Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase.
For the synthesis of polylactic acid (PLA) and its copolymers by one-step fermentation process, heterologous pathways involving Clostridium propionicum propionate CoA transferase (Pct(Cp)) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Ps6-19)) were introduced into Escherichia coli for the generation of lactyl-CoA endogenously and incorporation of lactyl-CoA into the polymer, respectively. Since the wild-type PhaC1(Ps6-19) did not efficiently accept lactyl-CoA as a substrate, site directed mutagenesis as well as saturation mutagenesis were performed to improve the enzyme. The wild-type Pct(Cp) was not able to efficiently convert lactate to lactyl-CoA and was found to exert inhibitory effect on cell growth, random mutagenesis by error-prone PCR was carried out. By employing engineered PhaC1(Ps6-19) and Pct(Cp), poly(3-hydroxybutyrate-co-lactate), P(3HB-co-LA), containing 20-49 mol% lactate could be produced up to 62 wt% from glucose and 3HB. By controlling the 3HB concentration in the medium, PLA homopolymer and P(3HB-co-LA) containing lactate as a major monomer unit could be synthesized. Also, P(3HB-co-LA) copolymers containing various lactate fractions could be produced from glucose alone by introducing the Cupriavidus necator beta-ketothiolase and acetoacetyl-CoA reductase genes. Fed-batch cultures were performed to produce P(3HB-co-LA) copolymers having 9-64 mol% of lactate, and their molecular weights, thermal properties, and melt flow properties were determined.
Yang
Taek Ho
TH
Corporate R&D, LG Chem Research Park, 104-1 Moonji-dong, Yuseong-gu, Daejeon 305-380, Republic of Korea.
Kim
Tae Wan
TW
Kang
Hye Ok
HO
Lee
Sang-Hyun
SH
Lee
Eun Jeong
EJ
Lim
Sung-Chul
SC
Oh
Sun Ok
SO
Song
Ae-Jin
AJ
Park
Si Jae
SJ
Lee
Sang Yup
SY
D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D003013:Clostridium / Q000201:enzymology*; D015256:Coenzyme A-Transferases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D019344:Lactic Acid / Q000096:biosynthesis*; D015394:Molecular Structure; D009154:Mutation; D011091:Polyesters; D011108:Polymers; D011994:Recombinant Proteins / Q000235:genetics; D015398:Signal Transduction
D011091:Polyesters; D011108:Polymers; D011994:Recombinant Proteins; D019344:Lactic Acid; C033616:poly(lactide); D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D015256:Coenzyme A-Transferases