Heterologous production of novel and rare C30-carotenoids using Planococcus carotenoid biosynthesis genes

초록

Background: Members of the genus Planococcus have been revealed to utilize and degrade solvents such as aromatic hydrocarbons and alkanes, and likely to acquire tolerance to solvents. A yellow marine bacterium Planococcus maritimus strain iso-3 was isolated from an intertidal sediment that looked industrially polluted, from the Clyde estuary in the UK. This bacterium was found to produce a yellow acyclic carotenoid with a basic carbon 30 (C-30) structure, which was determined to be methyl 5-glucosyl-5,6-dihydro-4,4 '-diapolycopenoate. In the present study, we tried to isolate and identify genes involved in carotenoid biosynthesis from this marine bacterium, and to produce novel or rare C-30-carotenoids with anti-oxidative activity in Escherichia coli by combinations of the isolated genes. Results: A carotenoid biosynthesis gene cluster was found out through sequence analysis of the P. maritimus genomic DNA. This cluster consisted of seven carotenoid biosynthesis candidate genes (orf1-7). Then, we isolated the individual genes and analyzed the functions of these genes by expressing them in E. coli. The results indicated that orf2 and orf1 encoded 4,4 '-diapophytoene synthase (CrtM) and 4,4 '-diapophytoene desaturase (CrtNa), respectively. Furthermore, orf4 and orf5 were revealed to code for hydroxydiaponeurosporene desaturase (CrtNb) and glucosyltransferase (GT), respectively. By utilizing these carotenoid biosynthesis genes, we produced five intermediate C-30-carotenoids. Their structural determination showed that two of them were novel compounds, 5-hydroxy-5,6-dihydro-4,4 '-diaponeurosporene and 5-glucosyl-5,6-dihydro-4,4 '-diapolycopene, and that one rare carotenoid 5-hydroxy-5,6-dihydro-4,4 '-diapolycopene is included there. Moderate singlet oxygen-quenching activities were observed in the five C-30-carotenoids including the two novel and one rare compounds. Conclusions: The carotenoid biosynthesis genes from P. maritimus strain iso-3, were isolated and functionally identified. Furthermore, we were able to produce two novel and one rare C-30-carotenoids in E. coli, followed by positive evaluations of their singlet oxygen-quenching activities.

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