Biosynthesis of polyhydroxyalkanoate (PHA) copolymer from fructose using wild-type and laboratory-evolved PHA synthases.
Eleven laboratory-evolved polyhydroxyalkanoate (PHA) synthases which originated from Pseudomonas sp. 61-3 enzyme (PhaC1(Ps)), together with the wild-type enzyme, were applied for PHA synthesis from fructose using Ralstonia eutropha PHB(-)4 as a host strain. The evolved PhaC1(Ps) mutants had amino acid substitution(s) at position 325 and/or position 481. In these mutants, serine-325 (S325) was replaced by cysteine (C) or threonine (T), while glutamine-481 (Q481) was replaced by lysine (K), methionine (M) or arginine (R). All recombinant strains harboring the genes of the evolved PhaC1(Ps) mutants produced a significantly increased amount of PHA (55-68 wt.-%) compared with the one harboring the wild-type gene (49 wt.-%). Particularly, those evolved PhaC1(Ps) mutants having multiple amino acid substitutions showed higher activities for PHA synthesis. Characterization of the PHA by NMR spectroscopy revealed that they were copolymers consisting of (R)-3-hydroxybutyrate (98-99 mol-%) and medium-chain-length comonomers (1-2 mol-%). This study also confirmed that amino acid substitution at position 481 in PhaC1(Ps) led to an increasing molecular weight of PHA. The number-average molecular weight (Mn) of PHA (Mn = 240,000) synthesized by the evolved PhaC1(Ps) (Q481K) mutant was 4.6-fold greater than that (Mn = 52,000) synthesized by the wild-type enzyme.
Tsuge
Takeharu
T
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8502, Japan. ttsuge@iem.titech.ac.jp
Yano
Kotaro
K
Imazu
Shin-ichi
S
Numata
Keiji
K
Kikkawa
Yoshihiro
Y
Abe
Hideki
H
Taguchi
Seiichi
S
Doi
Yoshiharu
Y
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D020118:Cupriavidus necator / Q000201:enzymology; D019020:Directed Molecular Evolution; D005632:Fructose / Q000378:metabolism*; D008970:Molecular Weight; D009154:Mutation; D011108:Polymers / Q000378:metabolism*
D011108:Polymers; D005632:Fructose; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase