Eicosapentaenoic acid (EPA) biosynthetic gene cluster ofShewanella oneidensis MR-1: Cloning, heterologous expression, and effects of temperature and glucose on the production of EPA inEscherichia coli

Abstract

The putative EPA synthesis gene cluster was mined from the entire genome sequence ofShewanella oneidensis MR-1. The gene cluster encodes a PKS-like pathway that consists of six open reading frames (ORFs): ORFSO1602 (multi-domain beta-ketoacyl synthase, KS-MAT-4ACPs-KR), ORFSO 1600 (acyl transferase, AT), ORFSO 1599 (multi-domain beta-ketoacyl synthase, KS-CLF-DH-DH), ORFSO 1597 (enoyl reductase, ER), ORFSO 1604 (phosphopentetheine transferase, PPT), and ORFSO1603 (transcriptional regulator). In order to prove involvement of the PKS-like machinery in EPA synthesis, a 20.195-kb DNA fragment containing the genes was amplified fromS. oneidensis MR-1 by the long-PCR method. Its identity was confirmed by the methods of restriction enzyme site mapping and nested PCR of internal genesorfSO1597 andorfSO1604. The DNA fragment was cloned intoEscherichia coli using cosmid vector SuperCos1 to form pCosEPA. Synthesis of EPA was observed in fourE. coli clones harboring pCosEPA, of which the maximum yield was 0.689% of the total fatty acids in a clone designated 9704-23. The production yield of EPA in theE. coli clone was affected by cultivation temperature, showing maximum yield at 20°C and no production at 30°C or higher. In addition, production yield was inversely proportional to glucose concentration of the cultivation medium. From the above results, it was concluded that the PKS-like modules catalyze the synthesis of EPA. The synthetic process appears to be subject to regulatory mechanisms triggered by various environmental factors. This most likely occurs via the control of gene expression, protein stability, or enzyme activity.