US 20150240271 A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0240271 A1 Marliere (43) Pub. Date: Aug. 27, 2015

(54) METHOD FOR THE PRODUCTION OF (30) Foreign Application Priority Data 3-HYDROXY-3-METHYLBUTYRIC ACID FROMACETONE AND AN ACTIVATED Sep. 15, 2009 (EP) ...... O917O312.4 ACETYL COMPOUND Publication Classification (71) Applicant: SCIENTIST OF FORTUNE, S.A., (51) Int. Cl. Luxembourg (LU) CI2P 7/52 (2006.01) (72) Inventor: Philippe Marliere, Mouscron (BE) CI2N 9/10 (2006.01) (52) U.S. Cl. CPC ...... CI2P 7/52 (2013.01); C12N 9/1025 (21) Appl. No.: 14/658,170 (2013.01); C12Y 203/0301 (2013.01) (57) ABSTRACT (22) Filed: Mar. 14, 2015 Described is a method for the production of 3-hydroxy-3- methylbutyric acid by enzyme-catalyzed covalent bond for mation between the carbon atom of the oxo group of acetone Related U.S. Application Data and the methyl group of a compound which provides an (63) Continuation of application No. 13/395.293, filed on activated acetyl group. Also described are recombinant May 16, 2012, now Pat. No. 9,017,977, filed as appli organisms which produce 3-hydroxy-3-methylbutyric acid, cation No. PCT/EP2010/063460 on Sep. 14, 2010. and related compositions and methods. Patent Application Publication Aug. 27, 2015 Sheet 1 of 7 US 2015/0240271 A1

OH

Figure 1

Figure 2 Patent Application Publication Aug. 27, 2015 Sheet 2 of 7 US 2015/0240271 A1

Accly E-CoA O OH O —2 - O O

C S-CoA HC ulus {C} CH EMG-CoA lyase Beta-hydroxy-beta-methylglutaryl-CoA Acetoacetic acid (HMG-CoA)

Figure 3

Q O SH Pisc s's 'g sis, Maleak - Pf-ack) s pH Juli. 5-l s: s 1s- S-i,Sir s'---ho * -- ishs's ; airo--- SH 5'-->

Pks PKSG ---Pks PksH PksL Pist Pks

Figure 4 Patent Application Publication Aug. 27, 2015 Sheet 3 of 7 US 2015/0240271 A1

H Hg" h–ch o=c p'=o HC X

H2O X

HC H 3. N A

HoHc st-chCl-O A HO

Figure 5 Patent Application Publication Aug. 27, 2015 Sheet 4 of 7 US 2015/0240271 A1

-Q1: 1.359 to 2.436 min from MT20090720143306, wiff, subtracted (0.656 to 0.937 min)

95% 90% 85% 80%

ss Na --

5

Figure 6 Patent Application Publication Aug. 27, 2015 Sheet 5 of 7 US 2015/0240271 A1

-Q1: 1.587 to 3.174 min from MT20090723161455, wiff, subtracted (0.703 to 1105 min) 100% 95% 90% 85% 80% 75% 70% 65% 60% 55% 50% 45% 40% 35% 30% 25% 20% 15%

10% 220.2O 5% 21.20 11330-130 58.60 2.38 "oo isoso200 "220 2.6 miz, amu

Figure 7 Patent Application Publication Aug. 27, 2015 Sheet 6 of 7 US 2015/0240271 A1

-Q1: 1,065 to 3,033 min from MT20090723160718, wiff, subtracted (0.502 to 0.703 min.) 100% 95% 90%

85% 80% 75% 70% 65% 60% 55% 50% 78.70 45%

40% 35% 30% 134.10 25% 20% 220.10 15% 34.90 10% 5% 177.20 21.20

0% O 1249. "L. al 250 2" 80 100 12O 140 60 80 200 220 240 26 m/z, amu

Figure 8 Patent Application Publication Aug. 27, 2015 Sheet 7 of 7 US 2015/0240271 A1

1,6 1,4

2s , * ! : X E : 33d5 0.6 - 95d5 4 Q ------O O4 C,6 O,8 i acetone, mM

Figure 9 US 2015/0240271 A1 Aug. 27, 2015

METHOD FOR THE PRODUCTION OF HIV and cancer and trauma victims with severe injuries. 3-HYDROXY-3-METHYLBUTYRIC ACID Thus, it is of commercial interest because of its use as a FROMACETONE AND AN ACTIVATED muscle enhancer for bodybuilding and as a medicament for ACETYL COMPOUND avoiding muscle wasting. U.S. Pat. No. 7,026,507 describes a process for preparing Solid formulations of sodium 3-hy CROSS REFERENCE TO RELATED droxy-3-methylbutyrate in which, in a first process step, 4.4- APPLICATIONS dimethyloxetan-2-one is reacted with aqueous Sodium 0001. This application is a continuation co-pending U.S. hydroxide to form a solution of sodium 3-hydroxy-3-meth patent application Ser. No. 13/395.293, filed May 16, 2012, ylbutyrate, and then, if appropriate after concentration, the which is the U.S. National Phase of International Application Solution is applied, in a further process step, to synthetic PCT/EP2010/063460, which was published in English on silica, and in which the resultant product is, if appropriate, Mar. 24, 2011, as WO 2011/032934, and claims the benefit of dried. the filing date of European Patent Application No. 09170312. 0008. It would be desirable to provide a process for the 4, filed Sep. 15, 2009, the entire disclosures of which are production of 3-hydroxy-3-methylbutyrate which would be incorporated herein by reference. independent of inorganic production steps and which could be effected in living organisms thereby being environmen FIELD OF THE INVENTION tally sound and inexpensive. In this context, Lee et al. (Appl. Environ. Microbiol. 63 (1997), 4191-4195) describes a 0002 The present invention relates to a method for the method for the production of 3-hydroxy-3-methylbutyrate by production of 3-hydroxy-3-methylbutyric acid (also referred converting 3-methylbutyric acid to 3-hydroxy-3-methylbu to as beta-hydroxyisovalerate or HIV) from acetone and a tyric acid using the microorganism Galactomyces reessii. compound which provides an activated acetyl group compris However, although this process allowed the production of ing the enzymatic conversion of acetone and a compound 3-hydroxy-3-methylbutyrate there is still a need to provide which provides an activated acetyl group into 3-hydroxy-3- alternative efficient and cost effective ways of producing methylbutyric acid. The conversion makes use of an enzyme 3-hydroxy-3-methylbutyrate in particular by biological pro which is capable of catalyzing the formation of a covalent CCSSCS. bond between the carbon atom of the oxo (i.e. the C=O) 0009. The present invention meets this demand for an group of acetone and the methyl group of the compound alternative process for the production of 3-hydroxy-3-meth which provides an activated acetyl group. Preferably, the ylbutyrate and provides a method which is based on biologi enzyme employed in the process is an enzyme with the activ cal resources and allows to produce 3-hydroxy-3-methylbu ity of a HMG CoA synthase (EC 2.3.3.10) and/or a PksG tyrate in vitro or in vivo in a microorganism and other species. protein and/or an enzyme with the activity of a C-C bond cleavage/condensation lyase, such as HMG CoA lyase (EC DETAILED DESCRIPTION OF THE INVENTION 4.1.3.4). The present invention also relates to organisms able to produce 3-hydroxy-3-methylbutyric acid from acetone and (0010 Method for the Production of 3-hydroxy-3-methyl a compound which provides an activated acetyl group and to butyric Acid the use of the above-mentioned enzymes and organisms for 0011. In particular, the present invention relates to a the production of 3-hydroxy-3-methylbutyric acid. Finally, method for the production of 3-hydroxy-3-methylbutyric the present invention relates to the use of acetone for the acid (also referred to as beta-hydroxyisovalerate or HIV) production of 3-hydroxy-3-methylbutyric acid. from acetone and a compound which provides an activated acetyl group comprising the enzymatic conversion of acetone BACKGROUND OF THE INVENTION and a compound which provides an activated acetyl group 0003 3-hydroxy-3-methylbutyric acid (also referred to as into 3-hydroxy-3-methylbutyric acid. beta-hydroxyisovalerate or HIV: see FIG. 1) is a metabolite of 0012 Acetone is represented by the following formula: the essential amino acid leucine and is synthesized in the CH (C=O)—CH. In a preferred embodiment the com human body. It can be found in Small quantities in grapefruit, pound which provides an activated acetyl group is character alfalfa and catfish. It is also known to occur in Some metabolic ized by the following formula (I): disorders of leucine catabolism, i.e. hypoValeric acidemia. It has been shown that 3-hydroxy-3-methylbutyric acid may have an effect on increasing muscle weight and strength (Nis sen et al., J. Appl. Physiol. 81 (1996), 2095-2104). Wilson et al. (Nutrition & Metabolism 5 (2008)) proposes as the mecha nisms of action the following: 0004 increased sarcolemmal integrity via conversion by HMG CoA reductase 0005 enhanced protein synthesis via the mTOR path 0013 , wherein X is selected from the group consisting of way S CH2-CH2-NH CO CH2-CH2-NH CO CH 0006 depression of protein degradation through inhibi (OH) C(CH3)2-CH2-O-PO2H C10H13N5O7P (coen tion of the ubiquitin pathway. Zyme A), S CH2-CH2-NH CO-CH2-CH2-NH CO 0007 3-hydroxy-3-methylbutyric acid is supposed to help CH(OH)–C(CH3)2-CH2-O-PO2H-polypeptide (acyl muscles combat protein breakdown, assist in muscle repair carrier protein), S CH2-CH2-NH CO-CH2-CH2 and Support increased endurance. It has been described to NH CO-CH(OH)–C(CH3)2-CH2-OH (pantetheine), help patients with chronic obstructive pulmonary disease in S CH-CH NH CO-CH (N-acetyl-cysteamine), hospital intensive care units, muscle wasting associated with S-CH (methane thiol), S CH2-CH(NH2)-CO2H (cys US 2015/0240271 A1 Aug. 27, 2015

teine), S CH2-CH2-CH(NH2)-CO2H (homocysteine), the PksG protein. The PksG protein is one of the proteins S CH2-CH(NH C5H8NO3)-CO. NH CH2-CO2H encoded by the pksX gene cluster from Bacillus subtilis. The (glutathione), S CH, CH, SOH (coenzyme M) and PksG protein is capable of catalyzing the transfer of a car OH (acetic acid). boxymethyl group —CH COH from acetyl-S-AcpK to a 0014. The conversion makes use of an enzyme which is B-ketothioester polyketide intermediate linked to one of the capable of catalyzing the formation of a covalent bond thiolation domains of the PksL protein, in a reaction which is between the carbon atom of the oxo (i.e. the C=O) group of analogous to that catalyzed by HMG CoA synthase. How acetone and the carbon atom (C) corresponding to the ever, it has been shown in the context of the present invention methyl group of the compound which provides an activated that the PksG protein can also use acetyl CoA instead of the acetyl group according to formula (I). According to this reac acetyl-S-AcpK protein as a donor of an activated acetyl tion scheme the oxo group of acetone reacts as an electrophile group. and the methyl group of the compound which provides an 0019. In one preferred embodiment the compound which activated acetyl group according to formula (I) reacts as a provides an activated acetyl group is acetyl CoA. Acetyl CoA nucleophile. The general reaction of the conversion of (also known as acetyl Coenzyme A) in chemical structure is acetone and a compound which provides an activated acetyl the thioester between coenzyme A (a thiol) and acetic acid. group according to formula (I) is shown in FIG. 5. 0020. In another preferred embodiment the compound 0015 The reaction can occur in one step, i.e. 3-hydroxy which provides an activated acetyl group has the formula (I) 3-methylbutyrate can be the direct product of a reaction cata in which X is an acyl-carrier-protein, Such as the acetyl-S- lyzed by the above described enzyme. Alternatively, the reac AcpK protein encoded by the pkSX gene cluster for produc tion may comprise two steps, in particular in the case where ing bacillaene in Bacillus subtilis. acetyl CoA is used as the compound which provides an acti 0021 Preferably, the enzyme employed in the process is vated acetyl group, in the sense that first an adduct of 3-hy an enzyme with the activity of a HMG CoA synthase (EC droxy-3-methylbutyrate and the compound which provides 2.3.3.10) and/or a PksG protein and/or an enzyme with the an activated acetyl group is produced, e.g. 3-hydroxy-3-me activity of a C-C bond cleavage/condensation lyase, Such as thylbutyryl-CoA, which is Subsequently hydrolyzed, e.g. to a HMG CoA lyase (EC 4.1.3.4). 3-hydroxy-3-methylbutyrate and CoA. Thus, in the first alter 0022. In one preferred embodiment, the method according native the enzyme catalyzes the complete reaction as shown to the present invention comprises the enzymatic conversion in FIG. 5. In the second alternative, the enzyme catalyzes the of acetone and acetyl CoA into 3-hydroxy-3-methylbutyrate formation of a covalent bond between the carbonatom of the with an enzyme which is capable of catalyzing the formation oxo (i.e. the C=O) group of acetone and the carbonatom(C) of a covalent bond between the carbon atom of the oxo (i.e. corresponding to the methyl group of the compound which the C=O) group of acetone and the carbonatom Cofacetyl provides an activated acetyl group but X stays in the mol CoA according to formula (I). ecule. X is then removed subsequently from the molecule by 0023. In a preferred embodiment, the enzyme employed in hydrolysis. the process according to the invention is an enzyme which has 0016. The present invention shows for the first time that it the activity of a HMG CoA synthase (EC 2.3.3.10) or an is possible to produce 3-hydroxy-3-methylbutyrate by mak enzyme which has the activity of a PksG protein oran enzyme ing use of an enzyme which can transfer an activated acetyl which has the activity of a C-C bond cleavage/condensation group to acetone. In the prior art production of 3-hydroxy-3- lyase, such as a HMG CoA lyase (EC 4.1.3.4). methylbutyrate from isovaleric acid through bioconversion 0024. In particular, it has been shown in the context of the using the fungus Galactomyces reessii has been reported. present invention that HMG CoA synthase can acceptacetone However, considering that isovaleric acid is obtained from instead of its normal substrate acetoacetyl-CoA thereby leucine through decarboxylation and that leucine itself allowing the conversion of acetyl-CoA (or a compound derives in metabolism from the overall condensation of two according to formula (I)) and acetone into 3-hydroxy-3-me molecules of pyruvate and one molecule of acetyl CoA, this thylbutyrate. production process is energetically unfavorable. The process (0025 HMG CoA Synthase of the present invention avoids this disadvantage. 0026. Moreover, it has been shown in the context of the 0017. In general, in the context of the present invention present invention that the PksG protein can use acetyl CoA as any enzyme could be used which accepts a compound which a Substrate instead of the Ac-S-AcpK protein and can catalyze provides an activated acetyl group as defined above as one the reaction which is normally catalyzed by HMG CoA syn Substrate as well as a Substrate which contains as a component thase. Thus, it is contemplated that also the PksG protein, an acetone group. In one preferred embodiment, the enzyme which catalyzes a reaction analogous to the reaction of HMG is an enzyme which accepts acetyl CoA as a Substrate. CoA synthase, will be able to catalyze the conversion of Examples for such enzymes are HMG CoA synthase, HMG acetone and a compound of formula (I) into 3-hydroxy-3- CoA lyase or other C-C bond cleavage/condensation lyases. methylbutyrate. Moreover, it is contemplated that C C bond However, as will be explained below, also enzymes which cleavage/condensation lyases, such as HMG CoA lyase, can normally use in the reaction that they catalyze in nature an catalyze the conversion of acetyl-CoA and acetone into 3-hy acetyl-donor different from acetyl CoA, may use acetyl CoA droxy-3-methylbutyryl-CoA which in turn can be hydrolysed or analogues thereof, e.g. the PkSG protein. to 3-hydroxy-3-methylbutyrate and CoA. 0018. In another preferred embodiment the enzyme is an 0027. In the context of the present application the term enzyme which accepts as a Substrate a compound which “HMG CoA synthase' or “a protein/enzyme having the activ provides an activated acetyl group according to formula (I) in ity of a HMG CoA synthase' refers to any enzyme which is which X is an acyl-carrier-protein, such as the acetyl-S-AcpK classified in the EC number EC 2.3.3.10 (formerly, HMG protein encoded by the pkSX gene cluster for producing bacil CoA synthase has been classified as EC 4.1.3.5 but has been laene in Bacillus subtilis. An example for Such an enzyme is transferred to EC 2.3.3.10), in particular it refers to any US 2015/0240271 A1 Aug. 27, 2015 enzyme which is able to catalyze the reaction where acetyl prot accession number Q9FD56), Streptococcus pyogenes CoA condenses with acetoacetyl-CoA to form 3-hydroxy-3- (Uniprot accession number Q9FD61) and Methanobacterium methylglutaryl-CoA (HMG-CoA) (see FIG. 2) and the term thermoautotrophicum (accession number AE000857), Borre also refers to any enzyme which is derived from such a HMG lia burgdorferi (NCBI accession number BB0683). CoA synthase and which is capable of catalyzing the conver 0033 Moreover, the following Table A lists some known sion of acetone and a compound which provides an activated HMG CoA synthases from prokaryotes: acetyl group as defined above, preferably acetyl CoA, into 3-hydroxy-3-methylbutyrate. TABLE A 0028. The enzymatic activity of condensing acetyl-CoA SwissprotTrEmbl with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl Accession number Organism CoA (HMG-CoA) can be measured by methods well known in the art. One possible and preferably used assay is Q9YASO Aeropyrim pernix A7ZAY2 Bacilius amyloiquefaciens described, e.g., in Clinkenbeard et al. (J. Biol. Chem. 250 P4083 0287.4037340 Bacilius subtiis (1975), 3108-3116). In this assay HMG-CoA synthase activ B8G-795 Chloroflexits aggregains ity is measured by monitoring the decrease in absorbance at ASEUV4 Dicheliobacter nodosus 303 nm that accompanies the acetyl-CoA-dependent disap ASFMS4 Flavobacterium johnsoniae Q18GC4 Halloquadratin waisbyi pearance of the enolate form of acetoacetyl-CoA. Preferably B9LS15 Haiorubrum lacusprofindi HMG CoA synthase activity is assayed as described in A9B8FO Herpetosiphon attrantiacus Example 3. A2BMY8 Hyperthermus butyllicus 0029 HMG CoA synthase is part of the mevalonate path Q5FLB7 Lactobacilius acidophilus Q03QRO Lactobacilius brevis way. Two pathways have been identified for the synthesis of Q1GAH5 Lactobacilius delbruecki isopentenyl pyrophosphate (IPP), i.e. the mevalonate path B2GBL1 Lactobacilius fermentum way and the glyceraldehyde 3-phosphate-pyruvate pathway. B1MZ51 LeticonoStoc Citretin HMG CoA synthase catalyzes the biological Claisen conden Q03WZO Leticonostoc mesenteroides A4YH99 Metaliosphaera Sedutia sation of acetyl-CoA with acetoacetyl-CoA and is a member ASUNI8 Meihanobrevibacter Smithii of a Superfamily of acyl-condensing enzymes that includes Q58941 Methanocaldococcus jannaschii beta-ketothiolases, fatty acid synthases (beta-ketoacyl carrier Q12UR3 Meihanococcoides burtonii protein synthase) and polyketide synthases. A6USZ1 Methanococci is aeolicits A4FWW6 Methanococcus maripaludis 0030 HMG CoA synthase has been described for various A6UPL1 Meihanosarcina mazei organisms. Also amino acid and nucleic acid sequences A2STY2 Methanocorpusculum labreanum encoding HMG CoA synthases from numerous sources are Q8TVLO Methanopyrus andleri Q8PYJO Meihanosarcina mazei available. Generally, the sequences only share a low degree of Q2NHU7 Methanosphaera Stadtmanae overall sequence identity. For example, the enzymes from Q2FPH4 Methanospirilium hungatei Staphylococcus or Streptococcus show only about 20% iden B2HGT6 Mycobacterium marinum tity to those of human and avian HMG CoA synthase. In some Q3IMZ7 Natronomonas pharaonis Q8EP69 Oceanobacillusiheyensis sources it is reported that the bacterial HMG CoA synthases Q04F95 Oenococci is oeni and their animal counterparts exhibit only about 10% overall Q03FU5 Pediococcuspentosaceus sequence identity (Sutherlin et al., J. Bacteriol. 184 (2002), Q6L233 Picrophilus torridus 4065-4070). However, the amino acid residues involved in A6G7N7 Plesiocystis pacifica A4WJ12 Pyrobaculum arsenaticum the acetylation and condensation reactions are conserved A7NHZ7 Roseiflexus castenholzii among bacterial and eukaryotic HMG CoA synthases (Cam Q8CNO6 Staphylococci is epidermidis pobasso et al., J. Biol. Chem. 279 (2004), 44883-44888). The Q4L958 Staphylococciis haemolyticus three-dimensional structure of three HMG CoA synthase Q4AOD6 Staphylococci is saprophyticus B4U364 Streptococci is equi enzymes has been determined and the amino acids crucial for Q8DUIS Streptococci is mutans the enzymatic reaction are in principle well characterized Q4J933 Sulfolobus acidocaidarius (Campobasso et al., loc. cit.; Chun et al., J. Biol. Chem. 275 Q971 K8 Sulfolobus tokodai (2000), 17946-17953; Nagegowda et al., Biochem. J. 383 Q9HI87 Thermoplasma acidophilum Q31EW2 Thionicrospira crinogena (2004), 517-527; Hegardt, Biochem.J.338 (1999), 569-582). Q51798 Pyrococcus furiosus In eukaryotes there exist two forms of the HMG CoA syn ASVJB7 Lactobacilius rentieri thase, i.e. a cytosolic and a mitochondrial form. The cytosolic Q7CF79 Streptococci is pyogenes form plays a key role in the production of cholesterol and Q9UWUO Sulfolobus solfataricus other isoprenoids and the mitochondrial form is involved in the production of ketone bodies. 0034 Eukaryotic HMG CoA synthases are described, 0031. In principle any HMG CoA synthase enzyme can be e.g., from fungi, Such as Schizosaccharomyces pombe (acces used in the context of the present invention, in particular from sion numbers U32187 and P54874), Saccharomyces cerevi prokaryotic or eukaryotic organisms. siae (accession number P54839), plants, such as Arabidopsis 0032. Prokaryotic HMG CoA synthases are described, thaliana (accession numbers X83882 and P54873), Pinus e.g., from Staphylococcus aureus (Campobasso et al., loc. Sylvestris (accession number X96386) and animals, such as cit.; Uniprot accession number Q9FD87), Staphylococcus Caenorhabditis elegans (accession number P54871), Mus epidermidis (Uniprot accession number Q9FD76), Staphylo musculus (mitochondrial; accession number P54869 and coccus haemolyticus (Uniprot accession number Q9FD82), Hegardt, Biochem. J.338 (1999), 569-582), Rattus norvegi Enterococcus faecalis (Sutherlin et al., loc. cit.; Unirprot cus (mitochondrial: accession number P22791 and Hegardt, accession number Q9FD7). Enterococcus faecium (Uniprot Biochem. J. 338 (1999); cytosolic: accession number accession number Q9FD66), Streptococcus pneumonia (Uni P17425), 569-582), Chinese hamster (Cricetulus griseus. US 2015/0240271 A1 Aug. 27, 2015

accession number P13704), Sus scrofa (mitochondrial; acces determined according to methods well known in the art using sion number U90884 and Hegardt, Biochem. J.338 (1999), preferably suitable computer algorithms such as CLUSTAL. 569-582), Homo sapiens (mitochondrial: accession number 0038. When using the Clustal analysis method to deter P54868 and Hegardt, Biochem. J.338 (1999), 569-582: cyto mine whether a particular sequence is, for instance, 80% solic: accession number Q01581), Blattella germanica (cyto identical to a reference sequence default settings may be used solic form 1; accession number P54961), Blattella germanica or the settings are preferably as follows: Matrix: blosum 30: (cytosolic form 2; accession number P54870) and Gallus Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay gallus (cytosolic; accession number P23228). divergent: 40; Gap separation distance: 8 for comparisons of 0035 Examples of HMG CoA synthases from different amino acid sequences. For nucleotide sequence comparisons, organisms are given in SEQID NO: 1 to 14. SEQID NO: 1 the Extend gap penalty is preferably set to 5.0. shows the sequence of the cytoplasmic HMG CoA synthase 0039. Preferably, the degree of identity is calculated over of Caenorhabditis elegans (P54871, gene bank F25B4..6), the complete length of the sequence. SEQID NO: 2 shows the sequence of the cytoplasmic HMG 0040. The HMG CoA synthase employed in the process CoA synthase of Schizosaccharomyces pombe (fission yeast; according to the invention can be a naturally occurring HMG P54874), SEQID NO:3 shows the sequence of the cytoplas CoA synthase or it can be a HMG CoA synthase which is mic HMG CoA synthase of Saccharomyces cerevisiae (bak derived from a naturally occurring HMG CoA synthase, e.g. er's yeast; P54839, gene bank CAA65437.1), SEQID NO: 4 by the introduction of mutations or other alterations which, shows the sequence of the cytoplasmic HMG CoA synthase e.g., alter or improve the enzymatic activity, the stability, etc. of Arabidopsis thaliana (Mouse-ear cress: P54873), SEQID 0041. The term “HMG CoA synthase” or “a protein?en NO: 5 shows the sequence of the cytoplasmic HMG CoA Zyme having the activity of a HMG CoA synthase' in the synthase of Dictyostelium discoideum (Slime mold; P54872, context of the present application also covers enzymes which gene bank L2114), SEQID NO: 6 shows the sequence of the are derived from a HMG CoA synthase, which are capable of cytoplasmic HMG CoA synthase of Blattella germanica producing 3-hydroxy-3-methylbutyrate by an enzymatic con (German cockroach: P54961, gene bank X73679), SEQ ID version of acetone and a compound which provides an acti NO: 7 shows the sequence of the cytoplasmic HMG CoA vated acetyl group as defined above, preferably acetyl-CoA, synthase of Gallus gallus (Chicken; P23228, gene bank but which only have a low affinity to acetoacetyl-CoA as a CHKHMGCOAS), SEQID NO: 8 shows the sequence of the Substrate or do no longer accept acetoacetyl-CoA as a Sub cytoplasmic HMG CoA synthase of Homo sapiens (Human; strate. Such a modification of the preferred substrate of a Q01581, gene bank X66435), SEQ ID NO: 9 shows the HMG CoA synthase allows to improve the conversion of sequence of the mitochondrial HMG CoA synthase of Homo acetone into 3-hydroxy-3-methylbutyrate and to reduce the sapiens (Human: P54868, gene bank X83618), SEQID NO: production of the by-product, e.g. HMG-CoA. Methods for 10 shows the sequence of the mitochondrial HMG CoA syn modifying and/or improving the desired enzymatic activities thase of Dictyostelium discoideum (Slime mold; Q86HL5, of proteins are well-known to the person skilled in the art and gene bank XM 638984), SEQ ID NO: 11 shows the include, e.g., random mutagenesis or site-directed mutagen sequence of the HMG CoA synthase of Staphylococcus epi esis and Subsequent selection of enzymes having the desired dermidis (Q9FD76), SEQID NO: 12 shows the sequence of properties or approaches of the so-called “directed evolu the HMG CoA synthase of Lactobacillus fermentum tion’. For example, for genetic engineering in prokaryotic (B2GBL1), SEQID NO: 13 shows the sequence of the HMG cells, a nucleic acid molecule encoding HMG CoA synthase CoA synthase of Hyperthermus butyllicus (A2BMY8), SEQ can be introduced into plasmids which permit mutagenesis or ID NO: 14 shows the sequence of the HMG CoA synthase of sequence modification by recombination of DNA sequences. Chloroflexus aggregains (B8G795), SEQ ID NO: 24 shows Standard methods (see Sambrook and Russell (2001), the sequence of the HMG CoA synthase of Lactobacillus Molecular Cloning: A Laboratory Manual, CSH Press, Cold delbrueckii (Q1GAH5) and SEQ ID NO: 25 shows the Spring Harbor, N.Y., USA) allow base exchanges to be per sequence of the HMG CoA synthase of Staphylococcus formed or natural or synthetic sequences to be added. DNA haemolyticus Q4L958 (198>V difference compared to wild fragments can be connected to each other by applying adapt type protein). ers and linkers to the fragments. Moreover, engineering mea 0036. In a preferred embodiment of the present invention sures which provide suitable restriction sites or remove sur the HMG CoA synthase is an enzyme comprising an amino plus DNA or restriction sites can be used. In those cases, in acid sequence selected from the group consisting of SEQID which insertions, deletions or Substitutions are possible, in NOs: 1 to 14 or a sequence which is at least n 9% identical to vitro mutagenesis, “primer repair, restriction or ligation can any of SEQID NOs: 1 to 14 and having the activity of a HMG be used. In general, a sequence analysis, restriction analysis CoA synthase with n being an integer between 10 and 100, and other methods of biochemistry and molecular biology are preferably 10, 15, 20, 25, 30,35, 40, 45, 50, 55,60, 65,70, 75, carried out as analysis methods. The resulting HMG CoA 80, 85,90,91, 92,93, 94, 95, 96, 97,98 or 99. synthase variants are then tested for their enzymatic activity 0037 Preferably, the degree of identity is determined by and in particular for their capacity to prefer acetone as a comparing the respective sequence with the amino acid Substrate rather than acetoacetylco A. An assay for measuring sequence of any one of the above-mentioned SEQID NOS. the capacity of a HMG CoA synthase to use acetone as a When the sequences which are compared do not have the substrate is described in Example 5. The formation of 3-hy same length, the degree of identity preferably either refers to droxy-3-methylbutyrate can be detected by comparison with the percentage of amino acid residues in the shorter sequence standard compound, e.g. after separation by thin-layer chro which are identical to amino acid residues in the longer matography, LC/MS and colorimetric assay after its deriva sequence or to the percentage of amino acid residues in the tization or by mass spectrometry. longer sequence which are identical to amino acid residues in 0042. In particular, a reaction is carried out in a reaction the shorter sequence. The degree of sequence identity can be mixture containing 40 mM Tris-HCl pH 8, 5 to 50 mMacetyl US 2015/0240271 A1 Aug. 27, 2015

CoA, 100 to 500 mMacetone, 1 MgCl2 (except formitochon substrate acetoacetyl-CoA of HMG CoA synthase. One dria HMG-CoA synthase), 0.5 mM DTT (dithiothreitol) and example of Such a cofactor would be coenzyme A or a struc enzyme varying in the range from 0.2 to 8 mg/ml. Control turally closely related molecule such as S-nitroso-CoA. reactions are carried in the absence of enzyme and one of the 0050. The modified version of the HMG CoA synthase Substrates. accepting acetone as a Substrate but having a low affinity to 0043. The progress of synthesis is followed by analyzing acetoacetyl-CoA as a Substrate or no longer accepting aliquots taken after increasing period of incubation at 30 or acetoacetyl-CoA as a Substrate may be derived from a natu 37°C. Typically, an aliquot of 50 ul is removed after 48 h of rally occurring HMG CoA synthase or from an already modi incubation, heated for 1 min at 100° C. to eliminate the fied, optimized or synthetically synthesized HMG CoA syn proteins, centrifuged and the Supernatant is transferred to a thase. clean vial for HIV detection by mass spectrometry. A solution 0051 PksG Protein of 3-hydroxy-3-methylbutyrate is prepared in 40 mM Tris 0.052 Another example for a protein which can be used in HCl pH 8, 1 mM MgCl, 0.5 mM DTT, heated as described a method according to the invention is a PksG protein. In the above and used as reference. context of the present application the term “PksG protein’ or 0044) The samples are analyzed on a PE SCIEX(R) API “a protein/enzyme having the activity of a PksG protein’ 2000 triple quadrupole mass spectrometer (mass spectrom refers to any enzyme which is able to catalyze the reaction eter, Perkin-Elmer) in negative ion mode with H2O/acetoni which is naturally catalyzed by the PksG protein, i.e the trile=60/40 containing 0.1% triethylamine as mobile phase, transfer of —CH2COO from acetyl-S-AcpK (Ac-S-AcpK) flow rate was 40 ul/min. 10 ul of each supernatant are mixed to a B-ketothioester polyketide intermediate linked to one of with an equal quantity of mobile phase and directly injected the thiolation domains of the PksL protein. This is a reaction into the mass spectrometer. The presence of 3-hydroxy-3- which is analogous to that catalyzed by HMG CoA synthase methylbutyrate-H ion is monitored. with the difference that the acetyl-thioester of the phospho 0045 3-hydroxy-3-methylbutyrate synthesis can also be pantetheyl moiety is attached to a carrier protein rather than to carried out in the presence of radiolabeled 2-C acetone. part of Coenzyme A. Although the PksG protein in the reac The formation of product is analyzed after separation of the tion which it naturally catalyzes transfers the acetyl group reaction mixture by TLC or HPLC. from acetyl-S-AcpK to an acceptor, it has been shown in the 0046. In a preferred embodiment the HMG CoA synthase context of the present invention that the PksG protein can also employed in the present invention is an enzyme which has a effect the reaction which is normally catalyzed by HMG CoA KM value for acetone of 300 mM or lower, preferably of 250 synthase, i.e. the synthesis of HMG CoA starting from mM or lower even more preferably of 200 mM or lower and acetoacetyl CoA and acetyl CoA (see Example 3 where it is particularly preferred of 150 mM or lower. It is preferred that shown in Table 1 that the enzyme from Mycobacterium mari the KM value is determined under the conditions described in num (B2HGT6) can act on acetoacetyl CoA and acetyl CoA). Example 7. In another preferred embodiment the HMG CoA 0053. The enzymatic activity of the PksG protein can be synthase employed in the present invention has akvalue for measured by methods known in the art. One possible and the described reaction of at least 0.1x10" sec', preferably at preferably used assay is described, e.g., in Calderone et al. least 0.2x10 sec' even more preferably at least 0.5x10 (Proc. Natl. Acad. Sci. USA 103 (2006), 8977-8982). In this secland particularly preferred at least 1x10" sec', at least assay acetoacetyl (Acac)-S-PkSL-T2 is used as a model Sub 2x10 sec', at least 3x10 sec' or at least 5x10 sec'. It strate and is incubated together with Ac-S-AcpK and the is preferred that the k value is determined under the condi PksG protein. The formation of HMG-S-PksL-T2 indicates tions described in Example 7 that the PksG protein is capable of transferring the carboxym 0047. It is known in the art that His264 of avian HMG CoA ethyl group —CH CO.H from Ac-S-AcpK to (Acac)-S- synthase plays a role in the interaction of the enzyme with PkSL-T2. The formation of HMG-S-PkSL-T2 can be deter acetoacetyl-CoA and that the Ala264 variant lacks interaction mined either by electrospray ionization (ESI)-FTMS or in an with the oxygen of the thioester moiety of acetoacetyl-CoA autoradiography. In a preferred embodiment the correspond (Misraa et al., Biochem. 35 (1996), 9610-9616). Thus, in ing assays are carried out as described on page 8982 of Cal order to develop variants of HMG CoA synthase which show derone et al. (Proc. Natl. Acad. Sci. USA 103 (2006), 8977 a lower acceptance of acetoacetyl-CoA as a Substrate but 8982). which accept acetone as a Substrate, it is conceivable to sys 0054 The PksG protein is part of the pksX pathway in tematically mutate in a HMG CoA synthase the histidine Bacillus subtilis which encodes the enzymes responsible for residue which corresponds to His264 of the avian HMG CoA the biosynthesis of bacillaene (Butcher et al., Proc. Natl. synthase described in Misraa et al. (loc. cit.) So as to reduce or Acad. Sci. USA 104 (2007), 1506-1509). The encoded pro disable the acceptance of acetoacetyl-CoA as Substrate. teins are AcpK, PksC, PksL, PksF, PksG, PksH and Pks.I. 0048. In addition, HMG CoA synthase variants can be According to Calderone et al. (Proc. Natl. Acad. Sci. USA provided which show an increased activity. Steussy et al. 103 (2006), 8977-8982) these enzymes act to incorporate an (Biochemistry 45 (2006), 14407-14414), for example, acetate derived B-methyl branch on an acetoacetyl-S-carrier describe a mutant of the Enterococcus faecalis HMG CoA protein. synthase in which Ala 110 was changed to Gly 110 and which 0055. In a preferred embodiment of the present invention shows an 140-fold increase of the overall reaction rate. the PkSG protein is an enzyme comprising an amino acid 0049 Methods for identifying variants with improved sequence as shown in SEQ ID NO: 15 or 16 or a sequence enzymatic properties as regards the production of 3-hydroxy which is at least n% identical to SEQID NO: 15 or 16 and 3-methylbutyrate may also be carried out in the presence of a having the activity of a PkSG protein with n being an integer cofactor which allows for a steric and/or electronic comple between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, mentation in the catalytic site of the enzymefenzymes due to 50, 55, 60, 65,70, 75,80, 85,90,91, 92,93, 94, 95, 96, 97,98 the fact that the substrate acetone is shorter than the natural or 99. SEQID NO: 15 shows the amino acid sequence of the US 2015/0240271 A1 Aug. 27, 2015

PksG protein of Bacillus subtilis (P40830) and SEQID NO: lowing reaction: acetyl-CoA+3-methyl-2-oxobutanoate-- 16 shows the amino acid sequence of the PksG protein of HOs (2S)-2-isopropylmalate--CoA. Examples for such Mycobacterium marinum (B2HGT6). enzymes are the corresponding enzyme from Brucella abor 0056. As regards the determination of the degree of tus (strain 2308; Q2YRT1) and the corresponding enzyme sequence identity the same applies as has been set forth above from Hahella cheiuensis (strain KCTC 2396; Q2SFA7). in connection with HMG CoA synthase. 0064. A homocitrate synthase (EC 2.3.3.14) is an enzyme 0057 The PksG protein employed in the process accord that catalyzes the chemical reaction acetyl-CoA-H2O+2- ing to the invention can be a naturally occurring PkSG protein oxoglutarates= (R)-2-hydroxybutane-1,2,4-tricarboxylate+ or it can be a PksG protein which is derived from a naturally CoA. The 4-hydroxy-2-ketovalerate aldolase catalyzes the occurring PkSG protein, e.g. by the introduction of mutations chemical reaction 4-hydroxy-2-oxopentanoates=s acetalde or other alterations which, e.g., alter or improve the enzy hyde--pyruvate. matic activity, the stability, etc. 0065. In the context of the present invention the term 0058. The term “PksG protein' or “a protein/enzyme hav “HMG CoA lyase' or “a protein/enzyme having the activity ing the activity of a PksG protein’ in the context of the present of a HMG CoA lyase' refers to any enzyme which is classi application also covers enzymes which are derived from a fied in the EC number EC 4.1.3.4, in particular it refers to any PksG protein, which are capable of producing 3-hydroxy-3- enzyme which is able to catalyze the cleavage of HMG CoA methylbutyrate by an enzymatic conversion of acetone and a into acetyl CoA and acetoacetate (see FIG.3) or the reverse of compound which provides an activated acetyl group as this reaction, i.e. the production of HMG CoA through the defined above, preferably acetyl-CoA, but which only have a condensation of acetyl CoA and acetoacetate, and the term low affinity to their natural Substrate or do no longer accept also refers to any enzyme which is derived from such a HMG their natural substrate. Such a modification of the preferred CoA lyase and which is capable of catalyzing the conversion substrate of a PksG protein allows to improve the conversion of acetone and a compound providing an activated acetyl ofacetone into 3-hydroxy-3-methylbutyrate and to reduce the group as defined above, preferably acetyl CoA, into 3-hy production of unwanted by-product. Methods for modifying droxy-3-methylbutyryl-CoA. In the context of the present and/or improving the desired enzymatic activities of proteins invention the produced 3-hydroxy-3-methylbutyryl-CoA can are well-known to the person skilled in the art and have been then be hydrolyzed to produce 3-hydroxy-3-methylbutyrate. described above. The resulting PksG protein variants are then This could be achieved by measures known to the person tested for their enzymatic activity and in particular for their skilled in the art, e.g. by making use of an acyl-CoA hydrolase capacity to prefer acetone as a substrate. An assay for mea (EC 3.1.2.20) or an acyl-CoA transferase (EC 2.8.3.8). Suring the capacity of a PkSG protein to use acetone as a 0066. The enzymatic activity of HMG CoA lyase can be substrate is the one described in Example 5 for HMG-CoA measured by methods well known in the art. One possible synthase. The formation of 3-hydroxy-3-methylbutyrate can assay is described, e.g., in Mellanby et al. (Methods of Enzy be detected as described above. matic Analysis; Bergmeyer Ed. (1963), 454-458). In particu 0059. Such methods for identifying variants with lar, the enzyme activity is measured by a spectrophotometric improved enzymatic properties as regards the production of assay using the NADH-dependent reduction of acetoacetate 3-hydroxy-3-methylbutyrate may also be carried out in the by 3-hydroxybutyrate dehydrogenase. presence of a cofactor which allows for a steric and/or elec 0067 Preferably HMG CoA lyase activity is assayed as tronic complementation in the catalytic site of the enzyme? described in Example 4. In Such an assay the reaction mixture enzymes due to the fact that the substrate acetone is shorter (1 ml) contains 40 mM Tris-HCl pH 8, 1 mMMgCl, 0.5 mM than the natural substrate of the PksG protein. DTT, 0.4 mM HMG-CoA, 0.2 mM NADH, 5 units of 3-hy 0060. The modified version of the PksG protein accepting droxybutyrate dehydrogenase and is incubated for 5 min acetone as a Substrate but having a low affinity to or no longer before adding 0.005 mg/ml of HMG-CoA lyase and then the accepting its natural Substrate may be derived from a natu progress of the reaction is monitored by the decrease in absor rally occurring PksG protein or from an already modified, bance at 340 nm. optimized or synthetically synthesized PksG protein. 0068. The reaction catalyzed by HMG CoA lyase is 0061 C C Bond Cleavage/Condensation Lyase, HMG described in Some instances to require the presence of a CoA Lyase divalent cation, such as Mg" or Mn". Thus, it is preferred 0062. In the context of the present invention the term that an assay for determining the activity of HMG CoA lyase “C–C bond cleavage/condensation lyase' or “a protein/en includes Such divalent cations and that the method according Zyme having the activity of a C-C bond cleavage/conden to the invention for the production of 3-hydroxy-3-methylbu sation lyase' refers to an enzyme which is capable of cleaving tyric acid, if it makes use of HMG CoA lyase, is carried out in or forming by condensation a C-C bond and which contains the presence of Such cations. a so-called TIM (triose-phosphate isomerase) barrel domain. 0069. HMG CoA lyase is part of the hepatic ketogenesis. This TIM barrel domain is found in a number of pyruvate It catalyses the terminal reaction in the hepatic ketogenesis binding enzymes and acetyl-CoA dependent enzymes which is a key step of this pathway. The reaction is also an (Forouhar et al. J. Biol. Chem. 281 (2006), 7533-7545). The important step in leucine catabolism. TIM barrel domain has the classification lineage 3.20.20.150 (0070 HMG CoA lyase has been described for various in the CATH protein classification database (www.cathdb. organisms. Amino acid and nucleic acid sequences encoding info/cathnode/3.20.20.150). HMG CoA lyases are available from numerous sources. Gen 0063. The term “C C bond cleavage/condensation erally, the sequences only share an intermediate degree of lyases’ in particular includes enzymes which are classified as overall sequence identity. For example, the enzymes from isopropylmalate synthase (EC 2.3.3.13), as homocitrate Syn Bacillus subtilis or Brucella melitensis show only about 45% thase (EC 2.3.3.14) or as 4-hydroxy-2-ketovalerate aldolase identity to those of human HMG CoA lyase (Forouhar et al., (EC 4.1.3.39). Isopropylmalate synthase catalyzes the fol J. Biol. Chem. 281 (2006), 7533-7545). The three-dimen US 2015/0240271 A1 Aug. 27, 2015

sional structure of various HMG CoA lyase enzymes has been TABLE B-continued determined and the amino acids crucial for the enzymatic reaction are in principle well characterized (Forouhar et al., SwissprotTrEmbl loc. cit.; Fu et al., J. Biol. Chem. 281 (2006), 7526-7532). In Accession number Organism eukaryotes the HMG CoA lyase is located in the mitochon A7NGX6 Roseiflexus castenholzii drial matrix. A7NPP8 Roseiflexus castenholzii 0071. In principle any HMG CoA lyase enzyme can be A7NPR9 Roseiflexus castenholzii Q163P7 Roseobacter denitrificans used in the context of the present invention, in particular from A4XOW1 Salinispora tropica prokaryotic or eukaryotic organisms. A9KVP4 Shewanelia baitica 0072 Prokaryotic HMG CoA lyases are described, e.g., Q12LZ6 Shewanella denitrificans A8FT92 Shewanelia sediminis from Brucella abortus (UniProt accession numbers Q2YPLO Q82CR7 Streptomyces avermitiis and B2S7S2), Bacillus subtilis (UniProt accession number Q72IHO Thermus thermophilus O34873), Bacillus licheniformis (Fu et al., loc. cit) A9WGE2 Chloroflexus aurantiacus Pseudomonas Syringae (UniProt accession numbers B7EH4C6 Acinetobacter battmannii Q4ZTL2 and Q4ZRW6), Pseudomonas mevalonii (UniProt accession number P13703), Shewanella piezotolerans (Uni Prot accession number B8CRY9), Cellvibrio japonicus (Uni 0074 Eukaryotic HMG CoA lyases are described, e.g., Prot accession number B3PCQ7), Azotobacter vinelandii from plants, such as radish (Raphanus sativus) and Zea mays (UniProt accession numbers C1DJK8 and C1DL53), Hermi (Accession number B6U7B9, gene bank ACG45252) and nimonas arsenicoxydans (UniProt accession number A4G1 animals, such as human (Homo sapiens, UniProt accession F2) and Burkholderia cenocepacia (UniProt accession num number P35914), Cynomolgus monkey (UniProt accession ber A2VUW7). number Q8XZ6), Sumatran orangutan (Pongo abelii, Uni 0073 Moreover, the following Table B lists some known Prot accession number Q5R9E1), rat (Rattus norvegicus, HMG CoA lyases from prokaryotes: UniProt accession number P97519: Fu et al., loc. cit.), Mus musculus (UniProt accession number P38060), duck (Anas spec.), cattle (Bos taurus, UniProt accession number TABLE B Q29448), goat (Capra hircus), pigeon (Columba livia), SwissprotTrEmbl chicken (Gallus gallus, UniProt accession number P35915), Accession number Organism sheep (Ovis aries), pig (Sus scrofa), Danio rerio (Brachy Q6MHG9 Bdeliovibrio bacteriovorus danio rerio. A8WG57, gene bank BC154587) and from the A2TNG9 Dokdonia donghaensis protozoa Tetrahymena pyriformis. Q0C392 Hyphomonas neptunium B2HGF8 Mycobacterium marinum (0075. Examples of HMG CoA lyases from different QOK3L2 Ralstonia eutropha organisms are given in SEQID NOs: 17 to 23. SEQID NO: A9IB40 Bordeteila petrii 17 shows the sequence of the HMG CoA lyase of Zea mays QOB179 Burkholderia ambifaria (Accession number B6U7B9, gene bank ACG45252), SEQ ASFHS2 Flavobacterium johnsoniae Q5X487 Legionella pneumophila ID NO: 18 shows the sequence of the HMG CoA lyase of A1VH1 Polaromonas naphthalenivorans Danio rerio (Brachydanio rerio. A8WG57, gene bank Q5WKL8 Bacilius clausii BC154587), SEQID NO: 19 shows the sequence of the HMG A9IFQ7 Bordeteila petrii CoA lyase of Bos taurus (Uniprot accession number Q29448) A6HOL4 Flavobacterium psychrophilum Q8F7U7 Leptospira interrogans and SEQ ID NO: 20 shows the sequence of the HMG CoA A1VLB1 Polaromonas naphthalenivorans lyase of Homo sapiens (mitochondrial, Uniprot accession A9IR28 Bordeteila petrii number P35914, gene bank HUMHYMEGLA), SEQID NO: B1HZX7 Lysinibacilius sphaericits 21 shows the sequence of the HMG CoA lyase of Pseudomo A1VT25 Polaromonas naphthalenivorans nasputida (Q88H25), SEQID NO: 22 shows the sequence of Q9KDS7 Bacilius halodurans A9HXH6 Bordeteila petrii the HMG CoA lyase of Acinetobacter baumannii (B7H4C6) Q39QG8 Geobacter metairednicens and SEQ ID NO: 23 shows the sequence of the HMG CoA Q2GBZ7 Novosphingobium aromaticivorans lyase of Thermus thermophilus (Q72IHO). QOKC96 Ralstonia eutropha Q7CSK6 Agrobacterium tunefaciens 0076. In a preferred embodiment of the present invention Q65IT6 Bacilius licheniformis the HMG CoA lyase is an enzyme comprising an amino acid Q7NX69 Chronobacterium violaceum sequence selected from the group consisting of SEQID NOs: B9LMV8 Halorubrum lacusprofundi A6F2LO Marinobacter algicola 17 to 23 or a sequence which is at least n°6 identical to any of Q8ERF9 Oceanobacillusiheyensis SEQIDNOs: 17 to 23 and having the activity of a HMG CoA Q88HG4 Pseudomonas puttida lyase with n being an integer between 10 and 100, preferably QOKF83 Ralstonia eutropha 10, 15, 20, 25, 30, 35,40, 45,50,55, 60, 65,70, 75,80, 85,90, QOVL35 Alcanivorax borkumensis B2ST8 Burkholderia phymatum 91, 92,93, 94, 95, 96, 97, 98 or 99. A9AX6 Herpetosiphon aurantiacus 0077. As regards the determination of the degree of B1ML74 Mycobacterium abscessus sequence identity the same applies as has been set forth above Q88H25 Pseudomonas puttida Q11V59 Cytophaga hitchinsonii in connection with HMG CoA synthase. QOBWU6 Hyphomonas neptunium (0078. The HMG CoA lyase employed in the process A1BBP4 Paracoccus denitrificans according to the invention can be a naturally occurring HMG Q3IGB2 Pseudoalteromonas haloplanktis Q21QR6 Rhodoferax ferrireducens CoA lyase or it can be a HMG CoA lyase which is derived Q21RTO Rhodoferax ferrireducens from a naturally occurring HMG CoA lyase, e.g. by the A4CMM6 Robiginitalea biformata introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc. US 2015/0240271 A1 Aug. 27, 2015

0079. The term “HMG CoA lyase” or “a protein/enzyme I0085. The process according to the present invention may having the activity of a HMG CoA lyase in the context of the be carried out in vitro or in vivo. An in vitro reaction is present application also covers enzymes which are derived understood to be a reaction in which no cells are employed, from a HMG CoA lyase, which are capable of producing i.e. an acellular reaction. 3-hydroxy-3-methylbutyryl-CoA by a condensation of I0086 For carrying out the process in vitro the substrates acetone and a compound which provides an activated acetyl for the reaction and the enzymefenzymes are incubated under group as defined above, preferably acetyl-CoA but which conditions (buffer, temperature, cofactors etc.) allowing the only have a low affinity to acetoacetate as a Substrate or do no enzyme? enzymes to be active and the enzymatic conversion longer accept acetoacetate as a substrate. Such a modification to occur. The reaction is allowed to proceed for a time suffi of the preferred substrate of a HMG CoA lyase allows to cient to produce 3-hydroxy-3-methylbutyrate. The produc improve the conversion of acetone into 3-hydroxy-3-methyl tion of 3-hydroxy-3-methylbutyrate and/or 3-hydroxy-3-me butyryl-CoA and to reduce the production of the by-product thylbutyryl-CoA can be detected by comparison with HMG-CoA. Methods for modifying and/or improving the standard compound after separation by thin-layer chromatog desired enzymatic activities of proteins are well-known to the raphy, LC/MS and colorimetric assay after its derivatization. person skilled in the art and have been described above. I0087. The enzyme/enzymes may be in any suitable form allowing the enzymatic reaction to take place. It/they may be 0080. The capacity of a given enzyme to catalyze the pro purified or partially purified or in the form of crude cellular duction of 3-hydroxy-3-methylbutyryl-CoA can be deter extracts or partially purified extracts. It is also possible that mined in an assay as described in Example 6. the enzyme? enzymes is immobilized on a Suitable carrier. I0081. The modified version of the HMG CoA lyase I0088 Since the substrate acetone is in general shorter than accepting acetone as a substrate but having a low affinity to the natural Substrate used by the enzyme, e.g. acetoacetyl acetoacetate as a Substrate or no longer accepting acetoac CoA/acetoacetate used by HMG CoA synthase and HMG etate as a Substrate may be derived from a naturally occurring CoA lyase, respectively, it may be advantageous to add to the HMG CoA lyase or from an already modified, optimized or reaction mixture a cofactor which allows for a steric and/or synthetically synthesized HMG CoA lyase. electronic complementation in the catalytic site of the enzyme? enzymes. One example of such a cofactor, in the case 0082 Reactions may be Conducted in Cellulo or In Vitro of HMG CoA synthase, would be coenzyme A or a structur 0083. In the process according to the invention it is pos ally closely related molecule such as S-nitroso-CoA. sible to employ only one enzyme as defined above, e.g. only I0089. For carrying out the process in vivo use is made of a a HMG CoA synthase or only a HMG CoA lyase or only a Suitable organism/microorganism(s) which is/are capable of PksG protein. However, it is of course also possible to employ providing the Substrates, i.e. acetone and a compound which more than one activity, i.e. different enzymes, in particular provides an activated acetyl group as defined above, and an any combination of a HMG CoA synthase and a HMG CoA enzyme which is capable of catalyzing the formation of a lyase and a PkSG protein. E.g., in the case of an in vitro covalent bond between the carbon atom of the oxo (i.e. the method, more than one enzyme activity can be added to the C=O) group of acetone and the carbon atom (C) corre reaction mixture, either simultaneously or Subsequently in sponding to the methyl group of the compound which pro any possible order. In an in vivo method employing organ vides an activated acetyl group. In a preferred embodiment isms, in particular microorganisms, it is, e.g., possible to use said enzyme is a HMG CoA synthase and/or PksG protein an organism, in particular microorganism, expressing an and/or a C-C bond cleavage/condensation lyase, Such as a enzyme as defined above. However, it is also conceivable to HMG CoA lyase. use an organism/microorganism expressing any possible 0090 Recombinant Microorganisms combination of the above mentioned enzymes. Moreover, it is 0091. Thus, in the case of this embodiment the method also possible to use a mixture of two or more types of organ according to the invention is characterised in that the conver isms/microorganisms with one type expressing one enzyme sion of acetone and a compound which provides an activated and another expressing another enzyme. These different acetyl group is realized in the presence of an organism, pref types can then be cocultivated. erably a microorganism capable of producing acetone and 0084. The enzyme, e.g. the HMG CoA synthase and/or expressing an enzyme which is capable of the formation of a PkSG protein and/or a C-C bond cleavage/condensation covalent bond between the carbon atom of the oxo (i.e. the lyase, such as a HMG CoA lyase, employed in the process C=O) group of acetone and the carbon atom (C) corre according to the present invention can be a natural version of sponding to the methyl group of the compound which pro the protein or a synthetic protein as well as a protein which vides an activated acetyl group, preferably expressing an has been chemically synthesized or produced in a biological enzyme with the activity of a HMG CoA synthase (EC 2.3. system or by recombinant processes. The enzyme, e.g. the 3.10) and/or expressing a PkSG protein and/or expressing an HMG CoA synthase and/or PksG protein and/or a C-C bond enzyme with the activity of a C-C bond cleavage/conden cleavage/condensation lyase. Such as a HMG CoA lyase, may sation lyase, such as a HMG CoA lyase (EC 4.1.3.4). also be chemically modified, for example in order to improve 0092. The term “which is capable of producing acetone' its/their stability, resistance, e.g. to temperature, for facilitat in the context of the present invention means that the organ ing its/their purification or its immobilization on a Support. ism/microorganism has the capacity to produce acetone The enzyme? enzymes may be used in isolated form, purified within the cell due to the presence of enzymes providing form, in immobilized form, as a crude or partially purified enzymatic activities allowing the production of acetone from extract obtained from cells synthesizing the enzymefen metabolic precursors. Zymes, as chemically synthesized enzyme(s), as recombi 0093 Acetone is produced by certain microorganisms, nantly produced enzyme(s), in the form of microorganisms Such as Clostridium acetobutylicum, Clostridium beijer producing them etc. inckii, Clostridium cellulolyticum, Bacillus polymyxa and US 2015/0240271 A1 Aug. 27, 2015

Pseudomonas putida. The synthesis of acetone is best char 0.098 Preferably, the promoter is a promoter heterologous acterized in Clostridium acetobutylicum. It starts out with a to the organism/microorganism, i.e. a promoter which does reaction (reaction step 1) in which two molecules of acetyl not naturally occur in the respective organism/microorgan CoA are condensed into acetoacetyl-CoA. This reaction is ism. Even more preferably, the promoter is an inducible pro catalyzed by acetyl-CoA acetyltransferase (EC 2.3.1.9). moter. Promoters for driving expression in different types of Acetoacetyl-CoA is then converted into acetoacetate by a organisms, in particular in microorganisms, are well known reaction with acetic acid or butyric acid resulting also in the to the person skilled in the art. production of acetyl-CoA or butyryl-CoA (reaction step 2). 0099. In another preferred embodiment the nucleic acid This reaction is catalyzed e.g. by acetoacetylCoA transferase molecule is foreign to the organism/microorganism in that the (EC 2.8.3.8). AcetoacetylCoA transferase is known from encoded enzyme(s), e.g. the HMG CoA synthase and/or the various organisms, e.g. from E. coli in which it is encoded by encoded C-C bond cleavage/condensation lyase. Such as the ato AD gene or from Clostridium acetobutyllicum in which HMG CoA lyase, and/or PksG protein, is/are not endogenous it is encoded by the ctfAB gene. However, also other enzymes to the organism/microorganism, i.e. are naturally not can catalyze this reaction, e.g. 3-oxoacid CoA transferase expressed by the organism/microorganism when it is not (EC 2.8.3.5) or succinate CoA ligase (EC 6.2.1.5). genetically modified. In other words, the encoded HMG CoA 0094. Finally, acetoacetate is converted into acetone by a synthase and/or the encoded C-C bond cleavage/condensa decarboxylation step (reaction step 3) catalyzed by acetoac tion lyase, such as HMG CoA lyase, and/or PksG protein etate decarboxylase (EC 4.1.1.4). is/are heterologous with respect to the organism/microorgan 0095. The above described reaction steps 1 and 2 and the 1S. enzymes catalyzing them are not characteristic for the 0100. The term “recombinant” in another embodiment acetone synthesis and can be found in various organism. In means that the organism is genetically modified in the regu contrast, reaction step 3 which is catalyzed by acetoacetate latory region controlling the expression of an enzyme as decarboxylase (EC 4.1.1.4) is only found in those organisms defined above which naturally occurs in the organism so as to which are capable of producing acetone. lead to an increase in expression of the respective enzyme in comparison to a corresponding non-genetically modified 0096. In one preferred embodiment the organism organism. The meaning of the term high “higher expression” employed in the method according to the invention is an organism, preferably a microorganism, which naturally has is described further below. the capacity to produce acetone. Thus, preferably the micro 0101 Such a modification of a regulatory region can be organism belongs to the genus Clostridium, Bacillus or achieved by methods known to the person skilled in the art. Pseudomonas, more preferably to the species Clostridium One example is to exchange the naturally occurring promoter acetobutyllicum, Clostridium beijerinckii, Clostridium cellu by a promoter which allows for a higher expression or to lolyticum, Bacillus polymyxa or Pseudomonas putida. modify the naturally occurring promoter so as to show a higher expression. Thus, in this embodiment the organism 0097. In a further preferred embodiment, the organism contains in the regulatory region of the gene encoding an employed in the method according to the invention is an enzyme as defined above a foreign nucleic acid molecule organism, preferably a microorganism, which naturally has which naturally does not occur in the organism and which the capacity to produce acetone and which is recombinant in leads to a higher expression of the enzyme in comparison to a the sense that it has further been genetically modified so as to corresponding non-genetically modified organism. express an enzyme as defined above. The term “recombinant in one embodiment means that the organism is genetically 0102 The foreign nucleic acid molecule may be present in modified so as to contain a foreign nucleic acid molecule the organism/microorganism in extrachromosomal form, e.g. encoding an enzyme as defined above. In a preferred embodi as plasmid, or stably integrated in the chromosome. A stable ment the organism has been genetically modified so as to integration is preferred. contain a foreign nucleic acid molecule encoding an enzyme 0103) In a further preferred embodiment the organism/ as defined above, e.g. a HMG CoA synthase, a C-C bond microorganism is characterized in that the expression/activity cleavage/condensation lyase. Such as a HMG CoA lyase, or a of an enzyme as defined above, e.g. of a HMG CoA synthase PkSG protein or a foreign nucleic acid sequence encoding any and/or a C-C bond cleavage/condensation lyase, such as possible combination of such proteins. The term “foreign' in HMG CoA lyase, and/or a PksG protein, is higher in the this context means that the nucleic acid molecule does not organism/microorganism genetically modified with the for naturally occur in said organism/microorganism. This means eign nucleic acid molecule in comparison to the correspond that it does not occur in the same structure or at the same ing non-genetically modified organism/microorganism. A location in the organism/microorganism. In one preferred “higher expression/activity means that the expression/activ embodiment, the foreign nucleic acid molecule is a recombi ity of the enzyme, in particular of the HMG CoA synthase nant molecule comprising a promoter and a coding sequence and/or a C-C bond cleavage/condensation lyase, such as encoding the respective enzyme, e.g. a HMG CoA synthase HMG CoA lyase, and/or a PksG protein, in the genetically and/or a C-C bond cleavage/condensation lyase, such as modified microorganism is at least 10%, preferably at least HMG CoA lyase, and/or a PksG protein, in which the pro 20%, more preferably at least 30% or 50%, even more pref moter driving expression of the coding sequence is heterolo erably at least 70% or 80% and particularly preferred at least gous with respect to the coding sequence. Heterologous in 90% or 100% higher than in the corresponding non-geneti this context means that the promoter is not the promoter cally modified organism/microorganism. In even more pre naturally driving the expression of said coding sequence but ferred embodiments the increase in expression/activity may is a promoter naturally driving expression of a different cod be at least 150%, at least 200% or at least 500%. In particu ing sequence, i.e., it is derived from another gene, or is a larly preferred embodiments the expression is at least 10-fold, synthetic promoter or a chimeric promoter. more preferably at least 100-fold and even more preferred at US 2015/0240271 A1 Aug. 27, 2015 least 1000-fold higher than in the corresponding non-geneti mitilis (Uniprot accession number Q82NF4), Legionella cally modified organism/microorganism. pneumophila (Uniprot accession number Q5ZXQ9). Lacto 0104. The term “higher expression/activity also covers bacillus salivarius (Uniprot accession number Q1WVG5), the situation in which the corresponding non-genetically Rhodococcus spec. (Uniprot accession number Q0S7W4), modified organism/microorganism does not express a corre Lactobacillus plantarum (Uniprot accession number sponding enzyme, e.g. a HMG CoA synthase and/or a C-C Q890GO), Rhizobium leguminosarum (Uniprot accession bond cleavage/condensation lyase, such as a HMG CoA number Q1 M911), Lactobacillus casei (Uniprot accession lyase, and/or a PkSG protein, so that the corresponding number Q03B66), Francisella tularensis (Uniprot accession expression/activity in the non-genetically modified organ number QOBLC9), Saccharopolyspora erythreae (Uniprot ism/microorganism is Zero. accession number A4FKR9), Korarchaeum cryptofilum (Uni 0105 Methods for measuring the level of expression of a prot accession number B1 L3N6), Bacillus amyloliquefaciens given protein in a cell are well known to the person skilled in (Uniprot accession number A7Z8K8), Cochliobolus heteros the art. In one embodiment, the measurement of the level of trophus (Uniprot accession number Q8NJO3), Sulfolobus expression is done by measuring the amount of the corre islandicus (Uniprot accession number C3ML22) and Fran sponding protein. Corresponding methods are well known to cisella tularensis subsp. holarctica (strain OSU18). the person skilled in the art and include Western Blot, ELISA 0.108 More preferably, the organism, preferably microor etc. In another embodiment the measurement of the level of ganism, is genetically modified so as to be transformed with expression is done by measuring the amount of the corre a nucleic acid molecule encoding an enzyme capable of cata sponding RNA. Corresponding methods are well known to lyzing the above mentioned reaction step 2 of the acetone the person skilled in the art and include, e.g., Northern Blot. synthesis, i.e. the conversion of acetoacetyl CoA into acetoac 0106 Methods for measuring the enzymatic activity of the etate. above-mentioned enzymes, in particular HMG CoA synthase 0109 Even more preferably, the organism, preferably and/or a C-C bond cleavage/condensation lyase, Such as a microorganism, is genetically modified so as to be trans HMG CoA lyase, and/or a PksG protein, respectively, are formed with a nucleic acid molecule encoding an enzyme known in the art and have already been described above. capable of catalyzing the above mentioned reaction step 1 of 0107. In another preferred embodiment, the organism the acetone synthesis, i.e. the condensation of two molecules employed in the method according to the invention is a geneti of acetyl CoA into acetoacetatyl CoA. cally modified organism, preferably a microorganism, 0110. In a particularly preferred embodiment the organ derived from an organism/microorganism which naturally ism/microorganism is genetically modified so as to be trans does not produce acetone but which has been genetically formed with a nucleic acid molecule encoding an enzyme modified so as to produce acetone, i.e. by introducing the capable of catalyzing the above mentioned reaction step 1 of gene(s) necessary for allowing the production of acetone in the acetone synthesis and with a nucleic acid molecule encod the organism/microorganism. In principle any microorgan ing an enzyme capable of catalyzing the above mentioned ism can be genetically modified in this way. The enzymes reaction step 2 of the acetone synthesis or with a nucleic acid responsible for the synthesis of acetone have been described molecule encoding an enzyme capable of catalyzing the above. Genes encoding corresponding enzymes are known in above mentioned reaction step 1 of the acetone synthesis and the art and can be used to genetically modify a given micro with a nucleic acid molecule encoding an enzyme capable of organism so as to produce acetone. As described above, the catalyzing the above mentioned reaction step 3 of the acetone reaction steps 1 and 2 of the acetone synthesis occur naturally synthesis or with a nucleic acid molecule encoding an in most organisms. However, reaction step 3 is characteristic enzyme capable of catalyzing the above mentioned reaction and crucial for acetone synthesis. Thus, in a preferred step 2 of the acetone synthesis and with a nucleic acid mol embodiment, a genetically modified organism/microorgan ecule encoding an enzyme capable of catalyzing the above ism derived from an organism/microorganism which natu mentioned reaction step 3 of the acetone synthesis or with a rally does not produce acetone is modified so as to contain a nucleic acid molecule encoding an enzyme capable of cata nucleotide sequence encoding an enzyme catalyzing the con lyzing the above mentioned reaction step 1 of the acetone version of acetoacetate into acetone by decarboxylation, e.g. synthesis and with a nucleic acid molecule encoding an an acetoacetate decarboxylase (EC 4.1.1.4). Nucleotide enzyme capable of catalyzing the above mentioned reaction sequences from several organisms encoding this enzyme are step 2 of the acetone synthesis and with a nucleic acid mol known in the art, e.g. the adc gene from Clostridium aceto ecule encoding an enzyme capable of catalyzing the above butyllicum (Uniprot accession numbers P23670 and P23673), mentioned reaction step 3 of the acetone synthesis. Clostridium beijerinckii (Clostridium MP; Q9RPK1), 0111 Methods for preparing the above mentioned geneti Clostridium pasteurianum (Uniprot accession number cally modified organism, preferably microorganisms, are P81336), Bradyrhizobium sp. (strain BTAi1/ATCC BAA well known in the art. Thus, generally, the organism/micro 1182: Uniprot accession number A5EBU7), Burkholderia organism is transformed with a DNA construct allowing mallei (ATCC 10399 A9LBS0), Burkholderia mallei (Uni expression of the respective enzyme in the microorganism. prot accession number A3MAE3), Burkholderia mallei FMH Such a construct normally comprises the coding sequence in A5XJB2, Burkholderia cenocepacia (Uniprot accession question linked to regulatory sequences allowing transcrip number AOB471), Burkholderia ambifaria (Uniprot acces tion and translation in the respective host cell, e.g. a promoter sion number Q0b5P1), Burkholderia phytofirmans (Uniprot and/enhancer and/or transcription terminator and/or ribo accession number B2T319), Burkholderia spec. (Uniprot Some binding sites etc. The prior art already describes micro accession number Q38ZUO), Clostridium botulinum (Uni organisms which have been genetically modified so as to be prot accession number B2TLN8), Ralstonia pickettii (Uni able to produce acetone. In particular genes from, e.g., prot accession number B2UIG7), Streptomyces nogalater Clostridium acetobutylicum have been introduced into E. coli (Uniprot accession number Q9EYI7), Streptomyces aver thereby allowing the synthesis of acetone in E. coli, a bacte US 2015/0240271 A1 Aug. 27, 2015

rium which naturally does not produce acetone (Bermejo et densation lyase, such as HMG CoA lyase, and/or a PksG al., Appl. Environ. Microbiol. 64 (1998); 1079-1085; Hanaiet protein, in the genetically modified organism/microorganism al., Appl. Environ. Microbiol. 73 (2007), 7814-7818). In par is at least 10%, preferably at least 20%, more preferably at ticular Hanai et al. (loc. cit.) shows that it is sufficient to least 30% or 50%, even more preferably at least 70% or 80% introduce a nucleic acid sequence encoding an acetoacetate and particularly preferred at least 90% or 100% higher than in decarboxylase (such as that from Clostridium acetobutyli the corresponding non-genetically modified organism/micro cum) in order to achieve acetone production in E. coli indi organism. In even more preferred embodiments the increase cating that the endogenous enzymes in E. coli catalyzing the in expression/activity may be at least 150%, at least 200% or above-mentioned reaction steps 1 and 2 (i.e. the expression at least 500%. In particularly preferred embodiments the products of the E. coliatoBandato AD genes) are sufficient to expression is at least 10-fold, more preferably at least 100 provide substrate for the acetone production. fold and even more preferred at least 1000-fold higher than in 0112. In a particularly preferred embodiment the organ the corresponding non-genetically modified organism/micro ism, preferably a microorganism, employed in the method organism. according to the invention is a recombinant organism/micro 0117 The term “higher expression/activity also covers organism derived from an organism/microorganism which the situation in which the corresponding non-genetically naturally does not produce acetone but which has been geneti modified organism/microorganism does not express said cally modified, as described above, so as to produce acetone enzyme, e.g. a HMG CoA synthase and/or a C-C bond and which expresses an enzyme which is capable of cata cleavage/condensation lyase. Such as a HMG CoA lyase, lyzing the formation of a covalent bond between the carbon and/or a PkSG protein, so that the corresponding expression/ atom of the oxo (i.e. the C=O) group of acetone and the activity in the non-genetically modified organism/microor carbon atom (C) corresponding to the methyl group of the ganism is Zero. As regards the methods for measuring the compound which provides an activated acetyl group as level of expression or activity, the same applies what has defined above. The term “recombinant in this context pref already been said above. erably means that the organism is recombinant in the sense 0118. The term “organism' as used in the context of the that it has further been genetically modified so as to express present invention refers in general to any possible type of an enzyme as defined above. The term “recombinant in one organism, in particular eukaryotic organisms, prokaryotic embodiment means that the organism is genetically modified organisms and archaebacteria. The term includes animal, So as to contain a foreign nucleic acid molecule encoding an plants, fungi, bacteria and archaebacteria. The term also enzyme as defined above, e.g. a HMG CoA synthase or a includes isolated cells or cell aggregates of such organisms, C-C bond cleavage/condensation lyase. Such as a HMG like tissue or calli. CoA lyase, or a PkSG protein, or a foreign nucleic acid mol 0119. In one preferred embodiment, the organism is a ecule encoding any possible combination of the above microorganism. The term “microorganism’ in the context of defined enzymes. the present invention refers to prokaryotic cells, in particular 0113. As regards the definition of the term “foreign bacteria, as well as to fungi. Such as yeasts, and also to algae nucleic acid molecule' the same applies what has already and archaebacteria. In one preferred embodiment, the micro been set forth above. organism is a bacterium. In principle any bacterium can be 0114. The term “recombinant” in another embodiment used. Preferred bacteria to be employed in the process accord means that the organism is genetically modified in the regu ing to the invention are bacteria of the genus Bacillus, latory region controlling the expression of an enzyme as Clostridium, Pseudomonas or Escherichia. In a particularly defined above which naturally occurs in the organism so as to preferred embodiment the bacterium belongs to the genus lead to an increase in expression of the respective enzyme in Escherichia and even more preferred to the species Escheri comparison to a corresponding non-genetically modified chia coli. organism. The meaning of the term high “higher expression” I0120 In another preferred embodiment the microorgan is described further below. ism is a fungus, more preferably a fungus of the genus Sac 0115 Such a modification of a regulatory region can be charomyces, Schizosaccharomyces, Aspergillus or Tricho achieved by methods known to the person skilled in the art. derma and even more preferably of the species One example is to exchange the naturally occurring promoter Saccharomyces cerevisiae, Schizosaccharomyces pombe, by a promoter which allows for a higher expression or to Aspergillus niger or of the species Trichoderma reesei. modify the naturally occurring promoter so as to show a I0121. In still another preferred embodiment the microor higher expression. Thus, in this embodiment the organism ganism is a photosynthetically active microorganism Such as contains in the regulatory region of the gene encoding an bacteria which are capable of carrying out photosynthesis or enzyme as defined above a foreign nucleic acid molecule micro-algae. which naturally does not occur in the organism and which I0122. In a particularly preferred embodiment the micro leads to a higher expression of the enzyme in comparison to a organism is an algae, more preferably an algae belonging to corresponding non-genetically modified organism. the diatomeae. 0116 Preferably such an organism/microorganism is I0123. If microorganism are used in the context of the characterized in that the expression/activity of said enzyme, method of the present invention, it is also conceivable to carry e.g. the HMG CoA synthase and/or a C-C bond cleavage/ out the method according to the invention in a manner in condensation lyase, such as a HMG CoA lyase, and/or a PksG which two types of microorganisms are employed, i.e. one protein, is higher in the recombinant organism/microorgan type which produces acetone and one type which uses the ism in comparison to the corresponding non-genetically acetone produced by the first type of microorganisms to con modified organism/microorganism. A "higher expression/ vert it with the help of an enzyme as defined herein above. activity means that the expression/activity of the enzyme, e.g. 0.124 When the process according to the invention is car the HMG CoA synthase and/or a C-C bond cleavage/con ried out in vivo by using microorganisms providing the US 2015/0240271 A1 Aug. 27, 2015 respective enzyme activity/activities, the microorganisms are produce acetone, i.e. by introducing the gene(s) necessary for cultivated under suitable culture conditions allowing the allowing the production of acetone in the organism/microor occurrence of the enzymatic reaction(s). The specific culture ganism. In principle any organism/microorganism can be conditions depend on the specific microorganism employed genetically modified in this way. The enzymes responsible for but are well known to the person skilled in the art. The culture the synthesis of acetone have been described above. Genes conditions are generally chosen in Such a manner that they encoding corresponding enzymes are known in the art and allow the expression of the genes encoding the enzymes for can be used to genetically modify a given organism, prefer the respective reactions. Various methods are known to the ably microorganism So as to produce acetone. person skilled in the art in order to improve and fine-tune the 0.132. In a preferred embodiment, a genetically modified expression of certain genes at certain stages of the culture organism/microorganism derived from an organism/microor Such as induction of gene expression by chemical inducers or ganism which naturally does not produce acetone is modified by a temperature shift. So as to contain a nucleotide sequence encoding an enzyme 0.125. In another preferred embodiment the organism catalyzing the conversion of acetoacetate into acetone by employed in the method according to the invention is an decarboxylation, e.g. an acetoacetate decarboxylase (EC 4.1. organism which is capable of photosynthesis, such as a plant 1.4). Nucleotide sequences from several organisms encoding or microalgae. In principle any possible plant can be used, i.e. this enzyme are known in the art, e.g. the adc gene from a monocotyledonous plant or a dicotyledonous plant. It is Clostridium acetobutyllicum. More preferably, the organism/ preferable to use a plant which can be cultivated on an agri microorganism is genetically modified so as to be trans culturally meaningful scale and which allows to produce formed with a nucleic acid molecule encoding an enzyme large amounts of biomass. Examples are grasses like Lolium, capable of catalyzing the above mentioned reaction step 2 of cereals like rye, barley, oat, millet, maize, other starch storing the acetone synthesis, i.e. the conversion of acetoacetyl CoA plants like potato or Sugar storing plants like Sugar cane or into acetoacetate. Sugar beet. Conceivable is also the use of tobacco or of veg 0.133 Even more preferably, the organism/microorganism etable plants such as tomato, pepper, cucumber, eggplant etc. is genetically modified so as to be transformed with a nucleic Another possibility is the use of oil storing plants such as rape acid molecule encoding an enzyme capable of catalyzing the seed, olives etc. Also conceivable is the use of trees, in par above mentioned reaction step 1 of the acetone synthesis, i.e. ticular fast growing trees Such as eucalyptus, poplar or rubber the condensation of two molecules of acetyl CoA into tree (Hevea brasiliensis). acetoacetatyl CoA. 0.126 The present invention also relates to an organism, 0134. In a particularly preferred embodiment the organ preferably a microorganism, which is characterized by the ism/microorganism is genetically modified so as to be trans following features: formed with a nucleic acid molecule encoding an enzyme I0127 (a) it is capable of producing acetone; and capable of catalyzing the above mentioned reaction step 1 of I0128 (b) it expresses an enzyme which is capable of the acetone synthesis and with a nucleic acid molecule encod catalyzing the formation of a covalent bond between the ing an enzyme capable of catalyzing the above mentioned carbonatom of the oxo (i.e. the C=O) group of acetone reaction step 2 of the acetone synthesis or with a nucleic acid and the carbon atom (C) corresponding to the methyl molecule encoding an enzyme capable of catalyzing the group of the compound which provides an activated above mentioned reaction step 1 of the acetone synthesis and acetyl group as defined above, preferably an enzyme with a nucleic acid molecule encoding an enzyme capable of with the activity of a HMG CoA synthase (EC 2.3.3.10) catalyzing the above mentioned reaction step 3 of the acetone and/or an enzyme with the activity of a C-C bond synthesis or with a nucleic acid molecule encoding an cleavage/condensation lyase. Such as a HMG CoA lyase enzyme capable of catalyzing the above mentioned reaction (EC 4.1.3.4) and/or a PksG protein. step 2 of the acetone synthesis and with a nucleic acid mol 0129. As regards the source, nature, properties, sequence ecule encoding an enzyme capable of catalyzing the above etc. of the enzyme, in particular the HMG CoA synthase, the mentioned reaction step 3 of the acetone synthesis or with a C C bond cleavage/condensation lyase, such as HMG CoA nucleic acid molecule encoding an enzyme capable of cata lyase, and/or a PkSG protein expressed in the organism lyzing the above mentioned reaction step 1 of the acetone according to the invention, the same applies as has been set synthesis and with a nucleic acid molecule encoding an forth above in connection with the method according to the enzyme capable of catalyzing the above mentioned reaction invention. step 2 of the acetone synthesis and with a nucleic acid mol 0130. In one preferred embodiment, the organism accord ecule encoding an enzyme capable of catalyzing the above ing to the invention is an organism, preferably a microorgan mentioned reaction step 3 of the acetone synthesis. ism which naturally has the capacity to produce acetone, i.e., 0.135 Methods for preparing the above mentioned geneti feature (a) mentioned above is a feature which the organism, cally modified orgnanisms/microorganisms are well known preferably microorganism, shows naturally. Thus, preferably in the art. Thus, generally, the organism/microorganism is the organism is a microorganism which belongs to the genus transformed with a DNA construct allowing expression of the Clostridium, Bacillus or Pseudomonas, more preferably to respective enzyme in the organism/microorganism. Such a the species Clostridium acetobutylicum, Clostridium beijer construct normally comprises the coding sequence in ques inckii, Clostridium cellulolyticum, Bacillus polymyxa or tion linked to regulatory sequences allowing transcription and Pseudomonas putida. translation in the respective host cell, e.g. a promoter and/ 0131. In another preferred embodiment, the organism, enhancer and/or transcription terminator and/or ribosome preferably microorganism, according to the invention is a binding sites etc. The prior art already describes organism, in genetically modified organism/microorganism derived from particular microorganisms which have been genetically an organism/microorganism which naturally does not pro modified so as to be able to produce acetone. In particular duce acetone but which has been genetically modified so as to genes from, e.g., Clostridium acetobutylicum have been US 2015/0240271 A1 Aug. 27, 2015

introduced into E. coli thereby allowing the synthesis of e.g. the HMG CoA synthase and/or the encoded C C bond acetone in E. coli, a bacterium which naturally does not cleavage/condensation lyase, such as HMG CoA lyase, and/ produce acetone (Bermejo et al., Appl. Environ. Microbiol. or the encoded PksG protein, is/are heterologous with respect 64 (1998); 1079-1085; Hanaietal. Appl. Environ. Microbiol. to the organism/microorganism. 73 (2007), 7814-7818). In particular Hanai et al. (loc. cit.) 0.138. The term “recombinant in another aspect means shows that it is sufficient to introduce a nucleic acid sequence that the organism is genetically modified in the regulatory encoding an acetoacetate decarboxylase (such as that from region controlling the expression of an enzyme as defined Clostridium acetobutyllicum) in order to achieve acetone pro above which naturally occurs in the organism so as to lead to duction in E. coli indicating that the endogenous enzymes in an increase in expression of the respective enzyme in com E. coli catalyzing the above-mentioned reaction steps 1 and 2 parison to a corresponding non-genetically modified organ (i.e. the expression products of the E. coli atoB and ato AD ism. The meaning of the term high “higher expression' is genes) are sufficient to provide Substrate for the acetone pro described further below. duction. 0.139. Such a modification of a regulatory region can be 0136. In a further preferred embodiment the organism, achieved by methods known to the person skilled in the art. preferably a microorganism, according to the invention is One example is to exchange the naturally occurring promoter genetically modified so as to express an an enzyme which is by a promoter which allows for a higher expression or to capable of catalyzing the formation of a covalent bond modify the naturally occurring promoter so as to show a between the carbon atom of the oxo (i.e. the C=O) group of higher expression. Thus, in this embodiment the organism acetone and the carbon atom (C) corresponding to the contains in the regulatory region of the gene encoding an methyl group of the compound which provides an activated enzyme as defined above a foreign nucleic acid molecule acetyl group. In this context, the term “recombinant’ means which naturally does not occur in the organism and which in a first aspect that the organism contains a foreign nucleic leads to a higher expression of the enzyme in comparison to a acid molecule encoding an enzyme which is capable of cata corresponding non-genetically modified organism. lyzing the formation of a covalent bond between the carbon 0140. In a further preferred embodiment the organism/ atom of the oxo (i.e. the C=O) group of acetone and the microorganism is characterized in that the expression/activity carbon atom (C) corresponding to the methyl group of the of said enzyme, e.g. the HMG CoA synthase and/or a C C compound which provides an activated acetyl group, prefer bond cleavage/condensation lyase, such as HMG CoA lyase, ably a foreign nucleic acid molecule encoding a HMG CoA and/or the PkSG protein, is higher in the organism/microor synthase or a foreign nucleic acid molecule encoding a C-C ganism genetically modified with the foreign nucleic acid bond cleavage/condensation lyase, such as a HMG CoA molecule in comparison to the corresponding non-genetically lyase, or a foreign nucleic acid molecule encoding a PkSG modified organism/microorganism. A "higher expression/ protein or a foreign nucleic acid molecule encoding any pos activity means that the expression/activity of the enzyme, e.g. sible combination of the enzymes having the above-men the HMG CoA synthase and/or a C-C bond cleavage/con tioned property. The term “foreign' in this context means that densation lyase, such as HMG CoA lyase, and/or the PksG the nucleic acid molecule does not naturally occur in said protein, in the genetically modified organism/microorganism organism/microorganism. This means that it does not occur in is at least 10%, preferably at least 20%, more preferably at the same structure or at the same location in the organism/ least 30% or 50%, even more preferably at least 70% or 80% microorganism. In one preferred embodiment, the foreign and particularly preferred at least 90% or 100% higher than in nucleic acid molecule is a recombinant molecule comprising the corresponding non-genetically modified organism/micro a promoter and a coding sequence encoding said enzyme, e.g. organism. In even more preferred embodiments the increase the HMG CoA synthase and/or a C-C bond cleavage/con in expression/activity may be at least 150%, at least 200% or densation lyase, such as a HMG CoA lyase, and/or a PksG at least 500%. In particularly preferred embodiments the protein, in which the promoter driving expression of the cod expression is at least 10-fold, more preferably at least 100 ing sequence is heterologous with respect to the coding fold and even more preferred at least 1000-fold higher than in sequence. Heterologous in this context means that the pro the corresponding non-genetically modified organism/micro moter is not the promoter naturally driving the expression of organism. said coding sequence but is a promoter naturally driving 0.141. The term “higher expression/activity also covers expression of a different coding sequence, i.e., it is derived the situation in which the corresponding non-genetically from another gene, or is a synthetic promoter or a chimeric modified organism/microorganism does not express a corre promoter. Preferably, the promoter is a promoter heterolo sponding enzyme, e.g. a HMG CoA synthase and/or a C-C gous to the organism/microorganism, i.e. a promoter which bond cleavage/condensation lyase, such as a HMG CoA does naturally not occur in the respective organism/microor lyase, and/or a PkSG protein, so that the corresponding ganism. Even more preferably, the promoter is an inducible expression/activity in the non-genetically modified organ promoter. Promoters for driving expression in different types ism/microorganism is Zero. of organisms, in particular microorganisms, are well known 0.142 Methods for measuring the level of expression of a to the person skilled in the art. given protein in a cell are well known to the person skilled in 0.137 In another preferred embodiment the nucleic acid the art. In one embodiment, the measurement of the level of molecule is foreignto the organism/microorganism in that the expression is done by measuring the amount of the corre encoded enzyme(s), e.g. the HMG CoA synthase and/or the sponding protein. Corresponding methods are well known to encoded C-C bond cleavage/condensation lyase, such as a the person skilled in the art and include Western Blot, ELISA HMG CoA lyase, and/or the encoded PksG protein, is/are not etc. In another embodiment the measurement of the level of endogenous to the organism/microorganism, i.e. are naturally expression is done by measuring the amount of the corre not expressed by the organism/microorganism when it is not sponding RNA. Corresponding methods are well known to genetically modified. In other words, the encoded enzyme(s), the person skilled in the art and include, e.g., Northern Blot. US 2015/0240271 A1 Aug. 27, 2015

0143 Methods for measuring the enzymatic activity of the bond cleavage/condensation lyase, Such as a HMG CoA above-mentioned enzyme, in particular of a HMG CoA syn lyase (EC 4.1.3.4), and/or a PksG protein for the pro thase and/or a HMG CoA lyase and/or a PksG protein, respec duction of 3-hydroxy-3-methylbutyric acid. tively, are known in the art and have already been described 0153. I.e., the present invention also relates to the use of an above. organism/microorganism according to the invention for the 0144. The term “organism' as used in the context of the production of 3-hydroxy-3-methylbutyric acid. present invention refers in general to any possible type of 0154 The present invention also relates to a composition organism, in particular eukaryotic organisms, prokaryotic comprising an organism according to the present invention. organisms and archaebacteria. The term includes animal, 0155 Moreover, the present invention also relates to a plants, fungi, bacteria and archaebacteria. The term also composition comprising (i) acetone; and (ii) a compound includes isolated cells or cell aggregates of Such organisms, which provides an activated acetyl group as defined herein like tissue or calli. above; and (iii) an enzyme which is capable of catalyzing the 0145. In one preferred embodiment, the organism is a formation of a covalent bond between the carbonatom of the microorganism. The term “microorganism’ in the context of oxo (i.e. the C=O) group of acetone and the carbonatom(C) the present invention refers to prokaryotic cells, in particular corresponding to the methyl group of the compound which bacteria, as well as to fungi. Such as yeasts, and also to algae provides an activated acetyl group as defined herein above. and archaebacteria. In one preferred embodiment, the micro 0156 For the preferred embodiments of the enzyme the organism is a bacterium. In principle any bacterium can be same applies as has already been set forth above in connection used. Preferred bacteria to be employed in the process accord with the method and the organism according to the invention. ing to the invention are bacteria of the genus Bacillus, 0157 Moreover, the present invention also relates to the Clostridium, Pseudomonas or Escherichia. In a particularly use of an enzyme which is capable of catalyzing the formation preferred embodiment the bacterium belongs to the genus of a covalent bond between the carbon atom of the oxo (i.e. Escherichia and even more preferred to the species Escheri the C=O) group of acetone and the carbon atom (C) corre chia coli. sponding to the methyl group of the compound which pro 0146 In another preferred embodiment the microorgan vides an activated acetyl group as defined herein above for the ism is a fungus, more preferably a fungus of the genus Sac production of 3-hydroxy-3-methylbutyric acid. For the pre charomyces, Schizosaccharomyces, Aspergillus or Tricho ferred embodiments of the enzyme the same applies as has derma and even more preferably of the species already been set forth above in connection with the method Saccharomyces cerevisiae, Schizosaccharomyces pombe, and the organism according to the invention. Aspergillus niger or of the species Trichoderma reesei. 0158 Finally, the present invention also relates to the use 0147 In still another preferred embodiment the microor of acetone for the production of 3-hydroxy-3-methylbutyric ganism is a photosynthetically active microorganism Such as acid, comprising the enzymatic conversion of acetone and a bacteria which are capable of carrying out photosynthesis or compound which provides an activated acetyl group as micro-algae. defined herein above. In a preferred embodiment the enzy 0148. In a particularly preferred embodiment the micro matic conversion is achieved by an enzyme as described organism is an algae, more preferably analgae from the genus above in connection with the method according to the inven belonging to the diatomeae. tion, more preferably with an enzyme having the enzymatic 0149. In another preferred embodiment the organism activity of a HMG CoA synthase and/or with an enzyme according to the invention is an organism which is capable of having the enzymatic activity of a C-C bond cleavage/con photosynthesis, such as a plant or micro-algae. In principle, it densation lyase, such as a HMG CoA lyase, and/or a PksG can be any possible plant, i.e. a monocotyledonous plant or a protein, and most preferably the conversion is achieved by the dicotyledonous plant. It is preferably a plant which can be use of an organism according to the invention. cultivated on an agriculturally meaningful scale and which allows to produce large amounts of biomass. Examples are BRIEF DESCRIPTION OF THE DRAWINGS grasses like Lolium, cereals like rye, barley, oat, millet, maize, 0159 FIG. 1: Chemical structure of 3-hydroxy-3-methyl other starch storing plants like potato or Sugar storing plants butyric acid (also referred to as beta-hydroxyisovalerate) like Sugar cane or Sugar beet. Conceivable is also the use of 0160 FIG. 2: Reaction scheme of the reaction catalysed tobacco or of vegetable plants such as tomato, pepper, cucum by HMG-CoA synthase ber, eggplant etc. In another preferred embodiment the plant 0.161 FIG. 3: Reaction scheme of the reaction catalysed is an oil storing plants such as rape seed, olives etc. Also by HMG-CoA lyase conceivable is the use of trees, in particular fast growing trees (0162 FIG. 4: Reaction schemes of the reactions of the Such as eucalyptus, poplar or rubber tree (Hevea brasiliensis). pksX pathway including the reaction catalysed by the PksG 0150. The present invention also relates to the use of an protein organism, preferably a microorganism, which is character (0163 FIG. 5: Reaction scheme of the reaction of the con ized by the following features: version of acetone and a compound containing an activated 0151 (a) it is capable of producing acetone; and acetyl group into 3-hydroxy-3-methylbutyric acid X stands 0152 (b) it expresses an enzyme which is capable of for S CH2-CH2-NH CO CH2-CH2-NH CO CH catalyzing the formation of a covalent bond between the (OH) C(CH3)2-CH2-O-PO2H C10H13N5O7P (coen carbonatom of the oxo (i.e. the C=O) group of acetone Zyme A), S CH2-CH2-NH CO-CH2-CH2-NH CO and the carbon atom (C) corresponding to the methyl CH(OH)–C(CH3)2-CH2-O-PO2H-polypeptide (acyl group of the compound which provides an activated carrier protein), S CH2-CH2-NH CO-CH2-CH2 acetyl group as defined herein above, preferably an NH CO-CH(OH)–C(CH3)2-CH2-OH (pantetheine), enzyme with the activity of a HMG CoA synthase (EC S CH-CH NH CO-CH (N-acetyl-cysteamine), 2.3.3.10) and/or an enzyme with the activity of a C C S-CH (methane thiol), S CH2-CH(NH2)-CO2H (cys US 2015/0240271 A1 Aug. 27, 2015

teine), S CH2-CH2-CH(NH2)-CO2H (homocysteine), (0174. Overexpression in E. Coli: S CH2-CH(NH C5H8NO3)-CO. NH CH2-CO2H 0.175 Plasmids are electroporated into E. coli BL21 bac (glutathione), S CH, CH, SOH (coenzyme M) and teria (Novagen) that are then spread on an amplicillin contain OH (acetic acid). ing LB-Agar Petri dish. The cultures are grown at 30° C. on 0164 FIG. 6: Mass spectra of commercial available 3-hy TB medium, containing 0.5 M sorbitol, 5 mM betaine, 100 droxy-3-methylbutyrate ug/ml amplicillin under moderate shaking. When OD (600 (0165 FIG. 7: Mass spectra of formation of 3-hydroxy-3- nm) reached 0.8, IPTG is added to a final concentration of 1 methylbutyrate from acetyl-CoA and acetone in the presence mM, and expression is run for 16 hours at 20° C. under of Hmg-CoA synthase from Gallus gallus (P23228). moderated shaking. The bacteria cells are then harvested by 0166 FIG. 8: Mass spectra of the control assay without centrifugation at 4°C., 10.000 rpm, 20 minutes and frozen at enzyme. -80° C. (0167 FIG. 9: Michaelis-Menten plot for the reaction with (0176 Cell Extract Preparation: the HMG CoA synthase of S. epidermidis described in 0177 Cell extracts are prepared by resuspending 1.6 g of Example 7 cell pellet in 5 ml 50 mM NaHPO, buffer, containing 300 mM NaCl, 5 mM MgCl, 1 mM DTT pH 8. 20 ul lysonase EXAMPLES (Novagen) is then added to the preparations, which are incu bated for 10 min at room temperature and 20 min on ice. The (0168 The following examples serve to illustrate the inven cell lysis is achieved by triple sonication treatment of 5 min tion. utes in ultrasonic water-bath on ice and homogenization of extract between each pulse. The crude extracts are then clari Example 1 fied by centrifugation at 4°C., 10.000 rpm, 20 minutes. Protein Purification: Bioinformatic Method Used to Create HMG-CoA 0178 Synthases and HMG-CoA Lyases Database 0179 The clear supernatants are loaded onto the PRO TINO-1000R Ni-IDA column (columns for the purification (0169. A panel of 12 HMG-CoA synthases and 8 HMG of proteins, Macherey-Nagel) which enables the specific CoA lyases were selected to create a non-redundant set of immobilization of proteins carrying 6-histidine tails. The col proteins aiming to represent the diversity of these enzyme umns are washed and the enzymes are eluted with 4 ml 50 classes as found across eukaryotic organisms. These proteins mM Na2HPO4 buffer, containing 300 mM. NaCl, 5 mM were identified by performing multiple sequence-based and MgCl, 1 mM DTT, 250 mM imidazole pH 8. The enzyme text-based searches on the Universal Protein Resource Data containing fractions are then concentrated and desalted on base Uniprot (www.uniprot.org). They all contain unique fea Amicon Ultra-4 10 kDa filter unit (membranes for filtration, tures such as conserved protein domains and motifs charac dialysis; Millipore) and resuspended in 250 ul 40 mM Tris teristic to the enzyme class of interest. In order to effectively HCl pH8, containing 0.5 mMDTT. The protein concentration cover the sequence diversity without having to Screen a large is determined by the Bradford method. set of proteins, the initial pool of enzymes was narrowed 0180. The homogeneity of purified enzymes varied from down by grouping them into clusters of sequences with more 20% to 75%. than 85% homology and then selecting one single candidate sequence representative of each cluster. Protein sequence Example 3 identity ranged from 30% to 80% and from 50% to 80% between any two proteins from the HMG-CoA synthases Measure of the HMG-CoA Synthase Activity. Using panel and the lyases panel respectively. Natural Substrates Acetoacetyl-CoA and Acetyl-CoA 0170 The same approach was applied to select the HMG CoA synthases and HMG-CoA lyases from prokaryotic 0181. The HMG-CoA synthase activity is measured organisms. The created set contained 50 proteins homologues according to Clinkenbeard et al. (J. Biol. Chem. 250 (1975), to HMG-CoA synthases, including pksG proteins, and 59 3.108-3116). The standard assay medium mixture for HMG proteins homologues to HMG-CoA lyases. CoA synthases contains 40 mM Tris-HCl pH 8, 1 mMMgCl, 100 uMacetoacetyl-CoA, 200 uMacetyl-CoA, 0.5 mMDTT Example 2 in a total volume of 1 ml. Mitochondria HMG-CoA synthases are assayed in the absence of MgCl, to avoid the inhibition Cloning, Expression and Purification of a Collection observed for this enzyme (Reed et al., J. Biol. Chem. 250 of HMG-CoA Lyases and HMG-CoA Synthases (1975), 3117-3123). Reaction is initiated by addition of 0.02 mg/mL enzyme. 0171 Gene Cloning: 0182. A Control assay was carried out in the absence of 0172. The nucleic acid sequences coding for HMG-CoA enzyme. HMG-CoA synthase activity was measured by synthase and lyase from eukaryotic organism were optimized monitoring the decrease in absorbance at 303 nm that accom for E. coli codon preference and the genes were obtained by panies the acetyl-CoA-dependent disappearance of the eno chemical synthesis (GeneArt(R), reagents). late form of acetoacetyl-CoA. To account for non-specific 0173 The genes encoding for HMG-CoA synthases and disappearance of acetoacetyl-CoA, results obtained in a con lyases from prokaryotic organisms were cloned from trol assay lacking enzyme were subtracted from results genomic DNA of different origins by routine recombinant obtained in test samples. The apparent absorption coefficient techniques. These genes were then inserted in a His-tag con for acetoacetyl-CoA under the assay conditions was 5600 taining plT 25b and plT22b vectors (Novagen, Inc), respec M''cm'. One enzyme unit represented the disappearance in tively, for eukaryotic and prokaryotic organisms. 1 min of 1 Jumol of acetoacetyl-CoA. US 2015/0240271 A1 Aug. 27, 2015

TABLE 1. TABLE 2-continued Physiological activity of some purified HMG-CoA synthases Physiological activity of some purified HMG-CoA lyases or enzymes honologous to HMG CoA Synthases Physiological Physiological Uniprot activity Uniprot activity Accession Imol/min mg Accession Imol/min mg number Organism protein number Organism protein A9IFQ7 Bordeteila petrii 9.84 PS4961 Blatteila germanica (German O.O2 A9IR28 Bordeteila petrii 1.74 cockroach) A1VT25 Polaromonas naphthalenivorans O.39 P23228 Gallus gallus (Chicken) O.O2 Q01581 Homo sapiens (Human) O.O3 P54873 Arabidopsis thaliana 1.19 P54871 Caenorhabditis elegans O.23 Example 5 P54874 Schizosaccharomyces pombe O.61 (Fission yeast) PS4839 Saccharomyces cerevisiae (Baker's O.28 3-hydroxy-3-methylbutyrate Production yeast) P54872 Dictyostelium discoideum (Slime O.09 0185. The complete reaction for 3-hydroxy-3-methylbu mold) tyrate synthesis contained 40 mM Tris-HCl pH 8, 5 to 50 mM Q86HL5 Dictyostelium discoideum (Slime O.O2 acetyl-CoA, 100 to 500 mM acetone, 1 MgCl, (except for mold) mitochondria HMG-CoA synthase), 0.5 mM DTT and Q9M6U3 Brassica iuncea O.O2 ASFMS4 Flavobacterium johnsoniae O.O2 enzyme varying in the range from 0.2 to 8 mg/ml. Control Q03WZO Leticonostoc mesenteroides O.28 reactions were carried in the absence of enzyme and one of Q2NHU7 Meihanosphaera Stadimanae O.O2 the substrates. Q8CNO6 Staphylococci is epidermidis O.O7 0186 The progress of synthesis was followed by analyz Q03QRO Lactobacilius brevis O.18 A6UPL1 Meihanosarcina mazei O.O1 ing aliquots taken after increasing period of incubation at 30 B2HGT6 Mycobacterium marinum O.O1 or 37°C. Typically, an aliquot of 50 ul was removed after 48 Q4L958 Staphylococciis haemolyticals O.18 h of incubation, heated for 1 min at 100° C. to eliminate the Q4AOD6 Staphylococci is saprophyticus O.08 proteins, centrifuged and the Supernatant was transferred to a Q1GAH5 Lactobacilius deilbruecki O.32 clean vial for HIV detection by mass spectrometry. A solution of 3-hydroxy-3-methylbutyrate was prepared in 40 mM Tris HCl pH 8, 1 mM MgCl, 0.5 mM DTT, heated as described Example 4 early and used as reference. 0187. The samples were analyzed on a PE SCIEX API Measuring of the HMG-CoA Lyase Activity. Using 2000 triple quadrupole mass spectrometer (mass spectrom Natural Substrate HMG-CoA eter, Perkin-Elmer) in negative ion mode with H2O/acetoni trile=60/40 containing 0.1% triethylamine as mobile phase, 0183 HMG-CoA lyase activity is measured according to flow rate was 40 ul/min. 10ul of each supernatant were mixed Mellanby J et al. (Methods of Enzymatic Analysis; Bergm with an equal quantity of mobile phase and directly injected eyer Ed. (1963), 454-458). The complete reaction mixture (1 into the mass spectrometer. The presence of 3-hydroxy-3- ml) containing 40 mM Tris-HCl pH 8, 1 mM MgCl2, 0.5 mM methylbutyrate-H ion was monitored. DTT, 0.4 mM HMG-CoA, 0.2 mM NADH, 5 units of 3-hy 0188 A peak corresponding to 3-hydroxy-3-methylbu droxybutyrate dehydrogenase is incubated for 5 min before tyrate was observed for the following enzymes: adding 0.005 mg/ml of HMG-CoA lyase and then the 0189 Blattella germanica (German cockroach) P54961 progress of the reaction is monitored by the decrease in absor (SEQID NO: 6) bance at 340 nm. A control assay was carried out in the (0190. Gallus gallus (Chicken) P23228 (SEQID NO: 7) absence of enzyme. (0191 Homo sapiens (Human) Q01581 (SEQID NO: 8) 0184. To account for non-specific disappearance of (0192 Arabidopsis thaliana P54873 (CAA58763) (SEQ NADH, results obtained in a control assay lacking enzyme ID NO: 4) were subtracted from results obtained in test samples. Spe (0193 Caenorhabditis elegans P54871 (SEQ ID NO: 1) cific activities were calculated as Aumol NADH/minimg pro 0194 Schizosaccharomyces pombe (Fission yeast) tein. P54874 (SEQID NO: 2) (0195 Saccharomyces cerevisiae (Baker's yeast) P54839 TABLE 2 (SEQ ID NO:3) 0196. Dictyostelium discoideum (Slime mold) Q86HL5 Physiological activity of some purified HMG-CoA lyases (SEQ ID NO: 10) Physiological (0197) Leuconostoc mesenteroides Q03WZ0 (SEQ ID Uniprot activity NO:) Accession Imol/min mg (0198 Staphylococcus epidermidis Q8CN06 (SEQID NO: number Organism protein 11) A8WGS7 Danio rerio (Zebrafish) 4.OS (0199 Lactobacillus delbrueckii Q1GAH5 (SEQ ID NO: (Brachydanio rerio) 24) Q29448 Bos taurus (Bovine) 5.79 B6U7B9 Zea mayS 13.31 (0200 Staphylococcus haemolyticus Q4L958 (198>V dif ASFHS2 Flavobacterium johnsoniae 2.89 ference compared to wild type protein) (SEQ ID NO:25) A1VH1 Polaromonas naphthalenivorans 34.92 0201 FIGS. 6 to 8 show representative results for com mercially available 3-hydroxy-3-methylbutyrate, for the US 2015/0240271 A1 Aug. 27, 2015 17 reaction using the HMG CoA synthase from Gallus gallus 0209. The reaction was initiated by the addition of 3 mg of (P23228) and for the control assay without enzyme. purified enzyme to the 1 ml reaction mixture. The mixture was then incubated without shaking at 37°C. for 40 h. Example 6 Analysis of 3-hydroxy-3-methylbutyrate Production 3-hydroxy-3-methylbutyryl-CoA Production Using 0210. Thermochemical conditions leading to the decom Lyases position of 3-hydroxy-3-methylbutyrate into isobutene were applied (Pressman et al., JACS, 1940, 2069-2080): the pH of 0202 3-hydroxy-3-methylbutyryl-CoA synthesis is car the reaction mixtures was first adjusted to pH4 using 6NHCl ried out in the presence of radiolabeled 2-CI acetone. The and the samples were then transferred into gas chromatogra complete reaction for 3-hydroxy-3-methylbutyryl-CoA syn phy vials (Interchim). The vials were sealed and incubated at thesis contains 40 mM Tris-HCl pH 8, 5 to 50 mM acetyl 110° C. for 4 hours, thus leading to the decomposition of CoA, 100 to 500 mM acetone, 1 to 10 mM MgCl, 0.5 mM 3-hydroxy-3-methylbutyrate into isobutene. DTT and enzyme varying in the range from 0.5 to 7 mg/ml. 0211. The calibration curve was prepared in the same con The formation of product is analyzed after separation of reac ditions using commercial 3-hydroxy-3-methylbutyrate. tion mixture by TLC or HPLC. 0212. One milliliter of headspace gas was collected and 0203 3-hydroxy-3-methylbutyryl-CoA is also analyzed injected into a HP5890 gas chromatograph (HP) equipped by TLC method (Stadtman E. R., J. Biol. Chem. 196 (1952), with a FID detector and a CP SilicaPlot(R) column (chroma 535-546). An aliquot of reaction is deposited on a cellulose tography column; Varian). Commercial isobutene was used as plate and chromatographied in the following solvent system: reference. From the isobutene signal the amount of 3-hy ethanol/0.1 M sodium acetate pH 4.5 (1/1). Co-A and acetyl droxy-3-methylbutyrate initially present in the sample was CoA are used as internal standards. R, reported for 3-hy calculated. droxy-3-methylbutyryl-CoA is 0.88. 0213. The kinetics parameters for several of the studied HMG-CoA synthases are presented in the following Table. Example 7

Kinetic Parameters for the Enzymatic Reaction Ka for Between Acetyl-CoA and Acetone in the Case of acetone, k/KX 10. HMG Synthases Organism mM k, x 10", sec' mM x sec Gallus gallus 250 5 2 0204 The kinetic parameters were measured using a vari Staphylococcits 2OO O6 O.3 able concentration of acetone and a constant concentration of epidermidis acetyl-CoA (10 mM) in following conditions: Schizosaccharomyces 2OO O.2 O.1 0205 40 mM Tris-HCl pH 8 pombe 0206. 2 mM MgCl, 0207 0-1 M acetone 0214. For the enzyme from S. epidermidis FIG.9 shows a 0208. The final pH was adjusted to 8. corresponding Michaelis-Menten plot.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 25

<21 Oc SEO ID NO 1 <211 LENGTH: 462 <212> TYPE PRT ORGANISM: Caenorhabditis elegans <4 OOs SEQUENCE: 1 Met Ser Lieu. Gly Glin Luell Ser Thir Pro Wall. Thir Asp Val Gly Ile 1. 5 15

Gly Ala Ile Glu Leu Tyr Phe Pro Glin Asn Phe Val Asp Glin Asn Asp 25 3 O

Luell Glu Lys Phe Asn. Asn Wall Ser Ser Gly Thir Ile Gly Lieu. 35 4 O 45

Gly Glin Glin Glin Met Gly Phe Ser Asp Asn Glu Asp Ile Wal Ser SO 55 60

Ile Ser Luell Thir Wall. Thir Arg Lell Ile Glu Thr Tyr Lys Ile Ser 65 70

Thr Asp Ser Ile Gly Cys Lieu. Wal Wall Gly Thr Gu Thir Met Ile Asp 85 90 95

Ser Ser Wall Lys Thir Ala Leul Met Asp Luell Phe Pro Gly Asn 105 110 US 2015/0240271 A1 Aug. 27, 2015 18

- Continued

Ser Asp Ile Glu Gly Val Asp Ile Lys Asn Ala Cys Phe Gly Gly Ala 115 12 O 125 Glin Ala Lieu. Lieu. His Ala Ile Asp Trp Val Thr Val Asn His Pro Lieu. 13 O 135 14 O Asp Llys Lys Asn Ala Ile Val Val Val Ala Asp Ile Ala Ile Tyr Glu 145 150 155 160 Glu Gly Pro Ala Arg Cys Thr Gly Gly Ala Gly Ala Ile Ala Phe Lieu. 1.65 17O 17s Ile Cys Pro Asp Ala Ser Ile Pro Ile Asp Arg Glin Phe Ser Ala Cys 18O 185 19 O His Met Lys Asn. Thir Trp Asp Phe Phe Llys Pro Ile Thr Pro Ile Pro 195 2OO 2O5 Ser Glu Tyr Pro Val Val Asp Gly Ser Leu Ser Leu Ser Ser Tyr Lieu. 21 O 215 22O Glu Ala Val Arg Met Thr Tyr Thr Tyr Phe Ile Ser Lys Val Asn Arg 225 23 O 235 24 O His Thr Thr Gly Ile Asp Gly Lieu. Asn Ser Phe Asp Gly Val Phe Leu 245 250 255 His Ser Pro Phe Thr Lys Met Val Glin Lys Gly Lieu Ala Val Met Asn 26 O 265 27 O Tyr Thr Asp Ser Glin Lieu. Arg His Lys Glin Lieu. Asn Gly Asn Gly Val 27s 28O 285 Asp His Llys Lieu. Asp Glu Asn Asp Arg Ala Gly Lieu Ala Lys Met Ile 29 O 295 3 OO Glu Lieu. Ser Ala Glin Val Trp Llys Glu Lys Thr Asp Pro Tyr Lieu Val 3. OS 310 315 32O Phe Asn Arg Arg Ile Gly Asn Met Tyr Thr Pro Ser Leu Phe Ala Glin 3.25 330 335 Lieu. Lieu Ala Tyr Lieu Ala Ala Asp Asp Cys Val Thr Gly Glu Lys Ser 34 O 345 35. O Ile Leu Phe Phe Ala Tyr Gly Ser Gly Lieu Ala Ser Ala Ile Phe Pro 355 360 365 Gly Arg Val Arg Glin Thir Ser Asn Lieu. Asp Llys Ile Arg Glin Val Ala 37 O 375 38O Ile Arg Ala Ile Lys Arg Lieu. Asp Asp Arg Ile Glin Phe Thr Pro Glu 385 390 395 4 OO Glu Phe Thr Glu Thir Lieu Gln Lys Arg Glu Val Phe Lieu. Arg Ser Lys 4 OS 41O 415 Glu Ile Pro Llys Ser Pro Ser Glu Thir Ser Leu Phe Pro Asn Thr Tyr 42O 425 43 O Phe Lieu. Asp Asn Met Asp Llys Lieu. Tyr Arg Arg Ser Tyr Thir Lieu. His 435 44 O 445 Glu Glu Pro Asn Gly Val Glin Asn Gly Asn Gly Ile His His 450 45.5 460

<210s, SEQ ID NO 2 &211s LENGTH: 447 212. TYPE: PRT <213> ORGANISM: Schizosaccharomyces pombe

<4 OOs, SEQUENCE: 2 Met Ser Phe Asp Arg Lys Asp Ile Gly Ile Lys Gly Lieu Val Lieu. Tyr US 2015/0240271 A1 Aug. 27, 2015 19

- Continued

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

Lieu. Thir Ala Lieu Lleu Ser Arg Val Pro Ala Asp Glu Lieu Lys Gly Lys 34 O 345 35. O

Arg Val Gly Ala Tyr Ser Tyr Gly Ser Gly Leu Ala Ala Ser Phe Phe 355 360 365

Ser Phe Val Val Lys Gly Asp Val Ser Glu Ile Ala Lys Llys Thr Asn 37 O 375 38O Lieu Val Asn Asp Lieu. Asp Asn Arg His Cys Lieu. Thr Pro Thr Glin Tyr 385 390 395 4 OO

Glu Glu Ala Ile Glu Lieu. Arg His Glin Ala His Lieu Lys Lys Asn. Phe 4 OS 41O 415 US 2015/0240271 A1 Aug. 27, 2015 20

- Continued

Thr Pro Lys Gly Ser Ile Glu Arg Lieu. Arg Ser Gly Thr Tyr Tyr Lieu. 42O 425 43 O Thr Gly Ile Asp Asp Met Phe Arg Arg Ser Tyr Ser Val Llys Pro 435 44 O 445

<210s, SEQ ID NO 3 &211s LENGTH: 491 212. TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae

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

Ala Lieu. Asp Glin Val Tyr Lys Ser Tyr Ser Lys Lys Ala Ile Ser Lys 26 O 265 27 O

Gly Lieu Val Ser Asp Pro Ala Gly Ser Asp Ala Lieu. Asn Val Lieu Lys 27s 28O 285 Tyr Phe Asp Tyr Asn Val Phe His Val Pro Thr Cys Llys Lieu Val Thr 29 O 295 3 OO

Llys Ser Tyr Gly Arg Lieu. Lieu. Tyr Asn Asp Phe Arg Ala Asn Pro Glin 3. OS 310 315 32O

Lieu. Phe Pro Glu Val Asp Ala Glu Lieu Ala Thr Arg Asp Tyr Asp Glu US 2015/0240271 A1 Aug. 27, 2015 21

- Continued

3.25 330 335 Ser Lieu. Thir Asp Lys Asn. Ile Glu Lys Thr Phe Val Asn Val Ala Lys 34 O 345 35. O Pro Phe His Lys Glu Arg Val Ala Glin Ser Lieu. Ile Val Pro Thr Asn 355 360 365 Thr Gly Asn Met Tyr Thr Ala Ser Val Tyr Ala Ala Phe Ala Ser Lieu. 37 O 375 38O Lieu. Asn Tyr Val Gly Ser Asp Asp Lieu. Glin Gly Lys Arg Val Gly Lieu. 385 390 395 4 OO Phe Ser Tyr Gly Ser Gly Lieu Ala Ala Ser Leu Tyr Ser Cys Lys Ile 4 OS 41O 415 Val Gly Asp Val Glin His Ile Ile Lys Glu Lieu. Asp Ile Thr Asn Lys 42O 425 43 O Lieu Ala Lys Arg Ile Thr Glu Thr Pro Lys Asp Tyr Glu Ala Ala Ile 435 44 O 445 Glu Lieu. Arg Glu Asn Ala His Lieu Lys Lys Asn. Phe Llys Pro Glin Gly 450 45.5 460 Ser Ile Glu. His Lieu. Glin Ser Gly Val Tyr Tyr Lieu. Thr Asn Ile Asp 465 470 47s 48O Asp Llys Phe Arg Arg Ser Tyr Asp Wall Lys Llys 485 490

<210 SEQ ID NO 4 &211s LENGTH: 461 212. TYPE: PRT <213> ORGANISM: Arabidopsis thaliana <4 OOs, SEQUENCE: 4 Met Ala Lys Asn Val Gly Ile Leu Ala Met Asp Ile Tyr Phe Pro Pro 1. 5 1O 15 Thir Cys Val Glin Glin Glu Ala Lieu. Glu Ala His Asp Gly Ala Ser Lys 2O 25 3O Gly Lys Tyr Thir Ile Gly Lieu. Gly Glin Asp Cys Lieu Ala Phe Cys Thr 35 4 O 45 Glu Lieu. Glu Asp Val Ile Ser Met Ser Phe Asn Ala Val Thr Ser Lieu. SO 55 6 O Phe Glu Lys Tyr Lys Ile Asp Pro Asn Glin Ile Gly Arg Lieu. Glu Val 65 70 7s 8O Gly Ser Glu Thr Val Ile Asp Llys Ser Lys Ser Ile Llys Thr Phe Leu 85 90 95 Met Glin Lieu. Phe Glu Lys Cys Gly Asn. Thir Asp Val Glu Gly Val Asp 1OO 105 11 O Ser Thr Asn Ala Cys Tyr Gly Gly Thr Ala Ala Lieu. Lieu. Asn. Cys Val 115 12 O 125

Asn Trp Val Glu Ser Asn. Ser Trp Asp Gly Arg Tyr Gly Lieu Val Ile 13 O 135 14 O

Cys Thr Asp Ser Ala Val Tyr Ala Glu Gly Pro Ala Arg Pro Thr Gly 145 150 155 160

Gly Ala Ala Ala Ile Ala Met Lieu. Ile Gly Pro Asp Ala Pro Ile Val 1.65 17O 17s Phe Glu Ser Lys Lieu. Arg Ala Ser His Met Ala His Val Tyr Asp Phe 18O 185 19 O US 2015/0240271 A1 Aug. 27, 2015 22

- Continued Tyr Llys Pro Asn Lieu Ala Ser Glu Tyr Pro Val Val Asp Gly Lys Lieu. 195 2OO 2O5 Ser Glin Thr Cys Tyr Lieu Met Ala Lieu. Asp Ser Cys Tyr Lys His Lieu. 21 O 215 22O Cys Asn Llys Phe Glu Lys Ile Glu Gly Lys Glu Phe Ser Ile Asn Asp 225 23 O 235 24 O Ala Asp Tyr Ile Val Phe His Ser Pro Tyr Asn Llys Lieu Val Glin Lys 245 250 255 Ser Phe Ala Arg Lieu. Lieu. Tyr Asn Asp Phe Lieu. Arg Asn Ala Ser Ser 26 O 265 27 O Ile Asp Glu Ala Ala Lys Glu Lys Phe Thr Pro Tyr Ser Ser Lieu. Thr 27s 28O 285 Lieu. Asp Glu Ser Tyr Glin Ser Arg Asp Lieu. Glu, Llys Val Ser Glin Glin 29 O 295 3 OO Ile Ser Llys Pro Phe Tyr Asp Ala Lys Val Glin Pro Thr Thr Lieu. Ile 3. OS 310 315 32O Pro Lys Glu Val Gly Asn Met Tyr Thr Ala Ser Lieu. Tyr Ala Ala Phe 3.25 330 335 Ala Ser Lieu. Ile His Asn Llys His Asn Asp Lieu Ala Gly Lys Arg Val 34 O 345 35. O Val Met Phe Ser Tyr Gly Ser Gly Ser Thr Ala Thr Met Phe Ser Leu 355 360 365 Arg Lieu. ASn Asp ASn Llys Pro Pro Phe Ser Ile Ser ASn Ile Ala Ser 37 O 375 38O Val Met Asp Val Gly Gly Llys Lieu Lys Ala Arg His Glu Tyr Ala Pro 385 390 395 4 OO Glu Lys Phe Val Glu Thr Met Lys Lieu Met Glu. His Arg Tyr Gly Ala 4 OS 41O 415 Lys Asp Phe Val Thir Thr Lys Glu Gly Ile Ile Asp Lieu. Lieu Ala Pro 42O 425 43 O Gly. Thir Tyr Tyr Lieu Lys Glu Val Asp Ser Lieu. Tyr Arg Arg Phe Tyr 435 44 O 445 Gly Lys Lys Gly Glu Asp Gly Ser Val Ala Asn Gly His 450 45.5 460

<210s, SEQ ID NO 5 &211s LENGTH: 482 212. TYPE: PRT <213> ORGANISM: Dictyostelium discoideum <4 OOs, SEQUENCE: 5 Met Thr Llys Pro Glu Asn Ile Gly Ile His Gly Ile Glu Val Tyr Phe 1. 5 1O 15

Pro Ser Thr Tyr Val Ala Glin Glu Asp Lieu. Glu, Llys Phe Asp Gly Val 2O 25 3O

Ser Glin Gly Lys Tyr Thr Lieu. Gly Lieu. Gly Glin Thr Asn Met Ala Phe 35 4 O 45

Cys Gly Asp Arg Glu Asp Ile Tyr Ser Lieu. Ser Lieu. Asn Ala Val Asn SO 55 6 O

Asn Lieu Met Asp Llys Phe Asn Val Asp Pro Asn. Ser Ile Gly Arg Lieu 65 70 7s 8O

Glu Val Gly Thr Glu Thr Val Ile Asp Llys Ser Lys Ser Val Lys Thr 85 90 95 US 2015/0240271 A1 Aug. 27, 2015 23

- Continued

Val Lieu Met Asp Lieu. Phe Ala Lys His Gly Asn. Thir Ser Ile Asp Gly 1OO 105 11 O Ile Asp Thir Ile Asn Ala Cys Tyr Gly Gly. Thir Ser Ala Lieu. His Asn 115 12 O 125 Ala Lieu Gln Trp Met Glu Ser Ser Tyr Trp Asp Gly Arg Asn Ala Ile 13 O 135 14 O Val Val Ala Gly Asp Ile Ala Val Tyr Glu Lys Gly Pro Ala Arg Pro 145 150 155 160 Thr Gly Gly Ala Gly Val Val Ala Met Lieu. Ile Gly Pro Asn Ala Pro 1.65 17O 17s Ile Thr Phe Glu Ser Gly Lieu. Arg Gly Val His Met Glu Asn Val Tyr 18O 185 19 O Asp Phe Tyr Llys Pro Asp Met Asp Ser Glu Tyr Pro Arg Val Asp Gly 195 2OO 2O5 Llys Lieu. Ser Ile Ser Cys Tyr Phe Arg Ala Ile Asp Asn. Cys Tyr Asn 21 O 215 22O Arg Tyr Ala Lys Ala Phe Glu Lys Llys Tyr Gly Llys Ser Phe Ser Lieu 225 23 O 235 24 O Asp Glin Val Asp Phe Ala Lieu. Phe His Ser Pro Tyr Asn Llys Lieu Val 245 250 255 Glin Llys Ser Phe Gly Arg Met Lieu. Tyr Asn Asp Phe Lieu. Asn. Asn Pro 26 O 265 27 O Asn Asp Ser Arg Tyr Ala Ser Lieu. Glu Ala Tyr Lys Asn Val Llys Pro 27s 28O 285 Glu Asp Thir Tyr Phe Asp Ser Val Lieu. Glu Lys Ala Lieu. Ser Ala Ile 29 O 295 3 OO Thir Lys Asn Asp Tyr Ala Thr Llys Val Ala Pro Thir Thr Lieu. Lieu Ala 3. OS 310 315 32O Lys Gln Leu Gly Asn Thr Tyr Cys Gly Ser Thr Tyr Ser Gly Lieu. Leu 3.25 330 335 Ser Lieu. Lieu. Asp Glu Lys Ser Asn Asp Lieu Val Gly Lys Arg Val Lieu. 34 O 345 35. O Thr Phe Ser Tyr Gly Ser Gly Lieu Ala Ala Ser Ala Phe Ser Phe Lys 355 360 365 Val Glu Lys Pro Ile Asn His Ile Val Glu Lys Val Asp Lieu Lys Asn 37 O 375 38O Arg Lieu Ala Lys Arg Val Arg Val Glu Pro Glu Ile Phe Thr Glu Lys 385 390 395 4 OO Lieu. Ser Lieu. Arg Glu Thir Arg His Asn Lieu Lys Asn Tyr Val Pro Ser 4 OS 41O 415 Asp Glu Thir Thr Asn Met Phe Pro Gly Ser Phe Tyr Lieu Ser Ser Val 42O 425 43 O

Asp Asn Ala Gly Ile Arg Llys Tyr Asp Arg Thr Tyr Ser Thir Ser Ala 435 44 O 445

Val Lieu. Gly Ala Phe Glin Arg Arg Glin Glin Ile Ser Glin Ser Thir Ile 450 45.5 460

Llys Ser Lieu. Asn Lieu. Phe Arg Ala Thir Lys Ser Val Lieu. Ser Ile Lieu. 465 470 47s 48O US 2015/0240271 A1 Aug. 27, 2015 24

- Continued

<210s, SEQ ID NO 6 &211s LENGTH: 453 212. TYPE: PRT <213> ORGANISM: Blattella germanica

<4 OOs, SEQUENCE: 6 Met Trp Pro Ser Asp Val Gly Ile Val Ala Lieu. Glu Lieu. Ile Phe Pro 1. 5 1O 15 Ser Glin Tyr Val Asp Glin Val Asp Lieu. Glu Val Tyr Asp Asin Val Ser 2O 25 3O Ala Gly Lys Tyr Thr Val Gly Lieu. Gly Glin Ala Arg Met Gly Phe Cys 35 4 O 45 Thir Asp Arg Glu Asp Ile Asn. Ser Lieu. Cys Lieu. Thr Val Val Ser Arg SO 55 6 O Lieu Met Glu Arg Trp Ser Ile Pro Tyr Ser Glin Ile Gly Arg Lieu. Glu 65 70 7s 8O Val Gly Thr Glu Thir Lieu. Lieu. Asp Llys Ser Lys Ser Wall Lys Thr Val 85 90 95 Lieu Met Glin Lieu. Phe Lys Asp Asn. Thir Asp Ile Glu Gly Val Asp Thr 1OO 105 11 O

Wall Asn. A a. Cy S Tyr Gly Gly Thr Ser Ala Leu Phe Asn Ala Ile Ser 115 12 O 125 Trp Val Glu Ser Ser Ser Trp Asp Gly Arg Tyr Ala Lieu Val Val Ala 13 O 135 14 O Gly Asp Ile Ala Val Tyr Ala Lys Gly Ser Ala Arg Pro Thr Gly Gly 145 150 155 160 Ala Gly Ala Val Ala Met Lieu Val Gly Ala Asn Ala Pro Lieu Val Phe 1.65 17O 17s Asp Arg Gly Val Arg Ser Ser His Met Gln His Ala Tyr Asp Phe Tyr 18O 185 19 O Llys Pro Asp Lieu. Ser Ser Lieu. Tyr Pro Thr Val Asp Gly Lys Lieu. Ser 195 2OO 2O5 Ile Glin Cys Tyr Lieu. Ser Ala Lieu. Asp His Cys Tyr Glin Lieu. Tyr Cys 21 O 215 22O Ser Lys Ile Glin Lys Glin Lieu. Gly Glu Lys Phe Asp Ile Glu Arg Lieu. 225 23 O 235 24 O Asp Ala Val Lieu. Phe His Ala Pro Tyr Cys Llys Lieu Val Glin Llys Ser 245 250 255 Lieu Ala Arg Lieu Val Lieu. Asn Asp Phe Val Arg Ala Ser Glu Glu Glu 26 O 265 27 O Arg Thir Thir Lys Tyr Ser Ser Lieu. Glu Ala Lieu Lys Gly Val Lys Lieu 27s 28O 285 Glu Asp Thr Tyr Phe Asp Arg Glu Val Glu Lys Ala Val Met Thr Tyr 29 O 295 3 OO

Ser Lys Asn Met Phe Glu Glu Lys Thr Llys Pro Ser Lieu. Lieu. Lieu Ala 3. OS 310 315 32O

Asn Glin Val Gly Asn Met Tyr Thr Pro Ser Leu Tyr Gly Gly Lieu Val 3.25 330 335

Ser Lieu. Lieu Val Ser Lys Ser Ala Glin Glu Lieu Ala Gly Lys Arg Val 34 O 345 35. O

Ala Leu Phe Ser Tyr Gly Ser Gly Lieu Ala Ser Ser Met Phe Ser Leu 355 360 365 US 2015/0240271 A1 Aug. 27, 2015 25

- Continued Arg Ile Ser Ser Asp Ala Ser Ala Lys Ser Ser Lieu. Glin Arg Lieu Val 37 O 375 38O Ser Asn Lieu. Ser His Ile Llys Pro Glin Lieu. Asp Lieu. Arg His Llys Val 385 390 395 4 OO Ser Pro Glu Glu Phe Ala Glin Thr Met Glu Thr Arg Glu. His Asn His 4 OS 41O 415 His Lys Ala Pro Tyr Thr Pro Glu Gly Ser Ile Asp Val Lieu. Phe Pro 42O 425 43 O Gly. Thir Trp Tyr Lieu. Glu Ser Val Asp Ser Leu Tyr Arg Arg Ser Tyr 435 44 O 445 Lys Glin Val Pro Gly 450

<210s, SEQ ID NO 7 &211s LENGTH: 522 212. TYPE: PRT <213> ORGANISM: Gallus gallus

<4 OO > SEQUENCE: 7 Met Pro Gly Ser Leu Pro Val Asn Thr Glu Ser Cys Trp Pro Lys Asp 1. 5 1O 15 Val Gly Ile Val Ala Lieu. Glu Ile Tyr Phe Pro Ser Glin Tyr Val Asp 2O 25 3O Glin Thr Glu Lieu. Glu Lys Tyr Asp Gly Val Asp Ala Gly Lys Tyr Thr 35 4 O 45 Ile Gly Lieu. Gly Glin Ser Llys Met Gly Phe Cys Ser Asp Arg Glu Asp SO 55 6 O Ile Asin Ser Lieu. Cys Lieu. Thr Val Val Glin Llys Lieu Met Glu Arg Asn 65 70 7s 8O Ser Lieu. Ser Tyr Asp Cys Ile Gly Arg Lieu. Glu Val Gly. Thr Glu Thir 85 90 95 Ile Ile Asp Llys Ser Lys Ser Val Lys Thr Val Lieu Met Glin Lieu. Phe 1OO 105 11 O Glu Glu Ser Gly Asn. Thir Asp Val Glu Gly Ile Asp Thir Thr Asn Ala 115 12 O 125 Cys Tyr Gly Gly Thr Ala Ala Lieu. Phe Asn Ala Ile Asn Trp Ile Glu 13 O 135 14 O Ser Ser Ser Trp Asp Gly Arg Tyr Ala Lieu Val Val Ala Gly Asp Ile 145 150 155 160 Ala Val Tyr Ala Thr Gly Asn Ala Arg Pro Thr Gly Gly Ala Gly Ala 1.65 17O 17s Val Ala Met Lieu Val Gly Ser Asn Ala Pro Lieu. Ile Phe Glu Arg Gly 18O 185 19 O Lieu. Arg Gly Thr His Met Gln His Ala Tyr Asp Phe Tyr Llys Pro Asp 195 2OO 2O5

Met Val Ser Glu Tyr Pro Val Val Asp Gly Lys Lieu Ser Ile Glin Cys 21 O 215 22O

Tyr Lieu. Ser Ala Lieu. Asp Arg Cys Tyr Ser Val Tyr Arg Asn Lys Ile 225 23 O 235 24 O His Ala Glin Trp Gln Lys Glu Gly Thr Asp Arg Gly Phe Thr Lieu. Asn 245 250 255 Asp Phe Gly Phe Met Ile Phe His Ser Pro Tyr Cys Llys Lieu Val Glin 26 O 265 27 O US 2015/0240271 A1 Aug. 27, 2015 26

- Continued

Llys Ser Val Ala Arg Lieu. Lieu. Lieu. Asn Asp Phe Lieu. Ser Asp Glin Asn 27s 28O 285 Ala Glu Thir Ala Asn Gly Val Phe Ser Gly Lieu. Glu Ala Phe Arg Asp 29 O 295 3 OO Val Llys Lieu. Glu Asp Thr Tyr Phe Asp Arg Asp Val Glu Lys Ala Phe 3. OS 310 315 32O Met Lys Ala Ser Ala Glu Lieu. Phe Asin Gln Lys Thr Lys Ala Ser Lieu. 3.25 330 335 Lieu Val Ser Asn Glin Asn Gly Asn Met Tyr Thr Pro Ser Val Tyr Gly 34 O 345 35. O Cys Lieu Ala Ser Lieu. Lieu Ala Glin Tyr Ser Pro Glu. His Lieu Ala Gly 355 360 365 Glin Arg Ile Ser Glu Phe Ser Tyr Gly Ser Gly Phe Ala Ala Thr Lieu. 37 O 375 38O Tyr Ser Ile Arg Val Thr Glin Asp Ala Thr Pro Gly Ser Ala Lieu. Asp 385 390 395 4 OO Lys Ile Thr Ala Ser Lieu. Ser Asp Lieu Lys Ala Arg Lieu. Asp Ser Arg 4 OS 41O 415 Lys Cys Ile Ala Pro Asp Val Phe Ala Glu Asn Met Lys Ile Arg Glin 42O 425 43 O Glu Thr His His Leu Ala Asn Tyr Ile Pro Gln Cys Ser Val Glu Asp 435 44 O 445 Lieu. Phe Glu Gly. Thir Trp Tyr Lieu Val Arg Val Asp Glu Lys His Arg 450 45.5 460 Arg Thr Tyr Ala Arg Arg Pro Wal Met Gly Asp Gly Pro Lieu. Glu Ala 465 470 47s 48O Gly Val Glu Val Val His Pro Gly Ile Val His Glu. His Ile Pro Ser 485 490 495 Pro Ala Lys Llys Val Pro Arg Ile Pro Ala Thr Thr Glu Ser Glu Gly SOO 505 51O Val Thr Val Ala Ile Ser Asn Gly Val His 515 52O

<210s, SEQ ID NO 8 &211s LENGTH: 52O 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 8 Met Pro Gly Ser Lieu Pro Lieu. Asn Ala Glu Ala Cys Trp Pro Lys Asp 1. 5 1O 15 Val Gly Ile Val Ala Lieu. Glu Ile Tyr Phe Pro Ser Glin Tyr Val Asp 2O 25 3O

Glin Ala Glu Lieu. Glu Lys Tyr Asp Gly Val Asp Ala Gly Lys Tyr Thr 35 4 O 45

Ile Gly Lieu. Gly Glin Ala Lys Met Gly Phe Cys Thr Asp Arg Glu Asp SO 55 6 O

Ile Asin Ser Lieu. Cys Met Thr Val Val Glin Asn Lieu Met Glu Arg Asn 65 70 7s 8O

Asn Lieu. Ser Tyr Asp Cys Ile Gly Arg Lieu. Glu Val Gly Thr Glu Thr 85 90 95

Ile Ile Asp Llys Ser Lys Ser Val Lys Thr Asn Lieu Met Glin Lieu. Phe US 2015/0240271 A1 Aug. 27, 2015 27

- Continued

1OO 105 11 O Glu Glu Ser Gly Asn. Thir Asp Ile Glu Gly Ile Asp Thir Thr Asn Ala 115 12 O 125 Cys Tyr Gly Gly Thr Ala Ala Val Phe Asn Ala Val Asn Trp Ile Glu 13 O 135 14 O Ser Ser Ser Trp Asp Gly Arg Tyr Ala Lieu Val Val Ala Gly Asp Ile 145 150 155 160 Ala Val Tyr Ala Thr Gly Asn Ala Arg Pro Thr Gly Gly Val Gly Ala 1.65 17O 17s Val Ala Lieu. Lieu. Ile Gly Pro Asn Ala Pro Lieu. Ile Phe Glu Arg Gly 18O 185 19 O Lieu. Arg Gly Thr His Met Gln His Ala Tyr Asp Phe Tyr Llys Pro Asp 195 2OO 2O5 Met Leu Ser Glu Tyr Pro Ile Val Asp Gly Lys Lieu Ser Ile Glin Cys 21 O 215 22O Tyr Lieu. Ser Ala Lieu. Asp Arg Cys Tyr Ser Val Tyr Cys Llys Lys Ile 225 23 O 235 24 O His Ala Glin Trp Gln Lys Glu Gly Asn Asp Lys Asp Phe Thr Lieu. Asn 245 250 255 Asp Phe Gly Phe Met Ile Phe His Ser Pro Tyr Cys Llys Lieu Val Glin 26 O 265 27 O Llys Ser Lieu Ala Arg Met Lieu. Lieu. Asn Asp Phe Lieu. Asn Asp Glin Asn 275 28O 285 Arg Asp Lys Asn. Ser Ile Tyr Ser Gly Lieu. Glu Ala Phe Gly Asp Wall 29 O 295 3 OO Llys Lieu. Glu Asp Thr Tyr Phe Asp Arg Asp Val Glu Lys Ala Phe Met 3. OS 310 315 32O Lys Ala Ser Ser Glu Lieu. Phe Ser Glin Llys Thr Lys Ala Ser Lieu. Lieu. 3.25 330 335 Val Ser Asn Glin Asn Gly Asn Met Tyr Thr Ser Ser Val Tyr Gly Ser 34 O 345 35. O Lieu Ala Ser Val Lieu Ala Glin Tyr Ser Pro Glin Glin Lieu Ala Gly Lys 355 360 365 Arg Ile Gly Val Phe Ser Tyr Gly Ser Gly Leu Ala Ala Thr Lieu. Tyr 37 O 375 38O Ser Lieu Lys Val Thr Glin Asp Ala Thr Pro Gly Ser Ala Lieu. Asp Llys 385 390 395 4 OO Ile Thr Ala Ser Lieu. Cys Asp Lieu Lys Ser Arg Lieu. Asp Ser Arg Thr 4 OS 41O 415 Gly Val Ala Pro Asp Val Phe Ala Glu Asn Met Lys Lieu. Arg Glu Asp 42O 425 43 O Thr His His Leu Val Asn Tyr Ile Pro Glin Gly Ser Ile Asp Ser Lieu. 435 44 O 445 Phe Glu Gly. Thir Trp Tyr Lieu Val Arg Val Asp Glu Lys His Arg Arg 450 45.5 460

Thir Tyr Ala Arg Arg Pro Thr Pro Asn Asp Asp Thir Lieu. Asp Glu Gly 465 470 47s 48O

Val Gly Lieu Val His Ser Asn Ile Ala Thr Glu. His Ile Pro Ser Pro 485 490 495

Ala Lys Llys Val Pro Arg Lieu Pro Ala Thr Ala Ala Glu Pro Glu Ala SOO 505 51O US 2015/0240271 A1 Aug. 27, 2015 28

- Continued

Ala Val Ile Ser Asn Gly Val Trp 515 52O

<210s, SEQ ID NO 9 &211s LENGTH: 508 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 9 Met Glin Arg Lieu. Lieu. Thr Pro Val Lys Arg Ile Lieu. Glin Lieu. Thir Arg 1. 5 1O 15 Ala Val Glin Glu Thir Ser Lieu. Thr Pro Ala Arg Lieu Lleu Pro Val Ala 2O 25 3O His Glin Arg Phe Ser Thr Ala Ser Ala Val Pro Lieu Ala Lys Thr Asp 35 4 O 45 Thir Trp Pro Lys Asp Val Gly Ile Leu Ala Lieu. Glu Val Tyr Phe Pro SO 55 6 O Ala Glin Tyr Val Asp Glin Thr Asp Lieu. Glu Lys Tyr Asn. Asn Val Glu 65 70 7s 8O Ala Gly Lys Tyr Thr Val Gly Lieu. Gly Glin Thr Arg Met Gly Phe Cys 85 90 95 Ser Val Glin Glu Asp Ile Asn. Ser Lieu. Cys Lieu. Thr Val Val Glin Arg 1OO 105 11 O Lieu Met Glu Arg Ile Gln Lieu Pro Trp Asp Ser Val Gly Arg Lieu. Glu 115 12 O 125 Val Gly Thr Glu Thir Ile Ile Asp Llys Ser Lys Ala Wall Lys Thr Val 13 O 135 14 O Lieu Met Glu Lieu. Phe Glin Asp Ser Gly Asn. Thir Asp Ile Glu Gly Ile 145 150 155 160 Asp Thir Thir Asn Ala Cys Tyr Gly Gly Thr Ala Ser Lieu. Phe Asn Ala 1.65 17O 17s Ala Asn Trp Met Glu Ser Ser Ser Trp Asp Gly Arg Tyr Ala Met Val 18O 185 19 O Val Cys Gly Asp Ile Ala Val Tyr Pro Ser Gly Asn Ala Arg Pro Thr 195 2OO 2O5 Gly Gly Ala Gly Ala Val Ala Met Lieu. Ile Gly Pro Lys Ala Pro Lieu. 21 O 215 22O Ala Lieu. Glu Arg Gly Lieu. Arg Gly. Thir His Met Glu Asn Val Tyr Asp 225 23 O 235 24 O Phe Tyr Llys Pro Asn Lieu Ala Ser Glu Tyr Pro Ile Val Asp Gly Lys 245 250 255 Lieu. Ser Ile Glin Cys Tyr Lieu. Arg Ala Lieu. Asp Arg Cys Tyr Thir Ser 26 O 265 27 O

Tyr Arg Llys Lys Ile Glin Asn Glin Trp Llys Glin Ala Gly Ser Asp Arg 27s 28O 285

Pro Phe Thr Lieu. Asp Asp Leu Gln Tyr Met Ile Phe His Thr Pro Phe 29 O 295 3 OO

Cys Llys Met Val Glin Llys Ser Lieu Ala Arg Lieu Met Phe Asin Asp Phe 3. OS 310 315 32O

Lieu. Ser Ala Ser Ser Asp Thr Glin Thir Ser Lieu. Tyr Lys Gly Lieu. Glu 3.25 330 335

Ala Phe Gly Gly Lieu Lys Lieu. Glu Asp Thr Tyr Thr Asn Lys Asp Lieu US 2015/0240271 A1 Aug. 27, 2015 29

- Continued

34 O 345 35. O Asp Lys Ala Lieu Lleu Lys Ala Ser Glin Asp Met Phe Asp Llys Llys Thr 355 360 365 Lys Ala Ser Leu Tyr Lieu Ser Thr His Asn Gly Asn Met Tyr Thr Ser 37 O 375 38O Ser Lieu. Tyr Gly Cys Lieu Ala Ser Lieu. Lieu. Ser His His Ser Ala Glin 385 390 395 4 OO Glu Lieu Ala Gly Ser Arg Ile Gly Ala Phe Ser Tyr Gly Ser Gly Lieu. 4 OS 41O 415 Ala Ala Ser Phe Phe Ser Phe Arg Val Ser Glin Asp Ala Ala Pro Gly 42O 425 43 O Ser Pro Lieu. Asp Llys Lieu Val Ser Ser Thir Ser Asp Lieu Pro Lys Arg 435 44 O 445 Lieu Ala Ser Arg Lys Cys Val Ser Pro Glu Glu Phe Thr Glu Ile Met 450 45.5 460 Asn Glin Arg Glu Glin Phe Tyr His Llys Val Asn Phe Ser Pro Pro Gly 465 470 47s 48O Asp Thr Asn Ser Leu Phe Pro Gly. Thir Trp Tyr Lieu. Glu Arg Val Asp 485 490 495 Glu Gln His Arg Arg Llys Tyr Ala Arg Arg Pro Val SOO 505

<210 SEQ ID NO 10 &211s LENGTH: 468 212. TYPE: PRT <213> ORGANISM: Dictyostelium discoideum <4 OOs, SEQUENCE: 10 Met Lys Llys Thir Lys Asp Ile Gly Ile Cys Ala Ile Asp Ile Tyr Phe 1. 5 1O 15 Pro Glin Thr Tyr Val Asn Glin Ser Glu Lieu Lys Llys Tyr Asp Llys Val 2O 25 3O Ser Asn Gly Lys Tyr Thr Ile Gly Lieu. Gly Glin Thr Asn Met Ser Phe 35 4 O 45 Val Gly Asp Arg Glu Asp Ile Val Ser Met Ala Met Thir Ser Val Lys SO 55 6 O Met Met Met Ser Lys Tyr Ser Ile Asp Tyr Glin Ser Ile Gly Arg Lieu. 65 70 7s 8O Glu Val Gly Thr Glu Thir Ile Ile Asp Llys Ser Lys Ser Wall Lys Ser 85 90 95 Ser Ile Met Ser Lieu. Phe Glin Glu Tyr Gly Asn Thr Ser Lieu. Glu Gly 1OO 105 11 O Val Asp Thir Lieu. Asn Ala Cys Tyr Gly Gly Thr Asn Ala Lieu. Phe Asn 115 12 O 125

Ser Lieu Gln Trp Ile Glu Ser Ser Tyr Trp Asp Gly Arg Tyr Ala Lieu. 13 O 135 14 O

Val Val Thr Gly Asp Ile Ala Val Tyr Ser Lys Gly Ala Ala Arg Pro 145 150 155 160

Thr Gly Gly Ala Gly Val Val Thr Met Lieu. Ile Gly Pro Asn Ala Thr 1.65 17O 17s Lieu. Ile Phe Asp Glin Ser Lieu. Arg Gly Thr His Met Glu Asn Val Asn 18O 185 19 O US 2015/0240271 A1 Aug. 27, 2015 30

- Continued Asp Phe Tyr Llys Pro Asp Leu Ser Ser Glu Tyr Pro Tyr Val Asp Gly 195 2OO 2O5 Llys Lieu. Ser Ile Glu. Cys Tyr Lieu. Arg Ala Lieu. Asp Llys Cys Tyr Lieu. 21 O 215 22O Glu Tyr Lys Llys Llys Phe Glu Ser Ile Asin Asp Asp Asn Llys Phe Ser 225 23 O 235 24 O Met Asp Ser Phe Asp Tyr Val Cys Phe His Ser Pro Tyr Asn Arg Lieu. 245 250 255 Val Glin Llys Ser Tyr Ala Arg Lieu. Ile Tyr Asn Asp Phe Lieu. Glin Asn 26 O 265 27 O Pro Asn. Asn Pro Llys Tyr Glin Asp Lieu. Lieu Pro Phe Lys Asp Lieu. Ser 27s 28O 285 Thr Gly Lys Asp Ser Tyr Ile Asin Ser Lys Lieu. Asp Glin Ile Thr Lieu. 29 O 295 3 OO Llys Lieu. Ser Lieu. Asp Asp Phe Llys Thr Llys Val Asn Pro Ser Thr Lieu. 3. OS 310 315 32O Lieu. Ser Lys Glu. Cys Gly Asn Ser Tyr Cys Gly Ser Val Tyr Ser Gly 3.25 330 335 Ile Lieu. Ser Lieu Lleu Ser Asn Val Asn Asp Lieu. Asn. Asn Llys Llys Val 34 O 345 35. O Lieu Val Phe Ser Tyr Gly Ser Gly Lieu Ala Ala Ser Leu Phe Ser Phe 355 360 365 Arg Ile ASn ASn ASn Lys Asn Arg ASn ASn ASn Asn ASn ASn ASn Asn 37 O 375 38O Cys Phe Phe Llys Thr Thr Asn Asp Ile Gly Lys Ile Ser Asn Ile Llys 385 390 395 4 OO Glu Arg Lieu. Ser Asn Arg Val Llys Val Ser Pro Glu Glu Phe Thr Arg 4 OS 41O 415 Ile Lieu. Asp Ile Arg Glu Lys Ser His Gln Met Val Gly Ala Arg Thr 42O 425 43 O Pro Ile Asp Thr Lieu. Asp Tyr Ile Ser Ala Gly Thr Phe Tyr Lieu. Glu 435 44 O 445 Lys Ile Asp Glu Lys Lieu. Ile Arg His Tyr Lys Ser Llys Pro Ile Ile 450 45.5 460

Ser Ser Lys Lieu. 465

<210s, SEQ ID NO 11 &211s LENGTH: 388 212. TYPE: PRT <213> ORGANISM: Staphylococcus epidermidis

<4 OOs, SEQUENCE: 11 Met Asin Ile Gly Ile Asp Lys Ile Ser Phe Tyr Val Pro Llys Tyr Tyr 1. 5 1O 15

Val Asp Met Ala Lys Lieu Ala Glu Ala Arg Glin Val Asp Pro Asn Lys 2O 25 3O

Phe Lieu. Ile Gly Ile Gly Glin Thr Glu Met Thr Val Ser Pro Val Asn 35 4 O 45

Glin Asp Ile Val Ser Met Gly Ala Asn Ala Ala Lys Asp Ile Ile Thr SO 55 6 O

Glu Glu Asp Llys Lys Asn. Ile Gly Met Val Ile Val Ala Thr Glu Ser 65 70 7s 8O US 2015/0240271 A1 Aug. 27, 2015 31

- Continued

Ala Ile Asp Asn Ala Lys Ala Ala Ala Val Glin Ile His His Lieu. Lieu 85 90 95 Gly Ile Glin Pro Phe Ala Arg Cys Phe Glu Met Lys Glu Ala Cys Tyr 1OO 105 11 O Ala Ala Thr Pro Ala Ile Glin Lieu Ala Lys Asp Tyr Lieu Ala Glin Arg 115 12 O 125 Pro Asn. Glu Lys Val Lieu Val Ile Ala Ser Asp Thr Ala Arg Tyr Gly 13 O 135 14 O Ile His Ser Gly Gly Glu Pro Thr Glin Gly Ala Gly Ala Val Ala Met 145 150 155 160 Met Ile Ser His Asp Pro Ser Ile Lieu Lys Lieu. Asn Asp Asp Ala Val 1.65 17O 17s Ala Tyr Thr Glu Asp Val Tyr Asp Phe Trp Arg Pro Thr Gly His Glin 18O 185 19 O Tyr Pro Lieu Val Ala Gly Ala Lieu. Ser Lys Asp Ala Tyr Ile Llys Ser 195 2OO 2O5 Phe Glin Glu Ser Trp Asn. Glu Tyr Ala Arg Arg His Asn Llys Thr Lieu. 21 O 215 22O Ala Asp Phe Ala Ser Lieu. Cys Phe His Val Pro Phe Thr Lys Met Gly 225 23 O 235 24 O Glin Lys Ala Lieu. Asp Ser Ile Ile Asn His Ala Asp Glu Thir Thr Glin 245 250 255 Asp Arg Lieu. Asn. Ser Ser Tyr Glin Asp Ala Val Asp Tyr Asn Arg Tyr 26 O 265 27 O Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Lieu Ser Lieu. Ile Ser Leu 27s 28O 285 Lieu. Glu Thir Arg Asp Lieu Lys Gly Gly Glin Thir Ile Gly Lieu. Phe Ser 29 O 295 3 OO Tyr Gly Ser Gly Ser Val Gly Glu Phe Phe Ser Gly Thr Lieu Val Asp 3. OS 310 315 32O Gly Phe Lys Glu Glin Lieu. Asp Val Glu Arg His Llys Ser Lieu. Lieu. Asn 3.25 330 335 Asn Arg Ile Glu Val Ser Val Asp Glu Tyr Glu. His Phe Phe Lys Arg 34 O 345 35. O Phe Asp Gln Lieu. Glu Lieu. Asn His Glu Lieu. Glu Lys Ser Asn Ala Asp 355 360 365 Arg Asp Ile Phe Tyr Lieu Lys Ser Ile Asp Asn. Asn. Ile Arg Glu Tyr 37 O 375 38O

His Ile Ala Glu 385

<210s, SEQ ID NO 12 &211s LENGTH: 389 212. TYPE: PRT <213> ORGANISM; Lactobacillus fermentum

<4 OOs, SEQUENCE: 12 Met Lys Ile Gly Ile Asp Llys Lieu Ala Phe Ala Thr Thr Pro Tyr Tyr 1. 5 1O 15

Lieu Ala Met Glu Asp Lieu Ala Glin Gly Arg Asn Val Asp Pro Asn Lys 2O 25 3O

Tyr Lieu. Ile Gly Ile Gly Glin Ser Lys Glin Ala Val Val Pro Pro Thr US 2015/0240271 A1 Aug. 27, 2015 32

- Continued

35 4 O 45 Glin Asp Val Val Thir Lieu Ala Ala Ala Ala Ala Asp Llys Lieu. Lieu. Asp SO 55 6 O Pro Val Glu Arg Asp Glin Val Ser Thr Val Ile Val Ala Thr Glu Ser 65 70 7s 8O Gly Ile Asp Asn. Ser Lys Ala Ala Ala Val Tyr Val Llys His Lieu. Lieu. 85 90 95 Llys Lieu. Ser Asp Phe Thr Arg Ala Val Glu Val Lys Glu Ala Cys Tyr 1OO 105 11 O Ser Ala Thr Ala Ala Lieu. Glin Phe Ala Arg Gly Lieu Val Ala Lieu. Asn 115 12 O 125 Pro Glin Glu Lys Ile Lieu Val Ile Ala Ser Asp Ile Ala Arg Tyr Gly 13 O 135 14 O Lieu. Glu Thr Gly Gly Glu Val Thr Glin Gly Ala Gly Ala Val Ala Met 145 150 155 160 Lieu. Ile Thr Ala Asn Pro Arg Val Lieu Ala Ile Glu Pro Thr Ser Val 1.65 17O 17s Ala Tyr Thir Lys Asp Wal Met Asp Phe Trp Arg Pro Lieu. Tyr Ala Glu 18O 185 19 O Glu Ala Lieu Val Asn Gly Llys Tyr Ser Thir Asn Val Tyr Ile Asp Phe 195 2OO 2O5 Phe Lys Glin Cys Trp Thr Arg Tyr Glin Glin Leu Ala Gly Tyr Gly Lieu. 210 215 220 Glu Asp Phe Ala Ala Leu Ala Phe His Leu Pro Phe Thr Lys Met Gly 225 23 O 235 24 O Llys Lys Ala Lieu. Glu Ala Glu Lieu. Gly Asp Arg Asp Asp Glin Val Ala 245 250 255 Thir Arg Lieu. Arg Ala Asn Lieu. Thir Ala Gly Glin Glu Ala Cys Arg Glin 26 O 265 27 O Val Gly Asn Lieu. Tyr Thr Gly Ser Leu Tyr Lieu. Gly Lieu Met Ser Leu 27s 28O 285 Lieu. Thr Glu Gly Asp Val Llys Pro Gly Glu Arg Ile Gly Lieu. Phe Ser 29 O 295 3 OO Tyr Gly Ser Gly Ala Glu Gly Glu Phe Phe Ala Gly Ile Leu Gln Pro 3. OS 310 315 32O Gly Tyr Glin Glu Gly Lieu. Gly Asp Lieu. Asn. Glu Glin Lieu Ala Ala Arg 3.25 330 335 Thr Glin Val Ser Lieu Ala Glu Tyr Glu Asp Lieu. Phe ASn Glin Glin Lieu. 34 O 345 35. O Gly Lieu Lys Glu Glu Asp Val Thr Phe Llys Thr Pro Ala Ala Gly Glin 355 360 365 Arg Phe Val Lieu Val Gly Glin Lys Asp His Glin Arg Glin Tyr Arg Asp 37 O 375 38O

Lieu Ala Glu Arg Asp 385

<210s, SEQ ID NO 13 &211s LENGTH: 351 212. TYPE: PRT <213> ORGANISM: Hyperthermus butyllicus

<4 OOs, SEQUENCE: 13 US 2015/0240271 A1 Aug. 27, 2015 33

- Continued Met Pro Arg Gly Ser Gly Ile Val Gly Trp Gly Gly Tyr Val Pro Arg 1. 5 1O 15 Tyr Arg Ile Lys Ala Ala Glu Ile Val Arg Val Trp Gly Trp Glu Pro 2O 25 3O Ser Val Pro Ala Gly Lieu. Gly Wall Lys Glu Lys Ala Val Glu Asn. Wall 35 4 O 45 Asp Glu Asp Ser Val Thr Met Gly Tyr Glu Ala Ala Arg Asn Ala Ile SO 55 6 O Ala Arg Ala Asn Val Asp Pro Arg Glu Ile Lys Ala Val Phe Phe Gly 65 70 7s 8O Thr Glu Ser Llys Pro Tyr Ala Val Llys Pro Ser Ala Thr Ile Ile Ala 85 90 95 Glu Ala Leu Gly Ile Thr Pro Glu Thr Met Ala Ser Asp Lieu. Glu Phe 1OO 105 11 O Ala Cys Arg Ala Ala Ser Glu Gly Lieu. Arg Ala Ser Lieu Ala Lieu Val 115 12 O 125 Glu Ala Gly Tyr Met Llys Tyr Ala Lieu Val Val Ala Ser Asp Thir Ala 13 O 135 14 O Glin Ala Asn Pro Gly Asp Val Lieu. Glu Phe Thr Ala Ala Ser Gly Ala 145 150 155 160 Ala Ala Phe Val Val Gly Pro Ala Ser Glu Ser Val Ala Val Lieu. Glu 1.65 17O 17s Gly Val Tyr Thr Tyr Val Thr Asp Thr Pro Asp Phe Trp Arg Gly Glin 18O 185 19 O His Ser Arg Tyr Pro Met His Gly Glu Ala Phe Thr Gly Glu Pro Ala 195 2OO 2O5 Tyr Phe His His Ile Glu Ser Ala Val Lys Gly Lieu Met Glu Lys Lieu. 21 O 215 22O Gly Lieu Lys Pro Glu Asp Phe Asp Tyr Ala Val Phe His Glin Pro Asn 225 23 O 235 24 O Gly Llys Phe Pro Lieu. Arg Val Gly Ala Arg Lieu. Gly Phe Pro Lys Glu 245 250 255 Lys Ile Leu Pro Gly Lieu Lleu. Thr Pro Ile Ile Gly Asn Thr Tyr Asn 26 O 265 27 O Ala Ser Ala Lieu. Lieu. Gly Phe Ala Arg Ile Lieu. Asp Glin Ala Lys Pro 27s 28O 285 Gly Glin Arg Ile Lieu Val Ala Pro Phe Gly Ser Gly Ala Gly Ser Asp 29 O 295 3 OO Ala Tyr Ser Phe Ile Val Thr Asp Arg Ile Glu Glu Ala Arg Asn Arg 3. OS 310 315 32O Ala Pro Llys Val Asp Asp Tyr Val Asn Trp Lys Arg Tyr Ile Asp Tyr 3.25 330 335 Ala Met His Ala Arg Met Arg Llys Lieu. Tyr Asp Arg Arg Pro Val 34 O 345 35. O

<210s, SEQ ID NO 14 &211s LENGTH: 349 212. TYPE: PRT <213> ORGANISM: Chloroflexus aggregains

<4 OOs, SEQUENCE: 14 Met Met Llys Pro Asn Glin Pro Val Gly Ile Ile Gly Tyr Gly Val Tyr 1. 5 1O 15 US 2015/0240271 A1 Aug. 27, 2015 34

- Continued

Ile Pro Arg Tyr Arg Ile Ala Ala Arg Glu Ile Ala Arg Ile Trp Thir 25

Asp Gly Glin Asn Gly Wall Pro Wall Glu Ala Lys Ser Wall Pro Gly Pro 35 4 O 45

Asp Glu Asp Thir Ile Thir Met Ala Ile Glu Ala Arg Asn Ala Luell SO 55

Wall Arg Ala Asp Ile Pro Ala Ser Ala Luell Gly Wall Trp Ile Gly 65 70

Ser Glu Ser His Pro Ser Wall Pro Ser Thir Wall Wall Ala 85 90 95

Asp Ala Luell Gly Ala Gly Pro Trp Wall Ser Ala Asp Trp Glu Phe 105 11 O

Ala Lys Ala Gly Ser Glu Ala Luell Thir Ala Met Ala Luell Wall 115 12 O 125

Gly Ser Gly Met Glin Arg Tyr Ala Luell Ala Ile Ala Asp Thir Ala 13 O 135

Glin Gly Arg Pro Gly Asp Ala Luell Glu Tyr Thir Ser Ala Gly Ala 145 150 155 160

Ala Ala Luell Ile Wall Gly Pro Ala Thir Glu Ala Lell Ala Thir Ile Asp 1.65 17O 17s

Ala Thir Wall Ser Tyr Wall Thir Asp Thir Pro Asp Phe Arg Arg Ala 18O 185 19 O

Asp Arg Pro Pro Wall His Gly Asn Arg Phe Thir Gly Glu Pro Ala 195

Phe His Glin Ile Glin Ser Ala Ala Ser Glu Lell Lell Arg Glin Luell 21 O 215

Asn Arg Thir Ala Ala Asp Phe Thir Ala Wall Phe His Glin Pro Asn 225 23 O 235 24 O

Phe Pro Glin Thir Wall Ala Arg Luell Gly Phe Thir Asp Ala 245 250 255

Ile Ala Pro Gly Lell Lell Ser Pro Glin Ile Gly Asn Thir Ser 26 O 265 27 O

Ala Ala Luell Lell Gly Lell Cys Ala Ile Luell Asp Wall Ala Pro 285

Asp Thir Ile Phe Wall Thir Ser Gly Ser Gly Ala Gly Ser Asp 29 O 295 3 OO

Ala Luell Thir Wall Thir Glu Ala Ile Wall Glu Arg Arg Glu Arg 310 315 32O

Pro Luell Thir Ala Ala Luell Ala Arg Lys Wall Met Ile Asp 3.25 330 335

Met Ala Lys Trp Arg Gly Lys Luell Wall Met Gly 34 O 345

SEO ID NO 15 LENGTH: TYPE : PRT ORGANISM; Bacillus subtilis

< 4 OOs SEQUENCE: 15 Met Val Ser Ala Gly Ile Glu Ala Met Asn Val Phe Gly Gly Thr Ala 1. 5 15 Tyr Lieu. Asp Wal Met Glu Lieu Ala Lys Tyr Arg His Lieu. Asp Thir Ala US 2015/0240271 A1 Aug. 27, 2015 35

- Continued

2O 25 3O Arg Phe Glu Asn Lieu. Lieu Met Lys Glu Lys Ala Val Ala Lieu Pro Tyr 35 4 O 45 Glu Asp Pro Val Thr Phe Gly Val Asn Ala Ala Lys Pro Ile Ile Asp SO 55 6 O Ala Lieu. Ser Glu Ala Glu Lys Asp Arg Ile Glu Lieu. Lieu. Ile Thr Cys 65 70 7s 8O Ser Glu Ser Gly Ile Asp Phe Gly Lys Ser Leu Ser Thr Tyr Ile His 85 90 95 Glu Tyr Lieu. Gly Lieu. Asn Arg Asn. Cys Arg Lieu. Phe Glu Wall Lys Glin 1OO 105 11 O Ala Cys Tyr Ser Gly Thr Ala Gly Phe Gln Met Ala Val Asin Phe Ile 115 12 O 125 Lieu. Ser Glin Thir Ser Pro Gly Ala Lys Ala Lieu Val Ile Ala Ser Asp 13 O 135 14 O Ile Ser Arg Phe Lieu. Ile Ala Glu Gly Gly Asp Ala Lieu. Ser Glu Asp 145 150 155 160 Trp Ser Tyr Ala Glu Pro Ser Ala Gly Ala Gly Ala Val Ala Val Lieu. 1.65 17O 17s Val Gly Glu Asn Pro Glu Val Phe Glin Ile Asp Pro Gly Ala Asn Gly 18O 185 19 O Tyr Tyr Gly Tyr Glu Val Met Asp Thr Cys Arg Pro Ile Pro Asp Ser 195 2OO 2O5 Glu Ala Gly Asp Ser Asp Lieu. Ser Lieu Met Ser Tyr Lieu. Asp Cys Cys 21 O 215 22O Glu Glin Thr Phe Lieu. Glu Tyr Gln Lys Arg Val Pro Gly Ala Asn Tyr 225 23 O 235 24 O Gln Asp Thr Phe Glin Tyr Lieu Ala Tyr His Thr Pro Phe Gly Gly Met 245 250 255 Val Lys Gly Ala His Arg Thr Met Met Arg Llys Val Ala Lys Wall Lys 26 O 265 27 O Thir Ser Gly Ile Glu Thir Asp Phe Lieu. Thr Arg Val Llys Pro Gly Lieu. 27s 28O 285 Asn Tyr Cys Glin Arg Val Gly Asn. Ile Met Gly Ala Ala Lieu. Phe Lieu 29 O 295 3 OO Ala Lieu Ala Ser Thir Ile Asp Glin Gly Arg Phe Asp Thr Pro Lys Arg 3. OS 310 315 32O Ile Gly Cys Phe Ser Tyr Gly Ser Gly Cys Cys Ser Glu Phe Tyr Ser 3.25 330 335 Gly Ile Thir Thr Pro Glin Gly Glin Glu Arg Glin Arg Thr Phe Gly Ile 34 O 345 35. O Glu Lys His Lieu. Asp Arg Arg Tyr Glin Lieu. Ser Met Glu Glu Tyr Glu 355 360 365

Lieu. Lieu. Phe Lys Gly Ser Gly Met Val Arg Phe Gly Thr Arg Asn. Wall 37 O 375 38O

Lys Lieu. Asp Phe Glu Met Ile Pro Gly Ile Met Glin Ser Thr Glin Glu 385 390 395 4 OO Llys Pro Arg Lieu. Phe Lieu. Glu Glu Ile Ser Glu Phe His Arg Llys Tyr 4 OS 41O 415

Arg Trp Ile Ser 42O US 2015/0240271 A1 Aug. 27, 2015 36

- Continued

<210s, SEQ ID NO 16 &211s LENGTH: 388 212. TYPE: PRT <213> ORGANISM: Mycobacterium marinum <4 OOs, SEQUENCE: 16 Met Val Ser Ile Gly Ile His Asp Leu Ser Ile Ala Thr Ala His Tyr 1. 5 1O 15 Val Lieu. Asp His Ala Thr Lieu Ala Glu. His His Gly Val Asp Val Asn 2O 25 3O Lys Tyr Lieu. Ile Gly Lieu. Gly Glin Glin Glin Met Ser Ile Val Ala Pro 35 4 O 45 Asp Glu Asp Ile Val Thr Lieu Ala Ala Ala Ala Ala Asp Pro Ile Ile SO 55 6 O Lys Arg His Gly Ser Glin Lys Ile Arg Thir Ile Val Ile Gly Thr Glu 65 70 7s 8O Thr Gly Val Asp Glin Ser Lys Ser Ala Gly Ile Trp Val Ser Ser Lieu. 85 90 95 Lieu. Gly Lieu Pro Ser Ser Ala Arg Val Lieu. Glu Val Lys Glin Ala Cys 1OO 105 11 O Tyr Gly Ala Thr Gly Ala Lieu. Glin Lieu Ala Lieu Ala Lieu Val His Arg 115 12 O 125 Asp Pro Thr Glin Glin Val Lieu Val Ile Ala Ala Asp Val Ala Arg Tyr 13 O 135 14 O Asp Lieu. Asp Ser Pro Gly Glu Pro Thr Glin Gly Ala Ala Ala Ala Ala 145 150 155 160 Met Lieu Val Ser Ala Asp Pro Ala Lieu. Lieu. Arg Lieu. Glu Glu Pro Thr 1.65 17O 17s Gly Ile Tyr Thr Ala Asp Ile Met Asp Phe Trp Arg Pro Asn Tyr Arg 18O 185 19 O Ser Thr Ala Lieu Val Asp Gly Lys Ala Ser Val Thr Ala Tyr Met Glu 195 2OO 2O5 Ala Ala Ser Gly Ala Trp Lys Asp Tyr Thr Glu Arg Gly Gly Arg Ala 21 O 215 22O Phe Gly Glu Phe Ala Ala Phe Cys Tyr His Gln Pro Phe Thr Lys Met 225 23 O 235 24 O Ala Tyr Lys Ala His Lys Glin Lieu Ala Ala Glu Ala Gly Glu Asp Ala 245 250 255 Ser Gly Ala Ala Val Glin Ala Ala Val Gly Asn. Thr Val Glu Tyr Asn 26 O 265 27 O Arg Arg Ile Gly Asn. Ser Tyr Thr Ala Ser Lieu. Tyr Lieu Ala Lieu Ala 27s 28O 285

Ala Lieu. Lieu. Asp Glin Ala Asp Asp Lieu. Ser Asp Gln Pro Ile Ala Met 29 O 295 3 OO

Lieu. Ser Tyr Gly Ser Gly Cys Val Ala Glu Lieu Phe Ala Gly Thr Val 3. OS 310 315 32O Thr Pro Gly Tyr Glin Gln His Lieu. Arg Thr Asp Glin His Arg Ala Ala 3.25 330 335 Lieu. Glu Thir Arg Ile Pro Lieu. Ser Tyr Glu. His Tyr Arg Arg Lieu. His 34 O 345 35. O

Asn Lieu. Thir Lieu Pro Thr Asn Gly Asn His His Ser Leu Pro Val Glu US 2015/0240271 A1 Aug. 27, 2015 37

- Continued

355 360 365 Thir Ser Arg Pro Phe Arg Lieu. Thir Ala Ile Ser Glu. His Lys Arg Met 37 O 375 38O Tyr Gly Ala Val 385

<210s, SEQ ID NO 17 &211s LENGTH: 435 212. TYPE: PRT <213> ORGANISM: Zea mays <4 OOs, SEQUENCE: 17 Met Lieu Ala Ala Ser Thr Llys Val Gly Ser Arg Lieu Ala Ser Pro His 1. 5 1O 15 Ala Ser Lieu. Ser Ala Gly Ala Ala Ala Ala Ala Lieu Ala Ser Ser Pro 2O 25 3O Val Lieu. Gly Ser Gly Met Leu Pro Gly Ala Gly Phe Gly Glu. Thr Gly 35 4 O 45 Asn His His Ala Ala Asp Ala Pro Pro Pro Leu Pro Cys Ser Ser Ser SO 55 6 O Gly Asp Ser Arg Glu Tyr Tyr Glin Trp Lys Arg Lieu Val Asin Glin Arg 65 70 7s 8O Gln Ser Thr Lieu. His Val Gly Glu Val Pro Ala Ala Lieu. Gly His His 85 90 95 Val Phe Gly Ala Gly Cys Ser Ser Arg Lys Gln His Ile Tyr Arg Tyr 1OO 105 11 O Phe Ser Ser Ser Ser His Glin Gly Ser Ile Trp Ala Arg Ser Lys Ile 115 12 O 125 Lieu. His Asp Lieu Pro Gly Tyr Val Lys Ile Val Glu Val Gly Pro Arg 13 O 135 14 O Asp Gly Lieu. Glin Asn. Glu Lys Asp Ile Val Pro Thr Pro Val Llys Val 145 150 155 160 Glu Lieu. Ile Arg Arg Lieu Ala Thir Ser Gly Lieu Pro Val Val Glu Ala 1.65 17O 17s Thir Ser Phe Val Ser Pro Llys Trp Val Pro Gln Leu Ala Asp Ala Lys 18O 185 19 O Asp Val Met Glu Ala Val Arg Thr Ile Gly Gly Val Arg Phe Pro Val 195 2OO 2O5 Lieu. Thr Pro Asn Lieu Lys Gly Phe Glu Ala Ala Ile Ala Ala Gly Ala 21 O 215 22O Lys Glu Ile Ala Ile Phe Ala Ser Ala Ser Glu Gly Phe Ser Lys Ser 225 23 O 235 24 O Asn. Ile Asn. Cys Thir Ile Lys Glu Ser Ile Ala Arg Tyr Asn Asp Wall 245 250 255

Ala Lieu Ala Ala Lys Glu Lys Glu Ile Pro Val Arg Gly Tyr Val Ser 26 O 265 27 O

Cys Val Val Gly Cys Pro Val Asp Gly Pro Val Pro Pro Ser Asn Val 27s 28O 285

Ala Tyr Val Ala Lys Glu Lieu. Tyr Asp Met Gly Cys Tyr Glu Val Ser 29 O 295 3 OO

Lieu. Gly Asp Thir Ile Gly Val Gly Thr Pro Gly Thr Val Val Pro Met 3. OS 310 315 32O US 2015/0240271 A1 Aug. 27, 2015 38

- Continued Lieu. Glu Ala Ala Ile Ser Val Val Pro Val Glu Lys Lieu Ala Val His 3.25 330 335 Phe His Asp Thr Tyr Gly Glin Ser Leu Ser Asn Ile Lieu. Ile Ser Lieu. 34 O 345 35. O Glin Met Gly Val Ser Val Val Asp Ser Ser Val Ala Gly Lieu. Gly Gly 355 360 365 Cys Pro Tyr Ala Lys Gly Ala Ser Gly Asn Val Ala Thr Glu Asp Wall 37 O 375 38O Val Tyr Met Lieu. Asn Gly Lieu. Gly Val Lys Thr Gly Val Asp Lieu. Gly 385 390 395 4 OO Llys Val Met Ala Ala Gly Glu Phe Ile Cys Arg His Lieu. Gly Arg Glin 4 OS 41O 415 Ser Gly Ser Lys Ala Ala Thr Ala Lieu. Ser Llys Val Thr Ala Asn Ala 42O 425 43 O

Ser Lys Lieu. 435

<210s, SEQ ID NO 18 &211s LENGTH: 335 212. TYPE: PRT <213> ORGANISM: Danio rerio (Brachydanio rerio) <4 OOs, SEQUENCE: 18 Met Gly Asn Val Ser Ser Ala Val Llys His Cys Lieu Ser Tyr Glu Thr 1. 5 1O 15 Phe Lieu. Arg Asp Tyr Pro Trp Lieu Pro Arg Lieu Lleu Trp Glu Glu Lys 2O 25 3O Cys Ser Glu Lieu Pro Llys Lieu Pro Val Tyr Val Lys Ile Val Glu Val 35 4 O 45 Gly Pro Arg Asp Gly Lieu. Glin Asn. Glu Lys Glu Ile Val Pro Thr Glu SO 55 6 O Val Lys Ile Glin Lieu. Ile Asp Lieu. Lieu. Ser Glin Thr Gly Lieu Pro Val 65 70 7s 8O Ile Glu Ala Thr Ser Phe Val Ser Ser Lys Trp Val Ala Gln Met Ala 85 90 95 Asp His Thir Ala Val Lieu Lys Gly Ile Lys Arg Ser Pro Asp Val Arg 1OO 105 11 O Tyr Pro Val Lieu. Thr Pro Asn Ile Glin Gly Phe Glin Ala Ala Val Ala 115 12 O 125 Ala Gly Ala Asn. Glu Val Ala Val Phe Gly Ser Ala Ser Glu Thir Phe 13 O 135 14 O Ser Arg Lys Asn. Ile Asn. Cys Ser Ile Glu Glu Ser Lieu. Glin Arg Phe 145 150 155 160

Glu Glin Val Val Ser Ala Ala Lys Glin Glu Gly Ile Pro Val Arg Gly 1.65 17O 17s

Tyr Val Ser Cys Ala Lieu. Gly Cys Pro Tyr Glu Gly Glin Val Llys Pro 18O 185 19 O

Ser Glin Val Thir Lys Val Ala Lys Arg Lieu. Phe Glu Lieu. Gly Cys Tyr 195 2OO 2O5

Glu Val Ser Leu Gly Asp Thir Ile Gly Val Gly Thr Ala Gly Ser Met 21 O 215 22O

Ala Glu Met Lieu. Ser Asp Val Lieu. Thr Glu Val Pro Ala Gly Ala Lieu 225 23 O 235 24 O US 2015/0240271 A1 Aug. 27, 2015 39

- Continued

Ala Wal His Cys His Asp Thr Tyr Gly Glin Ala Lieu Pro Asn. Ile Lieu 245 250 255 Ile Ala Lieu Gln Met Gly Val Ser Val Val Asp Ala Ser Val Ala Gly 26 O 265 27 O Lieu. Gly Gly Cys Pro Phe Ala Lys Gly Ala Ser Gly Asn Val Ser Thr 27s 28O 285 Glu Asp Lieu. Lieu. Tyr Met Lieu. His Gly Lieu. Gly Ile Glu Thr Gly Val 29 O 295 3 OO Asp Lieu. Lieu Lys Val Met Glu Ala Gly Asp Phe Ile Cys Lys Ala Lieu 3. OS 310 315 32O Asn Arg Llys Thr Asn. Ser Llys Val Ser Glin Ala Thr Arg Asn. Asn 3.25 330 335

<210s, SEQ ID NO 19 &211s LENGTH: 325 212. TYPE: PRT &213s ORGANISM: Bos taurus

<4 OOs, SEQUENCE: 19 Met Ala Thr Val Llys Llys Val Lieu Pro Arg Arg Lieu Val Gly Lieu Ala 1. 5 1O 15 Thr Lieu. Arg Ala Val Ser Thr Ser Ser Val Gly Thr Phe Pro Lys Glin 2O 25 3O Val Lys Ile Val Glu Val Gly Pro Arg Asp Gly Lieu Gln ASn Glu Lys 35 4 O 45 Asn. Ile Val Pro Thr Pro Wall Lys Ile Llys Lieu. Ile Asp Met Lieu. Ser SO 55 6 O Glu Ala Gly Lieu Pro Val Val Glu Ala Thr Ser Phe Val Ser Pro Llys 65 70 7s 8O Trp Val Pro Gln Met Ala Asp His Ala Glu Val Lieu Lys Gly Ile Glin 85 90 95 Llys Phe Pro Gly Val Asn Tyr Pro Val Lieu. Thr Pro Asn Phe Lys Gly 1OO 105 11 O Phe Glin Ala Ala Val Ala Ala Gly Ala Lys Glu Val Ala Ile Phe Gly 115 12 O 125 Ala Ala Ser Glu Lieu. Phe Thr Lys Lys Asn. Ile Asn. Cys Ser Ile Asp 13 O 135 14 O Glu Ser Lieu. Glin Arg Phe Asp Glu Ile Lieu Lys Ala Ala Arg Ala Ala 145 150 155 160 Gly Ile Ser Val Arg Gly Tyr Val Ser Cys Val Lieu. Gly Cys Pro Tyr 1.65 17O 17s Glu Gly Lys Ile Ser Pro Ala Lys Val Ala Glu Val Thir Lys Llys Lieu. 18O 185 19 O

Tyr Ser Met Gly Cys Tyr Glu Ile Ser Leu Gly Asp Thr Ile Gly Val 195 2OO 2O5

Gly Thr Pro Gly Ala Met Lys Asp Met Lieu. Ser Ala Val Lieu. Glin Glu 21 O 215 22O

Val Pro Val Thr Ala Lieu Ala Val His Cys His Asp Thr Tyr Gly Glin 225 23 O 235 24 O

Ala Lieu Ala Asn. Thir Lieu. Thir Ala Lieu Gln Met Gly Val Ser Val Met 245 250 255

Asp Ser Ser Val Ala Gly Lieu. Gly Gly Cys Pro Tyr Ala Glin Gly Ala US 2015/0240271 A1 Aug. 27, 2015 40

- Continued

26 O 265 27 O Ser Gly Asn Lieu Ala Thr Glu Asp Lieu Val Tyr Met Lieu Ala Gly Lieu. 27s 28O 285 Gly Ile His Thr Gly Val Asn Lieu Gln Lys Lieu. Lieu. Glu Ala Gly Ala 29 O 295 3 OO Phe Ile Cys Glin Ala Lieu. Asn Arg Arg Thr Asn. Ser Llys Val Ala Glin 3. OS 310 315 32O Ala Thr Cys Llys Lieu. 3.25

<210s, SEQ ID NO 2 O &211s LENGTH: 325 212. TYPE: PRT <213> ORGANISM: Homo sapiens

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

Ala Lieu. Thir Asn Thr Lieu Met Ala Leu Gln Met Gly Val Ser Val Val 245 250 255

Asp Ser Ser Val Ala Gly Lieu. Gly Gly Cys Pro Tyr Ala Glin Gly Ala 26 O 265 27 O

Ser Gly Asn Lieu Ala Thr Glu Asp Lieu Val Tyr Met Lieu. Glu Gly Lieu. 27s 28O 285 US 2015/0240271 A1 Aug. 27, 2015 41

- Continued Gly Ile His Thr Gly Val Asn Lieu Gln Lys Lieu. Lieu. Glu Ala Gly Asn 29 O 295 3 OO Phe Ile Cys Glin Ala Lieu. Asn Arg Llys Thir Ser Ser Llys Val Ala Glin 3. OS 310 315 32O Ala Thr Cys Llys Lieu. 3.25

<210s, SEQ ID NO 21 &211s LENGTH: 299 212. TYPE: PRT <213> ORGANISM: Pseudomonas putida Q88H25 <4 OOs, SEQUENCE: 21 Met Ser Lieu Pro Llys His Val Arg Lieu Val Glu Val Gly Pro Arg Asp 1. 5 1O 15 Gly Lieu. Glin Asn. Glu Ala Glin Pro Ile Ser Val Ala Asp Llys Val Arg 2O 25 3O Lieu Val Asn Asp Lieu. Thr Glu Ala Gly Lieu Ala Tyr Ile Glu Val Gly 35 4 O 45 Ser Phe Val Ser Pro Llys Trp Val Pro Glin Met Ala Gly Ser Ala Glu SO 55 6 O Val Phe Ala Gly Ile Glin Glin Arg Pro Gly Val Thr Tyr Ala Ala Lieu 65 70 7s 8O Ala Pro Asn Lieu. Arg Gly Phe Glu Asp Ala Lieu Ala Ala Gly Val Lys 85 90 95 Glu Val Ala Val Phe Ala Ala Ala Ser Glu Ala Phe Ser Glin Arg Asn 1OO 105 11 O Ile Asn Cys Ser Ile Ser Glu Ser Leu Lys Arg Phe Glu Pro Ile Met 115 12 O 125 Asp Ala Ala Arg Ser His Gly Met Arg Val Arg Gly Tyr Val Ser Cys 13 O 135 14 O Val Lieu. Gly Cys Pro Tyr Glu Gly Llys Val Ser Ala Glu Glin Val Ala 145 150 155 160 Pro Val Ala Arg Ala Lieu. His Asp Met Gly Cys Tyr Glu Val Ser Lieu. 1.65 17O 17s Gly Asp Thir Ile Gly Thr Gly Thr Ala Gly Asp Thr Arg Arg Lieu. Phe 18O 185 19 O Glu Val Val Ser Ala Glin Val Pro Arg Glu Glin Lieu Ala Gly His Phe 195 2OO 2O5 His Asp Thir Tyr Gly Glin Ala Lieu Ala Asn Val Tyr Ala Ser Lieu. Lieu. 21 O 215 22O Glu Gly Ile Ser Val Phe Asp Ser Ser Val Ala Gly Lieu. Gly Gly Cys 225 23 O 235 24 O

Pro Tyr Ala Lys Gly Ala Thr Gly Asn. Ile Ala Ser Glu Asp Val Val 245 250 255

Tyr Lieu. Lieu. Glin Gly Lieu. Gly Ile Glu Thr Gly Ile Asp Lieu. Gly Lieu. 26 O 265 27 O

Lieu. Ile Ala Ala Gly Glin Arg Ile Ser Gly Val Lieu. Gly Arg Asp Asn 27s 28O 285

Gly Ser Arg Val Ala Arg Ala Cys Ser Ala Glin 29 O 295

<210s, SEQ ID NO 22 US 2015/0240271 A1 Aug. 27, 2015 42

- Continued

&211s LENGTH: 312 212. TYPE: PRT <213> ORGANISM: Acinetobacter baumannii B7H4C6

<4 OOs, SEQUENCE: 22 Met Thr Ala Phe Ser Asp Lieu. Leu Val Val Glin Glu Val Ser Pro Arg 1. 5 1O 15 Asp Gly Lieu. Glin Ile Glu Pro Thir Trp Val Pro Thr Asp Llys Lys Ile 2O 25 3O Asp Lieu. Ile Asin Gln Lieu. Ser Thr Met Gly Phe Ser Arg Ile Glu Ala 35 4 O 45 Gly Ser Phe Val Ser Pro Lys Ala Ile Pro Asn Lieu. Arg Asp Gly Glu SO 55 6 O Glu Val Phe Thr Gly Ile Thr Arg His Lys Asp Ile Ile Tyr Val Gly 65 70 7s 8O Lieu. Ile Pro Asn Lieu Lys Gly Ala Lieu. Arg Ala Val Glu Ala Asn Ala 85 90 95

ASn Glu Lieu. Asn Lieu Val Lieu Ser Ala Ser Glin Thr His Asn Lieu Ala 1OO 105 11 O Asn Met Arg Met Thr Lys Ala Glin Ser Phe Ala Gly Phe Thr Glu Ile 115 12 O 125 Val Glu Gln Leu Gln Gly Lys Thr Glin Phe Asn Gly Thr Val Ala Thr 13 O 135 14 O Thr Phe Gly Cys Pro Phe Glu Gly Lys Ile Ser Glu Arg Glu Val Phe 145 150 155 160 Ser Lieu Val Glu. His Tyr Lieu Lys Lieu. Gly Ile His Asn. Ile Thr Lieu. 1.65 17O 17s Ala Asp Thir Thr Gly Met Ala Asn Pro Val Glin Val Lys Arg Ile Val 18O 185 19 O

Ser His Wall Leu Ser Lieu. Ile Ser Pro Glu Gln Lieu. Thir Lieu. His Phe 195 2OO 2O5 His Asn. Thir Arg Gly Lieu. Gly Lieu. Thir Asn Val Lieu Ala Ala Tyr Glu 21 O 215 22O Val Gly Ala Arg Arg Phe Asp Ala Ala Lieu. Gly Gly Lieu. Gly Gly Cys 225 23 O 235 24 O Pro Phe Ala Pro Gly Ala Ser Gly Asn Ile Cys Thr Glu Asp Leu Val 245 250 255 Asn Met Cys Glu Glu Ile Gly Ile Pro Thir Thir Ile Asp Lieu. Asp Ala 26 O 265 27 O Lieu. Ile Glin Lieu. Ser Arg Thr Lieu Pro Ala Lieu. Lieu. Gly. His Asp Thr 27s 28O 285 Pro Ser Glin Lieu Ala Lys Ala Gly Arg Asn. Thir Asp Lieu. His Pro Ile 29 O 295 3 OO

Pro Asp Tyr Ile Llys Ser Lieu. Asn 3. OS 310

<210s, SEQ ID NO 23 &211s LENGTH: 286 212. TYPE: PRT <213> ORGANISM: Thermus thermophilus Q72IHO

<4 OOs, SEQUENCE: 23 Met Lys Ala Ser Val Arg Trp Val Glu. Cys Pro Arg Asp Ala Trp Glin 1. 5 1O 15 US 2015/0240271 A1 Aug. 27, 2015 43

- Continued

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

<210s, SEQ ID NO 24 &211s LENGTH: 387 212. TYPE: PRT <213> ORGANISM; Lactobacillus delbrueckii

<4 OOs, SEQUENCE: 24 Met Asp Ile Gly Ile Asp Glin Ile Gly Phe Tyr Thr Pro Asn Llys Phe 1. 5 1O 15

Val Asp Met Val Asp Lieu Ala Asn Ala Arg Asn Glin Asp Pro Asn Lys 2O 25 3O

Phe Lieu. Ile Gly Ile Gly Glin Asp Arg Met Ala Val Ala Asp Llys Thr 35 4 O 45

Glin Asp Ala Val Ser Met Gly Ile Asn Ala Thr Ala Glu Tyr Lieu. Asp SO 55 6 O

Glin Val Asp Lieu. Glu Gln Lieu. Gly Lieu. Lieu. Ile Phe Ala Thr Glu Ser 65 70 7s 8O

Gly Ile Asp Glin Ser Llys Ser Ala Ser Lieu. Phe Val Lys Glu Ala Lieu. US 2015/0240271 A1 Aug. 27, 2015 44

- Continued

85 90 95 Asn Lieu Pro Ala Arg Ile Arg Thr Phe Glu Ile Llys Glu Ala Cys Phe 1OO 105 11 O Ala Lieu. Thir Ala Ser Lieu. Glin Val Ala Arg Asp Tyr Val Arg Ala His 115 12 O 125 Pro His His Ser Ala Met Ile Ile Gly Ser Asp Ile Ala Arg Tyr Gly 13 O 135 14 O Lieu Ala Thr Ala Gly Glu Val Thr Glin Gly Ala Gly Ala Ile Ser Met 145 150 155 160 Lieu. Ile Lys Glu Asn Pro Ala Ile Ile Ala Lieu. Glu Asp Gly His Thr 1.65 17O 17s Ser His Ser Glu Asn. Ile Asn Asp Phe Trp Arg Pro Asn. Asn Lieu Ala 18O 185 19 O Thir Ala Val Val Asp Gly His Tyr Ser Arg Asp Val Tyr Lieu. Asp Phe 195 2OO 2O5 Phe Llys Ser Thr Phe Llys Pro Phe Leu Ala Glu Lys Gln Leu Glin Val 21 O 215 22O Ser Asp Phe Ala Gly Ile Cys Tyr His Leu Pro Tyr Thr Lys Met Gly 225 23 O 235 24 O Tyr Lys Ala His Lys Ile Ala Ile Glu Gly Glin Asp Asp Glu Thr Val 245 250 255 Lys Arg Lieu. Ser Asp Asn. Phe Glin Lieu. Ser Ala Lys Tyr Ser Arg Glin 26 O 265 27 O Val Gly Asn Ile Tyr Thr Ala Ser Leu Tyr Met Ser Val Lieu. Ser Leu 27s 28O 285 Lieu. Glu Asn Gly Asp Lieu. Glu Ala Gly Asp Arg Ile Gly Phe Phe Ser 29 O 295 3 OO Tyr Gly Ser Gly Ala Met Ala Glu Phe Phe Ser Gly Llys Val Val Ala 3. OS 310 315 32O Gly Tyr Glin Lys Arg Lieu. Arg Pro Ala Lieu. His Ala Arg Met Lieu Lys 3.25 330 335 Glu Arg Ile Arg Lieu. Gly Val Gly Glin Tyr Glu Asp Ile Phe Thr Glu 34 O 345 35. O Gly Lieu. Glu Ala Lieu Pro Glu Asn Val Glu Phe Thir Ser Asp Ala Asn 355 360 365 His Gly. Thir Trp Tyr Lieu Ala Gly Glin Glu Gly Tyr Val Arg Glin Tyr 37 O 375 38O Lys Glin Lys 385

<210s, SEQ ID NO 25 &211s LENGTH: 388 212. TYPE: PRT <213> ORGANISM: Staphylococcus haemolyticus

<4 OOs, SEQUENCE: 25 Met Ser Ile Gly Ile Asp Lys Ile Asin Phe Tyr Val Pro Llys Tyr Tyr 1. 5 1O 15

Val Asp Met Ala Lys Lieu Ala Glu Ala Arg Glin Val Asp Pro Asn Lys 2O 25 3O

Phe Lieu. Ile Gly Ile Gly Glin Thr Gln Met Ala Val Ser Pro Val Ser 35 4 O 45 US 2015/0240271 A1 Aug. 27, 2015 45

- Continued

Glin Asp Ile Val Ser Met Gly Ala Asn Ala Ala Lys Asp Ile Ile Thr SO 55 6 O

Asp Asp Asp Llys Llys His Ile Gly Met Val Ile Val Ala Thr Glu Ser 65 70 7s 8O

Ala Ile Asp Asn Ala Lys Ala Ala Ala Val Glin Ile His Asn Lieu. Lieu 85 90 95

Gly Val Glin Pro Phe Ala Arg Cys Phe Glu Met Lys Glu Ala Cys Tyr 1OO 105 11 O

Ala Ala Thr Pro Ala Ile Glin Lieu Ala Lys Asp Tyr Ile Glu Lys Arg 115 12 O 125

Pro Asn. Glu Lys Val Lieu Val Ile Ala Ser Asp Thr Ala Arg Tyr Gly 13 O 135 14 O

Ile Glin Ser Gly Gly Glu Pro Thr Glin Gly Ala Gly Ala Val Ala Met 145 150 155 160

Lieu. Ile Ser Asn. Asn Pro Ser Ile Lieu. Glu Lieu. Asn Asp Asp Ala Val 1.65 17O 17s

Ala Tyr Thr Glu Asp Val Tyr Asp Phe Trp Arg Pro Thr Gly His Lys 18O 185 19 O

Tyr Pro Lieu Val Ala Gly Ala Lieu. Ser Lys Asp Ala Tyr Ile Llys Ser 195 2OO 2O5

Phe Glin Glu Ser Trp Asn. Glu Tyr Ala Arg Arg Glu Asp Llys Thr Lieu. 21 O 215 22O

Ser Asp Phe Glu Ser Lieu. Cys Phe His Val Pro Phe Thr Lys Met Gly 225 23 O 235 24 O

Llys Lys Ala Lieu. Asp Ser Ile Ile Asn Asp Ala Asp Glu Thir Thr Glin 245 250 255

Glu Arg Lieu. Thir Ser Gly Tyr Glu Asp Ala Val Tyr Tyr Asn Arg Tyr 26 O 265 27 O

Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Lieu Ser Lieu. Ile Ser Leu 27s 28O 285

Lieu. Glu Asn Arg Ser Lieu Lys Gly Gly Glin Thir Ile Gly Lieu. Phe Ser 29 O 295 3 OO

Tyr Gly Ser Gly Ser Val Gly Glu Phe Phe Ser Ala Thr Lieu Val Glu 3. OS 310 315 32O

Gly Tyr Glu Lys Glin Lieu. Asp Ile Glu Gly His Lys Ala Lieu. Lieu. Asn 3.25 330 335

Glu Arg Glin Glu Val Ser Val Glu Asp Tyr Glu Ser Phe Phe Lys Arg 34 O 345 35. O

Phe Asp Asp Lieu. Glu Phe Asp His Ala Thr Glu Glin Thr Asp Asp Asp 355 360 365

Llys Ser Ile Tyr Tyr Lieu. Glu Asn. Ile Glin Asp Asp Ile Arg Glin Tyr 37 O 375 38O

His Ile Pro Llys 385 US 2015/0240271 A1 Aug. 27, 2015 46

1. A method for the production of 3-hydroxy-3-methylbu and is genetically modified so as to be able to produce acetone tyric acid comprising an enzyme-catalyzed reaction between by the introduction of at least one gene necessary for the (a) acetone and (b) a compound of formula (I) which provides production of acetone by the organism. an activated acetyl group: 8. The recombinant organism of claim 7, wherein the organism is capable of photosynthesis. 9. A method of producing 3-hydroxy-3-methylbutyric acid, comprising incubating a recombinant organism which: (a) produces acetone; and (b) expresses an enzyme which has the enzymatic activity of a HMG CoA synthase (EC 2.3.3.10) and catalyzes the formation of a covalent bond between the carbon atom of the oxo (C=O) group of the acetone and the carbon atom (C) corresponding to the methyl group of the wherein X is S CH-CH NH CO CH-CH compound of formula (I) NH CO-CH(OH)–C(CH) CH-O-POH CHNO,P (coenzyme A), wherein the enzyme-cata lyzed reaction is performed by an enzyme having the activity of a HMG CoA synthase (EC 2.3.3.10) and which catalyzes the formation of a covalent bond between the carbon atom of the oxo (C=O) group of the acetone and the carbon atom (C) corresponding to the methyl group of said compound of formula (I). 2. The method of claim 1 wherein the enzyme-catalyzed reaction occurs in the presence of an organism capable of wherein X is coenzyme A: producing acetone and expressing the enzyme as defined in under conditions under which the acetone is produced and claim 1. the enzyme is expressed, wherein 3-hydroxy-3-methyl 3. A recombinant organism which: butyric acid is produced. (a) produces acetone; and 10. The method of claim 9, wherein the recombinant organ (b) expresses an enzyme which has the enzymatic activity ism is a recombinant Clostridium acetobutylicum, of a HMGCoA synthase (EC 2.3.3.10) and catalyzes the Clostridium beijerinckii, Clostridium cellulolyticum, Bacil formation of a covalent bond between the carbon atom lus polymyxa or Pseudomonas putida. of the oxo (C=O) group of the acetone and the carbon 11. A composition comprising atom (C) corresponding to the methyl group of the (i) acetone; and compound of formula (I) (ii) a compound of formula (I) which provides an activated acetyl group:

wherein X is coenzyme A. 4. The recombinant organism of claim 3 which is derived wherein X is S CH-CH NH CO-CH from an organism which naturally has the capacity to produce CH-NH CO-CH(OH)–C(CH), CH, acetOne. O—POH CHNO,P (coenzyme A); and 5. The recombinant organism of claim 4, wherein the (iii) an enzyme having the enzymatic activity of a HMO organism is of the genus Clostridium, Bacillus or Pseudomo CoA synthase (EC 2.3.3.10) and which is capable of C.S. catalyzing the formation of a covalent bond between the 6. The recombinant microorganism of claim 5, selected carbonatom of the oxo (C=O) group of the acetone and from the species Clostridium acetobutylicum, Clostridium the carbonatom (C) corresponding to the methyl group beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa, of the compound of formula (I). and Pseudomonas putida. 12. (canceled) 7. The recombinant organism of claim 3 which is derived 13. (canceled) from an organism which naturally does not produce acetone