US 2017.0137846A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0137846A1 ATSUMI et al. (43) Pub. Date: May 18, 2017 (54) BACTERIA ENGINEERED FOR Related U.S. Application Data CONVERSION OF ETHYLENE TO (60) Provisional application No. 62/009,857, filed on Jun. N-BUTANOL 9, 2014. (71) Applicant: The Regents of the University of California, Oakland, CA (US) Publication Classification (51) Int. Cl. (72) Inventors: Shota ATSUMI, Davis, CA (US); CI2P 7/06 (2006.01) Michael D. TONEY, Davis, CA (US); CI2P 7/16 (2006.01) Gabriel M. RODRIGUEZ, Davis, CA CI2N 15/52 (2006.01) (US); Yohei TASHIRO, Davis, CA (52) U.S. Cl. (US); Justin B. SIEGEL, Davis, CA CPC ................ CI2P 7/06 (2013.01): CI2N 15/52 (US); D. Alexander CARLIN, Davis, (2013.01); C12Y402/01053 (2013.01); C12Y CA (US); Irina KORYAKINA, Davis, 402/01095 (2013.01); C12Y 114/13 (2013.01); CA (US); Shuchi H. DESAI, Davis, CI2Y 102/01002 (2013.01); C12Y 114/13069 CA (US) (2013.01); C12Y 203/01009 (2013.01); C12Y 101/01157 (2013.01); C12Y402/01 (2013.01); (73) Assignee: The Regents of the University of CI2Y 103/01038 (2013.01); C12Y 102/01003 California, Oakland, CA (US) (2013.01); C12P 7/16 (2013.01) (21) Appl. No.: 15/317,656 (57) ABSTRACT (22) PCT Fed: Jun. 9, 2015 The present disclosure provides recombinant bacteria with elevated production of ethanol and/or n-butanol from eth (86) PCT No.: PCT/US 15/34942 ylene. Methods for the production of the recombinant bac S 371 (c)(1), teria, as well as for use thereof for production of ethanol (2) Date: Dec. 9, 2016 and/or n-butanol are also provided. Patent Application Publication May 18, 2017 Sheet 1 of 8 US 2017/O137846 A1 T"?IH Patent Application Publication May 18, 2017 Sheet 2 of 8 US 2017/O137846 A1 O#40 HOWNXZ c}{{{5%; Patent Application Publication May 18, 2017. Sheet 3 of 8 US 2017/O137846 A1 Product Substrate -- 1500 3.0 As 2.5 mm. As g g s El 1 OOO s 2.0 a d s O 3. old 1.5 D s . 2 500 is 1.0 O.5 ESCherichia COlli - -- -- -- -- - - Toluene monooxygenase Ethene added Oxirane added - -- - FG, 3A FIG. 3B Patent Application Publication May 18, 2017. Sheet 4 of 8 US 2017/O137846 A1 FIG. 4A FIG. 4B No C-Source % Glucose 88 sthanol 5O A.O 3 O 2O ~w 2%a 2%22% 10 O 1 2 3 No C-Source Glucose Ethanol Time (day) Patent Application Publication May 18, 2017. Sheet 5 of 8 US 2017/O137846 A1 FIG. 5A No FIG. 5B % NC r 8 in adh BEC as : sadh EEC w8 mhpF+adhP w8 mhpF+adhP O OO1 O 2 4. 6 8 1D Time (day) Time (day) Patent Application Publication May 18, 2017. Sheet 6 of 8 US 2017/O137846 A1 700 - 6 O O 5O O 4. O O 3 O O O-EdgE/AdhP +EdgE/AdhP 2O O O O O Glucose Ethanol Carbon Source F.G. 6 Patent Application Publication US 2017/O137846 A1 Patent Application Publication May 18, 2017 Sheet 8 of 8 US 2017/O137846 A1 -asemepauºnei?useoonp-z-)- ww.wawww. Š. %• asemeupauenealo-{ US 2017/O 137846 A1 May 18, 2017 BACTERIA ENGINEERED FOR Petersen et al., supra, 2008). The use of high temperatures CONVERSION OF ETHYLENE TO and pressures are also needed. These processes are N-BUTANOL extremely energy intense and also lack product specificity, requiring additional purification steps to separate the various CROSS-REFERENCE TO RELATED products. APPLICATIONS 0007 To circumvent these pitfalls, described herein are 0001. This application claims the priority benefit of U.S. biosynthetic pathways for the conversion of ethylene, which may be converted from methane with established methods, Provisional Application No. 62/009,857, filed Jun. 9, 2014, to acetyl-CoA. The biological assimilation of ethylene has which is hereby incorporated by reference in its entirety. only been reported in methanotrophs (Bull et al., Nature STATEMENT OF GOVERNMENT SUPPORT 405, 175-178, 2000; and Treude et al., Appl Environ Micro biol 73,2271-2283, 2007). Due to the difficulties in cultur 0002 This invention was made with Government support ing methanotrophs and very few genetic modification tools, under Grant No. DE-AR0000429 awarded by the Depart no large-scale applications of these organisms has been ment of Energy. The Government has certain rights in this demonstrated. Therefore, the present disclosure describes invention. the construction of ethylene assimilation pathway in recom binant bacteria, which already have a plethora of available FIELD genetic tools and has well-established large-scale applica 0003. The present disclosure provides recombinant bac tions. Furthermore, several examples of pathways for con teria with elevated production of ethanol and/or n-butanol verting basic metabolites to fuels and chemicals (e.g., acetyl from ethylene. Methods for the production of the recombi CoA to n-butanol) in E. coli have been extensively reported nant bacteria, as well as for use thereof for production of (Rabinovitch-Deere et al., Chemical reviews 113, 4611-32, ethanol and/or n-butanol are also provided. 2013). Thus, by engineering a high flux ethylene assimila tion pathway in recombinant bacteria, better performance BACKGROUND may be achieved than what has been demonstrated in methanotrophs. 0004. In the midst of declining fossil fuels reserves and a great expansion of natural gas production, increasing effort 0008 Lastly, since ethylene is already a high volume is seeking to commercialize the conversion of methane into chemical feedstock used in the chemical industry, a high chemical feedstocks and fuels as an alternative to petroleum. performance ethylene assimilation pathway in recombinant Large natural gas reservoirs exist throughout the world and, bacteria could enable immediate industrial applications. Due thus, have an enormous potential as a clean fuel and chemi to the broad uses of ethylene as a chemical feedstock, cal feedstock. Currently, the vast majority of natural gas is technological innovations for the conversion of methane to used for heating purposes. This is due largely to the prop ethylene will be developed. Therefore, a well-engineered erties of methane as a heating fuel as well as the difficulty ethylene assimilation pathway in a user-friendly host Such as in economically converting methane into larger, higher recombinant bacteria will enable the biological conversion value chemicals and liquid fuels. of ethylene into liquid fuels and other high value chemicals. 0005. Several methods to convert methane indirectly and directly into higher olefins have been report in literature. SUMMARY However, the two large-scale methods being used commer 0009. The present disclosure provides recombinant bac cially are methanol-to-olefins (MTO) and the Fischer-Trop teria with elevated production of ethanol and/or n-butanol sch synthesis (FT) (Alvarez-Galvan et al., Catalysis Today from ethylene. Methods for the production of the recombi 171, 15-23, 2011). Both these processes first involve the nant bacteria, as well as for use thereof for production of generation of synthesis gas (Syngas), a mixture of H and ethanol and/or n-butanol are also provided. CO (Alvarez-Galvan, supra, 2011). Syngas production is 0010. In particular, the present disclosure provides bac energy intense, requiring high temperatures (>600° C.), teria comprising a recombinant polynucleotide encoding an making it a costly process that typically represents 60% of ethylene hydratase (EH), wherein expression of the EH total capital costs (Aasberg-Petersen et al., available at: results in an increase in production of ethanol as compared www.topsoe.com/business areas/methanol/-/media/PDF to a corresponding bacterium (e.g., same genus and species) %20files/Methanol/Topsoe large scale methanol prod lacking the recombinant polynucleotide. In some embodi paper.ashx, 2008). Thus, direct routes for conversion of ments, the EH is an oleate hydratase. In certain embodi methane to olefins and other chemicals are conceptually ments, the oleate hydratase is a Lysinibacillus fusiformis preferable and many methods have been reported (Alvarez oleate hydratase. In some embodiments, the EH is a 2-ha Galvan et al., Supra, 2011). However, these reported meth loacrylate hydratase. In certain embodiments, the 2-halo ods suffer from low yield and low product specificity ren acrylate hydratase is a Pseudomonas species 2-haloacrylate dering them uneconomical thus far. Therefore, a need exists hydratase. In some embodiments, the EH is a kievitone for a novel, more cost effective route to produce chemical hydratase. In certain embodiments, the kievitone hydratase feedstocks and fuels from methane. is a Fusarium Solani kievitone hydratase. The present dis 0006. The challenges in the overall conversion of meth closure further provides bacteria comprising a recombinant ane to chemicals, either directly or indirectly, largely stem polynucleotide encoding an alkene monooxygenase (AMO) from the activation energy required to convert methane into and an ethylene oxide reductase (EOR), wherein expression large molecules. This is a result of stability and symmetry of of the AMO and the EOR results in an increase in production methane. Current solutions to activate methane involve the of ethanol as compared to a corresponding bacterium (e.g., use of inorganic catalysts such as palladium, which serve to same genus and species) lacking the recombinant polynucle reduce the energy required for methane activation (Aasberg otide. In some embodiments, the AMO is a toluene US 2017/O 137846 A1 May 18, 2017 monooxygenase. In certain embodiments, the toluene Some embodiments, the bacterium further comprises a yet monooxygenase is a Pseudomonas mendocina toluene still further recombinant polynucleotides encoding an monooxygenase. In certain embodiments, the toluene acetoacetyl-CoA thiolase (AT), a 3-hydroxybutyryl-CoA monooxygenase is a Burkholderia cepacia toluene dehydrogenase (HBD), a crotonase (CRT), a trans-enoyl monooxygenase.