WO 2014/066892 Al 1 May 2014 (01.05.2014) W P O P C T
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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/066892 Al 1 May 2014 (01.05.2014) W P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 1/21 (2006.01) C12P 5/02 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C12N 15/52 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (21) International Application Number: KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, PCT/US20 13/067079 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (22) International Filing Date: OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 28 October 2013 (28.10.201 3) SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (25) Filing Language: English ZW. (26) Publication Language: English 4 Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 61/719,198 26 October 2012 (26. 10.2012) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (72) Inventors; and TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (71) Applicants : COELHO, Pedro, S. [BR/US]; 801 South EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Grand Avenue, Suite 1609, Los Angeles, California 90017 MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, (US). FARROW, Mary, F. [US/US]; 4 11 S. Madison TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Ave., Apt 2 1 1, Pasadena, California 9 1101 (US). SMITH, KM, ML, MR, NE, SN, TD, TG). Matthew, A. [GB/US]; 4 11 S. Madison Ave., Apt 2 11, Published: Pasadena, California 9 1 101 (US). — with international search report (Art. 21(3)) (74) Agents: VEITENHEIMER, Erich, E. et al; Cooley LLP, 1299 Pennsylvania Ave, Suite 700, Washington, District of — before the expiration of the time limit for amending the Columbia 20004 (US). claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: DE NOVO METABOLIC PATHWAYS FOR ISOPRENE BIOSYNTHESIS 7 I I 0 0 © (57) Abstract: The invention provides non-naturally occurring metabolic pathways for anaerobic fermentation of isoprene from © glucose at 0.324 g g-1 theoretical yield. The invention additionally provides methods of cloning microorganisms with such pathways to produce isoprene and derivatives thereof. De novo metabolic pathways for isoprene biosynthesis PRIORITY This Application claims priority to U.S. Provisional Application No. 61/719,198, filed October 26, 2012, which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION The present invention relates generally to biosynthetic processes, and more specifically to the design of non-naturally occurring pathways for producing isoprene and the creation of organisms having such biosynthetic capability. Isoprene is naturally produced by bacteria, animals, humans, and plants; these organisms collectively release an estimated 600 million tons of isoprene into the atmosphere each year. Isoprene (2-methyl- 1,3-butadiene) is an important commodity chemical used in a wide range of industrial products, such as synthetic rubber for tires and coatings, adhesives, and specialty elastomers. Isoprene is also a versatile building block for the production of hydrocarbon fuels, including diesel, gasoline, and aviation fuels. Approximately 1 million tons of isoprene are made from petrochemical feedstocks every year. 1 Increasing global demand for isoprene and environmental concerns about greenhouse gas emissions have spurred interest in the development of a fermentative route for producing this chemical from renewable sugars. Isoprene consumers are also interested in isoprene bioproduction as a strategy to mitigate their vulnerability to uncertain supply and volatile prices. Isoprene synthases catalyze the elimination of pyrophosphate from dimethylallyl pyrophosphate (DMAPP) to yield isoprene. Despite isoprene 's pervasiveness in the environment, efforts to identify prokaryotic isoprene synthases have met with limited success.2 Thus far, only plant derived isoprene synthases have been well characterized. Sequence data for these enzymes exist for two plant families: kudzu (the Asian vine, Pueraria montana) and poplar (Populus). The catalytic efficiencies of the characterized isoprene synthases are sub-optimal for industrial applications (KM 1-10 mM and kcat 1 s ). ' Accordingly, protein engineering for superior kinetic parameters has been pursued by industrial groups.3 4 As a proof of concept, recombinant production of isoprene in microbial hosts has been achieved by expressing plant isoprene synthases in E. coli, S. cerevisiae, and photosynthetic bacteria. 5 There are two evolutionarily distinct pathways to biosynthesize the isoprenyl precursors isopentenyl pyrophosphate (IPP) and DMAPP (Fig 1). Archaea and non-plant eukaryotes use the mevalonate (MVA)-dependent pathway exclusively to convert the ubiquitous intermediate acetyl-coenzyme A (A-CoA) to IPP. Most prokaryotes use the l-deoxy-D-xylulose-5- phosphate/2-C-methyl-D-erythritol-4-phosphate (DXP/MEP) pathway to produce IPP and DMAPP from pyruvate and glyceraldehyde-3-phosphate derived from glycolysis. Plants use both the MVA and the DXP/MEP pathways for achieving terpenoid biosynthesis. As shown in Figure 2, the MVA and DXP/MEP pathways have different yields and co-factor requirements. (See Figure 3 for glucose to isoprene calculations.) The MVA pathway offers a lower theoretical yield of IPP from glucose due to its co-factor imbalance. However, the MVA pathway has proven amenable to genetic and metabolic engineering that has resulted in higher isoprenoid titers in both bacteria and yeast. Although the DXP/MEP pathway provides a higher theoretical yield of isoprene from glucose (Fig 2), it is linked to essential cell functions and contains iron-sulfur cluster enzymes that are poorly characterized on a biochemical level.6 The choice of which pathway to engineer for maximizing flux towards the universal precursors IPP and DMAPP is also dependent on the regulatory elements present in the desired fermentation host. It is important to note that accumulation of prenyl pyrophosphates is toxic to the host,7 such that metabolic engineering experiments have to include a terpene synthase that will produce the volatile hydrocarbon product. Genencor-DuPont and The Goodyear Tire & Rubber Co. are developing a high efficiency fermentative route for polymer grade isoprene. 1 Their metabolic engineering strategy imported the heterologous MVA pathway into E. coli to increase the flux towards IPP and DMAPP as well as plant-derived isoprene synthases. The mevalonate pathway was chosen in preference to the DXP/MEP pathway because it is better characterized and has been exploited industrially for isoprenoid production in yeast and bacteria. 7 8 The MVA pathway was cloned into E. coli as two synthetic operons - a top pathway converting A-CoA to MVA, and a bottom pathway producing DMAPP from MVA (this strategy is similar to that pursued by Martin et al. for the overproduction of amorphadiene in yeast7). The bottom operon was integrated into the chromosome under the control of a constitutive promoter. The top operon, the isoprene synthase and an additional copy of an archaeal mevalonate kinase were expressed on two different plasmids driven from the inducible Vtrc promoter. The reported parameters for this process consist of an isoprene yield of 0.1 1 g g , volumetric productivity of 2.0 g L h and a titer of 60 g L- . The volatile nature of isoprene (b.p. 34 °C) allows gas phase recovery of the product, which simplifies purification and eliminates potential feedback inhibition by virtue of product accumulation. All of these factors drive equilibrium in favor of isoprene synthesis, and enable in situ product removal. SUMMARY OF INVENTION Calculations of the maximal theoretical yield for fermentative isoprene production from glucose (Fig 3) reveal that both naturally occurring pathways offer sub-optimal yields [0.324 g g (maximum), 0.298 g g (DXP/MEP), 0.252 g g (MVA)]. Furthermore, as shown in Figure 2, both MVA and DXP/MEP pathways operate optimally under aerobic conditions, thus increasing process costs associated with expensive aeration of large fermentors. Because substrate cost is a significant fraction of the total cost of the desired fermentation product, pathways with superior yields have a better chance of reaching commercialization. Moreover, an anaerobic pathway for isoprene, whilst unprecedented, would enable lower fermentation costs compared to the MVA and DXP/MEP pathways that have thus far been pursued in industry. Since an anaerobic pathway for isoprene at 0.324 g g yield has not been reported in nature, we sought to formulate such a pathway based on existing reaction classes rather than limiting our search to enzymes working on their native substrates. We recognized that the common metabolite 2,3-dihydroxyisovalerate (DHIV) already contains the correct carbon skeleton for isoprene and is only two steps away from pyruvate derived from glycolysis (Fig 4). Herein, we propose 4 major isoprene biosynthetic pathways (Fig 5-1 1), comprising a series of reduction and dehydration steps to produce isoprene from DHIV. The proposed pathways are evaluated with respect to yield, redox balance, ATP balance, number of steps from glucose and number of unknown enzymes. This evaluation is summarized in Table 1. Table 1. Evaluation of isoprene biosynthetic routes.