title: In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from Pseudomonas sp. SG4502 with acquired LA-polymerizing activity.
abstract: Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a "thermostable LPE". The mutated PhaC1(SG) [PhaC1(SG)(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(SG)(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Hokkaido University, Sapporo, Japan. ktajima@eng.hokudai.ac.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Tajima
authors.forename: Xuerong
authors.initials: X
authors.lastname: Han
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Satoh
authors.forename: Ayako
authors.initials: A
authors.lastname: Ishii
authors.forename: Yuji
authors.initials: Y
authors.lastname: Araki
authors.forename: Masanobu
authors.initials: M
authors.lastname: Munekata
authors.forename: Seiichi
authors.initials: S
authors.lastname: Taguchi
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006358:Hot Temperature; D019344:Lactic Acid / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017354:Point Mutation; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D019344:Lactic Acid; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase