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Biosynthesis of Polyhydroxyalkanoate (PHA) Copolymer from Fructose Using Wild‐Type and Laboratory‐Evolved PHA Synthases
Author(s) -
Tsuge Takeharu,
Yano Kotaro,
Imazu Shinichi,
Numata Keiji,
Kikkawa Yoshihiro,
Abe Hideki,
Taguchi Seiichi,
Doi Yoshiharu
Publication year - 2005
Publication title -
macromolecular bioscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.924
H-Index - 105
eISSN - 1616-5195
pISSN - 1616-5187
DOI - 10.1002/mabi.200400152
Subject(s) - polyhydroxyalkanoates , biochemistry , ralstonia , amino acid , chemistry , mutant , methionine , cysteine , biosynthesis , fructose , wild type , lysine , enzyme , biology , gene , bacteria , genetics
Summary: 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 ( $\overline M _{\rm n}$ ) of PHA ( $\overline M _{\rm n}$ = 240 000) synthesized by the evolved PhaC1 Ps (Q481K) mutant was 4.6‐fold greater than that ( $\overline M _{\rm n}$ = 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.