Methanol dehydrogenase enzymes from Bacillus

Patent metadata

Field	Value
Publication number	US-9322000-B2
Application number	US-201314374878-A
Country	US
Kind code	B2
Filing date	Jan 25, 2013
Priority date	Jan 25, 2012
Publication date	Apr 26, 2016
Grant date	Apr 26, 2016

Abstract

The present invention relates to a nucleic acid molecule, which encodes a polypeptide having alcohol dehydrogenase activity, in particular methanol dehydrogenase activity, comprising having a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 (mdh2-MGA3), 3 (mdh3-MGA3), or 5 (mdh2-PB1); (ii) a nucleotide sequence having at least 90% sequence identity, more particularly at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity, with a nucleotide sequence as set forth in any one of SEQ ID NOs: 1, 3 or 5; (iii) a nucleotide sequence which is degenerate with any one of the nucleotide sequences of SEQ ID NOs: 1, 3 or 5; (iv) a nucleotide sequence which is a part of the nucleotide sequence of any one of SEQ ID NOs: 1, 3 or 5, or of a nucleotide sequence which is degenerate with a sequence of SEQ ID NOs: 1, 3 or 5; (v) a nucleotide sequence encoding all or part of a polypeptide whose amino acid sequence is set forth in any one of SEQ ID NOs: 2 (Mdh2-MGA3), 4 (Mdh3-MGA3) or 6 (Mdh2-PB1); and (vi) a nucleotide sequence encoding all or part of a polypeptide which has an amino acid sequence having at least 90% sequence identity, preferably at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity, with an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4 or 6; or a nucleic acid molecule comprising a nucleotide sequence which is complementary to the nucleotide sequence of any one of (i) to (vi). Also provided are recombinant constructs, vectors and host cells comprising such a nucleic acid molecule and polypeptides encoded thereby. Such molecules may advantageously be used in the genetic modification of host cells, for example to introduce or modify methanol dehydrogenase activity.

First claim

The invention claimed is:

1. A recombinant nucleic acid vector comprising a nucleotide sequence encoding, a polypeptide having methanol dehydrogenase activity, the nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 (mdh2-MGA3), 3 (mdh3-MGA3), or 5 (mdh2-PB1);
(ii) a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence as set forth in any one of SEQ ID NOs: 1, 3 or 5:
(iii) a nucleotide sequence which is degenerate with any one of the nucleotide sequences of SEQ ID NOs: 1, 3 or 5;
(iv) a nucleotide sequence encoding a polypeptide whose amino acid sequence is set forth in any one of SEQ ID NOs: 2 (Mdh2-MGA3), 4 (Mdh3-MGA3) or 6 (Mdh2-PB1);
(v) a nucleotide sequence encoding a polypeptide which has an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4 or 6; and
(vi) a nucleotide sequence which is complementary to the nucleotide sequence of any one of (i) to (v).

2. A recombinant polypeptide having methanol dehydrogenase activity and comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4 or 6.

3. The recombinant vector of claim 1 , wherein the at least 90% sequence identity is at least 95% sequence identity.

4. The recombinant vector of claim 1 , wherein the nucleotide sequence is operably linked to a heterologous expression control sequence.

5. A host microorganism comprising the recombinant vector of claim 1 .

6. The host microorganism of claim 5 , wherein the host microorganism is a bacterium selected from the genus Escherichia, Corynebacterium or Bacillus.

7. The host microorganism of claim 5 , wherein the host microorganism is E. coli, C. glutamicum, B. subtilis or B. methanolicus.

8. A method for introducing exogenous alcohol dehydrogenase activity in a host microorganism, the method comprising;
introducing the recombinant nucleic acid vector of claim 1 into the host microorganism; and
growing the host microorganism under conditions in which the nucleotide sequence is expressed.

9. The recombinant polypeptide according to claim 2 , wherein the amino acid sequence having at least 90% sequence identity with the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4 or 6 is an amino acid sequence having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4 or 6.

10. The recombinant nucleic acid vector of claim 1 , wherein the nucleotide sequence having at least 90% sequence identity with the nucleotide sequence as set forth in any one of SEQ ID NOs: 1, 3 or 5 is a nucleotide sequence having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the nucleotide sequence as set forth in any one of SEQ ID NOs: 1, 3 or 5.

11. The recombinant nucleic acid vector of claim 1 , wherein the amino acid sequence having at least 90% sequence identity with the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4 or 6 is an amino acid sequence having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4 or 6.

12. The method of claim 8 , wherein the host microorganism is E. coli, C. glutamicum, B. subtilis or B. methanolicus.

Assignees

• Sinvent As
• Univ Groningen
• Eth Zuerich
• Sintef Tto As

Inventors

• Brautaset Trygve
• Heggeset Tonje Marita Bjerkan
• Krog Anne
• Quax Wilhelmus Johannes
• Sibbald Mark Jan Jacobus Bernhard
• Vorholt Julia
• Müller Jonas
• Kiefer Patrick
• Potthoff Eva
• Wendisch Volker F
• Lessmeier Lennart
• Heux Stéphanie
• Portais Jean-Charles

Classifications

• C12Y101/01244

  Methanol dehydrogenase (1.1.1.244) · CPC title

• C12N2330/50

  Biochemical production, i.e. in a transformed host cell · CPC title

• C12N15/77

  for Corynebacterium; for Brevibacterium · CPC title

• C12N15/75

  for Bacillus · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title