Xylose utilization by recombinant yeasts

• US 5,866,382 A
• Filed: 11/03/1994
• Issued: 02/02/1999
• Est. Priority Date: 04/06/1990
• Status: Expired due to Term

• Current Assignee: Xyrofin Oy
• Original Assignee: Xyrofin Oy
• Inventors: Keranen, Sirkka, Airaksinen, Ulla, Penttila, Merja, Ojamo, Heikki, Hahn-Hagerdal, Barbel, Walfridsson, Mats, Hallborn, Johan
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/336,198
• Time in Patent Office: 1,552 Days
• Field of Search: 435/158, 435/161-165, 435/172.3, 435/155, 435/157
• US Class Current: 435/158
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/905 in yeast
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

This invention relates to recombinant-DNA-technology. Specifically, this invention relates to new recombinant yeast strains transformed with xylose reductase and/or xylitol dehydrogenase enzyme genes. A yeast strain transformed with the xylose reductase gene is capable of reducing xylose to xylitol and consequently of producing xylitol in vivo. If both of these genes are transformed into a yeast strain, the resultant strain is capable of producing ethanol on xylose containing medium during fermentation. Further, the said new yeast strains are capable of expressing the said two enzymes. Xylose reductase produced by these strains can be used in an enzymatic process for the production of xylitol in vitro.

Claims

1. A process for producing xylitol, which process comprises:

(a) cultivating in a xylose-containing medium a yeast strain selected from the group consisting of Saccharomyces spp., a Kluyveromyces spp. and a Schizosaccharomyces spp., transformed with a DNA molecule encoding a xylose reductase enzyme of a yeast or a mold, which DNA molecule, when transformed into said yeast strain, confers to said yeast strain the ability of reducing xylose to xylitol; and

(b) recovering xylitol formed from the medium.

2. The process of claim 1, wherein said transformed yeast strain belongs to the species Saccharomyces cerevisiae.

3. The process of claim 1, wherein said medium also contains glucose.

4. The process of claim 1, wherein said DNA molecule codes for a polypeptide comprising the amino acid sequence of SEQ. ID. NO. 3, or a fragment thereof wherein said polypeptide or fragment displays xylose reductase activity.

5. The process of claim 1, wherein said transformed yeast strain is selected from the group consisting of Saccharomyces cerevisiae H475, H477, H479 and H481.

6. The process of claim 1, wherein at least one additional carbon source is present in the medium to enhance regeneration of a cofactor required for xylose reductase activity.

7. The process of claim 1, wherein the said additional carbon source is selected from the group comprising ethanol, glucose and glycerol.

8. The process of claim 1, wherein said Saccharomyces species is Saccharomyces cerevisiae and wherein said Schizosaccharomyces species is Schizosaccharomyces pombe.

9. A process for producing xylulose, which process comprises:

(a) cultivating in a xylose-containing medium a yeast strain selected from the group consisting of Saccharomyces spp., a Kluyveromyces spp. and a Schizosaccharomyces spp., transformed with a DNA molecule encoding a xylose reductase enzyme of a yeast or a mold, which DNA molecule, when transformed into said yeast strain, confers the ability to said yeast strain of reducing xylose to xylitol; and

wherein said transformed yeast strain is further transformed with a second DNA molecule encoding a xylitol dehydrogenase enzyme of a yeast or a mold, which second DNA molecule, when transformed into said transformed yeast, confers to said yeast the ability to convert xylitol to xylulose.

10. The process of claim 9, wherein said second DNA molecule encodes a polypeptide comprising the amino acid sequence of SEQ. ID. NO. 7, or a fragment thereof wherein said polypeptide or fragment displays xylitol dehydrogenase activity.

11. The process of claim 9, wherein said transformed yeast strain is selected from the group consisting of Saccharomyces cerevisiae H492, H493 and H550.

12. The process of claim 9, wherein the xylitol dehydrogenase enzyme is effective to regenerate a cofactor required for xylitol reductase activity.

13. The process of claim 9, wherein said second DNA molecule has a nucleotide sequence of DNA encoding a xylitol dehydrogenase of an organism being a Kluyveromyces spp., a Candida spp., a Pichia spp., Pachysolen tannophilus, or Petromyces albertensis.

14. The process of claim 9, wherein said Saccharomyces species is Saccharomyces cerevisiae and wherein said Schizosaccharomyces species is Schizosaccharomyces pombe.

15. A process for producing xylitol, which process comprises:

(a) cultivating in a xylose-containing medium a yeast strain selected from the group consisting of Saccharomvces spp., a Kluyveromyces spp and a Schizosaccharomyces spp., transformed with a DNA molecule encoding a xylose reductase enzyme, which DNA molecule, when transformed into said yeast strain, confers to said yeast strain the ability of reducing xylose to xylitol; and

(b) recovering xylitol formed from the medium;

wherein said DNA molecule has a nucleotide sequence of DNA encoding a xylose reductase enzyme of an organism being a Candida spp., an Enterobacter spp., a Corynebacterium spp., a Kluyveromyces spp., a Pichia spp., Pachysolen tannophilus or Penicillium chrysogenum.