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EP-3362567-B1 - GENETICALLY ENGINEERED BACTERIUM COMPRISING ENERGY-GENERATING FERMENTATION PATHWAY

EP3362567B1EP 3362567 B1EP3362567 B1EP 3362567B1EP-3362567-B1

Inventors

  • KOEPKE, MICHAEL
  • JENSEN, Rasmus, Overgaard
  • BEHRENDORFF, James Bruce Yarnton Haycock
  • HILL, RYAN EDWARD
  • JUMINAGA, Darmawi
  • MUELLER, Alexander, Paul

Dates

Publication Date
20260506
Application Date
20161013

Claims (10)

  1. Use of a bacterium comprising heterologous phosphate butyryltransferase (Ptb) and butyrate kinase (Buk) (Ptb-Buk) to produce acetoacetate from acetoacetyl-CoA, 3-hydroxyisovalerate from 3-hydroxyisovaleryl-CoA, 3-hydroxybutyrate from 3-hydroxybutyryl CoA, 2-hydroxyisobutyrate from 2-hydroxyisobutyryl-CoA, adipic acid from adipyl-CoA, crotonate from crotonyl-CoA, acetobutyrate from acetobutyryl-CoA, 3-hydroxyhexanoate from 3-hydroxyhexanoyl-CoA, or isovalerate from isovaleryl-CoA; wherein the bacterium is selected from Clostridium autoethanogenum and Clostridium ragsdalei.
  2. The use of claim 1, wherein the bacterium does not produce butanol.
  3. The use of claim 1, wherein the bacterium produces one or more of an acid, an alkene, a ketone, an aldehyde, an alcohol, or a diol.
  4. The use of claim 1, wherein the bacterium produces one or more of acetone, isopropanol, isobutylene, 3-hydroxybutyrate, 1,3-butanediol, 2-hydroxyisobutyrate, adipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, or isoamyl alcohol.
  5. The use of claim 1, wherein the bacterium produces one or more of 1-propanol, 1-hexanol, and 1-octanol.
  6. The use of claim 1, wherein the bacterium is Clostridium autoethanogenum.
  7. The use of claim 1, wherein the bacterium further comprises: (a) heterologous or endogenous aldehyde:ferredoxin oxidoreductase (AOR); (b) a disruptive mutation in a phosphotransacetylase (Pta) and an acetate kinase (Ack); or (c) a disruptive mutation in a thioesterase.
  8. A method of producing a product comprising culturing the bacterium of any one of claims 1-7 in the presence of a gaseous substrate comprising one or more of CO, CO 2 , and H 2 , wherein the bacterium produces the product acetoacetate from acetoacetyl-CoA, 3-hydroxyisovalerate from 3-hydroxyisovaleryl-CoA, 3-hydroxybutyrate from 3-hydroxybutyryl CoA, 2-hydroxyisobutyrate from 2-hydroxyisobutyryl-CoA, adipic acid from adipyl-CoA, crotonate from crotonyl-CoA, acetobutyrate from acetobutyryl-CoA, 3-hydroxyhexanoate from 3-hydroxyhexanoyl-CoA, or isovalerate from isovaleryl-CoA.
  9. The method of claim 8, wherein the bacterium produces one or more of acetone, isopropanol, isobutylene, 3-hydroxybutyrate, 1,3-butanediol, 2-hydroxyisobutyrate, adipic acid, 1,3-hexanediol, 3-methyl-2-butanol, 2-buten-1-ol, isovalerate, or isoamyl alcohol.
  10. The method of claim 8, wherein the gaseous substrate is syngas or an industrial waste gas.

Description

CROSS REFERENCE TO RELATED APPLICATIONS 0001 This application claims the benefit of U.S. Provisional Patent Application No. 62/240,850 filed October 13, 2015. BACKGROUND OF THE INVENTION 0002 With recent advances in fermentation and metabolic engineering, fermentation routes to various products have been identified and developed (Clomburg, Appl Microbiol Biotechnol, 86: 419-434, 2010; Peralta-Yahya, Biotechnol J, 5: 147-162, 2010; Cho, Biotechnol Adv, pii: S0734-9750(14)00181-5, 2014. US7262037B2 describes a method for the production of D-(-)-3-hydroxybutyric acid by recombinant Escherichia coli. WO2014/198560A2 describes a method for producing organic compositions from oxyhydrogen and CO2 via acetoacetyl-coa as intermediate product. Ueki et al (MBIO, vol 5, no 5, 21 Oct 2014, pages e01636-14) describes the conversion of carbon dioxide to butyrate with an engineered strain of Clostridium ljungdahlii. Yang et al (Applied Microbiology and Biotechnology, vol 98, no 1, 11 Oct 2013, pages 95-104) describes the metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose. However, all of these fermentation routes are energy (ATP)-consuming or, at best, energy (ATP)-neutral, which restricts product yield in energy-limited systems and uncouples product production from microorganism growth. The present invention provides energy (ATP)-generating pathways that overcome these limitations by providing novel fermentation routes and pathways to a variety of products, including acids, alkenes, aldehydes, alcohols, and diols. These pathways are directly coupled to microorganism growth and offer high product yields. 0003 In particular, the invention relates to fermentation pathways involving Ptb-Buk. Phosphate butyryltransferase (Ptb) (EC 2.3.1.19) natively catalyzes the reaction of butanoyl-CoA and phosphate to form CoA and butanoyl phosphate. Butyrate kinase (Buk) (EC 2.7.2.7) natively catalyzes the reaction of butanoyl phosphate and ADP to form butyrate (butanoate) and ATP. Accordingly, these enzymes together (Ptb-Buk) natively catalyze the conversion of butanoyl-CoA to butyrate and generate one ATP via substrate level phosphorylation (SLP). 0004 The inventors have discovered that Ptb is promiscuous and is capable of accepting a variety of acyl-CoAs and enoyl-CoAs as substrates, such that Ptb-Buk may be used to convert a number of acyl-CoAs and enoyl-CoAs to their corresponding acids or alkenates, respectively, while simultaneously generating ATP via substrate level phosphorylation. 0005 Furthermore, in combination with an aldehyde:ferredoxin oxidoreductase (AOR) and an alcohol dehydrogenase, acids formed via the Ptb-Buk system can be further converted to their respective aldehydes, alcohols, or diols. AOR (EC 1.2.7.5) catalyzes the reaction of an acid and reduced ferredoxin (which can, for example, be generated from oxidation of CO or hydrogen) to form an aldehyde and oxidized ferredoxin. Alcohol dehydrogenase (EC 1.1.1.1 and EC 1.1.1.2) can convert an aldehyde and NAD(P)H to an alcohol and NAD(P). 0006 Introduction of Ptb-Buk and/or AOR into a heterologous species, therefore, provides a novel, alternate route to the formation of native and non-native products, such as acids, alkenes, ketones, aldehydes, alcohols, and diols at high yields, thus overcoming limitations of the current state of the art. SUMMARY OF THE INVENTION 0006a The present invention is defined by the appended claims. The technical information set out below may in some respects go beyond the disclosure of the invention defined by the appended claims. The additional technical information is provided to place the invention in a broader technical context and to illustrate possible related technical developments. Such additional technical information which does not fall within the scope of the appended claims is not part of the invention. Accordingly, any embodiments and examples of the present disclosure which do not fall under the scope of the appended claims do not form part of the invention and are merely provided for illustrative purposes. 0007 The invention provides the use of a bacterium comprising heterologous phosphate butyryltransferase (Ptb) and butyrate kinase (Buk) (Ptb-Buk) to produce acetoacetate from acetoacetyl-CoA, 3-hydroxyisovalerate from 3-hydroxyisovaleryl-CoA, 3-hydroxybutyrate from 3-hydroxybutyryl CoA, 2-hydroxyisobutyrate from 2-hydroxyisobutyryl-CoA, adipic acid from adipyl-CoA, crotonate from crotonyl-CoA, acetobutyrate from acetobutyryl-CoA, 3-hydroxyhexanoate from 3-hydroxyhexanoyl-CoA, or isovalerate from isovaleryl-CoA, wherein the bacterium is selected from Clostridium autoethanogenum and Clostridium ragsdalei. Generally, the Ptb-Buk acts on a non-native substrate, e.g., a substrate other than butanoyl-CoA and/or butanoyl phosphate, and produces a non-native product, e.g., a product other than butanoyl phosphate or butyrate. 0008 The bacterium may produce one or more