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(12) Patent Application Publication (10) Pub. No.: US 2017/0159083 A1 Valgepea Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2017/0159083 A1 Valgepea Et Al US 201701.59083A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0159083 A1 Valgepea et al. (43) Pub. Date: Jun. 8, 2017 (54) ARGININE SUPPLEMENTATION TO CI2N L/20 (2006.01) IMPROVE EFFICIENCY IN GAS CI2N 9/12 (2006.01) FERMENTING ACETOGENS CI2N 9/90 (2006.01) CI2N 9/06 (2006.01) (71) Applicant: LanzaTech New Zealand Limited, CI2N I/38 (2006.01) Skokie, IL (US) CI2N 9/78 (2006.01) CI2N 9/10 (2006.01) (72) Inventors: Kaspar Valgepea, Brisbane (AU): (52) U.S. Cl. Michael Koepke, Skokie, IL (US); CPC .................. CI2P 7/56 (2013.01); CI2N 9/78 James Bruce Yarnton Haycock (2013.01); C12Y305/03006 (2013.01); C12P Behrendorf, Skokie, IL (US); Esteban 7/54 (2013.01); C12P 7/065 (2013.01): CI2P Marcellin, Brisbane (AU), Lars K. 7/18 (2013.01); C12N 9/1018 (2013.01); C12Y Nielsen, Brisbane (AU); Renato de S.P. 201/03003 (2013.01); C12N 9/1217 (2013.01); Lemgruber, Brisbane (AU) CI2Y 207/02002 (2013.01); C12N 9/90 (2013.01); C12Y501/01012 (2013.01); C12Y (21) Appl. No.: 15/368,521 504/03005 (2013.01); C12N 9/0016 (2013.01); 1-1. CI2Y 104/01012 (2013.01); C12N 9/1029 (22) Filed: Dec. 2, 2016 (2013.01); C12N 1/38 (2013.01); C12N I/20 Related U.S. Application Data (2013.01) (60) Provisional application No. 62/262,886, filed on Dec. 57 ABSTRACT 3, 2015, provisional application No. 62/262,888, filed (57) on Dec. 3, 2015. The invention provides methods for improving efficiency of O O fermentation by arginine Supplementation, and genetically Publication Classification modified bacterium for use therefor. More particularly the (51) Int. Cl. invention provides methods for (i) increasing the production CI2P 7/56 (2006.01) ATP intensive products with arginine Supplementation, (ii) CI2P 7/54 (2006.01) increasing utilization of arginine by a C1-fixing bacterium: CI2P 7/06 (2006.01) and (iii) providing C1-fixing bacterium with optimized CI2P 7/18 (2006.01) arginine de-aminase pathways. Patent Application Publication Jun. 8, 2017. Sheet 1 of 14 US 2017/O159083 A1 20AA - OD & t) 2.5 . .progrg 80 i 60 i 50 i 30 20 10 Patent Application Publication Jun. 8, 2017. Sheet 2 of 14 US 2017/O159083 A1 Patent Application Publication Jun. 8, 2017. Sheet 3 of 14 US 2017/O159083 A1 3.O. : : - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -14AA & 8AAOD 1. 5 1.0 O.5 SS : s 0.0 & 16 O O 2O 3O 40 50 60 Time (h) Fig. 4 Patent Application Publication US 2017/O159083 A1 ar. Fig 6 Patent Application Publication Jun. 8, 2017. Sheet 5 of 14 US 2017/O159083 A1 12AA & 4AA - OED & t) 2.5 - AO : 3.2AA OD 2. ... 8 8 2AA to 3: *::: o 30 3.5 s eas S3 s S. 20 3.0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -a - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - O Fig. 7 78OO - 6000 a & O.O2 5000 B. :: O.O 4800 3000 : 2000 - 100C ASP G SER HS HR ARG AA WA ME E Fig. 8 Patent Application Publication Jun. 8, 2017. Sheet 6 of 14 US 2017/O159083 A1 88: 38 . E . S 5 3. 30 S f C 0.0 ---------------------------------------------------------------- icoor 0 Patent Application Publication Jun. 8, 2017. Sheet 7 of 14 US 2017/O159083 A1 Agiite - AA cessition a protiation & .23 Sis Arginine - Bugliab data --~~~~ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN Patent Application Publication Jun. 8, 2017. Sheet 8 of 14 US 2017/O159083 A1 20 E 15 ex o o was Vs g (s) 10. O 5 O With (A) and without (O) arginine supplementation (n = 3, meant SD) Fig. 16 Patent Application Publication Jun. 8, 2017. Sheet 9 of 14 US 2017/O159083 A1 : 3: 3. : s: With (A) arginine; without (O) arginine; arginine only () (n = 3, meant SD) Fig. 17 : irre : C. autoethanogenium DSM 23693 with (A) and without (O) arginine supplementation Fig. 18 Patent Application Publication Jun. 8, 2017. Sheet 10 of 14 US 2017/0159083 A1 83.8 - A 8. 6 8.8 is - 6 8:a: xx 3.2 - d 3. A 8. g A : : : : 3. : -agirire Citrue Carbamoyl 3. YN YY ghosphate HC annoniurn Phosphate -orfittire AD Atroit * AP Fig. 20 Patent Application Publication Jun. 8, 2017. Sheet 11 of 14 US 2017/0159083 A1 Arginie & interia: Exteria: hio 8x : x & a -s. --- - - - - - - - - - Arginite CAS33-33823. * deitase * : 83.38 ** * Argirire -Citrilise {{POs rise -3rite --- cart.8:8-yearsferase K. 28.3.3 CAET48.3823 : {A: 83.3883 :- : A$3338.332: : Carbaroyl-phosphate Offiti site Caractate kisse ... Fig. 21 Patent Application Publication Jun. 8, 2017. Sheet 12 of 14 US 2017/0159083 A1 & fix8:38: 8:::::::8 Ai383: 8: W3:e 8: 888:::::::: 3:33:3: Fig. 22A 2%. 75a 1, 8&88:38: 88:33:3SS :8388 8 A3:38 &etitia x 8.3:3:38 RS Fig. 22B Patent Application Publication Jun. 8, 2017. Sheet 13 of 14 US 2017/0159083 A1 -ornithine (2R,4S)-2,4-diaminopentanoate NAR}+ -2 --- NAD(pH Argir : 2-amino-4-oxopentanoate Coenzyme A Acetyl-coA D-alanine Fig. 23 Patent Application Publication Jun. 8, 2017. Sheet 14 of 14 US 2017/0159083 A1 US 2017/O 159083 A1 Jun. 8, 2017 ARGINNE SUPPLEMENTATION TO CO2 Fixation Pathway of Clostridium autoethanogenium by IMPROVE EFFICIENCY IN GAS Targeted Mutagenesis. mBio, 2016, 7: e()0427-16) since the FERMENTING ACETOGENS production of acetate provides the microorganism with an option to directly generate ATP from substrate level phos CROSS REFERENCE TO RELATED phorylation via Pta (phosphotransacetylase) (EC 2.3.1.8) APPLICATIONS and Ack (acetate kinase) (EC 2.7.2.1). The Pta-Ack system is highly specific to acetyl-CoA conversion to acetate and 0001. This application claims the benefit of U.S. Provi not utilizing other acyl-CoA.S. Although mechanisms such as sional Patent Application Nos. 62/262,886 filed Dec. 3, 2015 membrane gradients and electron bifurcation enzymes and 62/262,888 filed Dec. 3, 2015, the entirety of which is coupled to ion or proton translocating systems, e.g., the Rinf incorporated herein by reference. complex (Schuchmann, Nat Rev Microbiol, 12: 809-821, 2014), conserve ATP in these microorganisms, direct ATP BACKGROUND OF THE INVENTION generation remains critical for their Survival. As a result, 0002 Approximately 10% of the world’s energy demand when introducing heterologous pathways that do not allow and commodity chemicals are currently produced from for ATP generation, acetate is produced as a by-product renewable feedstocks, primarily using farmed Sugars. How (Schiel-Bengelsdorf, FEBS Lett, 586: 2191-2198, 2012). ever there is an increasing focus on the future use of non-food resources to meet climate targets. Gas fermenta SUMMARY OF THE INVENTION tion offers a route to use a wide range of readily available, 0007. The invention provides a method for improving the low cost C1 feedstocks such as industrial waste gases, efficiency of fermentation, the method comprising, flowing syngas or reformed methane into chemicals and fuels. a gaseous C1 containing Substrate to a bioreactor containing Believed to be one of the first biochemical pathways to a culture of one or more C1 fixing microorganisms in a emerge on earth, the Wood Ljungdahl pathway enables liquid nutrient media; and fermenting the culture to produce acetogenic Clostridia to fix these C1 gases into acetyl-CoA. at least one product. Arginine is provided to the culture in Clostridium autoethanogenium, in particular, offers a robust excess of the requirements for synthesis of biomass. Gen and flexible platform for gas fermentation and has been erally, the C1 fixing microorganism comprises an arginine deployed at industrial scale. Fermentation of gas by C. metabolism pathway. autoethanogenium is highly selective, tolerates contami 0008. In a second aspect, the invention provides a method nants, resolves refractiveness of the Fischer-Tropsch pro for increasing the production of at least one ATP intensive cesses and is economically viable even when Supplied with product, the method comprising; flowing a gaseous C1 Small Volume gas streams. containing Substrate to a bioreactor containing a culture of 0003. It is known, that whilst acetogens are capable of one or more C1 fixing microorganisms in a liquid nutrient producing many useful short-chain chemicals, the produc media; and fermenting the culture to produce at least one tion of longer-chain carbon molecules for use in biodiesel or product. In certain embodiments, arginine is provided to the jet fuels is outside the metabolic capacity of acetogenic culture in excess of the requirements for synthesis of bio bacteria on their own, due to ATP limitation. Fast et al mass and the C1 fixing microorganism comprises an argi determined that whilst the Wood Ljungdahl pathway was the nine metabolism pathway. best performing pathway for acetate and ethanol production, 0009. In particular embodiments, the arginine metabo butanol fermentation is an ATP-limited process, making lism pathway comprises at least one of an arginine deami anaerobic production of butanol from the Wood Ljungdahl nase pathway and an arginine decarboxylase pathway. The pathway inefficient. arginine deaminase pathway comprises one or more 0004 C. autoethanogenium natively produces acetate, enzymes selected from the group consisting of arginine ethanol, 2,3-Butanediol (2,3-BDO) and lactate. If energetic deiminase (EC 3.5.3.6), ornithine carbomyltransferase (pu impediments can be overcome, synthetic biology promises trescine carbomyltransferase) (EC 2.1.3.3) and a carbamate to enhance the product spectrum of C. autoethanogenium. kinase (EC 2.7.2.2). The arginine decarboxylase pathway Acetogenic bacteria are widespread in nature and play a comprises one or more enzymes selected from the group major role in global carbon cycle, but are considered to live consisting arginine decarboxylase (EC 4.1.1.19), putative on the thermodynamic edge of life.
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