Biosynthesis of Polyhydroxyalkanoate (PHA) Copolymer from Fructose Using Wild‐Type and Laboratory‐Evolved PHA Synthases

Summary: Eleven laboratory‐evolved polyhydroxyalkanoate (PHA) synthases which originated from Pseudomonas sp. 61‐3 enzyme (PhaC1Ps), together with the wild‐type enzyme, were applied for PHA synthesis from fructose using Ralstonia eutropha PHB‐4 as a host strain. The evolved PhaC1Ps 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 PhaC1Ps 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 PhaC1Ps 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 PhaC1Ps led to an increasing molecular weight of PHA. The number‐average molecular weight (M ₙ ) of PHA (M ₙ = 240 000) synthesized by the evolved PhaC1Ps (Q481K) mutant was 4.6‐fold greater than that (M ₙ = 52 000) synthesized by the wild‐type enzyme. PHA content in recombinant R. eutropha PHB‐4 expressing the wild‐type or the laboratory‐evolved PHA synthase. magnified image PHA content in recombinant R. eutropha PHB‐4 expressing the wild‐type or the laboratory‐evolved PHA synthase.