Overexpression of succinyl-CoA synthase for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production in engineered Escherichia coli BL21(DE3)
Overexpression of succinyl-CoA synthase for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production in engineered Escherichia coli BL21(DE3)
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DOI：
10.1111/jam.12880
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发表时间：
2015-09-01
影响因子：
4
通讯作者：
Yang, Y. -H.
中科院分区：
文献类型：
--
作者：
Bhatia, S. K.;Yi, D. -H.;Yang, Y. -H.
目的利用琥珀酰辅酶a合成酶提高聚（3-羟基丁酸酯-co-3-羟基戊酸酯）[P(HB-co-HV)]中3-羟戊酸酯（3HV）的含量。方法与结果利用大肠杆菌（escherichia coliYH090）对聚羟基烷酸（PHA）产生菌进行过表达琥珀酰辅酶a合成酶基因（sucCD）的工程设计，并利用混合物分析方法检测在多种前体分子存在下P（HB-co-HV）共聚物的产生。甘油、琥珀酸盐和丙酸盐被认为是控制胞内PHA积累和单体组成的重要因素。甘油浓度对总生物量浓度和胞内PHA含量的影响最大，而琥珀酸存在时丙酸浓度对共聚物的3HV含量影响最大。混合分析还表明，根据控制条件的不同，工程菌株有能力积累高达其细胞干重（CDW） 80%的PHA，其中3HV单体的可变分数（最大72wt %）。结论丙酸盐是P（HB-co-HV）生物聚合物中3HV单体的主要前体，其利用需要转化为丙酰辅酶a。工程E.coliYHY99过表达sucCD基因，导致琥珀酰辅酶a库的增加，从而通过向其他在丙酸利用中起作用的酰基转移酶提供辅酶a来提高丙酸的转化率。与对照菌株相比，大肠杆菌yhy99能够以45倍的速度利用丙酸，并合成了高3HV单体含量的共聚物。
AimThis study aims to increase the 3-hydroxyvalerate (3HV) fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] using succinyl-CoA synthase.Methods and ResultsEscherichia coliYH090, a polyhydroxyalkonate (PHA)-producing strain, was further engineered for overexpression of succinyl-CoA synthase genes (sucCD), and examined for P(HB-co-HV) copolymer production in the presence of various precursor molecules using mixture analysis. Glycerol, succinate and propionate were screened as important factors for controlling intracellular PHA accumulation and monomer composition. Glycerol concentrations exerted the greatest influence on the overall biomass concentration and the intracellular PHA content, while propionate concentrations in the presence of succinate influenced the 3HV content of the copolymer. Mixture analysis also demonstrated that the engineered strain has the capacity to accumulate up to 80% of its cell dry weight (CDW) as PHA with a variable fraction of 3HV monomer (maximum of 72 wt %) depending on the controlled conditions.ConclusionsPropionate is the principal precursor for 3HV monomer in P(HB-co-HV) biopolymer and its utilization requires conversion to propionyl-CoA. Engineered E.coliYHY99, overexpressing sucCD genes, leads to an increase of the succinyl-CoA pool, which enhances the conversion rate of propionate by providing a CoA supply to other acyltransferase enzymes that have a role in propionate utilization.Significance and Impact of the StudyEngineered E.coliYHY99 was able to utilize propionate with a 45-fold increase in rate, as compared to the control strain, and resulted in the synthesis of a copolymer with high 3HV monomer content.