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Methods and Organisms with Increased Carbon Flux Efficiencies

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(19) *EP003087174B1* (11) EP 3 087 174 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 1/21 (2006.01) C12P 7/52 (2006.01) (2006.01) (2006.01) 13.05.2020 Bulletin 2020/20 C12P 1/04 C12P 7/16 (21) Application number: 14875558.0 (86) International application number: PCT/US2014/072178 (22) Date of filing: 23.12.2014 (87) International publication number: WO 2015/100338 (02.07.2015 Gazette 2015/26) (54) METHODS AND ORGANISMS WITH INCREASED CARBON FLUX EFFICIENCIES VERFAHREN UND ORGANISMEN MIT ERHÖHTER KOHLENSTOFFFLUSSEFFIZIENZ MÉTHODES ET ORGANISMES À RENDEMENTS DE FLUX DE CARBONE ACCRUS (84) Designated Contracting States: • MARK SHEPHERD ET AL: "Compensations for AL AT BE BG CH CY CZ DE DK EE ES FI FR GB diminished terminal oxidase activity in GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Escherichia coli : cytochrome bd -II-mediated PL PT RO RS SE SI SK SM TR respiration and glutamate metabolism", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. (30) Priority: 27.12.2013 US 201361921292 P 285, no. 24, 11 June 2010 (2010-06-11), 17.06.2014 US 201462013390 P XP055372909, US ISSN: 0021-9258, DOI: 10.1074/jbc.M110.118448 (43) Date of publication of application: • JOONHOON KIM ET AL: "Large-Scale Bi-Level 02.11.2016 Bulletin 2016/44 Strain Design Approaches and Mixed-Integer Programming Solution Techniques", PLOS ONE, (73) Proprietor: Genomatica, Inc. vol. 6, no. 9, 9 September 2011 (2011-09-09), San Diego, CA 92121 (US) pages 1-13, XP055221322, DOI: 10.1371/journal.pone.0024162 (72) Inventors: • KIM ET AL.: ’Large-Scale Bi-Level Strain Design • BURGARD, Anthony P. Approaches and Mixed-Integer Programming San Diego, CA 92121 (US) Solution Techniques.’ PLOS ONE. vol. 6, no. 9, • OSTERHOUT, Robin E. 2011, pages 1 - 13, XP055221322 San Diego, CA 92121 (US) • HANKE ET AL.: ’Combined Fluxomics and • VAN DIEN, Stephen J. Transcriptomics Analysis of Glucose Catabolism San Diego, CA 92121 (US) via a Partially Cyclic Pentose Phosphate Pathway • PHARKYA, Priti in Gluconobacter oxydans 621 H.’ APPL San Diego, CA 92121 (US) ENVIRON MICROBIOL. vol. 79, no. 7, April 2013, • YANG, Tae Hoon pages 2336 - 2348, XP055221334 San Diego, CA 92121 (US) • GABRIEL ET AL.: ’Regulation of the Bacillus • CHOI, Jungik subtilis yciC gene and insights into the San Diego, CA 92121 (US) DNA-binding specificity of the zinc-sensing metalloregulator’ ZUR. J BACTERIOL. vol. 190, (74) Representative: Jones Day no. 10, May 2008, pages 3482 - 3488, XP055221340 Rechtsanwälte, Attorneys-at-Law, Patentanwälte • PRZYBYLA-ZAWISLAK ET AL.: ’Genes of Prinzregentenstrasse 11 succinyl-CoA ligase from Saccharomyces 80538 München (DE) cerevisiae.’ EUR J BIOCHEM vol. 258, no. 2, 01 December 1998, pages 736 - 43, XP055221345 (56) References cited: WO-A2-03/072785 US-A1- 2012 122 171 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 3 087 174 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 3 087 174 B1 • MCMAHON ET AL.: ’Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli.’ APPL ENVIRON MICROBIOL. vol. 80, no. 3, February 2014, pages 1042 - 1050, XP055221346 2 EP 3 087 174 B1 Description BACKGROUND OF THE INVENTION 5 [0001] The invention provides non-naturally occurring microbial organisms having reduced carbon flux from succinyl- CoA to succinate through an oxidative TCA cycle. The invention also provides methods of reducing carbon flux from succinyl-CoA to succinate through an oxidative TCA cycle using the microbial organisms. [0002] Carbon loss, through excess CO2 production, can come from three main routes in central metabolism: the pentose phosphate pathway, the glyoxylate shunt and the oxidative tricarboxylic acid (TCA) cycle. The main CO2 gen- 10 erating reaction of the pentose phosphate pathway is phosphogluconate dehydrogenase. Enzymes which can contribute to carbon loss in the TCA cycle and glyoxylate shunt include, for example, pyruvate dehydrogenase, pyruvate formate lyase, pyruvate oxidase, alpha-ketoglutarate dehydrogenase, isocitrate dehydrogenase, phosphoenolpyruvate carbox- ykinase, and malic enzyme. [0003] Carbon loss can also come from other metabolic reactions that include, for example, the glycine cleavage 15 system, formate hydrogen lyase, formate dehydrogenase, glutamate decarboxylase, pyruvate oxidase, acetolactate synthase and 2-oxo-4-methyl-3-carboxypentanoate decarboxylase, aspartate decarboxylase, lysine decarboxylase, di- aminopimelate decarboxylase and enzymes involved in fatty acid biosynthesis. [0004] Thus, there exists a need for alternative means for decreasing carbon loss and increasing carbon flux efficien- cies. The present invention satisfies this need and provides related advantages as well. 20 SUMMARY OF INVENTION [0005] The invention is directed to a non-naturally occurring microbial organism comprising a genetic alteration of attenuation of cydA or cydB, and one or more of a genetic alteration that increases expression of a protein encoded by 25 pntAB. The microbial organism also can include attenuation of a TCA cycle enzyme other than a succinyl-CoA synthetase or transferase. The microbial organism can include a metabolically engineered pathway for producing a bioderived compound from a TCA cycle intermediate or a TCA cycle substrate. The bioderived compound can be 4-hydroxybutyrate (4HB), 1,4-butanediol (1,4-BDO), 1,3-butanediol (1,3-BDO), polyhydroxylbutanoate (PHB), butadiene, adipate, 6-ami- nocaproate, caprolactam or methacrylic acid, or other compounds disclosed herein. The invention is directed to other 30 genetic alterations for enhancing the production of a bioderived compound. BRIEF DESCRIPTION OF THE DRAWINGS [0006] Figure 1 shows central metabolic pathways that generate CO2, including (1) the pentose phosphate pathway; 35 (2) the complete oxidative TCA cycle; and (3) the glyoxylate shunt. Abbreviations: Glc is glucose, G6P is glucose-6- phosphate, F6P is fructose-6-phosphage, FBP is fructose-1,6-bisphosphate, G3P is glyceraldehyde-3-phosphate, PEP is phosphoenolpyruvate, Pyr is pyruvate, cit is citrate, Icit is isocitrate, AKG is alpha-ketoglutarate, Succ is succinate, Fum is fumarate, Mal is malate, OAA is oxaloacetate, 6PGL is 6-phospogluconolactone, Ru5P is ribulose-5-phosphate, E4P is erythrose-4-phosphate, S7P is sedoheptulose-7-phosphate, and Glx is glyoxylate. 40 DETAILED DESCRIPTION OF THE INVENTION [0007] The invention provides non-naturally occurring microbial organisms having reduced carbon flux from succinyl- CoA to succinate through an oxidative TCA cycle, wherein the microbial organism includes one or more genetic disrup- 45 tions. The invention also provides methods of reducing carbon flux from succinyl-CoA to succinate through an oxidative TCA cycle using the microbial organisms. [0008] As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microor- ganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. 50 Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypep- tides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism’s genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterol- ogous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifi- cations include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or 55 operon. Exemplary metabolic polypeptides include, for example, enzymes or proteins that convert succinyl-CoA to succinate, tricarboxylic acid (TCA) cycle enzymes, pyridine nucleotide transhydrogenase, NADH dehyrogenase, ubiq- uinol oxidase, menaquinone biosynthetic enzymes, menaquinol biosynthetic enzymes, phosphoenoylpyruvate carbox- ykinase (PEPCK), phosphoenoylpyruvate carboxylase (PPC) and enzymes or proteins within a bioderived product bio- 3 EP 3 087 174 B1 synthetic pathway. [0009] A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein. 5 [0010] The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. 10 [0011] As used
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  • A Multi-Omics Approach to Liver Diseases: Integration of Single Nuclei Transcriptomics with Proteomics and Hicap Bulk Data in Human Liver

    A Multi-Omics Approach to Liver Diseases: Integration of Single Nuclei Transcriptomics with Proteomics and Hicap Bulk Data in Human Liver

    OMICS A Journal of Integrative Biology Volume 24, Number 4, 2020 Research Articles Mary Ann Liebert, Inc. DOI: 10.1089/omi.2019.0215 A Multi-Omics Approach to Liver Diseases: Integration of Single Nuclei Transcriptomics with Proteomics and HiCap Bulk Data in Human Liver Marco Cavalli,1,* Klev Diamanti,2,* Gang Pan,1 Rapolas Spalinskas,3 Chanchal Kumar,4,5 Atul Shahaji Deshmukh,6 Matthias Mann,6 Pelin Sahle´n,3 Jan Komorowski,2,7 and Claes Wadelius1 Abstract The liver is the largest solid organ and a primary metabolic hub. In recent years, intact cell nuclei were used to perform single-nuclei RNA-seq (snRNA-seq) for tissues difficult to dissociate and for flash-frozen archived tissue samples to discover unknown and rare cell subpopulations. In this study, we performed snRNA-seq of a liver sample to identify subpopulations of cells based on nuclear transcriptomics. In 4282 single nuclei, we detected, on average, 1377 active genes and we identified seven major cell types. We integrated data from 94,286 distal interactions ( p < 0.05) for 7682 promoters from a targeted chromosome conformation capture technique (HiCap) and mass spectrometry proteomics for the same liver sample. We observed a reasonable correlation between proteomics and in silico bulk snRNA-seq (r = 0.47) using tissue-independent gene-specific protein abundancy estimation factors. We specifically looked at genes of medical importance. The DPYD gene is involved in the pharmacogenetics of fluoropyrimidine toxicity and some of its variants are analyzed for clinical purposes. We identified a new putative polymorphic regulatory element, which may contribute to variation in toxicity.
  • Thioesterase-Mediated Control of Cellular Calcium Homeostasis Enables Hepatic ER Stress

    Thioesterase-Mediated Control of Cellular Calcium Homeostasis Enables Hepatic ER Stress

    Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress Baran A. Ersoy, … , Ipek Alpertunga, David E. Cohen J Clin Invest. 2018;128(1):141-156. https://doi.org/10.1172/JCI93123. Research Article Cell biology Metabolism The incorporation of excess saturated free fatty acids (SFAs) into membrane phospholipids within the ER promotes ER stress, insulin resistance, and hepatic gluconeogenesis. Thioesterase superfamily member 2 (Them2) is a mitochondria- associated long-chain fatty acyl-CoA thioesterase that is activated upon binding phosphatidylcholine transfer protein (PC- TP). Under fasting conditions, the Them2/PC-TP complex directs saturated fatty acyl-CoA toward β-oxidation. Here, we showed that during either chronic overnutrition or acute induction of ER stress, Them2 and PC-TP play critical roles in trafficking SFAs into the glycerolipid biosynthetic pathway to form saturated phospholipids, which ultimately reduce ER membrane fluidity. The Them2/PC-TP complex activated ER stress pathways by enhancing translocon-mediated efflux of ER calcium. The increased cytosolic calcium, in turn, led to the phosphorylation of calcium/calmodulin-dependent protein kinase II, which promoted both hepatic insulin resistance and gluconeogenesis. These findings delineate a mechanistic link between obesity and insulin resistance and establish the Them2/PC-TP complex as an attractive target for the management of hepatic steatosis and insulin resistance. Find the latest version: https://jci.me/93123/pdf The Journal of Clinical Investigation RESEARCH ARTICLE Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress Baran A. Ersoy, Kristal M. Maner-Smith, Yingxia Li, Ipek Alpertunga, and David E. Cohen Department of Medicine, Division of Gastroenterology and Hepatology, Weill Cornell Medical College, New York, New York, USA.