extract is dried mutants which are able to use oligoprenyl monophosphates as (MgSO4) and subjected to GC-MS and MDGC analysis. substrate (Dellas and Noel, ACS Chem. Biol. 5 (2010), 589 0036. As indicated above, it is also possible to employ a 601). The active site has been characterized and the amino monoterpene synthases in a method according to the inven acid residues crucial for binding and catalysis of the reaction tion. Particularly preferred are the monoterpene synthase have been identified. Because of the high structural similarity from Melaleuca alternifolia described in Shelton et al. (Plant of isopentenyl phosphate and crotyl phosphate and the fact Physiol. Biochem. 42 (2004), 875-882; an example for such that mutants of the isopentenyl phosphate kinase of Metha an enzyme is provided in SEQID NO: 11) or the monoterpene nocaldococcus jannaschii have already been shown to be able synthase from Eucalyptus globulus (UniProt accession num to use other oligoprenyl monophosphates as Substrates, it can ber QOPCI4; an example for such an enzyme is provided in be expected that this enzyme or mutants thereof will also be SEQID NO: 8). In a particularly preferred embodiment any able to convert crotyl phosphate into crotyl diphosphate. The protein showing an amino acid sequence as shown in any one sequence of the isopentenyl phosphate kinase from Metha of SEQID NO: 11 or 8 or showing an amino acid sequence nocaldococcus jannaschii is shown in SEQID NO: 6. which is at least 80% homologous to any of SEQID NO: 11 0043 Genes encoding an isopentenyl phosphate kinase or 8 and having the activity of a monoterpene synthase can be are also known from Methanothermobacter thermau employed in a method according to the present invention. totrophicus (MTH) and from Thermoplasma acidophilum 0037. The present inventors have shown that different (THA) (Chen and Poulter, Biochemistry 49 (2010), 207-217). types ofterpene synthases, e.g. isoprene synthases, (E)-beta For both these enzymes crystal structures have been deter ocimene and monoterpene synthase from different plant mined (Mabanglo et al., ACS Chem. Biol. 5 (2010),517-527). organisms are able to convert crotyl phosphate into butadiene The sequence of the isopentenyl phosphate kinase from (see Examples 12 and 13 and FIG. 11). Methanothermobacter thermautotrophicus is shown in SEQ 0038. The reactions catalyzed by the various terpene syn ID NO: 5. The sequence of the isopentenyl phosphate kinase thases, in particular the terpene synthases mentioned above, from Thermoplasma acidophilum is shown in SEQID NO: 4. show certain common features. For example, the reactions In a particularly preferred embodiment any protein showing catalyzed by isoprene synthases, by myrcene/ocimene Syn an amino acid sequence as shown in any one of SEQID NOs: thases, by farnesene synthases, by pinene synthase and by 4 to 6 or showing an amino acid sequence which is at least monoterpene synthases, respectively, are all believed to pro 80% homologous to any of SEQIDNOs: 4 to 6 and having the ceed through a common mechanism in which, in a first step a activity of an isopentenyl phosphate kinase can be employed carbocation is created by elimination of the diphosphate in a method according to the present invention. (PP), which is then followed by direct deprotonation so as to 0044 As regards step A(ii2b), the enzymatic conversion of form the corresponding diene. It could be shown by the crotyl diphosphate into butadiene involves the removal of a present inventors that enzymes which belong to the family of diphosphate group from crotyl diphosphate with the simulta terpene synthases are able to convert crotyl phosphate into neous production ofbutadiene. An enzyme which can cata butadiene. lyze this reaction is referred to as a crotyl diphosphate diphos 0039. In another embodiment of the method according to phate-lyase (butadiene forming). Examples of enzymes the invention step A(ii) consists of two enzymatic reactions which can catalyze the dephosphorylation of crotyl diphos comprising: phate into butadiene are enzymes which can be classified as (a) the enzymatic conversion of crotyl phosphate into crotyl belonging to the terpene synthase family. Preferably such an diphosphate; and enzyme belongs to the family of plant terpene synthases. (b) the enzymatic conversion of crotyl diphosphate into buta These enzymes have already been disclosed in detail herein diene. above in connection with the conversion of crotyl phosphate 0040. This option is in the following referred to as option into butadiene and the same applies here. A(ii2) and the different steps are referred to as steps A(ii2a) 0045 Preferably the terpene synthase is an isoprene syn and A(ii2b). thase (EC 4.2.3.27), a myrcene/ocimene synthase (EC 4.2.3. 0041 As regards step A(ii2a), the enzymatic conversion of 15), a farmesene synthase (EC 4.2.3.46 or EC 4.2.3.47) or a crotyl phosphate into crotyl diphosphate can be achieved by pinene synthase (EC 4.2.3.14). Also enzymes which are gen the use of an enzyme which can catalyze the transfer of a erally classified as monoterpene synthases can be used. Par phospho group onto a molecule. Such as kinases. Preferably, ticularly preferred the terpene synthase is an isoprene Syn ATP is the donor of the phospho group in such a reaction. The thase, an (E)-beta Ocimene synthase or a monoterpene conversion can in particular, e.g., beachieved by the use of an synthase. isopentenyl phosphate kinase. This enzyme has so far not yet 0046. In a particularly preferred embodiment the dephos been classified and, therefore, no EC number is available. It is phorylation of crotyl diphosphate into butadiene is achieved predicted to be a member of the amino acid kinase Superfam by the use of an isoprene synthase (EC 4.2.3.27). As regards ily, in particular the aspartokinase Superfamily. The enzyme the isoprene synthase to be employed in the method, the same isopentenyl phosphate kinase catalyzes the following reac applies as has been described herein above. In a particularly tion: preferred embodiment the isoprene synthase employed is an Isopentenyl phosphate+ATPv isopentenyl diphos isoprene synthase from P. montana var. lobata. As explained phate--ADP above, crotyl diphosphate is structurally closely related to US 2014/0256009 A1 Sep. 11, 2014 dimethylallyl diphosphate. Therefore, it is considered that an 0056. In principle, any known 2-amino-4-hydroxy-6-hy isoprene synthase can also use crotyl diphosphate as a Sub droxymethyldihydropteridine diphosphokinase can be strate and can convert it into butadiene. employed in the method according to the invention. 0047. In another particularly preferred embodiment, the 0057. In another embodiment the direct enzymatic conver dephosphorylation of crotyl diphosphate into butadiene is sion of crotyl alcohol into crotyl diphosphate in one step can achieved by the use of a myrcene/ocimene synthase (EC be achieved by the use of a thiamine diphosphokinase (EC 4.2.3.15). As regards the myrcene/ocimene synthase to be 2.7.6.2). This enzyme catalyzes the following reaction: employed in the method, the same applies as has been ATP+thiaminer AMP+thiamine diphosphate described herein above. In a particularly preferred embodi ment the myrcene/ocimene synthase employed is an (E)-beta 0058. The occurrence of this enzyme has been described ocimene synthase, most preferably an (E)-beta ocimene Syn for several organisms, e.g. for Salmonella enterica, Plasmo thase from Vitis vinifera or from L. japonicus or from P dium falciparum, Saccharomyces cerevisiae, Schizosaccha lunatus. romyces pombe, Candida albicans, Arabidopsis thaliana, 0048. In another particularly preferred embodiment, the Caenorhabditis elegans, Rattus norvegicus, Mus musculus dephosphorylation of crotyl diphosphate into butadiene is and Homo sapiens. In principle, any known thiamine diphos achieved by the use of a monoterpene synthase. As regards the phokinase can be employed in the method according to the monoterpene synthase to be employed in the method, the invention. same applies as has been described herein above. In a par 0059. In step B(II) the obtained crotyl diphosphate is then ticularly preferred embodiment the monoterpene synthase further converted enzymatically into butadiene. This enzy employed is a monoterpene synthase from Eucalyptus globu matic conversion of crotyl diphosphate into butadiene lus. involves the removal of a diphosphate group and can, e.g., be 0049. The present inventors have shown that different achieved by the use of terpene synthase as described herein types of terpene synthases, e.g. isoprene synthase, (E)-beta above. As regards the preferred embodiments, the same ocimene and monoterpene synthase from different plant applies as set forth herein above. organisms are able to convert crotyl diphosphate into butadi 0060. In a particularly, preferred embodiment an isoprene ene (see Examples 14 and 15 and FIG. 13). synthase (EC 4.2.3.27) is employed for the conversion of 0050. In another aspect (B), the method according to the crotyl diphosphate into butadiene. invention comprises the two enzymatic steps of 0061 The crotyl alcohol which is used as a substrate for (I) enzymatically converting crotyl alcohol into crotyl the enzymatic production of butadiene according to the diphosphate; and invention can either be supplied externally or it can itself be (II) enzymatically converting crotyl diphosphate into butadi provided by the reduction of crotonaldehyde (but-2-enal). This reduction can, e.g., beachieved by chemical reactions as C known to the person skilled in the art. However, according to 0051. This alternative is in the following referred to as the present invention it is preferable that the provision of Alternative Band the different steps are referred to as B(I) and crotyl alcohol is achieved by the enzymatic conversion of B(II). crotonyl-Coenzyme A (in the following crotonyl-CoA, see 0052. In this embodiment the enzymatic conversion of FIG. 9) or of crotonaldehyde into crotyl alcohol. Thus, in crotyl alcohol into crotyl diphosphate according to step B(I) another embodiment the method according to the invention consists of a single enzymatic reaction in which crotyl alco further comprises the step of providing crotyl alcohol by the hol is directly converted into crotyl diphosphate. enzymatic conversion of crotonaldehyde into crotyl alcohol 0053. The direct enzymatic conversion of crotyl alcohol aS described herein below. Crotonaldehyde into crotyl diphosphate in one step can, e.g., be achieved by (CH-CH=CHCHO; (2E)-but-2-enal) occurs naturally, e.g. the use of an enzyme which is able to catalyze the transfer of in Soybean oils, and can be synthesized chemically by the a diphosphate group, Such as a diphosphotransferase, for aldol condensation of acetaldehyde. Alternatively, the example enzymes which are classified as EC 2.7.6 (diphos method according to the invention further comprises the step photransferases). Examples are 2-amino-4-hydroxy-6-hy of providing crotyl alcohol by the enzymatic conversion of droxymethyldihydropteridine diphosphokinase (EC 2.7.6.3) crotonyl-CoA into crotyl alcohol as described below. Croto and thiamine diphosphokinase (EC 2.7.6.2). Preferably, ATP nyl-coenzyme A is a thioester between crotonic acid and is the donor of the diphosphate group in Such a reaction. Coenzyme A. It is an intermediate in the fermentation of 0054 Thus, in one embodiment the direct enzymatic con butyric acid, and in the metabolism of lysine and tryptophan. version of crotyl alcohol into crotyl diphosphate in one step During degradation of these amino acids, C.-ketoadipate is can be achieved by the use of a 2-amino-4-hydroxy-6-hy produced which is converted into glutaryl-CoA by oxidative droxymethyldihydropteridine diphosphokinase (EC 2.7.6.3). decarboxylation. Glutaryl-CoA is then converted by glutaryl This enzyme catalyzes the following reaction: CoA dehydrogenase into crotonyl-CoA, which can be con verted in two further steps into two molecules of acetyl-CoA. 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropte Crotonyl-CoA is also a metabolite in the fermentation of ridine+ATP 2-amino-7,8-dihydro-4-hydroxy glucose by Some obligatory anaerobe bacteria in which 6-(diphosphooxymethyl)pteridine--AMP butyric acid is produced. Such as Clostridium acetobutylicum. 0055. The occurrence of this enzyme has been described Moreover, crotonyl-CoA had been isolated in some microor for several organisms, e.g. for E. coli, Plasmodium falci ganisms which assimilate acetate via the so-called ethyl parum, Plasmodium chabaudi, Streptococcus pneumoniae, malonyl-CoA pathway. It also occurs as an intermediate in Toxoplasma gondii, Yersinia pestis, Pneumocystis carinii, Some metabolic pathways leading to the assimilation of car Haemophilus influenzae, S. cerevisiae, Arabidopsis thaliana bon dioxide, e.g. in the 3-hydroxyproprionate/4-hydroxybu and Pisum sativum. tyrate cycle or the dicarboxylate/4-hydroxybutyrate cycle. US 2014/0256009 A1 Sep. 11, 2014
0062. The present inventor also developed a method for clostridia, in several gamma-proteobacteria, in actinobacte enzymatically producing crotyl alcohol enzymatically start ria, in cyanobacteria and some amitochondriate protists (see ing from crotonaldehyde or from crotonyl-CoA. Atteia et al., loc. cit.). 0063 Thus, in a second aspect, the present invention 0069. Alternatively or in addition, the above described relates to a method for producing crotyl alcohol. Such a conversion of crotonyl-CoA into crotonaldehyde can also be method comprises the enzymatic conversion of crotonyl-CoA achieved by using enzymes referred to as acyl-CoA reduc into crotonaldehyde and the Subsequent enzymatic conver tases. Examples for Such enzymes are cinnamoyl-CoA reduc sion of crotonaldehyde into crotyl alcohol. The first reaction tase (EC 1.2.1.44), long-chain-fatty-acyl-CoA reductase (EC may occur according to the following schemes: 1.2.1.50) and malonyl-CoA reductase (malonate semialde Crotonyl-CoA+NADH--H' ve crotonaldehyde--CoA+ hyde-forming; EC 1.2.1.75). NAD 0070 According to the present invention the produced crotonaldehyde is further converted into crotyl alcohol. The O enzymatic conversion of crotonaldehyde into crotyl alcohol is Crotonyl-CoA+NADPH-i-H" se crotonaldehyde-- a reduction/hydrogenation and may occur according to the CoA-NADP following schemes: 0064. This reaction is a reduction and can be catalyzed by Crotonaldehyde--NADH+H" crotyl alcohol--NAD" various enzymes. In one aspect, it is possible to use for the above indicated conversion of crotonyl-CoA to crotonalde O hyde a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34). Crotonaldehyde:NADPH+H" crotyl alcohol This enzyme normally catalyzes the following reaction NADP (S)-3-hydroxy-methylglutaryl-CoA+2NADPH-i-H' 0071. This reaction can be catalyzed by various enzymes. s (R)-mevalonate+CoA+2NADP' In one aspect, it is possible to use for the above indicated 0065. Enzymes belonging to this class and catalyzing the conversion of crotonaldehyde to crotyl alcohol an enzyme above shown conversion occur in organisms of all kingdoms, which is known to be able to catalyze this reaction. One i.e. plants, animals, fungi, bacteria etc. and have extensively example is the aldo-keto reductase (AKR) encoded by the been described in the literature. Nucleotide and/oramino acid sakR1 gene. This enzyme had been identified in Synechococ sequences for Such enzymes have been determined for cus sp. PCC 7002 and has been described in Dongyi et al. numerous organisms, in particular bacterial organisms. In (Microbiol. 152 (2006), 2013-2021). It uses NADPH/NADP" principle, any hydroxymethylglutaryl-CoA reductase (EC as a cofactor. Another example is the aldo-keto reductase 1.1.1.34) can be used in the context of the present invention. (AKR) encoded by the At2g37770 gene, which had been 0066 Alternatively or in addition, the above described identified in Arabidopsis (Yamauchii et al. (J. Biol. Chem. conversion of crotonyl-CoA into crotonaldehyde can also be 286 (2011) 6999-7009). It uses NADPH/NADP+ as a cofac achieved by using an enzyme referred to as acetaldehyde tor. A further example is the 321-MB dehydrogenase from the dehydrogenase (EC 1.2.1.10). This enzyme normally cata soil bacterium Pseudomonas putida MB-1 (Malone et al., lyzes the following reaction Appl. Environm. Microbiol. 65 (1999), 2622-2630) which Acetyl-CoA+NADH+H" " acetaldehyde--CoA+ uses NADH/NAD as a cofactor. NAD 0072. In another embodiment it is also possible to use for 0067 Enzymes belonging to this class and catalyzing the the above indicated conversion of crotonaldehyde to crotyl above shown conversion occur in several types of bacteria, alcohol a hydroxymethylglutaryl-CoA reductase (EC 1.1.1. like e. coli, Acinetobacter sp., Leuconostoc mesenteroides, 34). This enzyme has already been described above and that Pseudomonas sp. Clostridium beijerinckii, Clostridium what had been said above holds also true for this reaction. Kluyveri, Giardia intestinalis, Propionibacterium feudenre 0073. Alternatively or in addition, the above described ichii and Thermoanaerobacter ethanolicus. In principle, any conversion of crotonaldehyde into crotyl alcohol can also be acetaldehyde dehydrogenase (EC 1.2.1.10) can be used in the achieved by using an alcohol dehydrogenase (EC 1.1.1.1). context of the present invention. Such an enzyme normally catalyzes the following reaction 0068 Alternatively or in addition, the above described Primary alcohol--NAD" aldehyde--NADH--H conversion of crotonyl-CoA into crotonaldehyde can also be achieved by using an enzyme referred to as aldehyde-alcohol 0074 Enzymes belonging to this class and catalyzing the dehydrogenase, such as the aldehyde-alcohol dehydrogenase above shown conversion occur in organisms of all kingdoms, as encoded by an adhE gene. Such an enzyme is bifunctional i.e. plants, animals, fungi, bacteria etc. and have extensively in that it shows at least the enzymatic activities of an alcohol been described in the literature. Nucleotide and/oramino acid dehydrogenase and an aldehyde dehydrogenase, such as an sequences for Such enzymes have been determined for acetaldehyde dehydrogenase. An example for Such an numerous organisms, in particular bacterial organisms. In enzyme is the aldehyde-alcohol dehydrogenase of E. coli principle, any alcohol dehydrogenase (EC 1.1.1.1) can be (adhE: UniProtKB/Swiss-Prot Accession number P0A9Q7: used in the context of the present invention. Jul. 27, 2011; Version 58). Corresponding enzymes are also 0075 Moreover, the conversion of crotonaldehyde to cro known from other organisms, such as, e.g. Leuconostoc tyl alcohol can also be achieved by the use of an enzyme mesenteroides (Koo et al., Biotechnology Letters 27, 505 referred to as an aldehyde reductase. Examples for such 510), Polytomella sp. and Chlamydomonas reinhardtii (At enzymes are alcohol dehydrogenase (NADP; EC 1.1.1.2). teia et al., Plant Mal. Biol. 53 (2003), 175-188). Genes encod allyl-alcohol dehydrogenase (EC 1.1.1.54), retinol dehydro ing Such enzymes have been found in the genomes of several genase (EC 1.1.1.105), sulcatone dehydrogenase (EC 1.1.1. Gram-positive bacteria belonging to the categories bacilliand 260) and 3-methylbutanal reductase (EC 1.1.1.265) US 2014/0256009 A1 Sep. 11, 2014
0076. The enzymatic conversion of crotonyl-CoA into Characteristically, proteins of this family possess two NAD crotyl alcohol may also occur according to the following (P)(H)-binding motifs, which have the conserved sequence schemes: GXGX(1-2x)G (Willis et al., Biochemistry 50 (2011), Crotonyl-CoA+2NADH+2H crotyl alcohol--CoA+ 10550-10558; Jornwall H. et al., Biochemistry 34 (1995), 2NAD 6003-6013). The first pattern, GTGFIG, is identified near the N-terminus and the second signature sequence, GXXXGXG. O is located between residues 384-390. Crotonyl-CoA+2NADPH+2H crotyl alcohol I0085. In principle any “short-chain dehydrogenase/fatty CoA-2NADP acyl-CoA reductase' or 'short-chain dehydrogenases/reduc 0.077 Similar to the above described conversions, this tases (SDR) can be applied in the method according to the reaction goes via crotonaldehyde as an intermediate. How invention. ever, in this embodiment of the invention, the conversion of I0086 Preferably, the short-chain dehydrogenase/fatty crotonyl-CoA into crotyl alcohol is catalyzed by one enzyme acyl-CoA reductase is a short-chain dehydrogenase/fatty which catalyses both reduction/hydrogenation steps. An acyl-CoA reductase from a marine bacterium, preferably enzyme which may be employed in this conversion is an from the genus Marinobacter or Hahella, even more prefer aldehyde-alcohol dehydrogenase, such as the aldehyde-alco ably from the species Marinobacter aquaeolei, more prefer holdehydrogenase as encoded by an adhE gene which had ably Marinobacter aquaeolei VT8, Marinobacter mangan already been described above. Oxydans, Marinobacter algicola, Marinobacter sp. ELB17 or 0078. Another enzyme which may be used in this conver Hahelly cheiuensis. Examples of Such enzymes are the short sion is a hydroxymethylglutaryl-CoA reductase (EC 1.1.1. chain dehydrogenase/fatty acyl-CoA reductase from Marino 34) which has already been described above. In a further bacter aquaeolei VT8 (Uniprot accession number A1U3L3; preferred embodiment the conversion of crotonyl-CoA into Willis et al., Biochemistry 50 (2011), 10550-10558), the crotyl alcohol is achieved by the use of a short-chain dehy short-chain dehydrogenase from Marinobacter manganoxy drogenase/fatty acyl-CoA reductase. dans (Uniprot accession number G6YQS9), the short-chain 007.9 The term “short-chain dehydrogenase/fatty acyl dehydrogenase from Marinobacter algicola (Uniprot acces CoA reductase' or “short-chain dehydrogenases/reductases sion number A6EUH6), the short-chain dehydrogenase from (SDR) in the context of the present invention refers to Marinobacter sp. ELB17 (Uniprot accession number enzymes which are characterized by the following features: A3JCC5) and the short-chain dehydrogenase from Hahella 0080) 1. They catalyze a two-step reaction in which fatty cheiuensis (Uniprot accession number Q2SCEO). acy-CoA is reduced to fatty alcohol. I0087. The sequence of the short-chain dehydrogenase/ 0081 2. They show a substrate specificity for acyl-CoA fatty acyl-CoA reductase from Marinobacter aquaeolei VT8 containing an aliphatic chain from 8 to 20 carbon atoms. is shown in SEQID NO: 13. The sequence of the short-chain 0082 Preferably such enzymes are furthermore character dehydrogenase/fatty acyl-CoA reductase from Marinobacter ized by the feature that they show a specific motif in their manganoxydans is shown in SEQIDNO: 14. The sequence of primary structure, i.e. amino acid sequence, namely they the short-chain dehydrogenase/fatty acyl-CoA reductase show two specific glycine motifs for NADP(H) binding. from Marinobacter sp. ELB17 is shown in SEQID NO: 15. 0083. The short-chain dehydrogenase/fatty acyl-CoA The sequence of the short-chain dehydrogenase/fatty acyl reductase or short-chain dehydrogenases/reductases (SDR) CoA reductase from Marinobacter algicola is shown in SEQ enzymes constitute a family of enzymes, most of which are ID NO: 16. The sequence of the short-chain dehydrogenase/ known to be NAD- or NADP-dependent oxidoreductase fatty acyl-CoA reductase from Hahella cheiuensis is shown (Jornwall H. et al., Biochemistry 34 (1995), 6003-6013). in SEQ ID NO: 17. In a particularly preferred embodiment Recently, a novel bacterial NADP-dependent reductase from any protein showing an amino acid sequence as shown in any Marinobacter aquaeolei VT8 was characterized (Willis et al., one of SEQ ID NOs: 13 to 17 or showing an amino acid Biochemistry 50 (2011), 10550-10558). This enzyme cata sequence which is at least 80% homologous to any of SEQID lyzes the four-electron reduction offatty acyl-CoA substrates NOs: 13 to 17 and having the activity of a short-chain dehy to the corresponding fatty alcohols. The enzymatic conver drogenase/fatty acyl-CoA reductase can be employed in a sion of fatty acyl-CoA into fatty alcohol occurs through an method according to the present invention. aldehyde intermediate according to the following scheme: I0088. The methods according to the first and second aspect of the present invention as described above may also be combined, i.e. it is possible that a method according to the NADPH NADP first aspect of the invention for the production of butadiene O from crotyl alcohol further comprises the steps of a method according to the second aspect of the invention for the provi --- sion of crotyl alcohol by enzymatic reactions as described CoA above. O NADPH NADP I0089. In another embodiment the methods according to the first and/or second aspect of the invention may also us N-4 R-CH2OH include the further step of enzymatically providing crotonyl R H CoA. This may be achieved by the enzymatic conversion of 3-hydroxybutyryl-Coenzyme A into crotonyl-Coenzyme A. 0084. The enzyme displays activity on fatty acyl-CoA This reaction may occur according to the following scheme: Substrates ranging from 8 to 20 carbons in length (both Satu 3-hydroxybutyryl-Coenzyme A a crotonyl-Coen rated and unsaturated) as well as on fatty aldehyde Substrates. Zyme A+H2O US 2014/0256009 A1 Sep. 11, 2014
0090 This reaction corresponds to a Michael elimination and/or amino acid sequences for Such enzymes have been and can, for example, be catalyzed by an enzyme called determined for numerous organisms, in particular bacterial 3-hydroxybutyryl-CoA dehydratase which is classified as EC organisms. In principle, any acetoacetyl-CoA reductase (EC 4.2.1.55. This enzyme belongs to the family of lyases, spe 1.1.1.36) can be used in the context of the present invention. cifically the hydro-lyases, which cleave carbon-oxygen In one embodiment the enzyme employed in the method bonds. The systematic name of this enzyme class is (3R)-3- according to the invention originates from E. coli. hydroxybutanoyl-CoA hydro-lyase (crotonoyl-CoA-form 0095. In yet a further embodiment the methods according ing). Other names in common use include D-3-hydroxybu to the first and/or second aspect of the invention may also tyryl coenzyme A dehydratase, D-3-hydroxybutyryl-CoA include the further step of enzymatically providing dehydratase, enoyl coenzyme A hydrase (D), and (3R)-3- acetoacetyl-CoA. This can be achieved by the enzymatic hydroxybutanoyl-CoA hydro-lyase. This enzyme partici conversion of two molecules acetyl-CoA into one molecule pates in butanoate metabolism. Enzymes belonging to this of acetoacetyl-CoA. Acetyl-CoA is a metabolic intermediate class and catalyzing the above shown conversion of 3-hy which occurs in all living organisms and plays a central role in droxybutyryl-Coenzyme A into crotonyl-Coenzyme A have metabolism. Thus, according to the present invention, acetyl been described to occur, e.g. in rat (Rattus norvegicus) and in CoA can, for example, be converted into acetoacetyl-CoA by Rhodospirillum rubrum. Nucleotide and/or amino acid the following reaction: sequences for Such enzymes have been determined, e.g. for Aeropyrum permix. In principle, any 3-hydroxybutyryl-CoA 2 acetyl-CoAv acetoacetyl-CoA CoA dehydratase (EC 4.2.1.55) can be used in the context of the 0096. This reaction is catalyzed by enzymes called acetyl present invention. CoA C-acetyltransferases which are classified as EC 2.3.1.9. 0091 Alternatively or in addition, the above described Enzymes belonging to this class and catalyzing the above conversion of 3-hydroxybutyryl-Coenzyme A into crotonyl shown conversion of two molecules of acetyl-CoA into Coenzyme A can also be achieved by using an enzyme acetoacetyl-CoA and CoA occur in organisms of all king referred to as enoyl-CoA hydratase (EC 4.2.1.17). Enoyl doms, i.e. plants, animals, fungi, bacteria etc. and have exten CoA hydratase is an enzyme that normally hydrates the sively been described in the literature. Nucleotide and/or double bond between the second and third carbons on acyl amino acid sequences for Such enzymes have been deter CoA. However, it can also be employed to catalyze the reac mined for a variety of organisms, like Homo sapiens, Arabi tion in the reverse direction. This enzyme, also known as dopsis thaliana, E. coli, Bacillus subtilis and Candida, to crotonase, is naturally involved in metabolizing fatty acids to name just some examples. In principle, any acetyl-CoA produce both acetyl-CoA and energy. Enzymes belonging to C-acetyltransferase (EC 2.3.1.9) can be used in the context of this class have been described to occur, e.g. in rat (Rattus the present invention. norvegicus), humans (Homo sapiens), mouse (Mus muscu 0097 Alternatively, the provision of acetoacetyl-CoA lus), wild boar (Sus scrofa), Bos taurus, E. coli, Clostridium may also be achieved by the enzymatic conversion of acetyl acetobutylicum and Clostridium aminobutyricum. Nucle CoA and malonyl-CoA into acetoacetyl-CoA. This reaction otide and/or amino acid sequences for Such enzymes have is catalyzed by an enzyme called acetoacetyl-CoA synthase. been determined, e.g. for rat, humans and Bacillus subtilits. In The gene encoding this enzyme was identified in the meva principle, any enoyl-CoA hydratase (EC 4.2.1.17) can be lonate pathway gene cluster for terpenoid production in a used in the context of the present invention. soil-isolated Gram-positive Streptomyces sp. Strain CL190 0092. In another embodiment it is also possible to use for (Okamura et al., PNAS USA 107 (2010) 11265-11270, the above described conversion of 3-hydroxybutyryl-Coen 2010). Moreover a biosynthetic pathway using this enzyme Zyme A into crotonyl-Coenzyme A an enoyl-CoA hydratase 2 for acetoacetyl-CoA production was recently developed in E. (EC 4.2.1.119) or a crotonyl-[acyl-carrier-protein hydratase coli (Matsumoto K et al., Biosci. Biotechnol. Biochem, 75 (EC 4.2.1.58). (2), 364-366, 2011, enclosed) 0093. In another embodiment the methods according to 0098. The methods according to the present invention may the first and/or second aspect of the invention may also be carried out in vitro or in vivo. An in vitro reaction is include the further step of enzymatically providing 3-hy understood to be a reaction in which no cells are employed, droxybutyryl-Coenzyme A. This can beachieved by the enzy i.e. an acellular reaction. Thus, in vitro preferably means in a matic conversion of acetoacetyl-CoA into 3-hydroxybutyryl cell-free system. The term “in vitro' in one embodiment Coenzyme A. This reaction may occur according to the means in the presence of isolated enzymes (or enzyme sys following scheme: tems optionally comprising possibly required cofactors). In acetoacetyl-CoA+NADH--H' vie 3 -hydroxybutyryl one embodiment, the enzymes employed in the method are Coenzyme A+NAD" used in purified form. For carrying out the process in vitro the Substrates for the reaction and the enzymes are incubated O underconditions (buffer, temperature, coSubstrates, cofactors acetoacetyl-CoA+NADPH-i-H' x 3 -hydroxybutyryl etc.) allowing the enzymes to be active and the enzymatic Coenzyme A+NADP' conversion to occur. The reaction is allowed to proceed for a 0094. This reaction is a reduction and can, e.g., be cata time sufficient to produce butadiene. The production ofbuta lyzed by an enzyme called acetoacetyl-CoA reductase which diene can be measured by methods known in the art, such as is classified as EC 1.1.1.36. Enzymes belonging to this class gas chromatography possibly linked to mass spectrometry and catalyzing the above shown conversion of acetoactyl detection. CoA into 3-hydroxybutyryl-Coenzyme A occur in organisms 0099. The enzymes may be in any suitable form allowing of all kingdoms, i.e. plants, animals, fungi, bacteria etc. and the enzymatic reaction to take place. They may be purified or have extensively been described in the literature. Nucleotide partially purified or in the form of crude cellular extracts or US 2014/0256009 A1 Sep. 11, 2014
partially purified extracts. It is also possible that the enzymes further genetically modified as described herein above so as are immobilized on a suitable carrier. to be able to produce crotyl alcohol. 0100. In one embodiment of the method according to the 0106. In alternative B it is, e.g., possible to use E. coli or S. invention the substrate which is used in such an in vitro cerevisiae, which both possess a gene encoding 2-amino-4- method is crotyl alcohol which is converted by the use of the hydroxy-6-hydroxymethyldihydropteridine diphosphoki above-mentioned enzymes to butadiene. In another embodi nase, and to introduce into Such a microorganism a gene, for ment, the Substrate used in Such an in vitro method is cro example from Bacillus subtilis encoding a terpene synthase, tonaldehyde which is first converted into crotyl alcohol as e.g. an isoprene synthase. Similarly, it is possible to use in described above which is then in turn converted into butadi alternative B as a microorganism B. subtilis and to genetically ene as described above. modify it with a gene encoding a 2-amino-4-hydroxy-6-hy 0101 The in vitro method according to the invention may droxymethyldihydropteridine diphosphokinase, e.g. from E. be carried out in a one-pot-reaction, i.e. the Substrate is com coli or from S. cerevisiae. Again, such microorganisms may bined in one reaction mixture with the above described be further genetically modified as described herein above so enzymes necessary for the conversion into butadiene and the as to be able to produce crotyl alcohol. reaction is allowed to proceed for a time sufficient to produce 0107 If a (micro)organism is used which naturally butadiene. Alternatively, the method may also be carried out expresses one of the required enzyme activities, it is possible by effecting one or more enzymatic steps in a consecutive to modify such a (micro)organism so that this activity is manner, i.e. by first mixing the Substrate with one or more overexpressed in the (mircro)organism. This can, e.g., be enzymes and allowing the reaction to proceed to an interme achieved by effecting mutations in the promoter region of the diate and then adding one or more further enzymes to convert corresponding gene So as to lead to a promoter which ensures the intermediate further either into an intermediate or into a higher expression of the gene. Alternatively, it is also pos butadiene. sible to mutate the gene as such so as to lead to an enzyme 0102 The in vitro method according to the invention fur showing a higher activity. thermore may comprise the step of collecting gaseous prod 0108) By using (micro)organisms which express the ucts, in particular butadiene, degassing out of the reaction, i.e. enzymes which are necessary according to alternative A or B recovering the products which degas, e.g., out of the culture. as described above, it is possible to carry out the method Thus, in one embodiment, the method is carried out in the according to the invention directly in the culture medium, presence of a system for collecting butadiene under gaseous without the need to separate or purify the enzymes. form during the reaction. 0109. In one embodiment, a (micro)organism is used hav 0103) As a matter of fact, butadiene adopts the gaseous ing the natural or artificial property of endogenously produc state at room temperature and atmospheric pressure. The ing crotyl alcohol, and also expressing or overexpressing the method according to the invention therefore does not require enzymes as described in connection with alternatives A and extraction of the product from the reaction mixture, a step B, above, so as to produce butadiene directly from a carbon which is always very costly when performed at industrial Source present in Solution. In another embodiment, the (mi scale. The evacuation and storage of butadiene and its pos cro)organism which is used has the natural or artificial prop sible Subsequent physical separation from other gaseous Sub erty of endogenously producing crotonaldehyde and to con stances as well as its chemical conversion can be performed vert it into crotyl alcohol which can then be further converted according to any method known to one of skill in the art. For into butadiene. example, butadiene can be separated from CO by the con 0110. In one embodiment the organism employed in the densation of CO at low temperatures. CO can also be method according to the invention is an organism, preferably removed by polar solvents, e.g. ethanolamine. Moreover, it a microorganism, which has been genetically modified to can be isolated by adsorption on a hydrophobic membrane. contain one or more foreign nucleic acid molecules encoding 0104. In another embodiment the method according to the one or more of the enzymes as described above in connection invention is carried out in culture, in the presence of an with alternatives A or B. The term “foreign' in this context organism, preferably a microorganism, producing at least the means that the nucleic acid molecule does not naturally occur enzymes described above which are necessary to produce in said organism/microorganism. This means that it does not butadiene according to a method of the invention according to occur in the same structure or at the same location in the the first aspect by using one of the alternative routes A or B organism/microorganism. In one preferred embodiment, the described above and starting either from crotyl alcohol or foreign nucleic acid molecule is a recombinant molecule from crotonaldehyde. Thus, in such an embodiment of the comprising a promoter and a coding sequence encoding the invention, an organism, preferably a microorganism, that pro respective enzyme in which the promoter driving expression duces the enzymes specified in the description of alternatives of the coding sequence is heterologous with respect to the A or B, above, is used. It is possible to use a (micro)organism coding sequence. Heterologous in this context means that the which naturally produces one or more of the required promoter is not the promoter naturally driving the expression enzymes and to genetically modify Such a (micro)organism of said coding sequence but is a promoter naturally driving so that it expresses also those enzymes which it does not expression of a different coding sequence, i.e., it is derived naturally express. Preferably a (micro)organism is used from another gene, or is a synthetic promoter or a chimeric which has been genetically modified as described herein promoter. Preferably, the promoter is a promoter heterolo above in connection with the second aspect of the invention so gous to the organism/microorganism, i.e. a promoter which as to be able to produce crotyl alcohol. does naturally not occur in the respective organism/microor 0105. In alternative A1 it is for example possible to use ganism. Even more preferably, the promoter is an inducible Bacillus subtilis which possesses a gene encoding the enzyme promoter. Promoters for driving expression in different types hydroxyethylthiazole kinase and a gene encoding, a terpene of organisms, in particular in microorganisms, are well synthase, e.g. an isoprene synthase. Such a bacterium may be known to the person skilled in the art. US 2014/0256009 A1 Sep. 11, 2014
0111 Inafurther embodiment the nucleic acid molecule is liungdahli, C. aceticum, Acetobacterium woodii, C. autoet foreign to the organism/microorganism in that the encoded hanogenium, and C. carboxydeviron, are frequently being enzyme is not endogenous to the organism/microorganism, used in Syngas fermentation (Munasingheet et al.; Biore i.e. is naturally not expressed by the organism/microorganism source Technology 101 (2010), 5013-5022). when it is not genetically modified. In other words, the 0119. It is also conceivable to use in the method according encoded enzyme is heterologous with respect to the organ to the invention a combination of (micro)organisms wherein ism/microorganism. The foreign nucleic acid molecule may different (micro)organisms express different enzymes as be present in the organism/microorganism in extrachromo described above. In a further embodiment at least one of the Somal form, e.g. as a plasmid, or stably integrated in the microorganisms is capable of producing crotyl alcohol or, in chromosome. A stable integration is preferred. Thus, the an alternative embodiment, a further microorganism is used genetic modification can consist, e.g. in integrating the cor in the method which is capable of producing crotyl alcohol. responding gene(s) encoding the enzyme(s) into the chromo I0120 In another embodiment the method according to the Some, or in expressing the enzyme(s) from a plasmid contain invention makes use of a multicellular organism expressing at ing a promoter upstream of the enzyme-coding sequence, the least the enzymes as described in connection with alternatives promoter and coding sequence preferably originating from A or B, above. Examples for Such organisms are plants or different organisms, or any other method known to one of animals. skill in the art. I0121. In a particular embodiment, the method according 0112. In a preferred embodiment the (micro)organism of to the invention involves culturing microorganisms in stan the present invention is also genetically modified so as to be dard culture conditions (30–37° C. at 1 atm, in a fermenter able to produce crotyl alcohol as described herein above. allowing aerobic growth of the bacteria) or non-standard con 0113. The organisms used in the invention can be prokary ditions (higher temperature to correspond to the culture con otes or eukaryotes, preferably, they are microorganisms such ditions of thermophilic organisms, for example). as bacteria, yeasts, fungi or molds, or plant cells or animal 0.122 Inafurther embodiment the method of the invention cells. In a particular embodiment, the microorganisms are is carried out under microaerophilic conditions. This means bacteria, preferably of the genus Escherichia or Bacillus and that the quantity of injected air is limiting so as to minimize even more preferably of the species Escherichia coli or Bacil residual oxygen concentrations in the gaseous effluents con lus subtilis. taining butadiene. 0114. In another embodiment, the microorganisms are I0123. In another embodiment the method according to the recombinant bacteria of the genus Escherichia or Bacillus, invention furthermore comprises the step of collecting the preferably of the species Escherichia coli or Bacillus subtilis, gaseous butadiene degassing out of the reaction. Thus in a having been modified so as to endogenously produce crotyl preferred embodiment, the method is carried out in the pres alcohol and to convert it into butadiene. ence of a system for collecting butadiene undergaseous form 0115. It is also possible to employ an extremophilic bac during the reaction. terium such as Thermus thermophilus, or anaerobic bacteria 0.124. As a matter of fact, butadiene adopts the gaseous from the family Clostridiae. state at room temperature and atmospheric pressure. The 0116. In one embodiment the microorganism is a fungus, method according to the invention therefore does not require more preferably a fungus of the genus Saccharomyces, extraction ofbutadiene from the liquid culture medium, a step Schizosaccharomyces, Aspergillus, Trichoderma, Pichia or which is always very costly when performed at industrial Kluyveromyces and even more preferably of the species Sac scale. The evacuation and storage of butadiene and its pos charomyces cerevisiae, Schizosaccharomyces pombe, sible Subsequent physical separation and chemical conver Aspergillus niger; Trichoderma reesei, Pichia pastoris or of sion can be performed according to any method known to one the species Kluyveromyces lactis. In a particularly preferred of skill in the art and as described above. embodiment the microorganism is a recombinant yeast 0.125. In a particular embodiment, the method also com capable of producing crotyl alcohol and converting it into prises detecting butadiene which is present in the gaseous butadiene due to the expression of the enzymes described in phase. The presence ofbutadiene in an environment of air or connection with alternatives A or B, above. another gas, even in Small amounts, can be detected by using 0117. In another embodiment, the method according to the various techniques and in particular by using gas chromatog invention makes use of a photosynthetic microorganism raphy systems with infrared or flame ionization detection, or expressing at least the enzymes as described in connection by coupling with mass spectrometry. with alternatives A or B, above. Preferably, the microorgan 0.126 When the process according to the invention is car ism is a photosynthetic bacterium, or a microalgae. In a fur ried out in vivo by using an organism/microorganism provid ther embodiment the microorganism is an algae, more pref ing the respective enzyme activities, the organism, preferably erably an algae belonging to the diatomeae. Even more microorganism, is cultivated under Suitable culture condi preferably Such a microorganism has the natural or artificial tions allowing the occurrence of the enzymatic reaction. The property of endogenously producing crotyl alcohol. In this specific culture conditions depend on the specific organism/ case the microorganism would be capable of producing buta microorganism employed but are well known to the person diene directly from CO present in solution. skilled in the art. The culture conditions are generally chosen 0118. In another embodiment, it is possible to use a micro in Such a manner that they allow the expression of the genes organism which belongs to the group of acetogenic bacteria encoding the enzymes for the respective reactions. which are capable of converting CO (or CO+H) to produce I0127. Various methods are known to the person skilled in acetyl-CoA via the so-called Wood-Ljungdahl pathway (Kö the art in order to improve and fine-tune the expression of pke et al.; PNAS 10 (2010), 13087-13092). A fermentation certain genes at certain stages of the culture such as induction process using such microorganisms is known as Syngas fer of gene expression by chemical inducers or by a temperature mentation. Strictly mesophilic anaerobes Such as C. shift. US 2014/0256009 A1 Sep. 11, 2014
0128. In another embodiment the organism employed in in the vectors. These expression control sequences may be the method according to the invention is a plant. In principle Suited to ensure transcription and synthesis of a translatable any possible plant can be used, i.e. a monocotyledonous plant RNA in bacteria or fungi. or a dicotyledonous plant. It is preferable to use a plant which I0135) In addition, it is possible to insert different muta can be cultivated on an agriculturally meaningful scale and tions into the polynucleotides by methods usual in molecular which allows to produce large amounts of biomass. Examples biology (see for instance Sambrook and Russell (2001), are grasses like Lolium, cereals like rye, wheat, barley, oat, Molecular Cloning: A Laboratory Manual, CSH Press, Cold millet, maize, other starch storing plants like potato or Sugar Spring Harbor, N.Y., USA), leading to the synthesis of storing plants like Sugar cane or Sugar beet. Conceivable is polypeptides possibly having modified biological properties. also the use of tobacco or of vegetable plants such as tomato, The introduction of point mutations is conceivable at posi pepper, cucumber, eggplant etc. Another possibility is the use tions at which a modification of the amino acid sequence for of oil storing plants such as rape seed, olives etc. Also con instance influences the biological activity or the regulation of ceivable is the use of trees, in particular fast growing trees the polypeptide. Such as eucalyptus, poplar or rubber tree (Hevea brasiliensis). 0.136 Moreover, mutants possessing a modified substrate Particularly preferred is the use of plants which naturally or product specificity can be prepared. Preferably, such produce crotonaldehyde, e.g. soybeans. Such plants are pref mutants show an increased activity. Furthermore, the intro erably further modified so as to be able to convert crotonal duction of mutations into the polynucleotides encoding an dehyde into crotyl alcohol. enzyme as defined above allows the gene expression rate 0129. In another embodiment, the method according to the and/or the activity of the enzymes encoded by said polynucle invention is characterized by the conversion of a carbon otides to be optimized. Source. Such as glucose, into crotyl alcohol (preferably via 0.137 For genetically modifying bacteria or fungi, the crotonyl-CoA and crotonaldehyde) followed by the conver polynucleotides encoding an enzyme as defined above or sion of crotyl alcohol into butadiene. parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by 0130. In another embodiment, the method according to the recombination of DNA sequences. Standard methods (see invention comprises the production ofbutadiene from atmo Sambrook and Russell (2001), Molecular Cloning: A Labo spheric CO, or from CO, artificially added to the culture ratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA) medium. In this case the method is implemented in an organ allow base exchanges to be performed or natural or synthetic ism which is able to carry out photosynthesis. Such as for sequences to be added. DNA fragments can be connected to example microalgae. each other by applying adapters and linkers to the fragments. 0131. As described above, it is possible to use in the Moreover, engineering measures which provide Suitable method according to the invention a (micro)organism which restriction sites or remove surplus DNA or restriction sites is genetically modified so as to contain a nucleic acid mol can be used. In those cases, in which insertions, deletions or ecule encoding at least one of the enzymes as described above Substitutions are possible, in vitro mutagenesis, “primer in connection with alternatives A or B. Such a nucleic acid repair, restriction or ligation can be used. In general, a molecule encoding an enzyme as described above can be used sequence analysis, restriction analysis and other methods of alone or as part of a vector. The nucleic acid molecules can biochemistry and molecular biology are carried out as analy further comprise expression control sequences operably sis methods. linked to the polynucleotide comprised in the nucleic acid 0.138. The polynucleotide introduced into a (micro)organ molecule. The term “operatively linked' or “operably ism is expressed so as to lead to the production of a polypep linked’, as used throughout the present description, refers to tide having any of the activities described above. An overview a linkage between one or more expression control sequences of different expression systems is for instance contained in and the coding region in the polynucleotide to be expressed in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. Such a way that expression is achieved under conditions com (Methods in Enzymology 153 (1987), 516-544) and in Saw patible with the expression control sequence. ers et al. (Applied Microbiology and Biotechnology 46 0132 Expression comprises transcription of the heterolo (1996), 1-9), Billman-Jacobe (Current Opinion in Biotech gous DNA sequence, preferably into a translatable mRNA. nology 7 (1996), 500-4), Hockney (Trends in Biotechnology Regulatory elements ensuring expression in fungi as well as 12 (1994), 456-463), Griffiths et al., (Methods in Molecular in bacteria, are well known to those skilled in the art. They Biology 75 (1997), 427-440). An overview of yeast expres encompass promoters, enhancers, termination signals, target sion systems is for instance given by Hensing et al. (Antonie ing signals and the like. Examples are given further below in van Leuwenhoek 67 (1995), 261-279). Bussineau et al. (De connection with explanations concerning vectors. velopments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93, 0133 Promoters for use in connection with the nucleic Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), acid molecule may be homologous or heterologous with Vedvick (Current Opinion in Biotechnology 2 (1991), 742 regard to its origin and/or with regard to the gene to be 745) and Buckholz (Bio/Technology 9 (1991), 1067-1072). expressed. Suitable promoters are for instance promoters 0.139 Expression vectors have been widely described in which lend themselves to constitutive expression. However, the literature. As a rule, they contain not only a selection promoters which are only activated at a point in time deter marker gene and a replication-origin ensuring replication in mined by external influences can also be used. Artificial and/ the host selected, but also a bacterial or viral promoter, and in or chemically inducible promoters may be used in this con most cases a termination signal for transcription. Between the text. promoter and the termination signal there is in general at least 0134. The vectors can further comprise expression control one restriction site or a polylinker which enables the insertion sequences operably linked to said polynucleotides contained of a coding DNA sequence. The DNA sequence naturally US 2014/0256009 A1 Sep. 11, 2014 controlling the transcription of the corresponding gene can be 0.148. As regards in particular the terpene synthase and the used as the promoter sequence, if it is active in the selected preferred embodiments of terpene synthases to be expressed host organism. However, this sequence can also be exchanged by the (micro)organism, the same applies as has been set forth for other promoter sequences. It is possible to use promoters above in connection with the method according to the inven ensuring constitutive expression of the gene and inducible tion. promoters which permit a deliberate control of the expression 0149 Thus, in one preferred embodiment the terpene syn of the gene. Bacterial and viral promoter sequences possess thase is ing these properties are described in detail in the literature. (a) an isoprene synthase (EC 4.2.3.27); or Regulatory sequences for the expression in microorganisms (b) a myrcene/ocimene synthase (EC 4.2.3.15); or (for instance E. coli, S. cerevisiae) are sufficiently described (c) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or in the literature. Promoters permitting a particularly high (d) a pinene synthase (EC 4.2.3.14); or expression of a downstream sequence are for instance the T7 (e) a monoterpene synthase. promoter (Studier et al., Methods in Enzymology 185 (1990), 0150. The present invention also relates to an organism, 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez preferably a microorganism, which is able to express the and Chamberlin (Eds), Promoters, Structure and Function; enzymes required for the conversion of crotonyl-CoA into Praeger, New York, (1982), 462-481; DeBoer et al., Proc. crotonaldehyde and/or crotyl alcohol as described in connec Natl. Acad. Sci. USA (1983), 21-25), Ip1, rac (Boros et al., tion with the second aspect of the invention. Thus, the present Gene 42 (1986), 97-100). Inducible promoters are preferably invention also relates to a (mirco)organism which expresses used for the synthesis of polypeptides. These promoters often (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); lead to higher polypeptide yields than do constitutive promot and/or ers. In order to obtain an optimum amount of polypeptide, a (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or two-stage process is often used. First, the host cells are cul (iii) an alcohol dehydrogenase (EC 1.1.1.1); and/or tured under optimum conditions up to a relatively high cell (iv) an aldehydefalcohol dehydrogenase; and/or density. In the second step, transcription is induced depend (v) an acyl-CoA reductase; and/or ing on the type of promoter used. In this regard, a tac promoter (vi) an aldo-keto reductase (AKR); and/or is particularly suitable which can be induced by lactose or (vii) an aldehyde reductase; and/or IPTG (isopropyl-B-D-thiogalactopyranoside) (deBoer et (viii) a short-chain dehydrogenase/fatty acyl-CoA reductase al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termina (ix) and which is capable of converting crotonyl-CoA into tion signals for transcription are also described in the litera crotonaldehyde and/or crotyl alcohol. ture. 0151. The present invention also relates to an organism, 0140. The transformation of the host cell with a polynucle preferably a microorganism, which is further able to express otide or vector according to the invention can be carried out the enzymes required for the conversion of 3-hydroxybu by standard methods, as for instance described in Sambrook tyryl-CoA into crotonyl-CoA. Thus, the present invention and Russell (2001), Molecular Cloning: A Laboratory also relates to a (mirco)organism which expresses a 3-hy Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Meth droxybutyryl-CoA dehydratase (EC 4.2.1.55) and/or an ods in Yeast Genetics, A Laboratory Course Manual, Cold enoyl-CoA hydratase (EC 4.2.1.17) and/or an enoyl-CoA Spring Harbor Laboratory Press, 1990. The host cell is cul hydratase 2 (EC 4.2.1.119) and/or a crotonyl-acyl-carrier tured in nutrient media meeting the requirements of the par protein hydratase (EC 4.2.1.58) and which is capable of ticular host cell used, in particular in respect of the pH value, converting 3-hydroxybutyryl-CoA into crotonyl-CoA. temperature, salt concentration, aeration, antibiotics, Vita 0152 The present invention also relates to an organism, mins, trace elements etc. preferably a microorganism, which is further able to express 0141. The present invention also relates to an organism, the enzymes required for the conversion of acetoacetyl-CoA preferably a microorganism, which is able to express the into 3-hydroxybutyryl-CoA. Thus, the present invention also enzymes required for the conversion of crotyl alcohol into relates to a (mirco)organism which further expresses an butadiene according to alternative A or B of the method of the acetoacetyl-CoA reductase (EC 1.1.1.36) and which is invention (according to the first aspect) as described above capable of converting acetoacetyl-CoA into 3-hydroxybu and which is able to convert crotyl alcohol into butadiene. tyryl-CoA. 0142. Thus, the present invention also relates to a (micro) 0153 Finally, the present invention also relates to an organism which expresses organism, preferably a microorganism, which is further able 0143 A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1. to express the enzymes required for the enzymatic production 50); or of acetoacetyl-CoA. This production may be achieved by the 0144 (ii) a thiamine kinase (EC 2.7.1.89); and conversion of acetyl-CoA into acetoacetyl-CoA or by the (b)(i) a terpene synthase, e.g. an isoprene synthase (EC conversion of acetyl-CoA and malonyl-CoA into acetoacetyl 4.2.3.27); or CoA. Thus, the present invention also relates to a (mirco) 0145 (ii) an isopentenylphosphate kinase and a terpene organism which further expresses an acetyl-CoA C-acetyl synthase, e.g. an isoprene synthase (EC 4.2.3.27); or transferase (EC 2.3.1.9) and which is capable of converting 0146 B) (a) (i) a 2-amino-4-hydroxy-6-hydroxymeth acetyl-CoA into acetoacetyl-CoA and/or which further yldihydropteridine diphosphokinase (EC 2.7.6.3); or expresses a acetoacetyl-CoA synthase and which is capable 0147 (ii) a thiamine diphosphokinase (EC 2.7.6.2); and of converting acetyl-CoA and malonyl-CoA into acetoacetyl (b) a terpene synthase, e.g. an isoprene synthase (EC 4.2. CoA. 3.27), and which is capable of converting crotyl alcohol 0154 In one embodiment an organism according to the into butadiene. As regards preferred embodiments, the present invention is a recombinant organism in the sense that same applies as has been set forth above in connection with it is genetically modified due to the introduction of at least one the method according to the invention. nucleic acid molecule encoding at least one of the above US 2014/0256009 A1 Sep. 11, 2014 mentioned enzymes. Preferably such a nucleic acid molecule 0166 The present invention also relates to a composition is heterologous with regard to the organism which means that comprising it does not naturally occur in said organism. (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); 0155 The microorganism is preferably a bacterium, a and/or yeast or a fungus. In another preferred embodiment the organ (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or ism is a plant or non-human animal. As regards other pre (iii) an alcohol dehydrogenase (EC 1.1.1.1); and/or ferred embodiments, the same applies as has been set forth (iv) an aldehydefalcohol dehydrogenase; and/or above in connection with the method according to the inven (v) an acyl-CoA reductase; and/or tion. (vi) an aldo-keto reductase (AKR); and/or 0156. In an embodiment according to the present inven (vii) an aldehyde reductase; and/or tion in which an organism, preferably a microorganism, is (viii) a short-chain dehydrogenase/fatty acyl-CoA reductase. employed which is capable of providing crotonyl-CoA, Such 0.167 Moreover, the present invention also relates to such a (micro)organism is advantageously further genetically a composition which further comprises a 3-hydroxybutyryl modified so as to avoid diverting of the crotonyl-CoA into CoA dehydratase (EC 4.2.1.55) and/or an enoyl-CoA other pathways. It is known, for example, that crotonyl-CoA hydratase (EC 4.2.1.17) and/or an enoyl-CoA hydratase 2 can be reduced by a variety of enzymes to lead to butyryl (EC 4.2.1.119) and/or a crotonyl-acyl-carrier-protein CoA. These enzymes generally belong to the EC classifica hydratase (EC 4.2.1.58). The present invention also relates to tion EC 1.3.1 and include acyl-CoA dehydrogenase (NADP", a composition which also comprises an acetoacetyl-CoA EC 1.3.1.8), enoyl-acyl-carrier-protein reductase (NADH: reductase (EC 1.1.1.36). Finally, the present invention also EC 1.3.1.9), enoyl-acyl-carrier-protein reductase (NADPH: relates to a composition which also comprises an acetyl-CoA EC 1.3.1.10), cis-2-enoyl-CoA reductase (NADPH: EC 1.3. C-acetyltransferase (EC 2.3.1.9) or an acetoacetyl-CoA syn 1.37) and trans-2-enoyl-CoA reductase (NADPH; EC 1.3.1. thase. 38). Thus, in one embodiment the organism is genetically 0.168. The present invention also relates to the use of a modified so as to decrease the activity of enzymes which may combination of enzymes comprising: lead to a reduction of crotonyl-CoA to butyryl-CoA and thus 0169 A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1. to a diversion of crotonyl-CoA into other pathways. Such a 50); or reduction of activity can be achieved by methods known to (0170 (ii) a thiamine kinase (EC 2.7.1.89); and the person skilled in the art and include, for example, the (b)(i) a terpene synthase, e.g. an isoprene synthase (EC decrease of the expression of the respective gene(s) coding 4.2.3.27); or for the respective enzyme(s) by known methods such as anti 0171 (ii) an isopentenylphosphate kinase and a terpene sense approaches, siRNA approaches or the like. In case the synthase, e.g. an isoprene synthase (EC 4.2.3.27); or respective enzyme activity is not necessary for Survival of the 0172 B) (a) (i) a 2-amino-4-hydroxy-6-hydroxymeth microorganism, it can also be knocked out completely, e.g. by yldihydropteridine diphosphokinase EC 2.7.6.3); or disrupting the gene or completely deleting the gene. 0173 (ii) a thiamine diphosphokinase (EC 2.7.6.2); and 0157. The present invention also relates to a composition (b) a terpene synthase, e.g. an isoprene synthase (EC 4.2. comprising 3.27); 0158 A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1. for the production of butadiene from crotyl alcohol. As 50); or regards preferred embodiments, the same applies as has been 0159 (ii) a thiamine kinase (EC 2.7.1.89); and set forth above in connection with the method according to (b)(i) a terpene synthase, e.g. an isoprene synthase (EC the invention. 4.2.3.27); or 0.174 As regards in particular the terpene synthase and the 0160 (ii) an isopentenylphosphate kinase and a terpene preferred embodiments of terpene synthases to be expressed synthase, e.g. an isoprene synthase (EC 4.2.3.27); or by the (micro)organism, the same applies as has been set forth 0161 B) (a) (i) a 2-amino-4-hydroxy-6-hydroxymeth above in connection with the method according to the inven yldihydropteridine diphosphokinase EC 2.7.6.3); or tion. 0162 (ii) a thiamine diphosphokinase (EC 2.7.6.2); and 0175 Thus, in one preferred embodiment the terpene syn (b) a terpene synthase, e.g. an isoprene synthase (EC 4.2. thase is 3.27). (k) an isoprene synthase (EC 4.2.3.27); or 0163 Such a composition may also comprise crotyl alco (1) a myrcene/ocimene synthase (EC 4.2.3.15); or hol. As regards preferred embodiments, the same applies as (m) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or has been set forth above in connection with the method (n) a pinene synthase (EC 4.2.3.14); or according to the invention. (o) a monoterpene synthase. 0164. As regards in particular the terpene synthase and the 0176 The present invention also relates to the use of preferred embodiments of terpene synthases to be expressed (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); by the (micro)organism, the same applies as has been set forth and/or above in connection with the method according to the inven (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or tion. (iii) an alcohol dehydrogenase (EC 1.1.1.1); and/or (0165 Thus, in one preferred embodiment the terpene syn (iv) an aldehydefalcohol dehydrogenase; and/or thase is (v) an acyl-CoA reductase; and/or (f) an isoprene synthase (EC 4.2.3.27); or (vi) an aldo-keto reductase (AKR); and/or (g) a myrcene/ocimene synthase (EC 4.2.3.15); or (vii) an aldehyde reductase; and/or (h) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or (viii) a short-chain dehydrogenase/fatty acyl-CoA reductase (i) a pinene synthase (EC 4.2.3.14); or for the conversion of crotonyl-CoA into crotonaldehyde and/ (i) a monoterpene synthase. or crotyl alcohol. US 2014/0256009 A1 Sep. 11, 2014
0177. Furthermore the present invention also relates to the 0201 Other aspects and advantages of the invention will use of a combination of enzymes comprising be described in the following examples, which are given for (a) an acetoacetyl-CoA reductase (EC 1.1.1.36); and purposes of illustration and not by way of limitation. (b)(i) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55); and or 0.178 (ii) an enoyl-CoA hydratase (EC 4.2.1.17); and/ EXAMPLES O (0179 (iii) an enoyl-CoA hydratase 2 (EC 4.2.1.119): Example 1 and/or 0180 (iv) a crotonyl-acyl-carrier-protein hydratase Cloning, Expression and Purification of Enzymes (EC 4.2.1.58) for the production of crotonyl-CoA from acetoacetyl-CoA. 0181. The present invention also relates to the use of a Cloning, Bacterial Cultures and Expression of Proteins combination of enzymes comprising (a)(i) an acetyl-CoA C-acetyltransferase (EC 2.3.1.9) and/or 0202 The genes encoding studied enzymes were cloned in 0182 (ii) an acetoacetyl-CoA synthase; and pET 25b vector (Novagen). A stretch of 6 histidine codons (b) an acetoacetyl-CoA reductase (EC 1.1.1.36): was inserted after the methionine initiation codon to provide (c) (i) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55); an affinity tag for purification. Competent E. coli BL21 (DE3) and/or cells (Novagen) were transformed with these vectors accord 0183 (ii) an enoyl-CoA hydratase (EC 4.2.1.17); and/ ing to the heat shock procedure. The transformed cells were O grown with shaking (160 rpm) on ZYM-5052 auto-induction 0.184 (iii) an enoyl-CoA hydratase 2 (EC 4.2.1.119): medium (Studier F.W. Prot. Exp. Pur. 41 (2005), 207-234) for and/or 6 hat 37°C. and protein expression was continued at 28°C. 0185 (iv) a crotonyl-acyl-carrier-protein hydratase or 20° C. overnight (approximately 16 h). The cells were (EC 4.2.1.58) collected by centrifugation at 4°C., 10,000 rpm for 20 min for the production of crotonyl-CoA from acetyl-CoA. and the pellets were frozen at -80° C. 0186 FIG. 1 shows the chemical structure of crotonyl alcohol. Protein Purification and Concentration 0187 FIG. 2 shows the chemical structure of crotyl phos phate. and crotyl diphosphate (0203 The pellets from 200 ml of culture cells were thawed 0188 FIG. 3 shows the chemical structure of crotonalde on ice and resuspended in 5 ml of NaHPO pH8 containing hyde. 300 mM. NaCl, 5 mM MgCl, and 1 mM DTT. Twenty micro 0189 FIG. 4 shows the chemical structure of butadiene. liters of lysonase (Novagen) were added. Cells were incu (0190 FIG. 5 shows a scheme of the ADP quantification bated 10 minutes at room temperature and then returned to ice assay, monitoring NADH consumption by the decrease of absorbance at 340 nm. for 20 minutes. Cell lysis was completed by sonication for 0191 FIG. 6 shows a mass spectrum of an enzymatic 3x15 seconds. The bacterial extracts were then clarified by assay with hydroxyethylthiazole kinase from E. coli. centrifugation at 4°C., 10,000 rpm for 20 min. The clarified 0.192 FIG. 7 shows a mass spectrum of a control assay bacterial lysates were loaded on PROTINO-1000 Ni-TED without enzyme. column (Macherey-Nagel) allowing adsorption of 6-His 0193 FIG. 8 shows a comparison between dimethylallyl tagged proteins. Columns were washed and the enzymes of diphosphate and crotyl diphosphate and their conversion into interest were eluted with 4 ml of 50 mM NaHPO pH 8 isoprene (2-methyl-buta-1,3-diene) and butadiene, respec containing 300 mMNaCl, 5 mMMgCl, 1 mM DTT,250 mM tively. imidazole. Eluates were then concentrated and desalted on 0194 FIG. 9 shows the formula of crotonyl-Coenzyme A Amicon Ultra-4 10 kDa filter unit (Millipore) and resus (0195 FIG. 10 shows the MS spectrum of the trans crotyl pended in 0.25 ml 50 mM Tris-HCl pH 7.4 containing 0.5 mM monophosphate phosphorylation reaction catalyzed by iso DTT and 5 mM MgCl. Protein concentrations were quanti pentenyl monophosphate kinase from M. jannaschii (a) and fied according to the Bradford method. The purity of proteins of a control assay without enzyme (b). 0.196 FIG. 11 shows 1,3-butadiene production from trans thus purified varied from 50% to 90%. crotyl monophosphate catalyzed by terpene synthases. 0.197 FIG. 12 shows the mass spectrum of commercial Example 2 1,3-butadiene (a) and 1,3-butadiene produced from trans cro tyl monophosphate in an enzymatic reaction catalyzed by Screening for Crotyl Alcohol Phosphorylation monoterpene synthase from E. globulus (b). Activity 0198 FIG. 13 shows 1,3-butadiene production from trans crotyl diphosphate catalyzed by terpene synthases. 0204 The release of ADP that is associated with crotyl (0199 FIG. 14 shows a time courses of NADPH oxidation alcohol phosphorylation was quantified using the pyruvate in crotonyl-CoA reduction assay with reductase from kinase/lactate dehydrogenase coupled assay (FIG. 5). The Hahella cheiuensis and varying concentrations of NADPH. purified 4-methyl-5-(2-hydroxyethyl)thiazole kinases from 0200 FIG. 15 shows a chromatogram of the crotonyl-CoA Escherichia coli (SEQ ID NO:2), Bacillus subtilis (SEQ ID reduction reaction catalyzed by acyl-CoA reductase from H. NO:1), Rhizobium leguminosarum (SEQ ID NO:3) were cheiuensis. evaluated for their ability to phosphorylate crotyl alcohol US 2014/0256009 A1 Sep. 11, 2014 releasing ADP. The studied enzymatic reaction was carried crotyl monophosphate, from the enzymatic sample but not out under the following conditions at 37°C.: from the controls (FIGS. 6 and 7). 50 mM Tris-HCl pH 7.5 Example 4 10 mM MgCl, Screening for 1,3-Butadiene Production from Crotyl Monophosphate Using Purified Isoprene Synthases 100 mM KC1 0212 Crotyl monophosphate is synthesized upon request 5 nM ATP by a company specialized in custom synthesis (Syntheval, France). O4 nM NADH 0213. The enzymatic assays are carried out under the fol lowing conditions at 37° C.: 1 mM Phosphoenolpyruvate 50 mM Tris-HC1 pH7.5 0205 3 U/ml Lactate dehydrogenase 1.5 U/ml Pyruvate kinase 0214) 1 to 200 mM cis or trans crotyl monophosphate 50 mM crotyl alcohol, mixture cis and trans The pH was adjusted to 7.5 1 mM DTT 0206 Each assay was started by addition of a particular enzyme at a concentration 0.05 mg/ml and the disappearance 1 to 20 mM MgCl, of NADH was monitored by following the absorbance at 340 0215 1 to 5 mg/ml isoprene synthase nM. 0216. The enzyme-free control reaction is carried out in 0207 Assays with hydroxyethylthiazole kinase from the parallel. The enzymatic mixture is incubated at 37°C. for 72 E. coli and Rh. leguminosarum gave rise to a reproducible and h in a sealed vial (Interchim). significant increase in ADP production in the presence of 0217 Volatile compounds in the headspace of the reaction crotyl alcohol (Table 1). Mass spectrometry was then used to mixture are collected using a gas Syringe equipped with an Verify the formation of crotyl monophosphate in the assay anti-backup mechanism and are directly injected into a with the E. coli enzyme. GC-430 gas chromatograph (Brucker) equipped with an FID detector and a GAS-PRO column (Agilent). The enzymatic TABLE 1. reaction product is identified by direct comparison with stan dard 1,3-butadiene (Sigma). 4-methyl-5-(2-hydroxyethyl) Activity, micromole? 0218. The identity of the gas is further confirmed in thiazole kinase min mg protein GC/MS analyses. E. coi O.220 Rh. legitiminosartin O.O87 Example 5 B. subtiis O.O14 Screening for Crotyl Monophosphate Phosphorylation Activity Example 3 0219 Sequences of isopentenyl monophosphate kinases inferred from the genomes of several members of the Mass Spectrometry Analysis of the Crotyl Alcohol Archaea, in particular Methanothermobacter (SEQ ID Phosphorylation Reaction NO:5), Methanocaldococcus (SEQ ID NO:6) and Thermo 0208. The desired enzymatic reactions were carried out plasma (SEQID NO:4) genus, are generated by oligonucle under the following conditions: otide concatenation to fit the codon usage of E. coli. A stretch of 6 histidine codons is inserted after the methionine initiation 50 mM Tris-HC1 pH7.5 codon to provide an affinity tag for purification. The genes thus synthesized are cloned in a pBT25b expression vector 10 mM MgCl2 and the proteins are produced according to the protocol described in Example 1. The enzymes are then assayed using 0209 50 mM cis or trans crotyl alcohol the method described in Example 2 with crotyl monophos phate concentrations varying from 0 to 50 mM. The release of 20 mM ATP ADP that is associated with crotyl monophosphate phospho 0210. 0.1 mg/ml purified hydroxythiazole kinase from E. rylation is quantified using the pyruvate kinase/lactate dehy coli (SEQID NO:2) drogenase coupled assay. Each assay is started by addition of 0211. The control reactions without enzyme, without sub particular enzyme (at a final concentration from 0.05 mg/ml strate and without ATP were run in parallel. The assays were to 1 mg/ml) and the disappearance of NADH is monitored by incubated overnight without shaking at 37°C. Typically, an following the absorbance at 340 nM. aliquot of 2001 reaction was removed, centrifuged and the supernatant was transferred into a clean vial. The MS spectra Example 6 were obtained on an ion trap mass spectrometer (Esquire 3000, Bruker) in negative ion mode by direct injection of the Mass Spectrometry Analysis of the Crotyl sample using a syringe pump operated at a flow rate of 2 ml/h. Monophosphate Phosphorylation Reaction The presence of crotyl monophosphate was evaluated. MS 0220 Enzymatic assays are run in 50 mM Tris-HCl pH analysis showed an M-H-ion at m/z, 151, corresponding to 7.5, contained 5 mM MgCl2, 20 mM ATP, 2 mM f-mercap US 2014/0256009 A1 Sep. 11, 2014 toethanol and crotyl monophosphate varying in the range Example 9 from 0 to 50 mM in a final volume of 0.25 ml. The reactions are initiated with the addition of purified isoprenol mono Kinetic Parameters of Crotyl Alcohol phosphate kinase and incubated overnight at 37–55°C. The Phosphorylation control reactions contain no enzyme. Following incubation 0229 Kinetic parameters of crotyl alcohol phosphoryla samples are processed by mass spectrometry analysis. An tion were determined using the spectrophotometric assay aliquot of 200 ul reaction is removed, centrifuged and the described in Example 2. Kinetic parameters obtained for puri supernatant is transferred to a clean vial. The MS spectra are fied 4-methyl-5-(2-hydroxyethyl)thiazole kinase from E. coli obtained on ion trapp mass spectrometer (Esquire 3000, Bruker) in negative ion mode by direct injection of sample are presented in Table 2. using a Syringe pump operated at a flow rate of 2 ml/h. TABLE 2 Example 7 Kinetic parameters Screening for 1,3-Butadiene Production from Crotyl Substrate K, mM kars' Diphosphate Using Purified Isoprene Synthases Ciscrotyl alcohol 13.6 O.19 Transcrotyl alcohol 30 O.11 0221 Crotyl diphosphate is synthesized upon request by a company specialized in custom synthesis, Syntheval (France). Example 10 0222. The enzymatic assays are carried out under the fol lowing conditions at 37° C.: Mass Spectrometry Analysis of the Crotyl Monophosphate Phosphorylation Reaction 50 mM Tris-HC1 pH7.5 0230. The enzymatic reactions were carried out under the 0223) 1 to 200 mM cis or trans crotyl diphosphate following conditions: 1 mM DTT 50 mM Tris-HCl pH 7.5 1 to 20 mM MgCl, 10 mM MgCl, 0224. 1 to 5 mg/ml isoprene synthase 100 mM KC1 0225. The enzyme-free control reaction is carried out in 0231 50 mM trans crotyl monophosphate parallel. The enzymatic mixture is incubated at 37°C. for 72 h in a sealed vial (Interchim). 20 mM ATP 0226 Volatile compounds in the headspace of the reaction mixture are collected using a gas Syringe equipped with an 0232 0.1 mg/ml purified isopentenyl monophosphate anti-backup mechanism and are directly injected into a kinase GC-430 gas chromatograph (Brucker) equipped with an FID 0233 Control assays were performed in which either no detector and a GAS-PRO column (Agilent). The enzymatic enzyme was added, or no Substrate was added. The assays reaction product is identified by direct comparison with stan were incubated overnight without shaking at 37°C. Typically, dard 1,3-butadiene (Sigma). an aliquot of 200 ul reaction was removed, centrifuged and the supernatant was transferred into a clean vial. The MS 0227. The identity of the gas is further confirmed in spectra were obtained onion trap mass spectrometer (Esquire GC/MS analyses. 3000, Bruker) in negative ion mode by direct injection of sample using a syringe pump operated at a flow rate of 2 ml/h. Example 8 The presence of crotyl diphosphate was evaluated. MS analy sis showed an M-H ion at m/Z 231.5, corresponding to Screening of Hydroxymethylglutaryl-CoA crotyl diphosphate, from the enzymatic samples but not from (Hmg-CoA) Reductases Using Crotonyl-CoA as a the controls. Examples of mass spectrums of enzymatic assay Substrate with isopentenyl monophosphate kinase from M. jannaschii 0228 Sequences of hydroxymethylglutaryl-CoA reduc and control assay without enzyme are shown in FIGS. 10a tases inferred from the genomes of prokaryotic and eukary and 10b. otic organisms are generated to fit the codon usage of E. coli. A stretch of 6 histidine codons is inserted after the methionine Example 11 initiation codon to provide an affinity tag for purification. The genes thus synthesized are cloned in a pET25b expression Kinetic Parameters of the Crotyl Monophosphate vector and the proteins are produced according to the protocol Phosphorylation Reaction described in Example 1. The reductase activity of the purified 0234 Cis crotyl monophosphate and trans crotyl mono enzymes using crotonyl-CoA as a Substrate is then deter phosphate were synthesized upon request by a company spe mined by measuring the initial decrease in absorbance at 340 cialized in custom synthesis (Syntheval, France). Kinetic nm due to the NADPH oxidation. The standard assay is per parameters for the phosphorylation of these substrates were formed at pH 7.5, 50 mM phosphate buffer, containing 10 determined using the spectrophotometric assay described in mM dithiothreitol, 0.1 mM NADPH and crotonyl-CoA at Example 2. Kinetic parameters obtained with purified isopen concentration varying from 0 to 10 mM. tenyl monophosphate kinases from different members of the US 2014/0256009 A1 Sep. 11, 2014
Archaea kingdom are presented in Table 3 (cis crotyl mono analyzed on a Varian 3400CX gas chromatograph equipped phosphate as a Substrate) and Table 4 (trans crotyl monophos with Varian Saturn 3 mass selective detector. A mass spectrum phate as a substrate). of 1,3-butadiene obtained by enzymatic conversion of trans crotyl monophosphate was similar to that of commercial 1.3- TABLE 3 butadiene (FIGS. 12a and 12b). Isopentenyl Example 13 monophosphate Kinetic parameters Kinetic Parameters of Enzyme Catalyzed Production kinase K, mM kees' of 1,3-Butadiene from Trans Crotyl Monophosphate Meihanocaidococcus O.20 3.4 iannaschii 0240. The kinetic parameters of enzyme catalyzed pro Meihanothermobacter O.94 5.7 duction of 1,3-butadiene from trans crotyl monophosphate thermautotrophicus Thermoplasma O.61 1.8 were measured under the following conditions: acidophilum 50 mM Tris-HCl pH 7.5 20 mM MgCl, TABLE 4 20 mMKC1 Kinetic parameters Enzyme K, mM kars' 2 mMDTT Meihanocaidococcus O49 3.0 0241 0-25 mM trans crotyl monophosphate iannaschii 0242. The reaction was initiated by addition of 0.25 mg of Meihanothermobacter O45 8.7 purified monoterpene synthase from Eucalyptus globulus to thermautotrophicus Thermoplasma 1 2.2 0.5 ml of reaction mixture. An enzyme-free control reaction acidophilum was carried out in parallel. Assays were incubated at 37° C. for 0.5-4h in a sealed vial of 1.5 ml (Interchim) with shaking. 0243) 1,3-butadiene production was analyzed using the GC/FID procedure described in Example 12. Monoterpene Example 12 synthase from E. globulus was found to have a K value of 6 mMandak. of at least 0.2x10 sec'. Enzyme Catalyzed Production of 1,3-Butadiene from egg Trans Crotyl Monophosphate with Purified Terpene Example 14 Synthases Enzyme Catalyzed Production of 1,3-Butadiene from 0235. The enzymatic assays were carried out under the Trans Crotyl Diphosphate with Purified Terpene following conditions at 37° C.: Synthases 50 mM Tris-HCl pH 7.5 0244. The enzymatic assays were carried out under the 0236 25 mM trans crotyl monophosphate following conditions at 37° C.: 2 mMDTT 50 mM Tris-HCl pH 7.5 0245 25 mM trans crotyl diphosphate 50 mM MgCl, 2 mMDTT 50 mM KC1 0237 2 mg of the purified terpene synthase was added to 50 mM MgCl, 0.5 ml of reaction mixture. An enzyme-free control reaction was carried out in parallel. Assays were incubated at 37° C. 50 mMKC1 for 24h in a sealed vial of 1.5 ml (Interchim) with shaking. 0246 2 mg of the purified terpene synthase was added to 0238. One ml of the gaseous phase was then collected and 0.5 ml of reaction mixture. An enzyme-free control reaction directly injected into a Varian GC-430 gas chromatograph was carried out in parallel. Assays were incubated at 37° C. equipped with a flame ionization detector (FID). Nitrogen for 24h in a sealed vial of 1.5 ml (Interchim) with shaking. was used as carrier gas with a flow rate of 1.5 ml/min. Volatile 0247 One ml of the gaseous phase was then collected and compounds were chromatographically separated on RT-Alu directly injected into a Varian GC-430 gas chromatograph mina Bond/Na2SO4 column (Restek) using an isothermal equipped with a flame ionization detector (FID). Nitrogen mode at 130°C. The enzymatic reaction product was identi was used as carrier gas with a flow rate of 1.5 ml/min. Volatile fied by direct comparison with 1,3-butadiene standard compounds were chromatographically separated on RT-Alu (Sigma). Several terpene synthases were shown to catalyze mina Bond/Na2SO4 column (Restek) using an isothermal butadiene production from transcrotyl monophosphate (FIG. mode at 130°C. The enzymatic reaction product was identi 11). fied by direct comparison with 1,3-butadiene standard 0239 Gas chromatography-mass spectrometry was then (Sigma). Several terpene synthases were shown to catalyze used to confirm the identity of the product detected by butadiene production from trans crotyl diphosphate (FIG. GC/FID. Assay with E.globulus enzyme (SEQIDNO: 8) was 13). US 2014/0256009 A1 Sep. 11, 2014 18
Example 15 TABLE 5-continued Kinetic Parameters of Enzyme Catalyzed Production Activity, Imol/min.img of 1,3-Butadiene from Trans Crotyl Diphosphate Enzyme protein 0248. The kinetic parameters of enzyme catalyzed pro Short chain alcohol dehydrogenase- 7.5 duction of 1,3-butadiene from trans crotyl monophosphate like protein from were measured under the following conditions: Marinobacter manganoxydians (SEQID NO: 14) Short chain alcohol dehydrogenase- 4 50 mM Tris-HCl pH 7.5 like protein from Marinobacter algicola 20 mM MgCl, (SEQID NO: 16) Short chain alcohol dehydrogenase- 4.9 20 mM KC1 like protein from Hahella cheuensis Short chain alcohol dehydrogenase- 1.2 2 mMDTT like protein from Marinobacter 0249 0-25 mM trans crotyl diphosphate sp. ELB17 (SEQID NO: 15) 0250. The reaction was initiated by addition of 0.25 mg of purified monoterpene synthase from Eucalyptus globulus to 0.5 ml of reaction mixture. An enzyme-free control reaction 0254 The products of enzymatic reduction of crotonyl was carried out in parallel. Assays were incubated at 37° C. CoA were next analyzed by high-performance liquid chro for 0.5-4h in a sealed vial of 1.5 ml (Interchim) with shaking. matography (HPLC). 0251 1,3-butadiene production was analyzed using the GC/FID procedure described in Example 12. Monoterpene Example 17 synthase from E. globulus was found to have a K value of 7 mMandak, of at least 0.3x10" sec'. HPLC Studies of Enzymatic Reduction of Crotonyl-CoA Example 16 0255. The enzymatic assays were carried out under the Screening of Short-Chain following conditions: Dehydrogenases/Reductases with Crotonyl-CoA as 50 mM Potassium phosphate pH 7.5 Substrate 0252 Sequences of short-chain dehydrogenases/reduc 100 mM KC1 tases inferred from the genomes of prokaryotic organisms were generated by oligonucleotide concatenation to fit the 0256 24 mM trans crotonyl-CoA codon usage of E. coli. A stretch of 6 histidine codons is inserted after the methionine initiation codon to provide an 80 nM NADPH affinity tag for purification. The genes thus synthesized were cloned in a pBT25b expression vector and the proteins were 0257 The reactions were initiated by addition of 150 ug of produced according to the protocol described in Example 1. purified dehydrogenase/reductase to 150 ul of reaction mix 0253 For the reductase assay, a reaction mixture contain ture. Assays were incubated at 37° C. for 0.5-6 h. The reac ing 50 mM potassium phosphate pH 7.5, 0.1-0.4 mM tions were stopped by heating at 65° C. for 5 minutes, reaction NADPH, 100 mMNaCl, 5 mM transcrotonyl-CoA and 0.5-1 mixtures were centrifuged and 120 ul of the clarified super mg/ml enzyme in a total Volume of 120 ul was used and the natant were transferred into a clean vial. The reaction prod reaction was carried out at 37°C. for 20 min. Control assays ucts were then extracted with an equal volume of ethyl were performed in which either no enzyme was added, or no acetate. 100 ul of the upper ethyl acetate phase was trans Substrate was added. Each sample was continuously moni ferred into a clean vial for HPLC analysis. Commercial cro tored for the decrease of NADPH at 340 nm on a SpectraMax tonaldehyde and crotyl alcohol were used as reference. Plus384 UV/Vis Microplate Reader (Molecular Device). HPLC-UV analysis was performed using a 1260 Inifinity LC Several enzymes demonstrated crotonyl-CoA reductase System (Agilent). 10ul of samples were separated on Zorbax SB-Aq column (250x4.6 mm, 3.5 um particle size) with a activity with NADPH as co-substrate (FIG. 14 which shows a mobile phase flow rate of 1.5 ml/min. The mobile phase time courses of NADPH oxidation in crotonyl-CoA reduction consisted of 95:5 (v/v) HO/Acetonitrile containing 8.4 mM assay with reductase from Hahella cheiuensis (SEQID NO: sulfuric acid. Retention time for trans crotyl alcohol and 17) and varying concentrations of NADPH, Table 5). crotonaldehyde in these conditions were 4.3 and 5.4 min, TABLE 5 respectively. (0258. The HPLC analysis showed that crotonaldehyde Activity, and crotyl alcohol were formed by enzyme catalyzed reduc Imol/min.img tion of crotonyl-CoA. A typical chromatogram obtained with Enzyme protein short chain alcohol dehydrogenase-like protein from H. Short-chain dehydrogenase/reductase 1.4 cheuensis is shown on FIG. 15. (Fatty alcohol forming acyl-CoA reductase) from Marinobacter 0259. These data indicate that the short-chain dehydroge aquaeolei VT8 (SEQ ID NO: 13) nase/reductase catalyzes the four-electron reduction of croto nyl-CoA to crotyl alcohol via the aldehyde intermediate. US 2014/0256009 A1 Sep. 11, 2014 19
Example 18 0261 The short chain alcohol dehydrogenase-like protein from H. cheuensis was found to have a K of 1 mMandak, of 6.0 sec' towards NADPH as substrate. Kinetic Parameters of Crotyl Alcohol Production 0262 Kinetic parameters values towards crotonyl-CoA from Trans Crotonyl-CoA were determined at fixed concentration of NADPH (80 mM) and varying crotonyl-CoA concentration from 0 to 32 mM. 0260 Kinetic parameters values towards NADPH were Kinetic parameters for the overall reaction were determined determined at fixed concentration of crotonyl-CoA (5 mM) by crotyl alcohol quantification using HPLC procedure and varying NADPH concentration from 0 to 0.8 mM. described in Example 17. The short chain alcohol dehydro NADPH oxidation was measured spectrophotometrically at genase-like protein from H. cheuensis was found to have a 340 nm according to the procedure described in Example 16. K of 5 mMandak of at least 0.05 sec'.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS : 17
<21 Os SEQ ID NO 1 &211s LENGTH: 272 212s. TYPE: PRT <213> ORGANISM; Bacillus subtilis
<4 OOs SEQUENCE: 1 Met Asp Ala Glin Ser Ala Ala Lys Cys Lieu. Thir Ala Val Arg Arg His 1. 5 1O 15
Ser Pro Leu Wal His Ser Ile Thir ASn Asn. Wal Wall Thir ASn Phe Thr 2O 25 3 O Ala Asn Gly Lieu. Lieu Ala Lieu. Gly Ala Ser Pro Wal Met Ala Tyr Ala 35 4 O 45 Lys Glu Glu Val Ala Asp Met Ala Lys Ile Ala Gly Ala Lieu Val Lieu. SO 55 60 Asn. Ile Gly. Thir Lieu. Ser Lys Glu Ser Val Glu Ala Met Ile Ile Ala
Gly Llys Ser Ala Asn. Glu. His Gly Val Pro Val Ile Lieu. Asp Pro Val 85 90 95 Gly Ala Gly Ala Thr Pro Phe Arg Thr Glu Ser Ala Arg Asp Ile Ile 1OO 105 110 Arg Glu Val Arg Lieu Ala Ala Ile Arg Gly Asn Ala Ala Glu Ile Ala 115 12O 125 His Thr Val Gly Val Thr Asp Trp Lieu. Ile Lys Gly Val Asp Ala Gly 13 O 135 14 O Glu Gly Gly Gly Asp Ile Ile Arg Lieu Ala Glin Glin Ala Ala Glin Lys 145 15 O 155 16 O Lieu. Asn Thr Val Ile Ala Ile Thr Gly Glu Val Asp Val Ile Ala Asp 1.65 17 O 17s Thir Ser His Val Tyr Thr Lieu. His Asn Gly His Llys Lieu Lleu. Thir Lys 18O 185 190 Val Thr Gly Ala Gly Cys Lieu Lleu. Thir Ser Val Val Gly Ala Phe Cys 195 2 OO 2O5
Ala Wall Glu Glu Asn. Pro Lieu. Phe Ala Ala Ile Ala Ala Ile Ser Ser 210 215 22 O Tyr Gly Val Ala Ala Glin Lieu Ala Ala Glin Glin Thr Ala Asp Llys Gly 225 23 O 235 24 O Pro Gly Ser Phe Glin Ile Glu Lieu. Lieu. Asn Llys Lieu Ser Thr Val Thr 245 25 O 255
Glu Gln Asp Val Glin Glu Trp Ala Thr Ile Glu Arg Val Thr Val Ser 26 O 265 27 O US 2014/0256009 A1 Sep. 11, 2014 20
- Continued
<210s, SEQ ID NO 2 &211s LENGTH: 262 212. TYPE: PRT <213> ORGANISM: E. coli
<4 OOs, SEQUENCE: 2 Met Glin Val Asp Lieu. Lieu. Gly Ser Ala Glin Ser Ala His Ala Lieu. His 1. 5 1O 15 Lieu. Phe His Gln His Ser Pro Leu Val His Cys Met Thr Asn Asp Val 2O 25 3O Val Glin Thr Phe Thr Ala Asn Thr Lieu. Leu Ala Lieu. Gly Ala Ser Pro 35 4 O 45
Ala Met Wall Ile Glu Thr Glu Glu Ala Ser Glin Phe Ala Ala Ile Ala SO 55 6 O Ser Ala Lieu. Lieu. Ile Asn Val Gly. Thir Lieu. Thr Glin Pro Arg Ala Glin 65 70 7s 8O Ala Met Arg Ala Ala Val Glu Glin Ala Lys Ser Ser Glin Thr Pro Trip 85 90 95 Thir Lieu. Asp Pro Val Ala Val Gly Ala Lieu. Asp Tyr Arg Arg His Phe 1OO 105 11 O Cys His Glu Lieu Lleu Ser Phe Llys Pro Ala Ala Ile Arg Gly Asn Ala 115 12 O 125 Ser Glu Ile Met Ala Lieu Ala Gly Ile Ala Asn Gly Gly Arg Gly Val 13 O 135 14 O Asp Thir Thir Asp Ala Ala Ala Asn Ala Ile Pro Ala Ala Glin Thir Lieu 145 150 155 160 Ala Arg Glu Thr Gly Ala Ile Val Val Val Thr Gly Glu Met Asp Tyr 1.65 17O 17s Val Thr Asp Gly His Arg Ile Ile Gly Ile His Gly Gly Asp Pro Lieu 18O 185 19 O Met Thr Llys Val Val Gly Thr Gly Cys Ala Leu Ser Ala Val Val Ala 195 2OO 2O5 Ala Cys Cys Ala Lieu Pro Gly Asp Thir Lieu. Glu Asn. Wall Ala Ser Ala 21 O 215 22O Cys His Trp Met Lys Glin Ala Gly Glu Arg Ala Val Ala Arg Ser Glu 225 23 O 235 24 O Gly Pro Gly Ser Phe Val Pro His Phe Lieu. Asp Ala Leu Trp Glin Leu 245 250 255
Thir Glin Glu Wall Glin Ala 26 O
<210s, SEQ ID NO 3 &211s LENGTH: 267 212. TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum
<4 OOs, SEQUENCE: 3 Met Glin Thr Arg Thr Thr Pro Gly Ala Met Leu Lys Ala Met Arg Glu 1. 5 1O 15
Llys Pro Pro Leu Val Glin Cys Ile Thr Asn Tyr Val Ala Met Asn Ile 2O 25 3O
Ala Ala Asn Val Lieu. Lieu Ala Ala Gly Ala Ser Pro Ala Met Val His 35 4 O 45
Ala Ala Glu Glu Ala Gly Glu Phe Ala Ala Ile Ala Ser Ala Lieu. Thr US 2014/0256009 A1 Sep. 11, 2014 21
- Continued
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
Thir Ala Luell Gly Ser Lell Thir Luell Wall Gly Ala Phe Ala Ala 195
Thir Ala Pro Glu Asp Ile Phe Gly Ala Thir Wall Ala Ala Luell Ser Thir 21 O 215
Phe Ala Ile Ala Gly Glu Glu Ala Ala Luell Gly Ala Ala Gly Pro Gly 225 23 O 235 24 O
Ser Phe Ser Trp Arg Phe Lell Asp Ala Luell Ala Ala Lell Asp Ala Glu 245 250 255
Thir Luell 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 Wall Ile Thir Asp Ser Ala 1. 5 15
Tyr Arg Thir Ala Arg Thir Ala Ile Arg Ser Ile Wall Lys Wall Luell 2O 25 3O
Ser Gly Ile Glu Asp Lell Wall Cys Wall Wall His Gly Gly Gly Ser Phe 35 4 O 45
Gly His Ile Ala Met Glu Phe Gly Luell Pro Gly Pro Asn Pro SO 55 6 O
Arg Ser Ser Ile Gly Tyr Ser Ile Wall His Arg Asp Met Glu Asn Luell 65 70
Asp Luell Met Wall Ile Asp Ala Met Ile Glu Met Gly Met Arg Pro Ile 85 90 95
Ser Wall Pro Ile Ser Ala Lell Arg Tyr Asp Gly Arg Phe Asp Thir 105 11 O
Pro Luell Ile Arg Tyr Ile Asp Ala Gly Phe Wall Pro Wall Ser Gly 115 12 O 125
Asp Wall Ile Asp Glu His Ser Gly Ile Ser Gly Asp 13 O 135 14 O US 2014/0256009 A1 Sep. 11, 2014 22
- Continued
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 Met Ile Ile Lieu Lys Lieu. Gly Gly Ser Val Ile Thr Arg Lys Asp Ser 1. 5 1O 15 Glu Glu Pro Ala Ile Asp Arg Asp Asn Lieu. Glu Arg Ile Ala Ser Glu 2O 25 30 Ile Gly Asn Ala Ser Pro Ser Ser Leu Met Ile Val 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 Lieu. Arg Arg Arg Arg Phe Gly Phe Ala Lieu. Thir 65 70 7s 8O Glin Asn Trp Val Llys Llys Lieu. Asn. Ser His Val Cys Asp Ala Lieu. Lieu. 85 90 95 Ala Glu Gly Ile Pro Ala Val Ser Met Gln Pro Ser Ala Phe Ile Arg 1OO 105 11 O Ala His Ala Gly Arg Ile Ser His Ala Asp Ile Ser Lieu. Ile Arg Ser 115 12 O 125 Tyr Lieu. Glu Glu Gly Met Val Pro Val Val Tyr Gly Asp Val Val Lieu. 13 O 135 14 O Asp Ser Asp Arg Arg Lieu Lys Phe Ser Val Ile Ser Gly Asp Gln Lieu 145 150 155 160 Ile Asn His Phe Ser Lieu. Arg Lieu Met Pro Glu Arg Val Ile Lieu. Gly 1.65 17O 17s Thir Asp Wall Asp Gly Val Tyr Thr Arg Asn Pro Llys Llys His Pro Asp 18O 185 19 O
Ala Arg Lieu. Lieu. Asp Val Ile Gly Ser Lieu. Asp Asp Lieu. Glu Ser Lieu 195 2OO 2O5
Asp Gly. Thir Lieu. Asn. Thir Asp Val Thr Gly Gly Met Val Gly Lys Ile 21 O 215 22O
Arg Glu Lieu. Lieu Lleu Lieu Ala Glu Lys Gly Val Glu Ser Glu Ile Ile 225 23 O 235 24 O
Asn Ala Ala Val Pro Gly Asn. Ile Glu Arg Ala Lieu. Lieu. Gly Glu Glu 245 250 255 US 2014/0256009 A1 Sep. 11, 2014 23
- Continued
Val Arg Gly Thr Arg Ile Thr Gly Llys His 26 O 265
<210s, SEQ ID NO 6 &211s LENGTH: 260 212. TYPE: PRT <213> ORGANISM: Methanocaldococcus jannaschii <4 OOs, SEQUENCE: 6 Met Lieu. Thir Ile Lieu Lys Lieu. Gly Gly Ser Ile Lieu. Ser Asp Lys Asn 1. 5 1O 15 Val Pro Tyr Ser Ile Llys Trp Asp Asn Lieu. Glu Arg Ile Ala Met Glu 2O 25 3O Ile Lys Asn Ala Lieu. Asp Tyr Tyr Lys Asn Glin Asn Lys Glu Ile Llys 35 4 O 45 Lieu. Ile Lieu Val His Gly Gly Gly Ala Phe Gly His Pro Val Ala Lys SO 55 6 O Llys Tyr Lieu Lys Ile Glu Asp Gly Lys Lys Ile Phe Ile Asn Met Glu 65 70 7s 8O Lys Gly Phe Trp Glu Ile Glin Arg Ala Met Arg Arg Phe Asin Asn. Ile 85 90 95 Ile Ile Asp Thr Lieu. Glin Ser Tyr Asp Ile Pro Ala Val Ser Ile Glin 1OO 105 11 O Pro Ser Ser Phe Val Val Phe Gly Asp Llys Lieu. Ile Phe Asp Thir Ser 115 12 O 125 Ala Ile Lys Glu Met Lieu Lys Arg Asn Lieu Val Pro Val Ile His Gly 13 O 135 14 O Asp Ile Val Ile Asp Asp Lys Asn Gly Tyr Arg Ile Ile Ser Gly Asp 145 150 155 160 Asp Ile Val Pro Tyr Lieu Ala Asn. Glu Lieu Lys Ala Asp Lieu. Ile Lieu 1.65 17O 17s Tyr Ala Thr Asp Wall Asp Gly Val Lieu. Ile Asp Asn Llys Pro Ile Llys 18O 185 19 O Arg Ile Asp Lys Asn. Asn. Ile Tyr Lys Ile Lieu. Asn Tyr Lieu. Ser Gly 195 2OO 2O5 Ser Asn Ser Ile Asp Val Thr Gly Gly Met Lys Tyr Lys Ile Asp Met 21 O 215 22O Ile Arg Lys Asn Lys Cys Arg Gly Phe Val Phe Asin Gly Asn Lys Ala 225 23 O 235 24 O Asn Asn. Ile Tyr Lys Ala Lieu. Lieu. Gly Glu Val Glu Gly Thr Glu Ile 245 250 255 Asp Phe Ser Glu 26 O
<210s, SEQ ID NO 7 &211s LENGTH: 608 212. TYPE: PRT <213> ORGANISM: Pueraria lobata
<4 OO > SEQUENCE: 7 Met Ala Thr Asn Lieu. Lieu. Cys Lieu. Ser Asn Llys Lieu. Ser Ser Pro Thr 1. 5 1O 15
Pro Thr Pro Ser Thr Arg Phe Pro Glin Ser Lys Asn Phe Ile Thr Glin 2O 25 3O US 2014/0256009 A1 Sep. 11, 2014 24
- Continued
Llys Thir Ser Lieu Ala ASn Pro Llys Pro Trp Arg Val Ile Cys Ala Thr 35 4 O 45 Ser Ser Glin Phe Thr Glin Ile Thr Glu. His Asn Ser Arg Arg Ser Ala SO 55 6 O Asn Tyr Glin Pro Asn Lieu. Trp Asin Phe Glu Phe Leu Gln Ser Lieu. Glu 65 70 7s 8O Asn Asp Lieu Lys Val Glu Lys Lieu. Glu Glu Lys Ala Thr Lys Lieu. Glu 85 90 95 Glu Glu Val Arg Cys Met Ile Asn Arg Val Asp Thr Glin Pro Lieu. Ser 1OO 105 11 O Lieu. Lieu. Glu Lieu. Ile Asp Asp Val Glin Arg Lieu. Gly Lieu. Thir Tyr Lys 115 12 O 125 Phe Glu Lys Asp Ile Ile Lys Ala Lieu. Glu Asn. Ile Val Lieu. Lieu. Asp 13 O 135 14 O Glu Asn Llys Lys Asn Llys Ser Asp Lieu. His Ala Thr Ala Lieu. Ser Phe 145 150 155 160 Arg Lieu. Lieu. Arg Gln His Gly Phe Glu Val Ser Glin Asp Val Phe Glu 1.65 17O 17s Arg Phe Lys Asp Llys Glu Gly Gly Phe Ser Gly Glu Lieu Lys Gly Asp 18O 185 19 O Val Glin Gly Lieu Lleu Ser Lieu. Tyr Glu Ala Ser Tyr Lieu. Gly Phe Glu 195 2OO 2O5 Gly Glu Asn Lieu. Lieu. Glu Glu Ala Arg Thr Phe Ser Ile Thr His Lieu. 21 O 215 22O Lys Asn. Asn Lieu Lys Glu Gly Ile Asn. Thir Lys Val Ala Glu Glin Val 225 23 O 235 24 O Ser His Ala Lieu. Glu Lieu Pro Tyr His Glin Arg Lieu. His Arg Lieu. Glu 245 250 255 Ala Arg Trp Phe Lieu. Asp Llys Tyr Glu Pro Lys Glu Pro His His Glin 26 O 265 27 O Lieu. Lieu. Lieu. Glu Lieu Ala Lys Lieu. Asp Phe Asn Met Val Glin Thr Lieu. 27s 28O 285 His Gln Lys Glu Lieu. Glin Asp Lieu. Ser Arg Trp Trp Thr Glu Met Gly 29 O 295 3 OO Lieu Ala Ser Lys Lieu. Asp Phe Val Arg Asp Arg Lieu Met Glu Val Tyr 3. OS 310 315 32O Phe Trp Ala Leu Gly Met Ala Pro Asp Pro Glin Phe Gly Glu. Cys Arg 3.25 330 335 Lys Ala Val Thir Lys Met Phe Gly Lieu Val Thir Ile Ile Asp Asp Wall 34 O 345 35. O Tyr Asp Val Tyr Gly Thr Lieu. Asp Glu Lieu Gln Lieu. Phe Thir Asp Ala 355 360 365
Val Glu Arg Trp Asp Val Asn Ala Ile Asn. Thir Lieu Pro Asp Tyr Met 37 O 375 38O
Llys Lieu. Cys Phe Lieu Ala Lieu. Tyr Asn Thr Val Asn Asp Thir Ser Tyr 385 390 395 4 OO Ser Ile Lieu Lys Glu Lys Gly His Asn. Asn Lieu. Ser Tyr Lieu. Thir Lys 4 OS 41O 415
Ser Trp Arg Glu Lieu. Cys Lys Ala Phe Lieu. Glin Glu Ala Lys Trp Ser 42O 425 43 O US 2014/0256009 A1 Sep. 11, 2014 25
- Continued
Asn Asn Lys Ile Ile Pro Ala Phe Ser Lys Tyr Lieu. Glu Asn Ala Ser 435 44 O 445 Val Ser Ser Ser Gly Val Ala Lieu. Leu Ala Pro Ser Tyr Phe Ser Val 450 45.5 460 Cys Glin Glin Glin Glu Asp Ile Ser Asp His Ala Lieu. Arg Ser Lieu. Thir 465 470 47s 48O Asp Phe His Gly Lieu Val Arg Ser Ser Cys Val Ile Phe Arg Lieu. Cys 485 490 495 Asn Asp Lieu Ala Thir Ser Ala Ala Glu Lieu. Glu Arg Gly Glu Thir Thr SOO 505 51O Asn Ser Ile Ile Ser Tyr Met His Glu Asn Asp Gly Thr Ser Glu Glu 515 52O 525 Glin Ala Arg Glu Glu Lieu. Arg Llys Lieu. Ile Asp Ala Glu Trp Llys Llys 53 O 535 54 O Met Asn Arg Glu Arg Val Ser Asp Ser Thir Lieu Lleu Pro Lys Ala Phe 5.45 550 555 560 Met Glu Ile Ala Val Asn Met Ala Arg Val Ser His Cys Thr Tyr Glin 565 st O sts Tyr Gly Asp Gly Lieu. Gly Arg Pro Asp Tyr Ala Thr Glu Asn Arg Ile 58O 585 59 O Llys Lieu. Lieu. Lieu. Ile Asp Pro Phe Pro Ile Asin Glin Lieu Met Tyr Val 595 6OO 605
<210s, SEQ ID NO 8 &211s LENGTH: 582 212. TYPE: PRT <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 US 2014/0256009 A1 Sep. 11, 2014 26
- Continued
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 Trp Tyr Asp Lieu. Cys Lys Ala Phe Lieu. Glin Glu Ala Lys Trp Ser Asn 4 OS 41O 415 Ser Arg Ile Ile Pro Gly Val Glu Glu Tyr Lieu. Asn. Asn Gly Trp Val 42O 425 43 O Ser Ser Ser Gly Ser Val Met Lieu. Ile His Ala Tyr Phe Leu Ala Ser 435 44 O 445 Pro Ser Ile Arg Lys Glu Glu Lieu. Glu Ser Lieu. Glu. His Tyr His Asp 450 45.5 460 Lieu. Lieu. Arg Lieu Pro Ser Lieu. Ile Phe Arg Lieu. Thir Asn Asp Ile Ala 465 470 47s 48O Ser Ser Ser Ala Glu Lieu. Glu Arg Gly Glu Thir Thr Asn. Ser Ile Arg 485 490 495 Cys Phe Met Glin Glu Lys Gly Ile Ser Glu Lieu. Glu Ala Arg Glu. Cys SOO 505 51O Val Lys Glu Glu Ile Asp Thr Ala Trp Llys Llys Met Asn Llys Tyr Met 515 52O 525
Val Asp Arg Ser Thr Phe Asn Glin Ser Phe Val Arg Met Thr Tyr Asn 53 O 535 54 O Lieu Ala Arg Met Ala His Cys Val Tyr Glin Asp Gly Asp Ala Ile Gly 5.45 550 555 560 Ser Pro Asp Asp Lieu. Ser Trp Asin Arg Val His Ser Lieu. Ile Ile Llys 565 st O sts
Pro Ile Ser Pro Ala Ala 58O US 2014/0256009 A1 Sep. 11, 2014 27
- Continued
<210s, SEQ ID NO 9 &211s LENGTH: 595 212. TYPE: PRT <213> ORGANISM: Lotus japonicus <4 OOs, SEQUENCE: 9
Met Ala Glin Ser Phe Ser Met Wall Lieu. Asn. Ser Ser Phe Thir Ser His 1. 5 1O 15 Pro Ile Phe Cys Llys Pro Gln Lys Lieu. Ile Ile Arg Gly. His Asn Lieu. 2O 25 3O Lieu. Glin Gly His Arg Ile Asn Ser Pro Ile Pro Cys Tyr Ala Ser Thr 35 4 O 45 Ser Ser Thr Ser Val Ser Glin Arg Llys Ser Ala Asn Tyr Glin Pro Asn SO 55 6 O Ile Trp Asn Tyr Asp Tyr Lieu. Glin Ser Lieu Lys Lieu. Gly Tyr Ala Asp 65 70 7s 8O Ala His Tyr Glu Asp Met Ala Lys Llys Lieu. Glin Glu Glu Val Arg Arg 85 90 95 Ile Ile Lys Asp Asp Lys Ala Glu Ile Trp Thir Thr Lieu. Glu Lieu. Ile 1OO 105 11 O Asp Asp Wall Lys Arg Lieu. Gly Lieu. Gly Tyr His Phe Glu Lys Glu Ile 115 12 O 125 Arg Glu Val Lieu. Asn Llys Phe Lieu. Ser Lieu. Asn. Thir Cys Val His Arg 13 O 135 14 O Ser Lieu. Asp Llys Thr Ala Lieu. Cys Phe Arg Lieu. Lieu. Arg Glu Tyr Gly 145 150 155 160 Ser Asp Wal Ser Ala Asp Ile Phe Glu Arg Phe Lieu. Asp Glin Asn Gly 1.65 17O 17s Asn Phe Llys Thir Ser Lieu Val Asn. Asn. Wall Lys Gly Met Lieu. Ser Lieu 18O 185 19 O Tyr Glu Ala Ser Phe Lieu. Ser Tyr Glu Gly Glu Glin Ile Lieu. Asp Llys 195 2OO 2O5 Ala Asn Ala Phe Thir Ser Phe His Lieu Lys Ser Ile His Glu Glu Asp 21 O 215 22O
Ile Asn. Asn. Ile Lieu. Lieu. Glu Glin Wall Asn His Ala Lieu. Glu Lieu Pro 225 23 O 235 24 O Lieu. His Arg Arg Ile His Arg Lieu. Glu Ala Arg Trp Tyr Thr Glu Ser 245 250 255 Tyr Ser Arg Arg Lys Asp Ala Asn Trp Val Lieu. Lieu. Glu Ala Ala Lys 26 O 265 27 O Lieu. Asp Phe Asn Met Val Glin Ser Thr Lieu Gln Lys Asp Lieu. Glin Glu 27s 28O 285 Met Ser Arg Trp Trp Lys Gly Met Gly Lieu Ala Pro Llys Lieu. Ser Phe 29 O 295 3 OO
Ser Arg Asp Arg Lieu Met Glu. Cys Phe Phe Trp Thr Val Gly Met Ala 3. OS 310 315 32O
Phe Glu Pro Llys Tyr Ser Asp Lieu. Arg Lys Gly Lieu. Thir Lys Val Thr 3.25 330 335 Ser Lieu. Ile Thr Thr Ile Asp Asp Ile Tyr Asp Val His Gly Thr Lieu. 34 O 345 35. O
Glu Glu Lieu. Glu Lieu. Phe Thr Ala Ile Val Glu Ser Trp Asp Ile Llys 355 360 365 US 2014/0256009 A1 Sep. 11, 2014 28
- Continued
Ala Met Glin Val Lieu Pro Glu Tyr Met Lys Ile Ser Phe Leu Ala Leu 37 O 375 38O Tyr Asn Thr Val Asn. Glu Lieu Ala Tyr Asp Ala Lieu. Arg Glu Glin Gly 385 390 395 4 OO His Asp Ile Lieu Pro Tyr Lieu. Thir Lys Ala Trp Ser Asp Met Lieu Lys 4 OS 41O 415 Ala Phe Lieu. Glin Glu Ala Lys Trp Cys Arg Glu Lys His Lieu Pro Llys 42O 425 43 O Phe Glu. His Tyr Lieu. Asn Asn Ala Trp Val Ser Val Ser Gly Val Val 435 44 O 445 Ile Lieu. Thr His Ala Tyr Phe Leu Lieu. Asn His Asn Thr Thr Lys Glu 450 45.5 460 Val Lieu. Glu Ala Lieu. Glu Asn Tyr His Ala Lieu Lleu Lys Arg Pro Ser 465 470 47s 48O Ile Ile Phe Arg Lieu. Cys Asn Asp Lieu. Gly. Thir Ser Thr Ala Glu Lieu. 485 490 495 Glin Arg Gly Glu Val Ala Asn. Ser Ile Lieu. Ser Cys Met His Glu Asn SOO 505 51O Asp Ile Gly Glu Glu Ser Ala His Gln His Ile His Ser Lieu. Lieu. Asn 515 52O 525 Glu Thir Trp Llys Lys Met Asn Arg Asp Arg Phe Ile His Ser Pro Phe 53 O 535 54 O Pro Glu Pro Phe Val Glu Ile Ala Thr Asn Lieu Ala Arg Ile Ala Glin 5.45 550 555 560 Cys Thr Tyr Glin Thr Gly Asp Gly His Gly Ala Pro Asp Ser Ile Ala 565 st O sts Lys Asn Arg Val Llys Ser Lieu. Ile Ile Glu Pro Ile Val Lieu. Asn Gly 58O 585 59 O Asp Ile Tyr 595
<210s, SEQ ID NO 10 &211s LENGTH: 593 212. TYPE: PRT <213s ORGANISM: Phaseolus lunatus
<4 OOs, SEQUENCE: 10 Met Leu Lieu. Asn Ser Ser Phe Ile Ser Arg Val Thr Phe Ala Lys Pro 1. 5 1O 15 Lieu Lys Pro Val Ala Pro Asn Lieu. Lieu. His Arg Arg Ile Ile Phe Pro 2O 25 3O Arg Cys Asn Gly. Thir Thir Ile Asin Val Asn Ala Ser Glu Arg Llys Ser 35 4 O 45
Ala Asn Tyr Glin Pro Asn Lieu. Trp Thr Tyr Asp Phe Leu Glin Ser Leu SO 55 6 O Llys His Ala Tyr Ala Asp Thr Arg Tyr Glu Asp Arg Ala Lys Glin Lieu. 65 70 7s 8O
Glin Glu Glu Val Arg Llys Met Ile Lys Asp Glu Asn. Ser Asp Met Trp 85 90 95
Lieu Lys Lieu. Glu Lieu. Ile Asn Asp Wall Lys Arg Lieu. Gly Lieu. Ser Tyr 1OO 105 11 O His Tyr Asp Llys Glu Ile Gly Glu Ala Lieu. Lieu. Arg Phe His Ser Ser US 2014/0256009 A1 Sep. 11, 2014 29
- Continued
115 12 O 125 Ala Thr Phe Ser Gly Thr Ile Val His Arg Ser Lieu. His Glu. Thir Ala 13 O 135 14 O Lieu. Cys Phe Arg Lieu. Lieu. Arg Glu Tyr Gly Tyr Asp Val Thir Ala Asp 145 150 155 160 Met Phe Glu Arg Phe Lys Glu Arg Asn Gly His Phe Lys Ala Ser Lieu. 1.65 17O 17s Met Ser Asp Wall Lys Gly Met Lieu. Ser Lieu. Tyr Glin Ala Ser Phe Lieu. 18O 185 19 O Gly Tyr Glu Gly Glu Glin Ile Lieu. Asp Asp Ala Lys Ala Phe Ser Ser 195 2OO 2O5 Phe His Lieu Lys Ser Val Lieu. Ser Glu Gly Arg Asn. Asn Met Val Lieu. 21 O 215 22O Glu Glu Val Asn His Ala Lieu. Glu Lieu Pro Lieu. His His Arg Ile Glin 225 23 O 235 24 O Arg Lieu. Glu Ala Arg Trp Tyr Ile Glu Tyr Tyr Ala Lys Glin Arg Asp 245 250 255 Ser Asn Arg Val Lieu. Lieu. Glu Ala Ala Lys Lieu. Asp Phe Asn. Ile Lieu. 26 O 265 27 O Glin Ser Thr Lieu. Glin Asn Asp Lieu. Glin Glu Val Ser Arg Trip Trp Llys 27s 28O 285 Gly Met Gly Lieu Ala Ser Llys Lieu. Ser Phe Ser Arg Asp Arg Lieu Met 29 O 295 3 OO Glu Cys Phe Phe Trp Ala Ala Gly Met Val Phe Glu Pro Glin Phe Ser 3. OS 310 315 32O Asp Lieu. Arg Lys Gly Lieu. Thir Lys Val Ala Ser Lieu. Ile Thir Thir Ile 3.25 330 335 Asp Asp Val Tyr Asp Val Tyr Gly. Thir Lieu. Glu Glu Lieu. Glu Lieu. Phe 34 O 345 35. O Thir Ala Ala Val Glu Ser Trp Asp Wall Lys Ala Ile Glin Val Lieu Pro 355 360 365 Asp Tyr Met Lys Ile Cys Phe Lieu Ala Lieu. Tyr Asn Thr Val Asn. Glu 37 O 375 38O Phe Ala Tyr Asp Ala Lieu Lys Glu Glin Gly Glin Asp Ile Lieu Pro Tyr 385 390 395 4 OO Lieu. Thir Lys Ala Trp Ser Asp Lieu. Lieu Lys Ala Phe Lieu. Glin Glu Ala 4 OS 41O 415 Llys Trp Ser Arg Asp Arg His Met Pro Arg Phe Asn Asp Tyr Lieu. Asn 42O 425 43 O Asn Ala Trp Val Ser Val Ser Gly Val Val Lieu Lleu. Thr His Ala Tyr 435 44 O 445 Phe Lieu. Lieu. Asn His Ser Ile Thr Glu Glu Ala Lieu. Glu Ser Lieu. Asp 450 45.5 460
Ser Tyr His Ser Lieu. Leu Gln Asn Thr Ser Leu Val Phe Arg Lieu. Cys 465 470 47s 48O
Asn Asp Lieu. Gly. Thir Ser Lys Ala Glu Lieu. Glu Arg Gly Glu Ala Ala 485 490 495
Ser Ser Ile Lieu. Cys Tyr Arg Arg Glu Ser Gly Ala Ser Glu Glu Gly SOO 505 51O Ala Tyr Lys His Ile Tyr Ser Lieu. Lieu. Asn. Glu Thir Trp Llys Llys Met 515 52O 525 US 2014/0256009 A1 Sep. 11, 2014 30
- Continued
Asn Glu Asp Arg Val Ser Glin Ser Pro Phe Pro Lys Ala Phe Val Glu 53 O 535 54 O Thr Ala Met Asn Lieu Ala Arg Ile Ser His Cys Thr Tyr Glin Tyr Gly 5.45 550 555 560 Asp Gly His Gly Ala Pro Asp Ser Thr Ala Lys Asn Arg Ile Arg Ser 565 st O sts Lieu. Ile Ile Glu Pro Ile Ala Leu Tyr Glu Thr Glu Ile Ser Thr 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 Lieu Lleu Ser Thr Pro His Lieu Pro Gln Lieu. Cys Ser 1. 5 1O 15 Arg Arg Val Ser Gly Arg Val His Cys Ser Ala Ser Thr Glin Val Ser 2O 25 3O Asp Ala Glin Gly Gly Arg Arg Ser Ala Asn Tyr Glin Pro Ser Val Trip 35 4 O 45 Thir Tyr Asn Tyr Lieu. Glin Ser Lieu Val Ala Asp Asp Ile Arg Arg Ser 50 55 60 Arg Arg Glu Val Glu Glin Glu Arg Glu Lys Ala Glin Ile Lieu. Glu Glu 65 70 7s 8O Asp Val Arg Gly Ala Lieu. Asn Asp Gly Asn Ala Glu Pro Met Ala Ile 85 90 95 Phe Ala Lieu Val Asp Asp Ile Glin Arg Lieu. Gly Lieu. Gly Arg Tyr Phe 1OO 105 11 O Glu Glu Asp Ile Ser Lys Ala Lieu. Arg Arg Cys Lieu. Ser Glin Tyr Ala 115 12 O 125 Val Thr Gly Ser Leu Gln Lys Ser Lieu. His Gly Thr Ala Leu Ser Phe 13 O 135 14 O Arg Val Lieu. Arg Gln His Gly Phe Glu Val Ser Glin Asp Val Phe Lys 145 150 155 160 Ile Phe Met Asp Glu Ser Gly Ser Phe Met Lys Thr Lieu. Gly Gly Asp 1.65 17O 17s Val Glin Gly Met Lieu. Ser Lieu. Tyr Glu Ala Ser His Lieu Ala Phe Glu 18O 185 19 O Glu Glu Asp Ile Lieu. His Lys Ala Lys Thir Phe Ala Ile Llys His Lieu. 195 2OO 2O5 Glu Asn Lieu. Asn His Asp Ile Asp Glin Asp Lieu. Glin Asp His Val Asn 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 Phe Ile Glu Ala Tyr Ser Arg Arg Ser Asn. Wall Asn Pro Arg 245 250 255
Ile Lieu. Glu Lieu Ala Val Met Llys Phe Asn. Ser Ser Glin Lieu. Thir Lieu. 26 O 265 27 O
Glin Arg Asp Lieu. Glin Asp Met Lieu. Gly Trp Trp Asn. Asn Val Gly Lieu. 27s 28O 285 US 2014/0256009 A1 Sep. 11, 2014 31
- Continued
Ala Lys Arg Lieu. Ser Phe Ala Arg Asp Arg Lieu Met Glu. Cys Phe Phe 29 O 295 3 OO Trp Ala Val Gly Ile Ala Arg Glu Pro Ala Lieu. Ser Asn. Cys Arg Llys 3. OS 310 315 32O Gly Val Thir Lys Ala Phe Ser Lieu. Ile Lieu Val Lieu. Asp Asp Val Tyr 3.25 330 335 Asp Val Phe Gly Thr Lieu. Asp Glu Lieu. Glu Lieu. Phe Thr Asp Ala Val 34 O 345 35. O Arg Arg Trp His Glu Asp Ala Val Glu Asn Lieu Pro Gly Tyr Met Lys 355 360 365 Lieu. Cys Phe Lieu Ala Lieu. Tyr Asn. Ser Val Asn Asp Met Ala Tyr Glu 37 O 375 38O Thr Lieu Lys Glu Thr Gly Glu Asn Val Thr Pro Tyr Lieu. Thir Lys Val 385 390 395 4 OO Trp Tyr Asp Lieu. Cys Lys Ala Phe Lieu. Glin Glu Ala Lys Trp Ser Tyr 4 OS 41O 415 Asn Lys Ile Thr Pro Gly Val Glu Glu Tyr Lieu. Asn. Asn Gly Trp Val 42O 425 43 O Ser Ser Ser Gly Glin Val Met Lieu. Thr His Ala Tyr Phe Leu Ser Ser 435 44 O 445 Pro Ser Lieu. Arg Lys Glu Glu Lieu. Glu Ser Lieu. Glu. His Tyr His Asp 450 45.5 460 Lieu. Lieu. Arg Lieu Pro Ser Lieu. Ile Phe Arg Lieu. Thir Asn Asp Lieu Ala 465 470 47s 48O Thir Ser Ser Ala Glu Lieu. Gly Arg Gly Glu Thir Thr Asn Ser Ile Leu 485 490 495 Cys Tyr Met Arg Glu Lys Gly Phe Ser Glu Ser Glu Ala Arg Lys Glin SOO 505 51O Val Ile Glu Glin Ile Asp Thr Ala Trp Arg Glin Met Asn Llys Tyr Met 515 52O 525 Val Asp His Ser Thr Phe Asn Arg Ser Phe Met Gln Met Thr Tyr Asn 53 O 535 54 O Lieu Ala Arg Met Ala His Cys Val Tyr Glin Asp Gly Asp Ala Ile Gly 5.45 550 555 560 Ala Pro Asp Asp Glin Ser Trp Asn Arg Val His Ser Lieu. Ile Ile Llys 565 st O sts 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 US 2014/0256009 A1 Sep. 11, 2014 32
- Continued
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 Arg Trp Asp Ile Asn Met Val Asn. Asn Lieu Pro Gly Tyr Met Lys Lieu 37 O 375 38O
Cys Phe Leu Ala Lieu. Tyr Asn Thr Val Asin Glu Met Ala Tyr Asp Thr 385 390 395 4 OO Lieu Lys Glu Glin Gly His Asn. Ile Lieu Pro Tyr Lieu. Thir Lys Ala Trp 4 OS 41O 415 Ala Asp Lieu. Cys Llys Val Phe Lieu Val Glu Ala Lys Trp Cys His Lys 42O 425 43 O
Glu Tyr Thr Pro Thr Phe Glu Glu Tyr Lieu. Glu Asn Gly Trp Arg Ser 435 44 O 445
Val Ser Gly Ala Ala Ile Lieu. Ile His Ala Tyr Phe Lieu Met Ser Lys 450 45.5 460 US 2014/0256009 A1 Sep. 11, 2014 33
- Continued
Asn. Ile Thir Lys Glu Ala Lieu. Glu. Cys Lieu. Glu Asn Asp His Glu Lieu 465 470 47s 48O Lieu. Arg Trp Pro Ser Thir Ile Phe Arg Lieu. Cys Asn Asp Lieu Ala Thr 485 490 495 Ser Lys Ala Glu Lieu. Glu Arg Gly Glu Ser Ala Asn. Ser Ile Ser Cys SOO 505 51O Tyr Met His Glin Thr Gly Val Ser Glu Glu Asp Ala Arg Glu. His Met 515 52O 525 Lys Ile Lieu. Ile Asp Glu Ser Trp Llys Llys Met Asn Llys Val Arg Glu 53 O 535 54 O Met Asp Ser Asp Ser Pro Phe Ala Lys Pro Phe Val Glu Thir Ala Ile 5.45 550 555 560 Asn Lieu Ala Arg Ile Ala Glin Cys Thr Tyr Glin Tyr Gly Asp Ser His 565 st O sts Gly Ala Pro Asp Ala Arg Ser Lys Lys Arg Val Lieu. Ser Lieu. Ile Val 58O 585 59 O Glu Pro Ile Pro Met Asn Lieu Lys Llys 595 6OO
<210s, SEQ ID NO 13 &211s LENGTH: 661 212. TYPE: PRT <213> ORGANISM: Marinobacter aquaeolei VT8 <4 OOs, SEQUENCE: 13 Met Asn Tyr Phe Lieu. Thr Gly Gly Thr Gly Phe Ile Gly Arg Phe Leu 1. 5 1O 15 Val Glu Lys Lieu. Lieu Ala Arg Gly Gly Thr Val Tyr Val Lieu Val Arg 2O 25 3O Glu Glin Ser Glin Asp Llys Lieu. Glu Arg Lieu. Arg Glu Arg Trp Gly Ala 35 4 O 45 Asp Asp Llys Glin Val Lys Ala Val Ile Gly Asp Lieu. Thir Ser Lys Asn SO 55 6 O Lieu. Gly Ile Asp Ala Lys Thr Lieu Lys Ser Lieu Lys Gly Asn. Ile Asp 65 70 7s 8O His Val Phe His Lieu Ala Ala Val Tyr Asp Met Gly Ala Asp Glu Glu 85 90 95 Ala Glin Ala Ala Thr Asn. Ile Glu Gly Thr Arg Ala Ala Val Glin Ala 1OO 105 11 O Ala Glu Ala Met Gly Ala Lys His Phe His His Val Ser Ser Ile Ala 115 12 O 125 Ala Ala Gly Lieu. Phe Lys Gly Ile Phe Arg Glu Asp Met Phe Glu Glu 13 O 135 14 O Ala Glu Lys Lieu. Asp His Pro Tyr Lieu. Arg Thr Llys His Glu Ser Glu 145 150 155 160
Llys Val Val Arg Glu Glu. Cys Llys Val Pro Phe Arg Ile Tyr Arg Pro 1.65 17O 17s Gly Met Val Ile Gly His Ser Glu Thr Gly Glu Met Asp Llys Val Asp 18O 185 19 O Gly Pro Tyr Tyr Phe Phe Lys Met Ile Glin Lys Ile Arg His Ala Leu 195 2OO 2O5
Pro Gln Trp Val Pro Thir Ile Gly Ile Glu Gly Gly Arg Lieu. Asn Ile US 2014/0256009 A1 Sep. 11, 2014 34
- Continued
21 O 215 22O Val Pro Val Asp Phe Val Val Asp Ala Lieu. Asp His Ile Ala His Lieu 225 23 O 235 24 O Glu Gly Glu Asp Gly Asn. Cys Phe His Lieu Val Asp Ser Asp Pro Tyr 245 250 255 Llys Val Gly Glu Ile Lieu. Asn. Ile Phe Cys 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 27s 28O 285 Phe Ile Arg Glin Ser Ile Lys Asn Lieu Pro Pro Val Lys Arg Ile Thr 29 O 295 3 OO Gly Ala Leu Lieu. Asp Asp Met Gly Ile Pro Pro Ser Val Met Ser Phe 3. OS 310 315 32O Ile Asn Tyr Pro Thr Arg Phe Asp Thr Arg Glu Lieu. Glu Arg Val Lieu. 3.25 330 335 Lys Gly Thr Asp Ile Glu Val Pro Arg Lieu Pro Ser Tyr Ala Pro Val 34 O 345 35. O Ile Trp Asp Tyr Trp Glu Arg Asn Lieu. Asp Pro Asp Lieu. Phe Lys Asp 355 360 365 Arg Thr Lieu Lys Gly Thr Val Glu Gly Llys Val Cys Val Val Thr Gly 37 O 375 38O Ala Thir Ser Gly Ile Gly Lieu Ala Thir Ala Glu Lys Lieu Ala Glu Ala 385 390 395 4 OO Gly Ala Ile Lieu Val Ile Gly Ala Arg Thir Lys Glu Thir Lieu. Asp Glu 4 OS 41O 415 Val Ala Ala Ser Lieu. Glu Ala Lys Gly Gly Asn. Wal His Ala Tyr Glin 42O 425 43 O Cys Asp Phe Ser Asp Met Asp Asp Cys Asp Arg Phe Val Lys Thr Val 435 44 O 445 Lieu. Asp Asn His Gly His Val Asp Val Lieu Val Asn. Asn Ala Gly Arg 450 45.5 460 Ser Ile Arg Arg Ser Lieu Ala Lieu. Ser Phe Asp Arg Phe His Asp Phe 465 470 47s 48O Glu Arg Thr Met Gln Lieu. Asn Tyr Phe Gly Ser Val Arg Lieu. Ile Met 485 490 495 Gly Phe Ala Pro Ala Met Lieu. Glu Arg Arg Arg Gly His Val 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 Ser Ser Lys Ser Ala Lieu. Asp Ala Phe Ser Arg Cys Ala Ala Ala 53 O 535 54 O
Glu Trp Ser Asp Arg Asn Val Thr Phe Thr Thr Ile Asn Met Pro Leu 5.45 550 555 560
Val Lys Thr Pro Met Ile Ala Pro Thr Lys Ile Tyr Asp Ser Val Pro 565 st O sts
Thir Lieu. Thr Pro Asp Glu Ala Ala Glin Met Val Ala Asp Ala Ile Val 58O 585 59 O
Tyr Arg Pro Lys Arg Ile Ala Thr Arg Lieu. Gly Val Phe Ala Glin Val 595 6OO 605
Lieu. His Ala Lieu Ala Pro Llys Met Gly Glu Ile Ile Met Asn Thr Gly 610 615 62O US 2014/0256009 A1 Sep. 11, 2014 35
- Continued
Tyr Arg Met Phe Pro Asp Ser Pro Ala Ala Ala Gly Ser Lys Ser 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 Tyr Trp 660
<210s, SEQ ID NO 14 &211s LENGTH: 540 212. TYPE: PRT <213> ORGANISM: Marinobacter manganoxydans
<4 OOs, SEQUENCE: 14 Met Asn Tyr Phe Lieu. Thr Gly Gly Thr Gly Phe Ile Gly Arg Phe Leu 1. 5 1O 15 Val Glu Lys Lieu. Lieu Ala Arg Gly Gly Thr Val His Val Lieu Val Arg 2O 25 3O Glu Glin Ser Glin Asp Llys Lieu. Asp Llys Lieu. Arg Glu Arg Trp Gly Ala 35 4 O 45 Asp Glu Thr Glin Val Lys Ala Val Ile Gly Asp Lieu. Thir Ser Lys Asn SO 55 6 O Lieu. Gly Ile Asp Ala Lys Thr Met Lys Ala Lieu Lys Gly Lys Ile Asp 65 70 7s 8O His Phe Phe His Lieu Ala Ala Val Tyr Asp Met Gly Ala Asp Glu Glu 85 90 95 Ala Glin Glin Ala Thr Asn. Ile Glu Gly Thr Arg Ala Ala Val Asn Ala 1OO 105 11 O Ala Glu Ala Met Gly Ala Lys His Phe His His Val Ser Ser Ile Ala 115 12 O 125 Ala Ala Gly Lieu. Phe Lys Gly Ile Phe Arg Glu Asp Met Phe Glu Glu 13 O 135 14 O Ala Glu Lys Lieu. Asp His Pro Tyr Lieu. Arg Thr Llys His Glu Ser Glu 145 150 155 160 Llys Val Val Arg Glu Glu. Cys Llys Val Pro Phe Arg Ile Tyr Arg Pro 1.65 17O 17s Gly Met Val Ile Gly His Thr Ala Thr Gly Glu Met Asp Llys Val Asp 18O 185 19 O Gly Pro Tyr Tyr Phe Phe Lys Met Ile Glin Lys Ile Arg His Ala Leu 195 2OO 2O5 Pro Gln Trp Val Pro Thir Ile Gly Val Glu Gly Gly Arg Lieu. Asn Ile 21 O 215 22O Val Pro Val Asp Phe Val Val Asn Ala Met Asp His Ile Ala His Lieu 225 23 O 235 24 O Glu Gly Glu Asp Gly Lys Cys Phe His Lieu Val Asp Thr Asp Pro Tyr 245 250 255
Llys Val Gly Glu Ile Lieu. 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 27s 28O 285
Phe Ile Arg Glin Ser Lieu Lys Asn Lieu Pro Pro Val Lys Arg Lieu. Thir 29 O 295 3 OO
Ser Ala Ile Lieu. Asp Asp Met Gly Ile Pro Pro Ser Val Met Ser Phe US 2014/0256009 A1 Sep. 11, 2014 36
- Continued
3. OS 310 315 32O Ile Asn Tyr Pro Thr Arg Phe Asp Ala Arg Glu Thr Glu Arg Val Lieu. 3.25 330 335 Lys Gly Thr Gly Ile Glu Val Pro Arg Lieu Pro Asp Tyr Ala Pro Val 34 O 345 35. O Ile Trp Asp Tyr Trp Glu Arg Asn Lieu. Asp Pro Asp Lieu. Phe Lys Asp 355 360 365 Arg Thr Lieu Lys Gly Thr Val Glu Gly Arg Val Cys Val Val Thr Gly 37 O 375 38O Ala Thir Ser Gly Ile Gly Lieu Ala Thir Ala Glin Llys Lieu Ala Asp Ala 385 390 395 4 OO Gly Ala Ile Lieu Val Ile Gly Ala Arg Llys Lieu. Glu Arg Lieu Lys Glu 4 OS 41O 415 Val Ala Ala Glu Lieu. Glu Ser Arg Gly Ala Ser Val His Ala Tyr Pro 42O 425 43 O Cys Asp Phe Ser Asp Met Asp Ala Cys Asp Glu Phe Val Lys Thr Val 435 44 O 445 Lieu. Asp Asn His Gly Glin Val Asp Val Lieu Val Asn. Asn Ala Gly Arg 450 45.5 460 Ser Ile Arg Arg Ser Lieu. Asp Lieu. Ser Phe Asp Arg Phe His Asp Phe 465 470 47s 48O Glu Arg Thr Met Gln Lieu. Asn Tyr Phe Gly Ser Val Arg Lieu. Ile Met 485 490 495 Gly Phe Ala Pro Llys Met Lieu. Glu Asn Arg Arg Gly His Val 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 Ser Ala Lieu. 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 Thr Gly Gly Thr Gly Phe Ile Gly Arg Phe Leu 1. 5 1O 15 Ile Ala Arg Lieu. Lieu Ala Arg Gly Ala Ile Val His Val Lieu Val Arg 2O 25 3O Glu Glin Ser Val Glin Llys Lieu Ala Asp Lieu. Arg Glu Lys Lieu. Gly Ala 35 4 O 45 Asp Glu Lys Glin Ile Lys Ala Val Val Gly Asp Lieu. Thir Ala Pro Gly SO 55 6 O
Lieu. Gly Lieu. Asp Llys Llys Thr Lieu Lys Glin Lieu. Ser Gly Lys Ile Asp 65 70 7s 8O His Phe Phe His Leu Ala Ala Ile Tyr Asp Met Ser Ala Ser Glu Glu 85 90 95
Ser Glin Glin Ala Ala Asn. Ile Asp Gly Thr Arg Ala Ala Val Ala Ala 1OO 105 11 O Ala Glu Ala Lieu. Gly Ala Gly Ile Phe His His Val Ser Ser Ile Ala 115 12 O 125 US 2014/0256009 A1 Sep. 11, 2014 37
- Continued Val Ala Gly Lieu. Phe Lys Gly Thr Phe Arg Glu Asp Met Phe Ala Glu 13 O 135 14 O Ala Gly Lys Lieu. Asp His Pro Tyr Phe Ser Thr Lys His Glu Ser Glu 145 150 155 160 Arg Val Val Arg Asp Glu. Cys Llys Lieu Pro Phe Arg Ile Tyr Arg Pro 1.65 17O 17s Gly Met Val Ile Gly Asp Ser Ala Thr Gly Glu Met Asp Llys Val Asp 18O 185 19 O Gly Pro Tyr Tyr Phe Phe Lys Met Ile Glin Lys Ile Arg Gly Ala Leu 195 2OO 2O5 Pro Gln Trp Val Pro Thir Ile Gly Lieu. Glu Gly Gly Arg Lieu. Asn Ile 21 O 215 22O Val Pro Val Asn. Phe Val Ala Asp Ala Lieu. Asp His Ile Ala His Lieu 225 23 O 235 24 O Pro Asp Glu Asp Gly Lys Cys Phe His Lieu Val Asp Ser Asp Pro Tyr 245 250 255 Llys Val Gly Glu Ile Lieu. Asn. Ile Phe Cys Glu Ala Gly. His Ala Pro 26 O 265 27 O Lys Met Gly Met Arg Ile Asp Ser Arg Met Phe Gly Phe Val Pro Pro 27s 28O 285 Phe Ile Arg Glin Ser Lieu Lys Asn Lieu Pro Pro Val Lys Arg Met Gly 29 O 295 3 OO Arg Ala Lieu Lieu. Asp Asp Lieu. Gly Ile Pro Ala Ser Val Lieu Ser Phe 3. OS 310 315 32O Ile Asn Tyr Pro Thr Arg Phe Asp Ala Arg Glu Thr Glu Arg Val Lieu. 3.25 330 335 Gln Gly Thr Gly Ile Glu Val Pro Arg Lieu Pro Asp Tyr Ala Pro Val 34 O 345 35. O Ile Trp Asp Tyr Trp Glu Arg Asn Lieu. Asp Pro Asp Lieu. Phe Thr Asp 355 360 365 Arg Thr Lieu. Arg Gly Thr Val Glu Gly Llys Val Cys Val Val Thr Gly 37 O 375 38O Ala Thir Ser Gly Ile Gly Lieu Ala Thir Ala Glu Lys Lieu Ala Asp Ala 385 390 395 4 OO Gly Ala Ile Lieu Val Ile Gly Ala Arg Thr Glin Glu Thir Lieu. Asp Glin 4 OS 41O 415 Val Ser Ala Glin Lieu. Asn Ala Arg Gly Ala Asp Wal His Ala Tyr Glin 42O 425 43 O Cys Asp Phe Ala Asp Met Asp Ala Cys Asp Arg Phe Ile Glin Thr Val 435 44 O 445 Ser Glu Asn His Gly Ala Val Asp Val Lieu. Ile Asn. Asn Ala Gly Arg 450 45.5 460 Ser Ile Arg Arg Ser Lieu. Asp Llys Ser 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 Ala Met Lieu. Glu Arg Arg Arg Gly His Ile Ile 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 Ser Phe Ser Arg Cys Ala Ala Ala US 2014/0256009 A1 Sep. 11, 2014 38
- Continued
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 Tyr 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
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
Pro Glin Trp Wall Pro Thir Ile Gly Ile Glu Gly Gly Arg Luell Asn Ile 21 O 215 22O US 2014/0256009 A1 Sep. 11, 2014 39
- Continued Val Pro Val Asp Phe Val Val Asn Ala Met Asp His Ile Ala His Lieu 225 23 O 235 24 O Lys Gly Glu Asp Gly Asn. Cys Phe His Lieu Val Asp Ser Asp Pro Tyr 245 250 255 Llys Val Gly Glu Ile Lieu. 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 Val Pro Pro 27s 28O 285 Phe Ile Arg Glin Ser Lieu Lys Asn Lieu Pro Pro Val Lys Arg Lieu. Thir 29 O 295 3 OO Thr Ala Leu Lieu. Asp Asp Met Gly Ile Pro Pro Ser Val Lieu. Ser Phe 3. OS 310 315 32O Ile Asn Tyr Pro Thr Arg Phe Asp Ala Arg Glu Thr Glu Arg Val Lieu. 3.25 330 335 Lys Asp Thr Gly Ile Val Val Pro Arg Lieu. Glu Ser Tyr Ala Ala Val 34 O 345 35. O Lieu. Trp Asp Phe Trp Glu Arg Asn Lieu. Asp Pro Asp Lieu. Phe Lys Asp 355 360 365 Arg Thr Lieu. Arg Gly Thr Val Glu Gly Llys Val Cys Val Ile Thr Gly 37 O 375 38O Gly. Thir Ser Gly Ile Gly Lieu Ala Thr Ala Glin Llys Lieu Ala Asp Ala 385 390 395 4 OO Gly Ala Ile Lieu Val Ile Gly Ala Arg Llys Lys Glu Arg Lieu Met Glu 4 OS 41O 415 Val Ala Ala Glu Lieu. Glu Ala Arg Gly Gly Asn. Wal His Ala Tyr Glin 42O 425 43 O Cys Asp Phe Ala Asp Met Asp Asp Cys Asp Arg Phe Val Lys Thr Val 435 44 O 445 Lieu. Asp Asn His Gly His Val Asp Val Lieu Val Asn. Asn Ala Gly Arg 450 45.5 460 Ser Ile Arg Arg Ser Lieu Ala Lieu. Ser Phe Asp Arg Phe His Asp Phe 465 470 47s 48O Glu Arg Thr Met Gln Lieu. Asn Tyr Phe Gly Ser Val Arg Lieu. Ile Met 485 490 495 Gly Phe Ala Pro Ala Met Lieu. Glu Arg Arg Arg Gly His Val 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 Ser Ala Lieu. Asp Thir Phe Ser Arg Cys Ala Ala Ala 53 O 535 54 O Glu Trp Ser Asp Arg Asn Val Thr Phe Thr Thr Ile Asn Met Pro Leu 5.45 550 555 560
Val Lys Thr Pro Met Ile Ala Pro Thr Lys Ile Tyr Asp Ser Val Pro 565 st O sts
Thir Lieu. Thr Pro Asp Glu Ala Ala Glu Met Val Ala Asp Ala Ile Val 58O 585 59 O
Tyr Arg Pro Lys Arg Ile Ala Thr Arg Lieu. Gly Ile Phe Ala Glin Val 595 6OO 605
Met Glin Ala Leu Ala Pro Llys Met Gly Glu Ile Val Met Asn Thr Gly 610 615 62O
Tyr Arg Met Phe Pro Asp Ser Pro Ala Ala Ala Gly Ser Arg Ser Gly US 2014/0256009 A1 Sep. 11, 2014 40
- Continued
625 630 635 64 O Ala Lys 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 <213> ORGANISM: Hahella cheuensis <4 OOs, SEQUENCE: 17 Met Asn Tyr Phe Val Thr Gly Gly Thr Gly Phe Ile Gly Arg Phe Leu 1. 5 1O 15 Val Pro Llys Lieu Lleu Lys Arg Gly Gly Thr Val Tyr Lieu. Lieu Val Arg 2O 25 3O Glu Ala Ser Lieu Pro Llys Lieu. Asp Glu Lieu. Arg Glu Arg Trp Asn Ala 35 4 O 45 Ser Asp Glu Glin Val Val Gly Val Val Gly Asp Lieu Ala Glin Pro Met SO 55 6 O Lieu. Gly Val Ser Glu Lys Asp Ala Ala Met Lieu. Arg Gly Llys Val Gly 65 70 7s 8O His Phe Phe His Lieu Ala Ala Ile Tyr Asp Met Glin Ala Ser Ala Glu 85 90 95 Ser Glin Glu Glin Ala Asn. Ile Glu Gly Thr Arg Asn Ala Wall Lys Lieu. 1OO 105 11 O Ala Asp Ser Lieu Lys Ala Ala Cys Phe His His Val Ser Ser Ile Ala 115 12 O 125 Ala Ala Gly Lieu. Tyr Arg Gly Ile Phe Arg Glu Asp Met Phe Glu Glu 13 O 135 14 O Ala Glu Lys Lieu. Asp Asn Pro Tyr Lieu. Arg Thr Llys His Glu Ser Glu 145 150 155 160 Llys Val Val Arg Glu Glu. Cys Glin Thr Pro Trp Arg Val Tyr Arg Pro 1.65 17O 17s Gly Met Val Val Gly His Ser Lys Thr Gly Glu Ile Asp Llys Ile Asp 18O 185 19 O Gly Pro Tyr Tyr Phe Phe Llys Lieu. Ile Glin Llys Lieu. Arg Ser Ala Lieu. 195 2OO 2O5 Pro Gln Trp Met Pro Thr Val Gly Lieu. Glu Gly Gly Arg Ile Asin Ile 21 O 215 22O Val Pro Val Asp Phe Val Val Asp Ala Met Asp His Ile Ala His Ala 225 23 O 235 24 O Glu Gly Glu Asp Gly Lys Cys Phe His Lieu. Thir Asp Pro Asp Pro Tyr 245 250 255 Llys Val Gly Glu Ile Lieu. Asn. Ile Phe Ala Glu Ala Gly. His Ala Pro 26 O 265 27 O
Lys Met Ala Met Arg Ile Asp Ala Arg Met Phe Gly Phe Ile Pro Pro 27s 28O 285
Met Ile Arg Glin Gly Ile Ala Arg Lieu Pro Pro Val Glin Arg Met Lys 29 O 295 3 OO
Asn Ala Val Lieu. Asn Asp Lieu. Gly Ile Pro Asp Glu Val Met Ser Phe 3. OS 310 315 32O US 2014/0256009 A1 Sep. 11, 2014 41
- Continued
Ile Asn Tyr Pro Thir Arg Phe Asp Asn Arg Glu Thir Glu Arg Luell Luell 3.25 330 335
Gly Thir Ala Ile Wall Pro Arg Luell Glin Asp Tyr Ser Pro Ala 34 O 345 35. O
Ile Trp Asp Arg His Luell Asp Pro Asp Lell His Asp 355 360 365
Arg Thir Luell Arg Gly Wall Glu Gly Arg Wall Wall Ile Thir Gly 37 O 375
Ala Thir Ser Gly Ile Lell Ser Ala Ala Arg Lell Ala Glu Ala 385 395 4 OO
Gly Ala Wall Wall Ala Ala Arg Thir Luell Glu Luell Glin Glu 4 OS 415
Wall Glu Lieu. Glu Luell Gly Gly Glu Wall Glu Tyr Ser 42O 425 43 O
Wall Asp Luell Ser Asp Lell Glu Asp Asp Arg Phe Wall Ala Asn Wall 435 44 O 445
Lell Lys Asp Lieu. Gly His Wall Asp Wall Luell Wall Asn Asn Ala Gly Arg 450 45.5 460
Ser Ile Arg Arg Ser Ile Glin His Ala Phe Asp Arg Phe His Asp Phe 465 470
Glu Arg Thir Met Glin Lell Asn Tyr Phe Gly Ser Lell Arg Luell Ile Met 485 490 495
Gly Phe Ala Pro Ser Met Lell Glu Arg Arg Arg Gly His Ile Wall Asn SOO 505
Ile Ser Ser Ile Gly Wall Lell Thir Asn Ala Pro Arg Phe Ser Ala 515 525
Wall Ala Ser Lys Ala Ala Lell Asp Ala Phe Ser Arg Ala Ala Ala 53 O 535 54 O
Glu Phe Ser Asp Llys Asn Wall Thir Phe Thir Thir Ile Asn Met Pro Luell 5.45 550 555 560
Wall Arg Thir Pro Met Ile Ser Pro Thir Ile Asp Ser Wall Pro 565 st O sts
Thir Luell Thir Pro Glu Glu Ala Ala Asp Luell Wall Ala Glu Ala Ile Ile 58O 585 59 O
His Arg Pro Ile Ala Thir Arg Luell Gly Wall Phe Ala Glin Wall 595 605
Lell His Ser Met Ala Pro Phe Ser Glu Ile Ile Met Asn Thir Gly 610 615
Phe Met Phe Pro Asp Ser Ser Ala Ala Thir Gly Gly Asp Gly 625 630 635 64 O
Glu Pro Llys Val Ser Thir Glu Glin Wall Ala Phe Ala Ala Ile Met 645 650 655
Arg Gly Ile His Trp 660 US 2014/0256009 A1 Sep. 11, 2014 42
1. A method for the production ofbutadiene comprising the 16. The method of claim 1 further comprising the step of enzymatic conversion of crotyl alcohol into butadiene via providing crotyl alcohol by the enzymatic conversion of cro crotyl phosphate or crotyl diphosphate. tonaldehyde into crotyl alcohol. 2. The method of claim 1 comprising the steps of 17. The method of claim 16 wherein the conversion of (i) enzymatically converting crotyl alcohol into crotyl crotonaldehyde into crotyl alcohol is achieved by the use of phosphate; and (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); (ii) enzymatically converting crotyl phosphate into butadi and/or CC. (ii) an alcohol dehydrogenase (EC 1.1.1.1); and/or 3. The method of claim 2 wherein step (ii) consists of a (iii) an aldehyde reductase; and/or single enzymatic reaction in which crotyl phosphate is (iv) an aldo-keto reductase. directly converted into butadiene. 18. An organism or microorganism which expresses 4. The method of claim 3 wherein the enzymatic conver A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1.50); or sion of crotyl phosphate into butadiene is achieved by the use (ii) a thiamine kinase (EC 2.7.1.89); and of a terpene synthase. (b)(i) a terpene synthase; or 5. The method of claim 4 wherein the terpene synthase is (ii) an isopentenyl phosphate kinase and a terpene Syn (a) an isoprene synthase (EC 4.2.3.27); or thase; or (b) a myrcene/ocimene synthase (EC 4.2.3.15); or B) (a) (i) a 2-amino-4-hydroxy-6-hydroxymethyldihy (c) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or dropteridine diphosphokinase (EC 2.7.6.3); or (d) a pinene synthase (EC 4.2.3.14); or (ii) a thiamine diphosphokinase (EC 2.7.6.2); and (e) a monoterpene synthase. (b) a terpene synthase, 6. The method of claim 2 wherein step (ii) consists of two and which is capable of converting crotyl alcohol into enzymatic reactions comprising: butadiene. (a) the enzymatic conversion of crotyl phosphate into cro 19. A composition comprising the organism or microor tyl diphosphate; and ganism of claim 18 and, optionally, crotyl alcohol. (b) the enzymatic conversion of crotyl diphosphate into 20. A composition comprising: butadiene. A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1.50); or 7. The method of claim 6 wherein the enzymatic conver (ii) a thiamine kinase (EC 2.7.1.89); and sion of crotyl phosphate into crotyl diphosphate is achieved (b)(i) a terpene synthase; or by the use of an isopentenyl phosphate kinase. (ii) an isopentenyl phosphate kinase and a terpene Syn 8. The method of claim 6 wherein the enzymatic conver thase; or sion of crotyl diphosphate into butadiene is achieved by the (B) (a) a 2-amino-4-hydroxy-6-hydroxymethyldihydrop use of a terpene synthase teridine diphosphokinase (EC 2.7.6.3); and 9. The method of claim 8 wherein the terpene synthase is (b) a terpene synthase. (a) an isoprene synthase (EC 4.2.3.27); or 21. The composition of claim 20 further comprising crotyl (b) a myrcene/ocimene synthase (EC 4.2.3.15); or alcohol. (c) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or 22. Use of a combination of enzymes comprising: (d) a pinene synthase (EC 4.2.3.14); or A) (a)(i) a hydroxyethylthiazole kinase (EC 2.7.1.50); or (ii) thiamine kinase (EC 2.7.1.89); and (e) a monoterpene synthase. (b)(i) a terpene synthase; or 10. The method of claim 2 wherein the enzymatic conver (ii) an isopentenyl phosphate kinase and a terpene Syn sion of crotyl alcohol into crotyl phosphate is achieved by the thase; or use of a hydroxyethylthiazole kinase (EC 2.7.1.50) or a thia B) (a) a 2-amino-4-hydroxy-6-hydroxymethyldihydropte mine kinase (EC 2.7.1.89). ridine diphosphokinase EC 2.7.6.3); and 11. The method of claim 1 comprising the steps of (b) a terpene synthase; (I) enzymatically converting crotyl alcohol into crotyl for the production of butadiene from crotyl alcohol. diphosphate; and 23. The organism of claim 18, wherein the terpene synthase (II) enzymatically converting crotyl diphosphate into buta 1S diene. (a) an isoprene synthase (EC 4.2.3.27); or 12. The method of claim 11 wherein the enzymatic con (b) a myrcene/ocimene synthase (EC 4.2.3.15); or version of crotyl alcohol into crotyl diphosphate consists of a (c) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or single enzymatic reaction in which crotyl alcohol is directly (d) a pinene synthase (EC 4.2.3.14); or converted into crotyl diphosphate. (e) a monoterpene synthase. 13. The method of claim 12 wherein the enzymatic con 24. A method for producing crotyl alcohol comprising the version of crotyl alcohol into crotyl diphosphate is achieved enzymatic conversion of crotonyl-CoA into crotonaldehyde by the use of a 2-amino-4-hydroxy-6-hydroxymethyldihy and the Subsequent enzymatic conversion of crotonaldehyde dropteridine diphosphokinase (EC 2.7.6.3) or a thiamine into crotyl alcohol. diphosphokinase (EC 2.7.6.2). 25. The method of claim 24, wherein the enzymatic con 14. The method of claims 11 wherein the enzymatic con version of crotonyl-CoA into crotonaldehyde is achieved by version of crotyl diphosphate into butadiene is achieved by the use of the use of a terpene synthase. (i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); 15. The method of claim 14 wherein the terpene synthase is and/or (a) an isoprene synthase (EC 4.2.3.27); or (ii) an acetaldehyde dehydrogenase (EC 1.2.1.10); and/or (b) a myrcene/ocimene synthase (EC 4.2.3.15); or (iii) an acyl-CoA reductase. (c) a farnesene synthase (EC 4.2.3.46 or EC 4.2.3.47); or 26. The method of claim 25 wherein the enzymatic con (d) a pinene synthase (EC 4.2.3.14); or version of crotonaldehyde into crotyl alcohol is achieved by (e) a monoterpene synthase. the use of US 2014/0256009 A1 Sep. 11, 2014 43
(i) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34); and/or (ii) an alcohol dehydrogenase (EC 1.1.1.1); and/or (iii) an aldehyde reductase; and/or (iv) an aldo-keto reductase. 27. The method of claim 24 wherein the enzymatic con version of crotonyl-CoA into crotyl alcohol is achieved by the use of an aldehyde/alcohol dehydrogenase or by the use of a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) or by the use of a short-chain dehydrogenase/fatty acyl-CoA reduc tase. 28. The method of claim 1 further comprising the enzy matic conversion of crotonyl-CoA into crotyl alcohol. k k k k k