Sunday, July 26, 2009
P123
Production of retinoids by using b-carotene monooxygenase from recombinant E. coli harboring β-carotene pathway
The retinoids are chemical compounds related chemically to vitamin A. Retinoids have important biological functions of regulation of cell proliferation and differentiation, growth of bone tissue, immune function, activation of tumor suppressor genes, and roles in vision. β-Carotene is cleaved by β-carotene monooxygenase to yield two molecules of retinal. β-Carotene is synthesized by using IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) as building blocks. IPP and DMAPP can be formed in E. coli via endogenous MEP pathway and exogenous Mevalonate pathway. The rate-limiting enzyme of DXP synthase (Dxs) of MEP pathway is overexpressed and an optimized artificial Mevalonate pathway is introduced in E. coli. In order to make E. coli produce β-carotene, it is also introduced the β-carotene pathway composed of 4 foreign enzymes; GGPP synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (CrtI), and lycopene β-cyclases (CrtY). Artificial E. coli codon optimized gene was synthesized, based on the amino acid sequence of blh gene of uncultured marine bacterium 66A03. The artificial β-Carotene 15,15'-monooxygenase gene was combined with β-carotene pathway genes to make retinal plasmid, pTDHBSR. Recombinant E. coli harboring pTDHBSR produced retinol (25 mg/L), retinal (19 mg/L), and retinyl acetate (9 mg/L). The β-Carotene 15,15'-monooxygenase expression led to production of retinal, retinol, and retinyl acetate at the expense of β-carotene in E. coli. This work was supported by the EB-NCRC (Grant No. R15-2003-012-02001-0), the 21C Frontier Microbial Genomics and Applications Center Program, and BK21 program of Korea .
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