Effective production and kinetic characterization of ultra-high-molecular-weight poly[()-3-hydroxybutyrate] in recombinant

An inducible and highly effective production of ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] in recombinant Escherichia coli XL1-Blue was investigated. Two expression plasmids harbouring Ralstonia eutropha P(3HB) biosynthesis genes (phaCABRe) downstream of an inducible trc promoter, pTrcphaCABRe and pJRDTrcphaCABRe, were constructed using the high copy number plasmid pTrc99a and the low copy number plasmid pJRDTrc1, respectively. These plasmids and the constitutive expression plasmid harbouring phaCABRe genes (pSYL105) were individually introduced into E. coli XL1-Blue, and a comparative study for P(3HB) production from glucose using a 10-l fermentor was carried out. The highest P(3HB) productivity (2.8g/lh) was obtained when the recombinant harbouring pJRDTrcphaCABRe was cultured with the induction of gene expression. The produced P(3HB) had extremely high weight-average molecular weights ranging from 3.5×106 to 5.0×106, with relatively low polydispersities of around 1.5. The molecular weights remained nearly unchanged during the course of cultivation from 12 to 54h, suggesting that a chain-transfer reaction took place to regulate the length of polymer chains within the E. coli cells.