Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 90, Issue 2, Pages 603-614Publisher
SPRINGER
DOI: 10.1007/s00253-010-3077-2
Keywords
PLA; P(3HB-co-LA); Pseudomonas PhaC1 variants; Site-directed mutagenesis
Categories
Funding
- LG Chem
- Ministry of Education, Science and Technology (MEST) [20090065571, R32-2008-000-10142-0]
- National Research Foundation of Korea [2005-2000364, 과C6A1907] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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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.
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