Method for the production of 1,3-propanediol by recombinant microorganisms

• US 6,013,494 A
• Filed: 11/12/1997
• Issued: 01/11/2000
• Est. Priority Date: 11/13/1996
• Status: Expired due to Term

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.), Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.), Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Nair, Ramesh V., Nakamura, Charles E., Diaz-Torres, Maria, La Reau, Richard D., Payne, Mark S., Picataggio, Stephen K., Hsu, Amy Kuang-Hua, Trimbur, Donald E., Gatenby, Anthony A., Nagarajan, Vasantha, Whited, Gregory M., Haynie, Sharon L.
• Primary Examiner(s): RAILEY, JOHNNY F
• Application Number:
  US08/968,563
• Time in Patent Office: 790 Days
• Field of Search: 435/69.1, 435/158, 435/252.3, 435/252.33, 435/254.21
• US Class Current: 435/158
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: Y10S 530/824 Yeasts

Abstract

Recombinant organisms are provided comprising genes encoding glycerol-3-phosphate dehydrogenase, glycerol-3-phosphatase, glycerol dehydratase and 1,3-propanediol oxidoreductase activites useful for the production of 1,3-propanediol from a variety of carbon substrates.

Claims

1. A method for the production of 1,3-propanediol from a recombinant microorganism comprising:

(i) transforming a suitable host microorganism with one or more transformation cassettes each of which comprises at least one of

(a) a gene encoding a glycerol-3-phosphate dehydrogenase activity;

(b) a gene encoding a glycerol-3-phosphatase activity;

(c) genes encoding a dehydratase activity; and

(d) a gene encoding 1,3-propanediol oxidoreductase activity,

wherein all of the genes of (a)-(d) are introduced into the host microorganism;

(ii) culturing the transformed host microorganism under suitable conditions in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, or a one-carbon substrate whereby 1,3-propanediol is produced; and

(iii) recovering the 1,3-propanediol.

2. The method of claim 1 wherein the suitable host microorganism is selected from the group consisting of bacteria, yeast, and filametous fungi.

3. The method of claim 2 wherein the suitable host microorganism is selected from the group of genera consisting of Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces and Pseudomonas.

4. The method of claim 3 wherein the suitable host microorganism is selected from the group consisting of E. coli, Klebsiella spp., and Saccharomyces spp.

5. The method of claim 1 wherein the transformed host microorganism is a Klebsiella spp. transformed with a transformation cassette comprising the genes GPD1 and GPP2.

6. The method claim 1 wherein the carbon source is glucose.

7. The method of claim 1 wherein the gene encoding a glycerol-3-phosphate dehydrogenase activity is selected from the group consisting of

(a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:

11, in SEQ ID NO;

12, and in SEQ ID NO;

13, or an enzymatically active fragment thereof;

(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;

0.1×

SSC, 0.1% SDS at 65°

C.; and

(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).

8. The method of claim 1 wherein the gene encoding a glycerol-3-phosphatase activity is selected from the group consisting of

(a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:

33 and in SEQ ID NO;

17, or an enzymatically active fragment thereof;

(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;

0.1×

SSC, 0.1% SDS at 65°

C.; and

(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).

9. The method of claim 1 wherein the gene encoding a glycerol-3-phosphatase activity is a glycerol kinase gene selected from the group consisting of

(a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:

18, or an enzymatically active fragment thereof;

(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;

0.1×

SSC, 0.1% SDS at 65°

C.; and

(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).

10. The method of claim 1 wherein the genes encoding a dehydratase activity comprise dhaB1, dhaB2 and dhB3, and are selected from the group consisting of

(a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:

34, SEQ ID NO;

35, and SEQ ID NO;

36, or an enzymatically active fragment thereof;

(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;

0.1×

SSC, 0.1% SDS at 65°

C.; and

(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).

11. The method of claim 1 wherein the gene encoding a 1,3-propanediol oxidoreductase activity selected from the group consisting of

(a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:

37, or an enzymatically active fragment thereof;

(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;

0.1×

SSC, 0.1% SDS at 65°

C.; and

(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).

12. A method for the production of 1,3-propanediol from a recombinant microorganism comprising:

(i) culturing, under suitable conditions in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, or a one-carbon substrate, a transformed host microorganism comprising

(a) a gene encoding a glycerol-3-phosphate dehydrogenase activity;

(b)a gene encoding a glycerol-3-phosphatase activity;

(c)genes encoding a dehydratase activity; and

(d) a gene encoding 1,3-propanediol oxidoreductase activity,

wherein all of the genes (a)-(d) are exogenous to the host microorganism, whereby 1,3-propanediol is produced; and

(ii) recovering the 1,3-propanediol.

13. A host cell transformed with a group of genes comprising:

(1) a gene encoding a glycerol-3-phosphate dehydrogenase enzyme corresponding to the amino acid sequence given in SEQ ID NO;

11;

(2) a gene encoding a glycerol-3-phosphatase enzyme corresponding to the amino acid sequence given in SEQ ID NO;

17;

(3) a gene encoding the a subunit of the glycerol dehydratase enzyme corresponding to the amino acid sequence given in SEQ ID NO;

34;

(4) a gene encoding the β

subunit of the glycerol dehydratase enzyme corresponding to the amino acid sequence given in SEQ ID NO;

35;

(5) a gene encoding the γ

subunit of the glycerol dehydratase enzyme corresponding to the amino acid sequence given in SEQ ID NO;

36; and

(6) a gene encoding the 1,3-propanediol oxidoreductase enzyme corresponding to the amino acid sequence given in SEQ ID NO;

37,

whereby the transformed host cell produces 1,3-propanediol on at least one substrate selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides or from a one-carbon substrate.