Biosynthesis of novel terpolymers poly(lactate-co-3-hydroxybutyrate-co-3-hydroxyvalerate)s in lactate-overproducing mutant Escherichia coli JW0885 by feeding propionate as a precursor of 3-hydroxyvalerate
Biosynthesis of novel terpolymers poly(lactate-co-3-hydroxybutyrate-co-3-hydroxyvalerate)s in lactate-overproducing mutant Escherichia coli JW0885 by feeding propionate as a precursor of 3-hydroxyvalerate
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DOI：
10.1007/s00253-009-2100-y
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发表时间：
2010-01-01
影响因子：
5
通讯作者：
Taguchi, Seiichi
中科院分区：
文献类型：
--
作者：
Shozui, Fumi;Matsumoto, Ken'ichiro;Taguchi, Seiichi
在LA过量产生突变体大肠杆菌JW0885中产生了新型乳酸（LA）基三元聚合物P[LA-co-3-羟基丁酸酯(3HB)-co-3-羟基戊酸酯（3HV）]s (PLBVs)，该突变体被发现是高效生产LA基聚酯的优越宿主。重组大肠杆菌JW0885基因编码la -聚合酶（Ser325Thr/Gln481Lys突变体，来自假单胞菌sp. 61-3）和三个单体供应酶[丙酰辅酶a转移酶，β -酮硫酶和烟酰胺腺嘌呤二核苷酸磷酸（还原形式）（NADPH）依赖乙酰乙酰辅酶a还原酶]在葡萄糖上有氧生长，并饲养丙酸盐作为3-羟戊酰辅酶a （3HV-CoA）的前体。气相色谱和核磁共振（NMR）分析表明，细胞内积累的聚合物由LA、3HB和3HV单元组成，因此被鉴定为三聚体，即plbv。此外，h -1核磁共振分析表明该三元共聚物中存在LA-3HV序列。当丙酸钠添加量为100 mg/l时，三元共聚物中的3HV分数线性升高至7.2 mol%， LA分数呈反比降低。这种现象可能是由于乳酸辅酶a （LA-CoA）和3HV-CoA的代谢通量随培养基中丙酸浓度的变化而变化。
Novel lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate(3HB)-co-3-hydroxyvalerate(3HV)]s (PLBVs), were produced in LA-overproducing mutant, Escherichia coli JW0885, which was found to be a superior host for the efficient production of LA-based polyesters. Recombinant E. coli JW0885 harboring the genes encoding LA-polymerizing enzyme (Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3) and three monomer supplying enzymes [propionyl-CoA transferase, beta-ketothiolase, and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-dependent acetoacetyl-CoA reductase] was aerobically grown on glucose with feeding of propionate as a precursor of 3-hydroxyvaleryl-CoA (3HV-CoA). Gas chromatography and nuclear magnetic resonance (NMR) analyses revealed that polymers accumulated in the cells were composed of LA, 3HB, and 3HV units, thus being identified as terpolymers, PLBVs. In addition, H-1-NMR analysis suggested the existence of LA-3HV sequence in the terpolymer. When 100 mg/l of sodium propionate was added into the medium, 3HV fraction in the terpolymer linearly reached up to 7.2 mol%, while LA fraction was inversely decreased. This phenomenon could be due to the change in metabolic fluxes of lactyl-CoA (LA-CoA) and 3HV-CoA depending on the concentration of propionate fed into the medium.