Engineering of class I lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature.
Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaCRe) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaCRe was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1PsSTQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323-17327, 2008). The LA-polymerizing activity of the PhaCReA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaCRe(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaCReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The (13)C and (1)H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 10(5) Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA -LA triad sequence, suggesting that the polymer synthesized by PhaCRe mutant may not be a random copolymer, but presumably had a block sequence.
Division of Biotechnology and Molecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo 060-8628, Japan.
Anna
A
Ochi
Ken'ichiro
K
Matsumoto
Takashi
T
Ooba
Kohei
K
Sakai
Takeharu
T
Tsuge
Seiichi
S
Taguchi
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering
D050505:Mutant Proteins; D054813:Polyhydroxyalkanoates; D019344:Lactic Acid; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase