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Non-Natural Microbial Organisms with Improved (19) *EP003194604B1* (11) EP 3 194 604 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12P 7/42 (2006.01) C12P 7/16 (2006.01) 26.02.2020 Bulletin 2020/09 (86) International application number: (21) Application number: 15775307.0 PCT/US2015/050923 (22) Date of filing: 18.09.2015 (87) International publication number: WO 2016/044713 (24.03.2016 Gazette 2016/12) (54) NON-NATURAL MICROBIAL ORGANISMS WITH IMPROVED ENERGETIC EFFICIENCY NICHT-NATÜRLICHE MIKROBIELLE ORGANISMEN MIT VERBESSERTER ENERGIEEFFIZIENZ ORGANISMES MICROBIENS NON NATURELS PRÉSENTANT UNE MEILLEURE EFFICACITÉ ÉNERGÉTIQUE (84) Designated Contracting States: • V. Monedero ET AL: "Mutations lowering the AL AT BE BG CH CY CZ DE DK EE ES FI FR GB phosphatase activity of HPr kinase/phosphatase GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO switch off carbon metabolism.", The EMBO PL PT RO RS SE SI SK SM TR journal, vol. 20, no. 15 1 August 2001 (2001-08-01), 1 August 2001 (2001-08-01), pages 3928-3937, (30) Priority: 18.09.2014 US 201462052341 P XP055233568, Retrieved from the Internet: URL:http://emboj.embopress.org/content/20/ (43) Date of publication of application: 15/3928.full.pdf [retrieved on 2015-12-03] 26.07.2017 Bulletin 2017/30 • MARTA PAPINI ET AL: "Physiological characterization of recombinant Saccharomyces (73) Proprietor: Genomatica, Inc. cerevisiae expressing the phosphoketolase San Diego, CA 92121 (US) pathway: validation of activity through 13C-based metabolic flux analysis", APPLIED (72) Inventors: MICROBIOLOGY AND BIOTECHNOLOGY, • PHARKYA, Priti SPRINGER, BERLIN, DE, vol. 95, no. 4, 26 San Diego February 2012 (2012-02-26), pages 1001-1010, California 92121 (US) XP035090894, ISSN: 1432-0614, DOI: • BURGARD, Anthony P. 10.1007/S00253-012-3936-0 San Diego • PANAGIOTOU G ET AL: "Monitoring novel Minnesota 92121 (US) metabolic pathways using metabolomics and • NUNEZ VAN NAME, Eric Roland machine learning: induction of the San Diego phosphoketolase pathway in Aspergillus California 92121 (US) nidulans cultivations", 20070613, vol. 3, no. 4, 13 June 2007 (2007-06-13), pages 503-516, (74) Representative: Schiweck Weinzierl Koch XP002477272, Patentanwälte Partnerschaft mbB • PANAGIOTOU G ET AL: "INDUCTION OF Ganghoferstraße 68 B XYLULOSE-5-PHOSPHATE 80339 München (DE) PHOSPHOKETOLASE IN ASPERGILLUS NIDULANS: THE KEY FOR EFFICIENT (56) References cited: PRODUCTION OF THE ACETYL-COA EP-A1- 2 062 967 WO-A1-2011/159853 PRECURSOR MOLECULE", INTERNET WO-A2-2005/042756 WO-A2-2011/063055 CITATION, [Online] page 203, XP002477270, WO-A2-2013/184602 WO-A2-2014/144135 01-04-2006 Retrieved from the Internet: WO-A2-2014/169144 WO-A2-2014/176514 URL:http://www.fgsc.net/ecfg8/symposiumVIp osters.pdf> [retrieved on 2008-04-16] Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 3 194 604 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 3 194 604 B1 • THYKAER JETTE ET AL: "Evidence, through C13-labelling analysis, of phosphoketolase activity in fungi", 20070701; 20070700, vol. 42, no. 7, 1 July 2007 (2007-07-01), pages 1050-1055, XP002477271, 2 EP 3 194 604 B1 Description SUMMARY OF INVENTION 5 [0001] The invention provides non-natural microbial organisms containing enzymatic pathways for enhancing carbon flux to acetyl-CoA, or oxaloacetate and acetyl-CoA, and methods for their use to produce bio-products, and bio-products made using such microbial organisms. [0002] Generally, microbial organisms are provided that make acetyl-CoA, or oxaloacetate and acetyl-CoA, have a phosphoketolase pathway (PK pathway) and has (i) a genetic modification that enhances the activity of the non-phos- 10 photransferase system (non-PTS) for sugar uptake, and/or (ii) a genetic modification(s) to the organism’s electron transport chain (ETC) that enhances efficiency of ATP production, that enhances availability of reducing equivalents or both. The modifications enhance energetic efficiency of the modified microbial organism. Optionally, the organism can include (iii) a genetic modification that maintains, attenuates, or eliminates the activity of a phosphotransferase system (PTS) for sugar uptake. 15 [0003] Through experimental studies associated with the current disclosure, it has been discovered that the PK pathway in combination with (i) and/or (ii), and optionally (iii) enhanced carbon flux through acetyl-CoA, or both oxaloacetate and acetyl-CoA. In turn, this increased the pool of acetyl-CoA and oxaloacetate useful for enhancing the production of a desired product or its intermediate (a bioderived compound) while advantageously minimizing undesired compounds. Therefore, the non-natural microbial organisms containing enzymatic pathways for enhancing carbon flux through acetyl- 20 CoA, or both oxaloacetate and acetyl-CoA, with the modifications as described herein can increase the production of intermediates or products such as alcohols (e.g., propanediol or a butanediol), glycols, organic acids, alkenes, dienes (e.g., butadiene), isoprenoids (e.g. isoprene), organic amines, organic aldehydes, vitamins, nutraceuticals, and phar- maceuticals. [0004] Therefore, in one aspect (e.g., a first aspect) the invention provides a non-natural microbial organism that 25 includes (a) a pathway to acetyl-CoA, or both oxaloacetate and acetyl-CoA, comprising a phosphoketolase pathway, and (b) a genetic modification that increases non-PTS activity for sugar uptake. Optionally, the organism can include (c) a genetic modification that maintains, attenuates, or eliminates a PTS activity for sugar uptake. The genetic modification includes those that change an enzyme or protein of the PTS or non-PTS, its activity, a gene-encoding that enzyme or protein, or the gene’s expression. The organism can also have a pathway to a bioderived compound, and a modification 30 to the non-PTS to increase non-PTS activity that improves production of the bioderived compound via improvements in synthesis of acetyl-CoA, or both oxaloacetate and acetyl-CoA, which serve as intermediates. Modification to the non- PTS can balance the fluxes from phosphoenolpyruvate (PEP) into oxaloacetate and pyruvate, which offers an improve- ment over organisms that rely on an endogenous PTS system for sugar uptake, and which can advantageously lead into the bioderived compound pathway. 35 [0005] The PTS and non-PTS can allow for uptake of primarily C5, C6 or C12 sugars and their oligomers. Non-natural microbial organism having a PTS for sugar (e.g., C6, C12, sugar alcohols, and amino sugars) uptake are able to phos- phorylate sugars by conversion of PEP into pyruvate.. The non-PTS allows for uptake of sugars via a facilitator or a permease and subsequent phosphorylation via a kinase. The non-PTS further allows uptake of C5 sugars such as xylose, disaccharides such as lactose, melibiose, and maltose. Other substrates such as ascorbate may be recognized 40 by a specific PTS or non-PTS enzyme or protein. Phosphorylated sugar then goes through the majority of reactions in glycolysis to generate reducing equivalents and ATP that are associated with the organism’s electron transport chain (ETC). [0006] Illustrative PK pathways, can include the following enzymes: 45 both fructose-6-phosphate phosphoketolase (1T) and a phosphotransacetylase (1V); all three of fructose-6-phosphate phosphoketolase (IT), an acetate kinase (1W), and an acetyl-CoA transferase, an acetyl-CoA synthetase, or an acetyl-CoA ligase (1X); both xylulose-5-phosphate phosphoketolase (1U) and a phosphotransacetylase (1V); or all three of xylulose-5-phosphate phosphoketolase (1U), an acetate kinase (1W), and an acetyl-CoA transferase, 50 an acetyl-CoA synthetase, or an acetyl-CoA ligase (1X). [0007] A non-natural microbial organism of the first aspect with the (a) a pathway to oxaloacetate, acetyl-CoA or both, comprising a phosphoketolase, and (b) a genetic modification to enhance non-PTS activity, can optionally further include one or more modification(s) to the organism’s electron transport chain to enhance efficiency of ATP production,, to 55 enhance availability of reducing equivalents, or both. [0008] A further (second) aspect described herein provides a non-natural microbial organism that includes (a) a pathway to oxaloacetate, acetyl-CoA or both, comprising a phosphoketolase pathway, and (b) one or more modification(s) to the organism’s electron transport chain to enhance efficiency of ATP production, to enhance availability of reducing equiv- 3 EP 3 194 604 B1 alents, or both. For example, modifications as described herein that increase the number of protons translocated per electron pair that reaches cytochrome oxidases or complex IV of the electron transport chain provide increased protons that are used by ATP synthase to produce ATP. Consequently, by increasing the amount of ATP generated per pair of electrons channeled through the electron transport chain, the energetic efficiency (also referred to as P/O ratio) of the 5 cell is increased. Similarly, modifications that attenuate or eliminate NADH dehydrogenases that do not transport or inefficiently transport protons increases the NADH pool available for the more efficient NADH dehydrogenases, e.g. nuo. Again, the energetic efficiency of the cell is increased. [0009] Organisms of the second aspect may include a pathway for assimilation of an alternate carbon source (e.g., methanol, syngas, glycerol, formate, methane), for example, if the PTS and non-PTS are modified, not present in the 10 organism, or otherwise do not provide the desired influx of a hydrocarbon energy source. Accordingly, organisms making oxaloacetate and/or acetyl-CoA and that contain a phosphoketolase pathway can also comprise a pathway for using non-sugar carbon substrates such as glycerol, syngas, formate, methane and methanol.
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