US 2015 0037860A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0037860 A1 B0tes et al. (43) Pub. Date: Feb. 5, 2015 (54) METHODS FOR BOSYNTHESIS OF Publication Classification ISOPRENE (51) Int. C. (71) Applicant: INVISTA North America S.ár.l., CI2P 5/00 (2006.01) Wilmington, DE (US) CI2N 15/70 (2006.01) (52) U.S. C. (72) Inventors: Adriana Leonora Botes, Rosedale East CPC ................. CI2P5/007 (2013.01); C12N 15/70 (GB); Alex Van Eck Conradie, (2013.01) Eaglescliffe (GB) USPC ................... 435/167; 435/252.33: 435/252.3: 435/252.32; 435/252.34; 435/252.31; (21) Appl. No.: 14/452,201 435/254.3; 435/254.21: 435/254.23; 435/254.2: 435/254.11: 435/254.22 (22) Filed: Aug. 5, 2014 (57) ABSTRACT This document describes biochemical pathways for produc ing isoprene by forming two vinyl groups in a central precur Related U.S. Application Data sor produced from isobutyryl-CoA, 3-methyl-2-oxopen (60) Provisional application No. 61/862,401, filed on Aug. tanoate, or 4-methyl-2-oxopentanoate as well as recombinant 5, 2013. hosts for producing isoprene. Patent Application Publication Feb. 5, 2015 Sheet 1 of 19 US 2015/0037860 A1 FIGURE 1. O O CoA Acetyl-CoA --sca {YN 1 -sc.AO Acetoacetyl-CoA EC 2.3.1.9 Acetyl-CoA Acetyl-CoA t HO -->Cs.O OH Q CoA 3-Hydroxy 3-methylglutaryl-CoA gy 2.H a. CoA Co a 2NAD(P)* Jyu HO OH (R)-mevalonate t ATP c N N e d ADP O HO o–F–o OH (R)-5-phosphomevalonate t ATP c N s na No ADP JyuO HO O-P-O-P-OH OH OH (R)-5-Diphosphomevalonate t ATP c a g ADP PP CO2 N-1 -- P-O-P-OH HEos3.32 -- . EC 42.327 ) Nr. OH, OH Dimethylallyl diphosphate isoprene Isopentenyl diphosphate Patent Application Publication Feb. 5, 2015 Sheet 8 of 19 US 2015/0037860 A1 FIGURE 8 O O CoA Acetyl-CA ulus YN-1 S-CoA { S-CoA Acetoacetyl-CoA EC 2.3.1.9 acetyl-CoA T Acetyl-CoA S HO go s s - O X-sc.OH O CoA 3-Hydroxy 3-methylglutaryl-CoA TT 2:NAD(P)H QQ 2.H ADP a a + CO s CoA -- ATP + Pi 2NAD(P) HO N-17 Yu N-Y HQ, EC 2.7.1- a- ) HO S H) 21 HO OH EC 4.2.1- EC 4.1.1.33 (R)-mevalonate 3-methyl-pent-4-enoate1N3-hydroxy- isoprene ATP ADP + CO + Pi Patent Appl ication Publica ion Feb. 5, 2015 Sheet 9 of 19 US 2015/0037860 A1 uusque3.JO Patent Application Publication Feb. 5, 2015 Sheet 10 of 19 US 2015/0037860 A1 uusque3.JO Patent Application Publication Feb. 5, 2015 Sheet 11 of 19 US 2015/0037860 A1 30uenb?sVLSVH snopopoldau?S apjuouunaud ppuu ppuu OT II Patent Appl ication Publiicat ion Feb. 5, 2015 Sheet 12 of 19 US 2015/0037860 A1 30uanbasVLSVH T’IÇIZIMEV/ · spuoluOuayb?b?ound |- |- |- Patent Application Pub ication Feb. 5, 2015 Sheet 13 Of 19 US 2015/0037860 A1 apuanbasVLSVH Patent Application Publication Feb. 5, 2015 Sheet 14 of 19 US 2015/0037860 A1 apuanbasvLSV) uusque3.JO Patent Application Publication Feb. 5, 2015 Sheet 15 of 19 US 2015/0037860 A1 FIGURE 10 0.6 outsourous O 50 1OO 150 2OO 250 300 350 Time (Seconds) -0-Substrate only control -H0.4 (uM) PhaJac Patent Applicat O Pub ication Feb. 5, 2015 Sheet 16 of 19 US 2015/0037860 A1 squauuuuoo (z/uu)ssewpanuesqo|uuu09Z@ eeuwMead (nyuu) CII?Iduues Patent Application Publication Feb. 5, 2015 Sheet 17 Of 19 US 2015/0037860 A1 VOO Patent Application Publication Feb. 5, 2015 Sheet 18 of 19 US 2015/0037860 A1 S. S. S. S. S. S. S. S. S. S. S. S S. S S. S S. S S. S S. S. S. S. S. S. S. S. S. S. S. S S S S S S S S S S 8S S S S S 99.99WWO S S S S S S S S S S S S S S S HS 60099WO S S S S S S S S 788 OHW S S S S S S S S S S S S S S S S S S 161cc)w S S S S S LO OO Lo N. Lo O Lo Co N. O epuepunce SN-5) JO Olfo Patent Application Publication Feb. 5, 2015 Sheet 19 of 19 US 2015/0037860 A1 |00081 0009L |000vi 000ZT 0000T 0008 0009 |000v Wd eeue seed eueudos US 2015/0037860 A1 Feb. 5, 2015 METHODS FOR BOSYNTHESIS OF a theoretical maximum yield of 25.2% (w/w) for the meva ISOPRENE lonate pathway, isoprene has been produced biocatalytically at a volumetric productivity of 2 g/(Lh) with a yield of 11% CROSS-REFERENCE TO RELATED (w/w) from glucose (Whited et al., 2010, supra). Particularly, APPLICATIONS the phosphate activation of mevalonate to 5-diphosphomeva lonate is energy intensive metabolically, requiring two moles 0001. This application claims priority to U.S. Application of ATP per mole of isoprene synthesis (FIG. 1). Accordingly, Ser. No. 61/862,401, filed Aug. 5, 2013, the disclosure of reducing the ATP consumption can improve the efficiency of which is incorporated by reference in its entirety. the pathway. TECHNICAL FIELD SUMMARY 0002 This invention relates to methods for biosynthesiz ing isoprene using one or more isolated enzymes Such as one 0012. The inventors have determined that it is possible to or more of a dehydratase, a monooxygenase, a cytochrome construct a biochemical pathway to synthesize isoprene from P450, an acyl-acp dehydrogenase, a mevalonate diphos (R)-mevalonate, 3-methyl-2-oxopentanoate, 4-methyl-2- phate decarboxylase, an acyl-acp decarboxylating oXopentanoate or isobutyryl-CoA, by introducing two vinyl groups without the need for terminal alcohol phosphoryla thioesterase, and a mevalonate-3-kinase; or using recombi tion. Such pathways rely on a dehydratase, monooxygenase, nant host cells expressing one or more Such enzymes. cytochrome P450, or dehydrogenase enzyme to introduce the BACKGROUND first vinyl group; and a MDD, mevalonate-3-kinase, acyl acp decarboxylating thioesterase (e.g., CurMTE) or a lina 0003) Isoprene is an important monomer for the produc lool dehydratase to introduce the second vinyl group into the tion of specialty elastomers including motor mounts/fittings, precursors leading to isoprene synthesis. The methods Surgical gloves, rubber bands, golfballs and shoes. Styrene described herein can include introducing the first vinyl group, isoprene-styrene block copolymers form a key component of introducing the second vinyl group, or introducing both the hot-melt pressure-sensitive adhesive formulations and cis first and second vinyl groups. poly-isoprene is utilised in the manufacture oftires (Whitedet 0013 Prior to the present invention, it was not known that al., Industrial Biotechnology, 2010, 6(3), 152-163). enzymes capable of introducing two vinyl groups, without the 0004. Manufacturers of rubber goods depend on either need for terminal alcohol phosphorylation, could be used to imported natural rubber from the Brazilian rubber tree or generate non-phosphorylated intermediates for the synthesis petroleum-based synthetic rubber polymers (Whited et al., of isoprene. Thus the invention provides enzymes that can 2010, supra). convert the central precursors mevalonate, 3-methyl-2-oxo 0005 Given a reliance on petrochemical feedstocks and pentanoate, 4-methyl-2-oxopentanoate or isobutyryl-CoA the harvesting of trees, biotechnology offers an alternative into isoprene. approach viabiocatalysis. Biocatalysis is the use of biological 0014. In some embodiments, 3-methyl-pent-2-enoyl-CoA catalysts, such as enzymes, to perform biochemical transfor or 4-methyl-pent-2-enoyl-CoA is formed by a 2-hydroxya mations of organic compounds. cyl-CoA dehydratase classified, for example, under EC 4.2. 0006. Accordingly, against this background, it is clear that 1.-, such as the gene products of HadBC (SEQID NOs: 3 and there is a need for Sustainable methods for producing inter 4) and its initiator Had I (SEQID NO: 2), or the gene products mediates, in particular isoprene, wherein the methods are of HgdAB (SEQ ID NOs: 6 and 7) and its initiator HagC biocatalysis based. (SEQID NO. 5). In some embodiments, the 2-hydroxyacyl 0007 Both bioderived feedstocks and petrochemical feed CoA dehydratase is the result of enzyme engineering. The stocks are viable starting materials for the biocatalysis pro 2-hydroxyacyl-CoA dehydratase enzymes isolated from CCSSCS. anaerobic bacteria possess a common catalytic mechanism 0008. The introduction of vinyl groups into medium car employed in amino acid degradation pathways. For example, bon chain length enzyme Substrates is a key consideration in the gene products of HadBC/HadI from Clostridium difficile synthesising isoprene via biocatalysis processes. catalyse the conversion of (R)-2-hydroxyisocaproyl-CoA to 0009. There are known metabolic pathways leading to the isocaprenoyl-CoA. Similarly, the gene products of HgdAB/ synthesis ofisoprene in prokaryotes such as Bacillis Subtillis HdgC catalyse the conversion of 2-hydroxyglutaryl-CoA to and eukaryotes such as Populus alba (Whited et al., 2010, glutaconyl-CoA (Kim et al., FEMS Microbiol. Reviews, Supra). 2004, 28,455-468). See FIGS. 2-5. 0010) Isoprene may be synthesized via two routes leading 0015. In some embodiments, the first vinyl group is intro to the precursor dimethylvinyl-PP, such as the mevalonate duced into 3-methyl-pent-2-enoyl-ACP, derived from the and the non-mevalonate pathway (Kuzuyama, Biosci. Bio central metabolite 3-methyl-2-oxopentanoate, which may be technol. Biochem., 2002, 66(8), 1619-1627). The mevalonate enzymatically converted in one or more steps to 3-methyl-3- pathway incorporates a decarboxylase enzyme, mevalonate hydroxypent-4-enoate or 3-methyl-3-sulphoryl-pent-4- diphosphate decarboxylase (hereafter MDD), that introduces enoyl-ACP (as shown, for example, in FIG.