Zymomonas with improved ethanol production in medium containing concentrated sugars and acetate

• US 7,803,623 B2
• Filed: 10/30/2008
• Issued: 09/28/2010
• Est. Priority Date: 10/30/2007
• Status: Expired due to Fees

• Current Assignee: Alliance For Sustainable Energy LLC, Sustainable Technology Corporation
• Original Assignee: Alliance For Sustainable Energy LLC, E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Zhang, Yuying, Knoke, Kyle, Chou, Yat-Chen, Caimi, Perry G., Franden, Mary Ann, Tao, Luan, Zhang, Min, Viitanen, Paul V.
• Primary Examiner(s): Mondesi; Robert B
• Assistant Examiner(s): Portner; Ginny
• Application Number:
  US12/261,166
• Publication Number:
  US 20090221078A1
• Time in Patent Office: 698 Days
• Field of Search: None
• US Class Current: 435/471
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• 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: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/92 Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
• CPC Class Codes: C12P 7/065 with microorganisms other than yeasts
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Through screening of a Zymomonas mutant library the himA gene was found to be involved in the inhibitory effect of acetate on Zymomonas performance. Xylose-utilizing Zymomonas further engineered to reduce activity of the himA gene were found to have increased ethanol production in comparison to a parental strain, when cultured in medium comprising xylose and acetate.

Claims

1. A recombinant microorganism of the genus Zymomonas which utilizes xylose to produce ethanol by fermentation in a mixed sugar medium, said microorganism comprising at least one genetic modification which reduces expression of the endogenous integration host factor alpha subunit (HimA) protein encoded by a himA gene.

2. The recombinant microorganism of claim 1, wherein the genetic modification is a modification of the himA gene selected from the group consisting of insertion, deletion, mutation, cosuppression, and antisense RNA expression.

3. The genetic modification of claim 2, wherein the himA gene is made non-functional.

4. The genetic modification of claim 3, wherein an insertion is introduced into the himA gene by homologous recombination.

5. The microorganism of claim 1, comprising heterologous genes encoding xylose isomerase, xylulokinase, transaldolase and transketolase.

6. A microorganism according to claim 5, wherein the heterologous genes encoding xylose isomerase, xylulokinase, transaldolase and transketolase are from bacteria selected from the group consisting of Xanthomonas, Klebsiella, Escherichia, Rhodobacter, Flavobacterium, Acetobacter, Gluconobacter, Rhizobium, Agrobacterium, Salmonella and Pseudomonads.

7. A microorganism according to claim 6, wherein the heterologous genes are from E. coli.

8. A microorganism according to claim 7, identified as strain ZW801-4:

;

Δ

himA or strain AcR#3 which are derived from strain ATCC PTA 7858.

9. The microorganism of claim 1, wherein the microorganism has improved fermentation performance in the presence of acetate as compared to a microorganism with no genetic modification which reduces expression of the endogenous integration host factor alpha subunit (HimA) protein.

10. The microorganism of claim 9, wherein the microorganism produces at least about 4% more ethanol in the presence of acetate than an isogenic strain without reduced expression of the endogenous integration host factor alpha subunit (HimA) protein.

11. A process for generating the microorganism of claim 1 comprising:

a) providing a recombinant Zymomonas strain which utilizes xylose to produce ethanol under suitable conditions wherein the genome of said strain expresses endogenous integration host alpha subunit (HimA) protein; and

b) modifying the genome of said strain wherein said modifying reduces expression of the endogenous integration host factor alpha subunit (HimA) protein.

12. A process according to claim 11, wherein the recombinant Zymomonas strain of (a) is selected from the group consisting of ATCC31821/pZB5, ATCC PTA7858/ZW658, ZW800, ZW801-4, and ZW801-6, where strains [ZW658,] ZW800, ZW801-4 and ZW801-6 are derived from strain ATCC PTA 7858/ZW658.

13. A process according to claim 11, wherein the genetic modification is a modification of the himA gene selected from the group consisting of insertion, deletion, mutation, cosuppression, and antisense RNA expression.