Method for the recombinant production of 1,3-propanediol

• US 6,136,576 A
• Filed: 11/13/1997
• Issued: 10/24/2000
• Est. Priority Date: 11/13/1996
• Status: Expired due to Term

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Trimbur, Donald, Chase, Matthew W., Dunn-Coleman, Nigel S, Diaz-Torres, Maria
• Primary Examiner(s): Nashed, Nashaat T.
• Application Number:
  US08/969,683
• Time in Patent Office: 1,076 Days
• Field of Search: 435/158, 435/183, 435/189, 435/232, 435/320.1, 435/257.3, 435/252.33, 536/23.2, 536/23.1, 536/23.7, 530/350
• 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

The present invention provides an improved method for the production of 1,3-propanediol from a variety of carbon sources in an organism capable of 1,3-propanediol production and comprising DNA encoding protein X of a microorganismal dehydratase or protein X in combination with at least one of protein 1, protein 2 and protein 3, which proteins are comparable to those encoded by orfY, orfX and orfW, respectively from a microorganismal dha regulon. The protein X may be isolated from a diol dehydratase or a glycerol dehydratase. The present invention also provides host cells comprising protein X that are capable of increased production of 1,3-propanediol.

Claims

1. A method for the production of 1,3-propanediol from a recombinant microorganism comprising the step of culturing a recombinant microorganism capable of producing 1,3 propanediol and comprising at least nucleic acid encoding a dehydratase activity and a nucleic acid encoding protein X in the presence of at least one carbon source capable of being converted to 1,3 propanediol in said recombinant microorganism and under conditions suitable for the production of 1,3 propanediol, wherein the gene encoding protein X is isolated from a glycerol dehydratase gene cluster from an organism selected from the group of genera consisting of Klebsiella and Citrobacter or wherein the gene encoding protein X is isolated from a diol dehydratase gene cluster from an organism selected from the group of genera consisting of Klebsiella Clostridium and Salmonella, wherein production of 1,3-propanediol is greater in the microorganism than in the absence of said nucleic acid encoding protein X and wherein-protein X has no enzymatic activity, and wherein the carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and a one carbon substrate.

2. The method of claim 1 further comprising the step of introducing at least one gene encoding a protein selected from the group consisting of protein 1, protein 2 and protein 3 into the microorganism, wherein said protein 1 has an amino acid sequence corresponding to SEQ ID NO:

60 or SEQ ID NO;

61, wherein protein 2 has an amino acid sequence corresponding to SEQ ID NO;

62 or SEQ ID NO;

63, and wherein protein 3 has an amino acid sequence corresponding to SEQ ID NO;

64 or SEQ ID NO;

65 and, wherein the production of 1,3-propanediol is higher in the microorganism than in the absence of said gene.

3. The method of claim 1 further comprising the step of recovering the 1,3 propanediol.

4. The method of claim 1 wherein the gene encoding a dehydratase activity is heterologous to the microorganism.

5. The method of claim 1 wherein the gene encoding a dehydratase activity is homologous to the microorganism.

6. The method of claim 1 wherein the 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.

7. The method of claim 6 wherein the microorganism is selected from the group consisting of E. coli and Klebsiella ssp.

8. The method of claim 1 wherein the gene encoding protein X is integrated into the chromosomal DNA of the host genome.

9. The method of claim 2 wherein at least one gene encoding a protein selected from protein 1, protein 2 and protein 3 is integrated into the chromosomal DNA of the host genome.

10. The method of claim 1 wherein the carbon source is glucose.

11. The method of claim 1 wherein the gene encoding protein X has the sequence as shown in SEQ ID NO:

59.

12. The method of claim 2 wherein protein 1 has the sequence as shown in SEQ ID NO:

60 or SEQ ID NO;

61.

13. The method of claim 2 wherein protein 2 has the sequence as shown in SEQ ID NO:

62 or SEQ ID NO;

63.

14. The method of claim 2 wherein protein 3 has the sequence as shown in SEQ ID NO:

64 or SEQ ID NO;

65.

15. The method of claim 1, wherein said protein X of K. pneumoniae glycerol dehydratase is encoded by the nucleic acid sequence of residues 9749-11572 of SEQ ID NO:

19.

16. The method of claim 1, wherein said protein X is from C. freundii.

17. The method of claim 1, wherein said protein X is encoded by the ORF Z of the dha regulon of the genus Citrobacter.