Gene coding for eicosapentaenoic acid synthesizing enzymes and process for production of eicosapentaenoic acid

• US 5,683,898 A
• Filed: 01/20/1995
• Issued: 11/04/1997
• Est. Priority Date: 05/15/1992
• Status: Expired due to Fees

• Current Assignee: Sagami Chemical Research Center
• Original Assignee: Sagami Chemical Research Center
• Inventors: Yamada, Akiko, Kato, Seishi, Yazawa, Kazunaga, Kondo, Kiyosi
• Primary Examiner(s): Jagannathan, Vasu S.
• Assistant Examiner(s): KIM, HYO
• Application Number:
  US08/375,709
• Time in Patent Office: 1,019 Days
• Field of Search: 435/69.1, 435/134, 435/183, 435/240.2, 435/252.3, 435/320.1, 435/136, 536/22.1, 536/23.1, 536/23.2, 536/23.7
• US Class Current: 435/136
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

There is provided an advantageous process for production of EPA by a gene recombinant technique wherein genes coding for biosynthesis enzymes for eicosapentaenoic acid (EPA) useful as pharmaceuticals, agrochemicals, foods, feeds or the like is obtained from microorganisms.

EPA is produced by obtaining genes coding for eicosapentaenoic acid (EPA) biosynthesis enzymes, constructing a plasmid by joining the genes to a vector, transforming E. coli with the plasmid, and culturing the transformed E. coli.

Claims

1. An isolated DNA encoding a group of eicosapentaenoic acid synthesis enzymes derived from a microorganism belonging to the genus Shewanella.

3. A plasmid comprising the isolated DNA of claim 1.

5. A bacterium transformed with a plasmid according to claim 3.

7. A process for producing eicosapentaenoic acid, comprising the step of culturing a bacterium according to claim 5.

2. An isolated DNA encoding a group of eicosapentaenoic acid synthesis enzymes having the nucleotide sequence shown in SEQ ID NO:

1.

4. A plasmid comprising the isolated DNA of claim 2.

6. A bacterium transformed with a plasmid according claim 4.

8. A process for producing eicosapentaenoic acid, comprising the step of culturing a bacterium according to claim 6.

9. A DNA encoding a group of eicosapentaenoic acid synthesis enzymes, which is obtainable by the steps of:

(1) culturing a microorganism belonging to the genus Shewanella capable of producing eicosapentaenoic acid to obtain cultured cells;

(2) extracting chromosomal DNA from the cultured cells;

(3) partially digesting the chromosomal DNA with restriction enzyme Sau3A1 to obtain DNA fragments;

(4) inserting the DNA fragments into cosmid pWE15 completely digested with restriction enzyme BamH1 to obtain recombinant expression vectors;

(5) transforming Escherichia coli cells with the recombinant expression vectors to obtain transformants;

(6) culturing the transformants to obtain cultured transformed cells;

(7) detecting eicosapentaenoic acid in the cultured transformed cells to select a positive transformed cell capable of producing eicosapentaenoic acid; and

(8) recovering a DNA encoding a group of eicosapentaenoic acid synthesis enzymes by cleaving the recombinant expression vector extracted from the positive transformant with restriction enzyme Sau3A1.