| Shewanella marinintestina MCCC 1A01703, a marine bacterium producing EPA (eicosapentaenoic acid), was isolated from the intestine of marine animal. A total length of 18.4 kb of the EPA biosynthetic gene cluster, including pfaA,pfaB, pfaC and pfaD, were cloned by PCR, Overlap-PCR and Gibson Assemble methods. The similarity of this gene cluster with the EPA gene cluster from S. pneunatophri SCRC-2738 was 88%. And sequences analysis revealed that all encoded polyketide synthases. The complete gene cluster was inserted into the low copy number expression vector pACYC-Trc as a framework by Gibson Assemble method, to construct plasmid pLYSCY03. The entD gene, which performs the same function as pfaE, was fished from E. coli DH5α, cloned into expression vector pTrc99a, to construct recombinant plasmid pLYSCY01. The two expression plasmids were co-transformed into E. coli DH5α, and obtained an EPA engineered strain. The results showed that the EPA polyketide biosynthesis gene cluster contained in marine animal intestine S. marinintestina, and entD gene in E. coli DH5α could replace pfaE gene to synthesize EPA cooperate with the fished EPA synthase. |
