Enhanced dihydroxy-acid dehydratase activity in lactic acid bacteria

• US 8,637,281 B2
• Filed: 09/29/2009
• Issued: 01/28/2014
• Est. Priority Date: 09/29/2008
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Paul, Brian James, Suh, Wonchul
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US12/569,168
• Publication Number:
  US 20100081183A1
• Time in Patent Office: 1,582 Days
• Field of Search: None
• US Class Current: 435/160
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Lactic acid bacterial (LAB) cells were modified such that they have a specific activity of dihydroxy-acid dehydratase enzyme activity that is increased to about 0.1 μmol min⁻¹ mg⁻¹. LAB cells with even higher activities of 0.2 to 0.6 μmol min⁻¹ mg⁻¹ of DHAD activity were obtained.

These modified cells may be used to produce isobutanol when additional isobutanol biosynthetic pathway enzymes are expressed.

Claims

1. A recombinant lactic acid bacterial cell comprising at least one gene encoding a heterologous polypeptide having dihydroxy-acid dehydratase activity and at least about 95% sequence identity to the polypeptide encoded by the polynucleotide sequence of SEQ ID NO:

167 or SEQ ID NO;

564 wherein the bacterial cell is substantially free of lactate dehydrogenase activity and wherein the heterologous polypeptide having dihydroxy-acid dehydratase activity has a specific activity of at least about 0.1 μ

mol min<sup>−

1</sup> mg<sup>−

1</sup> total soluble protein in a crude cell extract.

2. The recombinant lactic acid bacterial cell of claim 1 wherein the heterologous polypeptide having dihydroxy-acid dehydratase activity has a specific activity of at least about 0.6 μ

mol min<sup>−

1</sup> mg<sup>−

1</sup> total soluble protein in a crude cell extract.

3. The recombinant lactic acid bacterial cell of claim 1 wherein the dihydroxy-acid dehydratase polypeptide has an amino acid sequence that matches the Profile HMM of table 7 with an E value of <

10<sup>−

5</sup> wherein the polypeptide additionally comprises all three conserved cysteines, corresponding to positions 56, 129, and 201 in the amino acids sequences of the Streptococcus mutans DHAD enzyme corresponding to SEQ ID NO;

168.

4. The recombinant lactic acid bacterial cell of claim 1 comprising a disruption in at least one endogenous gene encoding a polypeptide having lactate dehydrogenase activity.

5. The recombinant lactic acid bacterial cell of claim 4 wherein the gene encoding lactate dehydrogenase is selected from the group consisting of ldhL, ldhD, ldhL1, and ldhL2.

6. The recombinant lactic acid bacterial cell of claim 1 wherein the lactic acid bacteria is selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.

7. The recombinant lactic acid bacterial cell of claim 1 wherein the bacteria produces isobutanol.

8. The recombinant lactic acid bacterial cell of claim 7 wherein the bacteria comprises an isobutanol biosynthetic pathway.

9. The recombinant lactic acid bacterial cell of claim 8 wherein the isobutanol biosynthetic pathway comprises genes encoding acetolactate synthase, acetohydroxy acid isomeroreductase, dihydroxy-acid dehydratase, branched-chain α

-keto acid decarboxylase, and branched-chain alcohol dehydrogenase.

10. A method of making isobutanol comprising providing the recombinant lactic acid bacteria of claim 8 and growing the lactic acid bacteria under conditions wherein isobutanol is produced.

11. The recombinant lactic acid bacterial cell of claim 1 wherein the heterologous polypeptide having dihydroxy-acid dehydratase activity comprises cysteines at the amino acid positions corresponding to positions 57, 130, and 203 in the amino acid sequence of the Streptococcus mutans DHAD enzyme corresponding to SEQ ID NO:

168.

12. The recombinant lactic acid bacterial cell of claim 1 wherein the heterologous polypeptide having dihydroxy-acid dehydratase activity has at least about 98% sequence identity to the polypeptide encoded by the polynucleotide sequence of SEQ ID NO:

167.

13. The recombinant lactic acid bacterial cell of claim 1 wherein the heterologous polypeptide having dihydroxy-acid dehydratase activity has at least about 98% sequence identity to the polypeptide encoded by the polynucleotide sequence of SEQ ID NO:

564.

14. The recombinant lactic acid bacterial cell of claim 1 wherein the heterologous polypeptide having dihydroxy-acid dehydratase activity comprises the polypeptide sequence of SEQ ID NO:

168.

15. The recombinant lactic acid bacterial cell of claim 14 wherein the lactic acid bacteria is Lactobacillus plantarum.

16. The recombinant lactic acid bacterial cell of claim 1 wherein the bacteria comprises a valine biosynthetic pathway comprising genes encoding acetohydroxy acid reductoisomerase, dihydroxy-acid dehydratase and branched-chain amino acid aminotransferase.

17. The recombinant lactic acid bacterial cell of claim 1 wherein the bacteria comprises a leucine biosynthetic pathway comprising genes encoding acetohydroxy acid reductoisomerase, dihydroxy-acid dehydratase, 2-isopropylmalaate synthase, isopropylmalate isomerase, 3-isopropylmalate dehydrogenase and aromatic amino acid transaminase.

18. The recombinant lactic acid bacterial cell of claim 1 wherein the bacteria comprises a panthotenate biosynthetic pathway comprising genes encoding acetohydroxy acid reductoisomerase, dihydroxy-acid dehydratase, 3-methyl-2-oxobutanoate hydroxymethyltransferase, 2-dehydropantoate reductase and pantoate-beta-alanine ligase.

19. The recombinant lactic acid bacterial cell of claim 1 wherein the heterologous polypeptide having dihydroxy-acid dehydratase activity comprises the polypeptide sequence of SEQ ID NO:

232.

20. The isolated recombinant lactic acid bacterial cell of claim 19 wherein the lactic acid bacteria is Lactobacillus plantarum.

21. The recombinant lactic acid bacterial cell of claim 14 wherein the lactic acid bacteria is Lactobacillus plantarum.