Xylose utilizing zymomonas mobilis with improved ethanol production in biomass hydrolysate medium

• US 8,476,048 B2
• Filed: 12/12/2011
• Issued: 07/02/2013
• Est. Priority Date: 12/17/2010
• Status: Active Grant

• Current Assignee: Sustainable Technology Corporation
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Caimi, Perry G, Hitz, William D, Stieglitz, Barry, Viitanen, Paul V
• Primary Examiner(s): NOAKES, SUZANNE MARIE
• Application Number:
  US13/316,597
• Publication Number:
  US 20120329114A1
• Time in Patent Office: 568 Days
• Field of Search: None
• US Class Current: 435/161
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12P 7/065 with microorganisms other than yeasts
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Xylose-utilizing, ethanol producing strains of Zymomonas mobilis with improved performance in medium comprising biomass hydrolysate were isolated using an adaptation process. Independently isolated strains were found to have independent mutations in the same coding region. Mutation in this coding may be engineered to confer the improved phenotype.

Claims

1. A recombinant, xylose-utilizing, ethanol-producing microorganism of the genus Zymomonas, having at least one genetic modification in the zmo1432 open reading frame, wherein said zmo1432 open reading frame having at least one genetic modification encodes a polypeptide having an amino acid sequence with at least about 95% identity to the amino acid sequence as set forth in SEQ ID NO:

2, wherein the presence of the mutant protein increases ethanol production by the Zymomonas during fermentation in biomass hydrolysate medium as compared to production by a Zymomonas lacking the genetic modification under the same conditions.

2. The recombinant Zymomonas of claim 1 wherein the genetic modification is a mutation in the zmo1432 coding region that results in an amino acid substitution selected from the group consisting of:

1) a substitution at position 366 in SEQ ID NO;

2 of arginine for threonine; and

     2) a substitution at position 117 in SEQ ID NO;

2 of phenylalanine for serine.

3. The recombinant Zymomonas of claim 1 having a higher tolerance to biomass hydrolysate as compared with a Zymomonas lacking the at least one genetic modification in the zmo1432 open reading frame.

6. A method of the production of ethanol comprising:

a) providing the recombinant Zymomonas of claim 1;

b) providing a biomass hydrolysate medium comprising xylose; and

c) growing the Zymomonas of a) in the biomass hydrolysate medium of b) wherein ethanol is produced.

4. A polynucleotide encoding a protein having the amino acid sequence selected from the group consisting of SEQ ID NO:

3 and SEQ ID NO;

4.

5. A method for the production of a recombinant Zymonomas ethanologen comprising:

a) providing a xylose-utilizing, ethanol-producing microorganism of the genus Zymomonas comprising a zmo1432 open reading frame encoding a polypeptide having the amino acid sequence as set forth in SEQ ID NO;

2; and

b) introducing a mutation in the zmo1432 open reading frame of a) such that expression of the mutated open reading frame expresses a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO;

3 and SEQ ID NO;

4.