tailieunhanh - Saika et al. AMB Express 2011, 1:6 http://www.amb-express.com/content/1/1/6 ORIGINAL Open

Saika et al. AMB Express 2011, 1:6 ORIGINAL Open Access Enhanced Incorporation of 3-Hydroxy-4Methylvalerate Unit into Biosynthetic Polyhydroxyalkanoate Using Leucine as a Precursor Azusa Saika1, Yoriko Watanabe1, Kumar Sudesh2, Hideki Abe3 and Takeharu Tsuge1* Abstract Ralstonia eutropha PHB-4 expressing Pseudomonas sp. 61-3 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps) synthesizes PHA copolymer containing 3-hydroxybutyrate (3HB) and a small amount ( mol%) of 3-hydroxy-4methylvalerate (3H4MV) from fructose as a carbon source. In this study, enhanced incorporation of 3H4MV into PHA was investigated using branched amino acid leucine as a precursor of 3H4MV. Leucine has the same carbon backbone as 3H4MV and is expected to be a natural. | Saika et al. AMB Express 2011 1 6 http content 1 1 6 o AMB Express a SpringerOpen Journal ORIGINAL Open Access Enhanced Incorporation of 3-Hydroxy-4-Methylvalerate Unit into Biosynthetic Polyhydroxyalkanoate Using Leucine as a Precursor Azusa Saika1 Yoriko Watanabe1 Kumar Sudesh2 Hideki Abe3 and Takeharu Tsuge1 Abstract Ralstonia eutropha PHB-4 expressing Pseudomonas sp. 61-3 polyhydroxyalkanoate PHA synthase 1 PhaC1Ps synthesizes PHA copolymer containing 3-hydroxybutyrate 3HB and a small amount mol of 3-hydroxy-4-methylvalerate 3H4MV from fructose as a carbon source. In this study enhanced incorporation of 3H4MV into PHA was investigated using branched amino acid leucine as a precursor of 3H4MV. Leucine has the same carbon backbone as 3H4MV and is expected to be a natural and self-producible precursor. We found that the incorporation of 3H4MV was enhanced by the supplementation of excess amount 10 g L of leucine in the culture medium. This finding indicates that 3H4MV can be derived from leucine. To increase metabolic flux to leucine biosynthesis in the host strain by eliminating the feedback inhibition the cells were subjected to N-methyl-N -nitro-N-nitrosoguanidine NTG mutagenesis and leucine analog resistant mutants were generated. The mutants showed statistically higher 3H4MV fraction than the parent strain without supplementing leucine. Additionally by supplying excess amount of leucine the mutants synthesized 3HB-based PHA copolymer containing mol 3H4MV and mol 3-hydroxyvalerate 3HV as minor constituents which significantly affected the thermal properties of the copolymer. This study demonstrates that it is possible to enhance the monomer supply of 3H4MV into PHA by manipulating leucine metabolism. Keywords polyhydroxyalkanoate copolymer 3H4MV precursor leucine analog resistant mutant Introduction Polyhydroxyalkanoate PHA is a kind of aliphatic polyester synthesized by a wide variety of microorganisms as intracellular storage