Biosynthesys Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) With High 3-Hydroxyvalerate Content By Over-expressing Genes PrpE, PrpP From Ralstonia Eutropha H16

Polyhydroxyalkanoates (PHA) are a family of microbially synthesized biologically degrable thermoplastics composed of 3-hydroxyalkanoates building blocks. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one kind of PHA copolymers and is less brittle and less crystalline than poly-3-hydroxybutyrate homopolymer. High cost and complicated fermentation technique hold back PHBV industrialized production. The control of copolymer composition is important because the physical and mechanical material properties of the copolymer depend on the fraction of 3-hydroxyvalerate(3HV).In this study, the prpP and prpE, encoding propionate permease and propoinic-CoA synthase,respectively, and the PHB operon (phCAB) from Ralstonia eutropha were together cloned into a plasmid,resulting in a new plasmid pBHR68-prpP and pBHR68-prpE. using the recombinant plasmid pBHR68-prpP, an Escherichia coli was transformed by a calcium chloride method to prepare recombinant Escherichia coli XL10-Gold. The recombinant Escherichia coli XL10-Gold was cultured from a mixed carbon soures of glucose and propionic acid. in 60 hours, PHBV with high 3HV fraction(14.3 mol%),high molecular weight(~1100000) and high PHA content (62 wt%), was obtained. the result indicated that expression of prpP stimulated the uptake and utilization of propionic acid and increase 3HV fracion of copolymer.A plasmid PCS2-L01 was also constructed by inserting prpP gene into plasmid pBBR1MCS-2 in this study. the recombinant vector was introduced in the host Ralstonia eutropha H16. in flaker experiment,cell dry weight, pHBV content(PHBV/CDW) and 3HV content obtained in 72 hours were 4.2 g/l, 78.5% and 21.6 mol%. The result showed that the 3HV fraction was increased 1.5-2.5 fold,compared with 3HV content of PHBV produced the wildtype Ralstonia eutropha H16.The above results proposed that both of over-expression gene prpP in Escherichia coli XL10-Gold and Ralstonia eutropha H16 stimulated the uptake and utilization of propionic acid and increased 3HV fracion of copolymer.When the feedstock composition is not adjustable, such as waste carbon sources, and use the glucose and propionic acid as a mixed carbon sources for PHBV produce industrially, the two systems will be useful.