| Polylactate?PLA?is a bio-based plastic synthesized by the chemo-bio process under metal catalyst mediated chemical polymerization of lactate?LA?supplied by microbial fermentation.Meanwhile,polyhydroxyalkanoates?PHA?are diverse polyesters with different physical properties to suit various applications.Copolymerization of LA with different hydroxyalkanoate?HA?monomers is an effective way to improve the physical properties of PLA or PHA.Besides,the development of high-cell-density cultivation methods is a very promising area to reduce the cost of the final products.In this study,a mutant PHA synthase PhaC1Ps?E130D S325T S477G Q481K?was constructed which could incorporate LA as well as other 3-hydroxyalkanoates?3-HAs?substrates at the same time.Thereafter,a novel LA-containing terpolyesters P?LA-co-3HB-co-3HP?,short as PLBP,were successfully synthesized for the first time by a recombinant Escherichia coli from unrelated carbon source.Furthermore,the monomer ratios of PLBP could be regulated by the control of the substrates ratio and metabolic engineering of the host strain.The thermal and mechanical properties of PLBP terpolyster can be manipulated by adjusting the monomer ratios.To facilitate low cost production of PLBP and other related biopolysters,an evolutionary system based on a genome-wide random mutagenesis associated with an error-prone DNA polymerase III?subunit was established and used in conjunction with an artificial high cell density culture environment during the evolution selection process.The high-cell-density strain Halomonas bluephagenesis TDHCD-R3 was obtained after 3 rounds of selection with an enhancd resistance towards toxic metabolites.By overexpression of an optimized phaCAB operon,the strain was able to grow to 14.83 g/L in cell dry weight?CDW?along with a 94.46%PHA content in shake flask studies cultivation.While it was grown to more than 90 g/L CDW containing 80%PHA in a 7-L fermentor. |
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