title: Tailor-made type II Pseudomonas PHA synthases and their use for the biosynthesis of polylactic acid and its copolymer in recombinant Escherichia coli.
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.
authors.affiliation: Corporate R&D, LG Chem Research Park, 104-1 Moonji-dong, Yuseong-gu, Daejeon 305-380, Republic of Korea. aprilkenzo@kaist.ac.kr
authors.forename: Taek Ho
authors.initials: TH
authors.lastname: Yang
authors.forename: Yu Kyung
authors.initials: YK
authors.lastname: Jung
authors.forename: Hye Ok
authors.initials: HO
authors.lastname: Kang
authors.forename: Tae Wan
authors.initials: TW
authors.lastname: Kim
authors.forename: Si Jae
authors.initials: SJ
authors.lastname: Park
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lee
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D015256:Coenzyme A-Transferases / Q000378:metabolism; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011091:Polyesters; D011108:Polymers; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D000214:Acyl Coenzyme A; D004274:DNA, Recombinant; D011091:Polyesters; D011108:Polymers; C047009:lactoyl-coenzyme A; D019344:Lactic Acid; C033616:poly(lactide); D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D015256:Coenzyme A-Transferases; C031212:acyl-CoA transferase