DataSheet1_Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) From Glucose by Escherichia coli Through Butyryl-CoA Formation Driven by Ccr-Emd Combination.PDF

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is a practical kind of bacterial polyhydroxyalkanoates (PHAs). A previous study has established an artificial pathway for the biosynthesis of P(3HB-co-3HHx) from structurally unrelated sugars in Ralstonia eutropha, in which crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) are a key combination for generation of butyryl-CoA and the following chain elongation. This study focused on the installation of the artificial pathway into Escherichia coli. The recombinant strain of E. coli JM109 harboring 11 heterologous genes including Ccr and Emd produced P(3HB-co-3HHx) composed of 14 mol% 3HHx with 41 wt% of dry cellular weight from glucose. Further investigations revealed that the C6 monomer (R)-3HHx-CoA was not supplied by (R)-specific reduction of 3-oxohexanoyl-CoA but by (R)-specific hydration of 2-hexenoyl-CoA formed through reverse β-oxidation after the elongation from C4 to C6. While contribution of the reverse β-oxidation to the conversion of the C4 intermediates was very limited, crotonyl-CoA, a precursor of butyryl-CoA, was generated by dehydration of (R)-3HB-CoA. Several modifications previously reported for enhancement of bioproduction in E. coli were examined for the copolyester synthesis. Elimination of the global regulator Cra or PdhR as well as the block of acetate formation resulted in poor PHA synthesis. The strain lacking RNase G accumulated more PHA but with almost no 3HHx unit. Introduction of the phosphite oxidation system for regeneration of NADPH led to copolyester synthesis with the higher cellular content and higher 3HHx composition by two-stage cultivation with phosphite than those in the absence of phosphite.

聚(（R）-3-羟基丁酸酯-co-（R）-3-羟基己酸酯) [P(3HB-co-3HHx)] 是一类具备实用价值的细菌源聚羟基脂肪酸酯（polyhydroxyalkanoates, PHAs）。既往研究已在真养罗尔斯通氏菌（Ralstonia eutropha）中构建了一条以结构无关的糖类为底物生物合成P(3HB-co-3HHx)的人工途径，其中巴豆酰辅酶A羧化酶/还原酶（crotonyl-CoA carboxylase/reductase, Ccr）与乙基丙二酰辅酶A脱羧酶（ethylmalonyl-CoA decarboxylase, Emd）是生成丁酰辅酶A及后续碳链延长过程的关键组合。本研究聚焦于将该人工途径导入大肠杆菌（Escherichia coli）。携带包括Ccr与Emd在内的11个异源基因的重组大肠杆菌JM109菌株，可利用葡萄糖合成P(3HB-co-3HHx)，该共聚酯中3HHx单体摩尔占比为14 mol%，细胞干重中该聚合物的含量达41 wt%。后续研究进一步揭示，C6单体（R）-3-羟基己酰辅酶A并非通过3-氧代己酰辅酶A的（R）特异性还原反应生成，而是经由从C4到C6的碳链延长后通过逆向β-氧化生成的2-己烯酰辅酶A的（R）特异性水合作用产生。尽管逆向β-氧化对C4中间体的转化贡献极低，但丁酰辅酶A的前体巴豆酰辅酶A可通过（R）-3-羟基丁酰辅酶A的脱水反应生成。本研究还针对该共聚酯的合成，考察了既往报道中用于提升大肠杆菌生物合成效率的多种改造策略：敲除全局调控因子Cra或PdhR，以及阻断乙酸生成途径，均会导致PHA合成能力显著下降；缺失核糖核酸酶G（RNase G）的菌株虽可积累更多PHA，但几乎无法合成3HHx单元；引入用于再生烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）的亚磷酸氧化系统后，通过添加亚磷酸的两步培养工艺，可使共聚酯的细胞含量与3HHx单体占比均显著高于未添加亚磷酸的对照组。