Recombinant host cells comprising phosphoketolases

Patent metadata

Field	Value
Publication number	US-9550999-B2
Application number	US-201414335734-A
Country	US
Kind code	B2
Filing date	Jul 18, 2014
Priority date	Jun 18, 2010
Publication date	Jan 24, 2017
Grant date	Jan 24, 2017

Abstract

The present invention is related to recombinant host cells comprising: (i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate, or acetyl-CoA; and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. The present invention is also related to recombinant host cells further comprising (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

First claim

What is claimed:

1. A recombinant host cell comprising:
(i) at least one genetic modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, wherein the at least one genetic modification reduces or eliminates pyruvate decarboxylase activity; and
(ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity.

2. The recombinant host cell of claim 1 , wherein said host cell further comprises:
(iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

3. The recombinant host cell of claim 1 , wherein said host cell has a reduced requirement for an exogenous two-carbon substrate for its growth in culture compared to a recombinant eukaryotic host cell comprising (i) and not (ii).

4. The recombinant host cell of claim 1 , wherein said host cell has improved growth as compared to a host cell comprising (i) and not (ii) in culture media that is not supplemented with an exogenous two-carbon carbon substrate.

5. The recombinant host cell of claim 4 , wherein said exogenous to-carbon substrate is ethanol or acetate.

6. The recombinant host cell of claim 1 , wherein said endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is PDC1, PDC5, PDC6, or combinations thereof and said host cell is S. cerevisiae.

7. The recombinant host cell of claim 1 , wherein said heterologous polynucleotide encoding a polypeptide having phosphoketolase activity is xpk1 from Lactobacillus plantarum , xpkA from Lactobacillus pentosus MD363 or 6-phosphate phosphoketolase from B. lactis.

8. The recombinant host cell of claim 1 wherein said polypeptide having phosphoketolase activity comprises at least 85% identity to SEQ ID NO 481 or an active fragment thereof.

9. The recombinant host cell of claim 2 , wherein said heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity is EutD from Lactobacillus plantarum or phosphotransacetylase from Bacillus subtilis.

10. The recombinant host cell of claim 2 , wherein said, polypeptide having phosphotransacetylase activity comprises at least 85% identity to SEQ ID NO: 1472 or an active fragment thereof.

11. The recombinant host cell of claim 1 , wherein said host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula , or Saccharomyces.

12. The recombinant host cell of claim 1 wherein said host, cell is Saccharomyces cerevisiae.

13. The recombinant host cell of claim 1 further comprising a pyruvate-utilizing biosynthetic pathway, wherein said pyruvate-utilizing biosynthetic pathway forms a product selected from the group consisting of: 2,3-butanediol, 2-butanol, 2-butanone, valine, leucine, lactic acid, malate, and isoamyl alcohol.

14. The recombinant host cell of claim 1 , further comprising a pyruvate-utilizing biosynthetic pathway herein said pyruvate-utilizing biosynthetic pathway is an isobutanol biosynthetic pathway comprising the substrate to product conversions:
(i) pyruvate to acetolactate;
(ii) acetolactate to 2,3-dihydroxyisovalerate;
(iii) 2,3-dihydroxyisovalerate to 2-ketoisovalerate;
(iv) 2-ketoisovalerate to isobutyraldehyde;
(v) isobutyraldehyde to isobutanol; and
wherein said recombinant host cell produces isobutanol.

15. The recombinant host cell of claim 13 , wherein said pyruvate-utilizing biosynthetic pathway is a 2-butanone biosynthetic pathway comprising the substrate to product conversions:
(i) pyruvate to acetolactate;
(ii) acetolactate to acetoin;
(iii) acetoin to 2,3-butanediol;
(iv) 2,3-butanediol to 2-butanone;
wherein said recombinant host cell produces 2-butanone.

16. The recombinant host cell of claim 13 , wherein said pyruvate-utilizing biosynthetic pathway is a 2-butanol biosynthetic pathway comprising the substrate to product conversions:
(i) pyruvate to acetolactate;
(ii) acetolactate to acetoin;
(iii) acetoin to 2,3-butanediol;
(iv) 2,3-butanediol to 2-butanone;
(v) 2-butanone to 2-butanol;
wherein said recombinant host cell produces 2-butanol.

17. The recombinant host cell of claim 1 wherein the phosphoketolase matches the Profile HMM given in Table 6 with an E value of less than 7.5E-242.

18. The recombinant host cell of claim 1 wherein the phosphoketolase matches the Profile HMMs given in Tables 6, 7, 8, and 9 with E values of less than 7.5E-242, 1.1E-124, 2.1E-49, 7.8E-37, respectively.

19. The recombinant host cell of claim 1 further comprising a phosphotransacetylase which matches the Profile HMM given in Table 14 with an E value of less than 5E-34.

20. The recombinant host cell of claim 13 , wherein said pyruvate-utilizing biosynthetic pathway is a 2,3-butanediol biosynthetic pathway comprising the substrate to product conversations:
(i) pyruvate to acetolactate;
(ii) acetolactate to acetoin;
(iii) acetoin 2,3-butanediol; and
wherein said recombinant host cell produces 2,3 butanediol.

21. A method for the production of 2,3-butanediol, 2-butanol, 2-butanone, valine, leucine, lactic acid, malic acid, alanine, fumaric acid, succinic acid or isoamyl alcohol comprising growing the recombinant host cell of claim 13 under conditions wherein the product is produced and optionally recovering the product.

22. A method for the production of isobutanol comprising growing the recombinant host cell of claim 14 under conditions wherein the product is produced and optionally recovering the product.

Assignees

• Butamax Advanced Biofuels Llc

Inventors

• Dauner Michael
• Maggio-Hall Lori Ann
• Tomb Jean-Francois

Classifications

• C12N9/88

  Lyases (4.) · CPC title

• C12Y401/01001

  Pyruvate decarboxylase (4.1.1.1) · CPC title

• C12P13/06

  Alanine; Leucine; Isoleucine; Serine; Homoserine · CPC title

• C12P7/46

  Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title