Transformed eukaryotic cells that directly convert xylose to xylulose

• US 7,622,284 B2
• Filed: 01/23/2003
• Issued: 11/24/2009
• Est. Priority Date: 01/23/2002
• Status: Expired due to Term

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Royal Nedalco B.V (Koninklijke Cooperatie Cosun U.A.)
• Inventors: Pronk, Jacobus Thomas, Van Der Drift, Christiaan, Harhangi, Harry Ramanoedj, Op Den Camp, Hubertus Johannes Marie
• Primary Examiner(s): Fronda; Christian L
• Application Number:
  US10/500,872
• Publication Number:
  US 20080014620A1
• Time in Patent Office: 2,497 Days
• Field of Search: 435/183, 435/233, 435/252.3, 435/320.1, 536/23.2
• US Class Current: 435/161
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12N 9/92 Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
• CPC Class Codes: C12P 13/001 Amines; Imines
• CPC Class Codes: C12P 17/10 Nitrogen as only ring hetero atom
• CPC Class Codes: C12P 35/00 Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
• CPC Class Codes: C12P 5/026 Unsaturated compounds, i.e. alkenes, alkynes or allenes
• CPC Class Codes: C12P 7/00 Preparation of oxygen-containing organic compounds
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12P 7/20 Glycerol
• CPC Class Codes: C12P 7/46 Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
• CPC Class Codes: C12P 7/54 Acetic acid vinegar C12J
• CPC Class Codes: C12P 7/56 Lactic acid
• CPC Class Codes: C12Y 207/01017 Xylulokinase (2.7.1.17)
• CPC Class Codes: C12Y 503/01005 Xylose isomerase (5.3.1.5)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention relates to host cells transformed with a nucleic acid sequence encoding a eukaryotic xylose isomerase obtainable from an anaerobic fungus. When expressed, the sequence encoding the xylose isomerase confers to the host cell the ability to convert xylose to xylulose which may be further metabolised by the host cell. Thus, the host cell is capable of growth on xylose as carbon source. The host cell preferably is a eukaryotic microorganism such as a yeast or a filamentous fungus. The invention further relates to processes for the production of fermentation products such as ethanol, in which a host cell of the invention uses xylose for growth and for the production of the fermentation product. The invention further relates to nucleic acid sequences encoding eukaryotic xylose isomerases and xylulose kinases as obtainable from anaerobic fungi.

Claims

1. A process for producing ethanol, comprising the steps of:

(a) fermenting medium containing a source of xylose with a cultured yeast cell that

(i) is transformed with a nucleic acid expression construct comprising a nucleotide sequence that encodes xylose isomerase protein, the amino acid sequence of which;

(A) is at least 950% identical with SEQ ID NO;

1,

(B) comprises a first xylose isomerase signature pattern;

(C) comprises a second xylose isomerase signature pattern;

(D) comprises a catalytic triad including the following four residues at the indicated positions in SEQ ID NO;

1;

His 102 plus Asp 105, and Asp 340 and Lys 235;

(E) comprises at least one Mg-binding site that is residue Glu 233 of

SEQ ID NO;

1; and

(ii) operative linked to the nucleotide sequence of (i), a promoter that drives active expression of the xylose isomerase coding sequence in the transformed cell,

wherein the expression construct is expressible in said cell, and expression thereof confers on the cell the ability to directly isomerize xylose to xylulose, and thereby, to produce ethanol; and

(b) optionally, recovering the ethanol from said medium.

2. The process according to claim 1, wherein the medium also contains a source of glucose.

3. The process according to claim 1, wherein the production of ethanol occurs at a rate of at least 0.5 g ethanol per liter per hour.

4. The process according to claim 1, wherein the ethanol yield is at least 50%.

5. A process for producing a non-ethanolic fermentation product fermentation product, which process comprises the steps of:

(a) fermenting a medium containing a source of xylose with a cultured yeast cell that;

(i) is transformed with a nucleic acid expression construct comprising a nucleotide sequence that encodes xylose isomerase protein, the amino acid sequence of which

(A) is at least 950% identical with SEQ ID NO;

1,

(B) comprises a first xylose isomerase signature pattern;

(C) comprises a second xylose isomerase signature pattern;

(D) comprises a catalytic triad including the following four residues at the indicated positions in SEQ ID NO;

1;

His 102 plus Asp 105, and Asp 340 and Lys 235;

(E) comprises at least one Mg-binding site that is residue Glu 233 of SEQ ID NO;

1; and

which nucleotide sequence is operative linked to a promoter that drives active expression of the xylose isomerase coding sequence in the transformed cell,

(ii) expresses one or more enzymes that confers on the cell the ability to produce a non-ethanolic fermentation product,

wherein, expression of the construct confers on the cell the ability to directly ferment and isomerize xylose to xylulose and thereby produce said non-ethanolic fermentation product; and

(b) optionally, recovering the non-ethanolic fermentation product from said medium.

6. The process of claim 5 wherein the fermentation product is selected from the group consisting of lactic acid, acetic acid, succinic acid, an amino acid, 1, 3-propanediol, ethylene, and glycerol.

7. The process of claim 5 wherein the fermentation product is a β

-lactam antibiotic or a cephalosporin.

8. A process according to claim 5, wherein the medium also contains a source of glucose.

9. The process of claim 5, wherein the cell further comprises a genetic modification that results in decreased alcohol dehydrogenase activity so as to reduce ethanol production by said cell.

10. A cultured yeast cell transformed with a nucleic acid expression construct which construct comprises:

(a) a nucleotide sequence that encodes xylose isomerase protein, the amino acid sequence of which

(i) is at least 950o identical with SEQ ID NO;

1,

(ii) comprises a first xylose isomerase signature pattern;

(iii) comprises a second xylose isomerase signature pattern;

(iv) comprises a catalytic triad including the following four residues at the indicated positions in SEQ ID NO;

1;

His 102 plus Asp 105, and Asp 340 and Lys 235;

(v) comprises at least one Mg-binding site that is residue Glu 233 of SEQ ID NO;

1; and

(b) operative linked to the nucleotide sequence of (i), a promoter that drives active expression of the xylose isomerase coding sequence in the transformed cell,

wherein, said expression construct is expressible in said cell and expression thereof confers on the cell the ability to directly isomerize xylose to xylulose.

11. The cell according to claim 10, wherein the nucleotide sequence encodes a xylose isomerase the amino acid sequence of which is SEQ ID NO:

1.

12. The yeast cell of claim 10 that is a member of a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia.

13. The yeast cell according to claim 12 that is a member of a species selected from the group consisting of S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, and K. fragills.

14. The cell according to claim 10, wherein the promoter is insensitive to catabolite repression in the cell.

15. The cell according to claim 10 that has been further genetically modified to confer on the cell one or more of the following properties:

(1) increased transport of xylose into the host cell;

(2) increased xylulose kinase activity;

(3) increased flux of the pentose phosphate pathway;

(4) decreased sensitivity to catabolite repression;

(5) increased tolerance to ethanol, osmolarity or organic acids;

or

(6) decreased production of by-products, in comparison to a similar cell that has not undergone said genetic modification.

16. The cell according to claim 15, wherein the nucleotide sequence encodes a xylose isomerase the amino acid sequence of which is SEQ ID NO:

1.

17. The cell according to claim 15, wherein the genetic modification that results in said properties (1) - (6) is

(A) overexpression of an endogenous gene,

(B) expression of a heterologous gene, or

(ii) a pentose transporter;

(iii) a xylulose kinase;

(iv) an enzyme from the pentose phosphate pathway,

(v) a glycolytic enzyme, or

(vi) an ethanologenic enzyme.

18. The cell according to claim 15 wherein the genetic modification that results in said properties (1) - (6) is one that causes inactivation of one of the following endogenous genes:

(a) a gene encoding a hexose kinase

(b) Saccharomyces MIGJ1 gene;

(c) Saccharomyces MIG2 gene;

or

(d) a gene homologous to (a), (b) or (c) and which hybridizes thereto.

19. The cell according to claim 10, that further expresses one or more enzymes that confers on the cell the ability to produce a non-ethanolic fermentation product.

20. The cell according to claim 19 wherein said fermentation product is selected from the group consisting of lactic acid, acetic acid, succinic acid, amino acids, 1 ,3-propanediol, ethylene, and glycerol.

21. The cell according to claim 19 wherein said fermentation product is a β

-lactam antibiotic or a cephalosporin.

22. The cell according to claim 19 in which alcohol dehydrogenase activity is genetically decreased so as to reduce ethanol production by said cell.