Biosynthesis of polyhydroxyalkanoate (PHA) copolymer from fructose using wild-type and laboratory-evolved PHA synthases
Biosynthesis of polyhydroxyalkanoate (PHA) copolymer from fructose using wild-type and laboratory-evolved PHA synthases
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DOI：
10.1002/mabi.200400152
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发表时间：
2005-02-23
影响因子：
4.6
通讯作者：
Doi, Y
中科院分区：
文献类型：
--
作者：
Tsuge, T;Yano, K;Doi, Y
以真养罗尔斯通氏菌（Ralstonia eutropha）PHB（-）4为宿主菌，将11种来源于假单胞菌61-3（PhaC 1（Ps））的聚羟基链烷酸（PHA）脱氢酶与野生型酶一起用于从果糖合成PHA。进化的PhaCl（Ps）突变体在位置325和/或位置481处具有氨基酸取代。在这些突变体中，丝氨酸-325（S325）被半胱氨酸（C）或苏氨酸（T）取代，而谷氨酰胺-481（Q481）被赖氨酸（K）、甲硫氨酸（M）或精氨酸（R）取代。携带进化PhaCl（Ps）突变体基因的所有重组菌株产生显著增加量的PHA（55-68重量%）。与含有野生型基因的（49重量%）相比。特别地，具有多个氨基酸取代的那些进化的PhaCl（Ps）突变体显示出更高的PHA合成活性。通过NMR光谱对PHA的表征表明，它们是由（R）-3-羟基丁酸酯（98-99摩尔%）和中链长度共聚单体（1-2摩尔%）组成的共聚物。本研究还证实了PhaC 1（Ps）中481位氨基酸的取代导致PHA分子量的增加。PhaC 1（Ps）（Q481 K）突变体合成的PHA数均分子量（M/bar（n）=240000）是野生型酶合成的PHA数均分子量（M/bar（n）= 52000）的4.6倍。
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 ((M) over bar (n)) of PHA ((M) over bar (n)=240000) synthesized by the evolved PhaC1(Ps) (Q481K) mutant was 4.6-fold greater than that ((M) over bar (n) = 52 000) synthesized by the wild-type enzyme.