Applied Microbiology and Biotechnology

, Volume 90, Issue 2, pp 603–614 | Cite as

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

Applied Genetics and Molecular Biotechnology

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 (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1Ps6-19 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 

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Corporate R&D, LG Chem Research ParkDaejeonRepublic of Korea
  2. 2.Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program)Center for Systems and Synthetic Biotechnology, and Institute for the BioCenturyDaejeonRepublic of Korea
  3. 3.Department of Bio and Brain Engineering, Department of Biological SciencesBioProcess Engineering Research Center, and Bioinformatics Research CenterDaejeonRepublic of Korea
  4. 4.Chemical Biotechnology Research Center, Green Chemistry DivisionKorea Research Institute of Chemical TechnologyDaejeonRepublic of Korea

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