US 2016O160223A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0160223 A1 Koepke et al. (43) Pub. Date: Jun. 9, 2016 (54) RECOMBINANT MICROORGANISMS CI2N 9/02 (2006.01) EXHIBITING INCREASED FLUX THROUGH CI2N 9/10 (2006.01) A FERMENTATION PATHWAY CI2P 7/18 (2006.01) CI2N 15/52 (2006.01) (71) Applicant: LanzaTech New Zealand Limited, (52) U.S. Cl. Skokie, IL (US) CPC, C12N 15/74 (2013.01); C12P 7/18 (2013.01); CI2N 15/52 (2013.01); C12N 9/0008 (72) Inventors: Michael Koepke, Skokie, IL (US); (2013.01); CI2N 9/1022 (2013.01): CI2N 9/88 Alexander Paul Mueller, Skokie, IL (2013.01); C12Y 102/07001 (2013.01); C12Y (US); Loan Phuong Tran, Skokie, IL 202/01006 (2013.01); C12Y 401/01005 (US) (2013.01) (21) Appl. No.: 14/961,146 (57) ABSTRACT (22) Filed: Dec. 7, 2015 The invention provides a recombinant, carboxydotrophic Related U.S. Application Data Clostridium bacterium that expresses one or more of pyru (60) Provisional application No. 62/168.969, filed on Jun. vate:ferredoxin oxidoreductase (EC 1.2.7.1), acetolactate 1, 2015, provisional application No. 62/089,053, filed synthase (EC 2.2.1.6). and acetolactate decarboxylase (EC On Dec. 8, 2014. 4.1.1.5). The invention further provides a method of produc s ing a fermentation product by fermenting the recombinant Publication Classification bacterium in the presence of a gaseous Substrate comprising CO to produce one or more of ethanol, butanol, isopropanol, (51) Int. Cl. isobutanol, higher alcohols, butanediol. 2,3-butanediol. Suc CI2N 15/74 (2006.01) cinate, isoprenoids, fatty acids, biopolymers, and mixtures CI2N 9/88 (2006.01) thereof. Patent Application Publication Jun. 9, 2016 Sheet 1 of 8 US 2016/O160223 A1 CO CO getsycrogenase 3. irgic CO+h CO Fornate:ydrogeniyase Forstate cehydrogesse 2.33;g 3i Jing Fa NADPH: Fornate Forty-33systhetase W eth eny -HF stylene-F dehydrogstase Fof sy-EF cyclohydrase Methylene-THF vethseise-recitcase CO, Fierytransferase N- CQ dehydrogerase 2.3 Ring Fi Methyl-CoFeS-P CO C3 dehydrogenssei Acetyl-CoA sys3sase Acetyl-CoA CoA alaisaldhyde keycrogeiase Phospickaisacetase N. 3.sg .33 ifing with AE:a:0.38 ris with NAP Acetate kinase y Atetaidehyde Acetate 2.35 is iccio dehydrogeitase N E.2 frig with ADH and, 15 fing with NAEF 3.8 irrig Fo: Acehydetersecaxsicsitoirectase Ethanoi F.G. Patent Application Publication Jun. 9, 2016 Sheet 2 of 8 US 2016/O160223 A1 CO cy defydrogenase N- 2. ifrig Fg: CO+hi, CO Format:ydrogeniyass Fortedly dragstase 3.4 ifing it ifrig Filikia;5-i} Forate Forriyi-syntheasts At hettyiene-f dehydrogenasef Wethe nyi HF Forty-EF cyclohydrolase 3.33 sing NAD? iViethylene-fiF h&atterests excease CO, yethyltransferase N- 2.7CO dehydrogenase3:ng Ed: Methyi-CoFeS-P CO (3 dehydrogenase Acetyl-CoA synthase Acetyl-CoA Pyruvate:Ferredoxis 8xidoreductase 3.3 frig Fa Pyruvate Acetoacatate systisse Acetoiacatate decarboxylase Acetoin 8tase:33:Secciary acco dehydrogerase i.2) fing 3.8 irrig with 8A38 anxi (, 3 ifrig with NA388} 2,3-Butanediol FG, 2. Patent Application Publication Jun. 9, 2016 Sheet 3 of 8 US 2016/O160223 A1 pyruvate ferredoxin oxidoreductase PFOR Acetyl-CoA ~- Pyruvate acetolactate synthase acetolactate synthase catabolic aiss anabolic iv6N, ifyi- 2-Oxo-3-hydroxyisovalerate C-Acetolactate -61w dihydroxy-acid reductoisonease dehydratase acetoactate iw decarboxylase also 2-Oxoisovalerate ACetoin anino- vmultiple transferase 2,3-butanedio primary-secondary dehydrogenase alcohol dehydrogenase \ 23.Bch CaAD Waine Leucine 2,3-Butanediot F.G. 3 Patent Application Publication Jun. 9, 2016 Sheet 4 of 8 US 2016/O160223 A1 acz alpha pMTL83159 4630 bp COE repH e F.G. 4 Patent Application Publication Jun. 9, 2016 Sheet 5 of 8 US 2016/O160223 A1 . :38.33333cs Siege: 83to: Patent Application Publication Jun. 9, 2016 Sheet 6 of 8 US 2016/O160223 A1 firsts::sesssss car: tp/jos) tionaripog ch s 3. & na x:s: 3 w s: & s s : s s s s .. s s: s: f8 autop:3e3e3-y sets: We 8ff:08:03:3g. Coio) C fixossessia ciga ipiliouiaomingold zh, s: x r: & S. 3. S. & & : i 3 s ; : ax g 3 : s y t; : a * *s x-x & a: as s : s 8c. 3. 3. s SS3. S f8::top;32:33, avies: 3.ar affitis: its posifs ki Patent Application Publication Jun. 9, 2016 Sheet 7 of 8 US 2016/O160223 A1 ipffautus gnanpagat 38 s w8 s w s' 8 t * 8 & w ... a s 3 5 : 3 & fifetistia:33poist of - C es f838038888c:g':3e3:38'ogs e ge & : x: s: : ;: 3 : : M. & cy R N & x s N Patent Application Publication Jun. 9, 2016 Sheet 8 of 8 US 2016/O160223 A1 1.4 r. 3 1.2 - Aa 2.5 1 r; A. s A is ? - O.8 s % 6 1.5 0.4 A 0.2 - O.5 O 2 A. 6 8 Days FG, 8 US 2016/0160223 A1 Jun. 9, 2016 RECOMBINANT MICROORGANISMS ism for 2,3-butanediol formation via pyruvate, which allows EXHIBITING INCREASED FLUX THROUGH for the identification of rate-limiting pathway reactions. A FERMENTATION PATHWAY 0008 FIG. 3 is a diagram showing the 2,3-butanediol pathway and the associated branched-chain amino acid bio CROSS REFERENCE TO RELATED synthesis pathway. Pyruvate is converted to O-acetolactate, APPLICATIONS the intermediate for both the 2,3-butanediol and the branched-chain amino acid biosynthesis pathways. Experi 0001. This application claims the benefit of U.S. Patent ments were performed to overexpress PFOR, alsS and alsD. Application 62/089,053 filed Dec. 8, 2014 and U.S. Patent 0009 FIG. 4 is a schematic representation of the Application 62/168,969 filed Jun. 1, 2015, the entirety of pMTL83159 plasmid. The plasmid contains a Gram negative which are incorporated herein by reference. origin of replication (ColE1) gene, a Gram positive origin of replication (repH) gene, the transfer gene tra.J., the catP gene BACKGROUND encoding for the chloramphenicol/thiamphenicol resistance, 0002 Carboxydotrophic microorganisms may be engi a multiple cloning site locating within lacZ alpha coding neered to produce products, such as fuels and chemicals, sequence and, a ferredoxin gene promoter (Pa.). through fermentation of a gaseous Substrate. Efforts to 0010 FIG. 5 is a set of graphs showing of the growth and improve product concentration and Substrate utilization have metabolite profiles (biomass, 2,3-butanediol (BDO), acetic historically focused on Strain selection and optimization of acid, and ethanol) versus time of five strains grown in Schott fermentation conditions (Abubackar, Bioresour Technol, bottles. 114: 518-522, 2012). The metabolism of natural microorgan 0011 FIG. 6 is a set of graphs showing the metabolite isms, however, did not evolve to achieve commercial objec profile (top graphs) and gas profile (bottom graphs) of the tives of high yields, rates, and titers, such that certain com combined PFOR, alsS and alsDoverexpression strain and the mercial objectives cannot be achieved through mere strain plasmid control strain at a 4 mol/L/d CO uptake over the selection and optimization of fermentation conditions. course of 20 days. Accordingly, there remains a need for improved microorgan 0012 FIG. 7 is a set of graphs showing the metabolite and isms and methods for the production of useful products. Such the gas profiles of the overexpression culture at an 8 mol/L/d as fuels and chemicals. CO uptake over the course of 11 days. 0013 FIG. 8 is a set of graphs showing biomass and SUMMARY OF THE INVENTION butanediol production of cultures expressing A. hydrophila alsD (open squares), L. lactis alsD (closed squares), and 0003. The invention provides a recombinant, carboxy empty plasmid control (triangles). Values are the average of dotrophic Clostridium bacterium comprising one or more three replicates and error bars represent one standard devia enzymes selected from the group consisting of pyruvate: tion. ferredoxin oxidoreductase (EC 1.2.7.1), acetolactate syn thase (EC 2.2.1.6), and acetolactate decarboxylase (EC 4.1. DETAILED DESCRIPTION OF THE INVENTION 1.5), wherein each enzyme is an overexpressed endogenous enzyme, a mutated endogenous enzyme, or an exogenous 0014. A fermentation pathway is a cascade of biochemical enzyme. The recombinant bacterium may express one, two, reactions (pathway reactions) by which a substrate, prefer or all three of these enzymes. ably a gaseous Substrate, is converted to a fermentation prod 0004. The recombinant bacterium may be derived from uct. Pathway reactions typically involve enzymes that catal any Clostridium microorganism. In one embodiment, the yse or increase the rate of the pathway reaction. recombinant bacterium is derived from C. autoethanogenium, (0015 “Flux” refers to the flow of metabolites through one C. liungdahli, or C. ragsdalei. In a preferred embodiment, or more reactions in a fermentation pathway. The flux through the recombinant bacterium is derived from C. autoethanoge individual pathway reactions has an upper and lower limit. num deposited under DSMZ Accession No. DSM23693 (C. Therefore, the flux may be changed by adjusting conditions or autoethanogenium LZ1561). factors that affect enzymatic activity. Adjustment of the flux 0005. The invention further provides a method of produc through one pathway reaction may alter the overall flux of the ing a fermentation product, comprising fermenting the fermentation pathway. Flux may be measured according to recombinant bacterium in the presence of a gaseous Substrate any method known in the art. By way of example, flux may be comprising CO to produce one or more of ethanol, butanol, measured using flux-balance analysis (FBA) (Gianchandani, isopropanol, isobutanol, higher alcohols, butanediol. 2,3-bu Systems Biol Medicine, 2:372-382, 2010). Flux through the tanediol. Succinate, isoprenoids, fatty acids, biopolymers, pathway may also be measured by the level of metabolites and and mixtures thereof.