ENHANCED DIHYDROXY-ACID DEHYDRATASE ACTIVITY IN LACTIC ACID BACTERIA

• US 20100081183A1
• Filed: 09/29/2009
• Published: 04/01/2010
• 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
• Granted Patent: US 8,637,281 B2
• Time in Patent Office: Days
• 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 wherein the bacterial cell is substantially free of lactate dehydrogenase activity.

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 0.1 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 2 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.

4. The lactic acid bacterial cell of claim 1 wherein the dihydroxy-acid dehydratase enzyme is expressed by a nucleic acid molecule that is heterologous to the bacteria.

5. The lactic acid bacterial cell of claim 1 wherein the dihydroxy-acid dehydratase is a [2Fe-2S] dihydroxy-acid dehydratase.

6. The lactic acid bacterial cell of claim 5 wherein the dihydroxyacid 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.

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

8. The lactic acid bacterial cell of claim 7 wherein the gene encoding lactate dehydrogenase is selected from the group consisting of IdhL, IdhD, IdhL1, and IdhL2.

9. The 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.

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

11. The lactic acid bacterial cell of claim 10 wherein the bacteria comprises an isobutanol biosynthetic pathway.

12. The lactic acid bacterial cell of claim 11 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.

13. A method of making isobutanol comprising providing the lactic acid bacteria of claim 11 and growing the lactic acid bacteria under conditions wherein isobutanol is produced.