Applied Genetics and Molecular Biotechnology

Applied Microbiology and Biotechnology

, Volume 90, Issue 2, pp 603-614

Tailor-made type II Pseudomonas PHA synthases and their use for the biosynthesis of polylactic acid and its copolymer in recombinant Escherichia coli

  • Taek Ho YangAffiliated withCorporate R&D, LG Chem Research Park Email author 
  • , Yu Kyung JungAffiliated withMetabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury
  • , Hye Ok KangAffiliated withCorporate R&D, LG Chem Research Park
  • , Tae Wan KimAffiliated withCorporate R&D, LG Chem Research Park
  • , Si Jae ParkAffiliated withChemical Biotechnology Research Center, Green Chemistry Division, Korea Research Institute of Chemical Technology
  • , Sang Yup LeeAffiliated withMetabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), Center for Systems and Synthetic Biotechnology, and Institute for the BioCenturyDepartment of Bio and Brain Engineering, Department of Biological Sciences, BioProcess Engineering Research Center, and Bioinformatics Research Center Email author 

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Abstract

Previously, we have developed metabolically engineered Escherichia coli strains capable of producing polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] by employing evolved Clostridium propionicum propionate CoA transferase (Pct Cp ) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 Ps6-19). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1 Ps6-19 have been found to affect the polymer content, lactate mole fraction, and molecular weight of P(3HB-co-LA). In this study, we have further engineered type II Pseudomonas PHA synthases 1 (PhaC1s) from Pseudomonas chlororaphis, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas resinovorans, and Pseudomonas aeruginosa PAO1 to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates by site-directed mutagenesis of four sites (E130, S325, S477, and Q481). All PhaC1s having mutations in these four sites were able to accept lactyl-CoA as a substrate and supported the synthesis of P(3HB-co-LA) in recombinant E. coli, whereas the wild-type PhaC1s could not accumulate polymers in detectable levels. The contents, lactate mole fractions, and the molecular weights of P(3HB-co-LA) synthesized by recombinant E. coli varied depending upon the source of the PHA synthase and the mutants used. PLA homopolymer could also be produced at ca. 7 wt.% by employing the several PhaC1 variants containing E130D/S325T/S477G/Q481K quadruple mutations in wild-type E. coli XL1-Blue.

Keywords

PLA P(3HB-co-LA) Pseudomonas PhaC1 variants Site-directed mutagenesis