Production of ethanol from xylose

• US 6,582,944 B1
• Filed: 01/08/1999
• Issued: 06/24/2003
• Est. Priority Date: 04/06/1990
• Status: Expired due to Fees

• Current Assignee: Xyrofin Oy
• Original Assignee: Xyrofin Oy
• Inventors: Ojamo, Heikki, Keränen, Sirkka, Walfridsson, Mats, Hallborn, Johan, Airaksinen, Ulla, Penttilä, Merja, Hahn-Hägerdal, Bärbel
• Primary Examiner(s): Slobodyansky, Elizabeth
• Application Number:
  US09/184,965
• Time in Patent Office: 1,628 Days
• Field of Search: 435/41, 435/155, 435/157, 435/161, 435/163, 435/440, 435/483, 435/489, 435/254.11, 435/254.2, 435/254.21, 435/254.22, 435/254.23, 435/254.4, 536/23.2
• US Class Current: 435/161
• 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 ethanol comprising:

(a) isolating from a donor yeast or fungus a first polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

3, or a fragment thereof, wherein the polypeptide or the fragment has xylose reductase activity, and a second polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

7, or a fragment thereof, wherein the polypeptide or the fragment has xylitol dehydrogenase activity;

(b) constructing yeast vectors comprising said polynucleotides;

(c) transforming a yeast host cell with the vectors obtained in step (b) to obtain a recombinant yeast strain;

(d) cultivating said recombinant yeast strain in a xylose containing medium permitting the expression of the first polynucleotide and the second polynucleotide; and

(e) isolating and purifying ethanol formed in the medium.

2. The process of claim 1, wherein

said first polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 2 or a fragment thereof, and

wherein the second polynucleotide comprises the nucleic acid sequence of SEQ ID NO;

6 or a fragment thereof.

3. The process of claim 1, wherein the yeast host is selected from the group consisting of Saccharomyces cerevisiae, a Kluyveromyces spp., Schizosaccharomyces pombe and a Pichia spp.

4. The process of claim 3, wherein the yeast host is of the species Saccharomyces cerevisiae.

5. A process for producing ethanol comprising:

(a) cultivating a recombinant yeast strain transformed with a first polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

3, or a fragment thereof, wherein the polypeptide or the fragment has xylose reductase activity, and a second polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

7, or a fragment thereof, wherein the polypeptide or the fragment has xylitol dehydrogenase activity, in a xylose-containing medium; and

(b) isolating and purifying ethanol formed in the medium;

wherein said first polynucleotide and second polynucleotide are isolated from a donor yeast or fungus.

8. The process of any one of claim 5, 6, or 7, wherein said first polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

2 or a fragment thereof, and wherein the second polynucleotide comprises the nucleic acid sequence of SEQ ID NO;

6 or a fragment thereof.

9. The process of any one of claim 5, 6, or 7, wherein the yeast host is selected from the group consisting of Saccharomyces cerevisiae, a Kluyveromyces spp., Schizosaccharomyces pombe and a Pichia spp.

10. The process of claim 9, wherein the yeast host is of the species Saccharomyces cerevisiae.

6. A process for producing ethanol comprising:

(a) isolating from a donor yeast or fungus a first polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

3, or a fragment thereof, wherein the polypeptide or the fragment has xylose reductase activity, and a second polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

7, or a fragment thereof, wherein the polypeptide or the fragment has xylitol dehydrogenase activity;

(b) constructing yeast vectors comprising said polynucleotides;

(c) transforming a yeast cell having xylose reductase activity and/or xylitol dehydrogenase activity with one of the vectors obtained in step (b) to obtain a recombinant yeast strain having an enhanced ability to utilize xylose;

(d) cultivating said recombinant yeast strain in a xylose containing medium permitting the expression of the polynucleotide as transformed; and

(e) isolating and purifying ethanol formed in the medium.

7. A process for producing ethanol comprising:

(a) transforming a host yeast strain with a first polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

3, or a fragment thereof, wherein the polypeptide or the fragment has xylose reductase activity, and/or with a second polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

7, or a fragment thereof, wherein the polypeptide or the fragment has xylitol dehydrogenase activity;

(b) cultivating the recombinant yeast strain transformed as described in step (a) in a xylose-containing medium; and

(c) isolating and purifying ethanol formed in the medium.

11. A process for producing ethanol comprising:

(a) cultivating Saccharomyces cerevisiae H550 in a xylose containing medium; and

(b) isolating and purifying ethanol formed in the medium.