DHA Production in Escherichia coli by Expressing Reconstituted Key Genes of Polyketide Synthase Pathway from Marine Bacteria

• Published in: PloS one 2016-09, Vol.11 (9), p.e0162861-e0162861
• Main Authors: Peng, Yun-Feng, Chen, Wen-Chao, Xiao, Kang, Xu, Lin, Wang, Lian, Wan, Xia
• Format: Article
• Language: English
• Subjects: Agriculture; Alteromonadaceae - enzymology; Alteromonadaceae - genetics; Amino Acid Sequence; Amino acids; Arachidonic acid; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biology; Biology and Life Sciences; Biosynthesis; Biosynthetic Pathways - genetics; Cerulenin; Cerulenin - pharmacology; Cloning; Colwellia; Deoxyribonucleic acid; DNA; Docosahexaenoic acid; Docosahexaenoic Acids - biosynthesis; E coli; Eicosapentaenoic acid; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli - metabolism; Fatty Acid Synthesis Inhibitors - pharmacology; Fatty acids; Gene expression; Gene Expression Regulation, Bacterial; Genes; Genetic aspects; Laboratories; Lipids; Low temperature; Metabolic Engineering - methods; Monounsaturated fatty acids; Multigene Family; Omega 3 fatty acids; Physical Sciences; Plasmids; Polyketide synthase; Polyketide Synthases - genetics; Polyketide Synthases - metabolism; Proteins; Research and Analysis Methods; Reverse Transcriptase Polymerase Chain Reaction; Saturated fatty acids; Seawater - microbiology; Sequence Homology, Amino Acid; Shewanella baltica; Strains (organisms); Supplements; Transferases (Other Substituted Phosphate Groups) - genetics; Transferases (Other Substituted Phosphate Groups) - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0162861

The gene encoding phosphopantetheinyl transferase (PPTase), pfaE, a component of the polyketide synthase (PKS) pathway, is crucial for the production of docosahexaenoic acid (DHA, 22:6ω3), along with the other pfa cluster members pfaA, pfaB, pfaC and pfaD. DHA was produced in Escherichia coli by co-expressing pfaABCD from DHA-producing Colwellia psychrerythraea 34H with one of four pfaE genes from bacteria producing arachidonic acid (ARA, 20:4ω6), eicosapentaenoic acid (EPA, 20:5ω3) or DHA, respectively. Substitution of the pfaE gene from different strain source in E. coli did not influence the function of the PKS pathway producing DHA, although they led to different DHA yields and fatty acid profiles. This result suggested that the pfaE gene could be switchable between these strains for the production of DHA. The DHA production by expressing the reconstituted PKS pathway was also investigated in different E. coli strains, at different temperatures, or with the treatment of cerulenin. The highest DHA production, 2.2 mg of DHA per gram of dry cell weight or 4.1% of total fatty acids, was obtained by co-expressing pfaE(EPA) from the EPA-producing strain Shewanella baltica with pfaABCD in DH5α. Incubation at low temperature (10-15°C) resulted in higher accumulation of DHA compared to higher temperatures. The addition of cerulenin to the medium increased the proportion of DHA and saturated fatty acids, including C12:0, C14:0 and C16:0, at the expense of monounsaturated fatty acids, including C16:1 and C18:1. Supplementation with 1 mg/L cerulenin resulted in the highest DHA yield of 2.4 mg/L upon co-expression of pfaE(DHA) from C. psychrerythraea.