Modification of acetoacetyl-CoA reduction step in Ralstonia eutropha for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from structurally unrelated compounds
Modification of acetoacetyl-CoA reduction step in Ralstonia eutropha for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from structurally unrelated compounds
复制标题
修饰真养罗尔斯通氏菌中的乙酰乙酰辅酶A还原步骤，用于从结构不相关的化合物生物合成聚(3-羟基丁酸酯-co-3-羟基己酸酯)
作者：
Mengxiao Zhang;Shunsuke Kurita;Izumi Orita;Satoshi Nakamura;T. Fukui
聚(（R)-3-羟基丁酸酯-co-(R)-3-羟基己酸酯）[P(3HB-co-3HHx)]是一种具有高生物降解性的细菌聚酯，即使在海洋环境中也是如此。富营养化Ralstonia eutropha已被设计用于从植物油中生物合成P(3HB-co-3HHx)，但其从结构不相关的碳源生产仍然令人不满意。结果整合已有的工程策略，构建了既能从果糖合成P(3HB-co-3HHx)，又能从葡萄糖和甘油合成P（3HB-co-3HHx）的富营养化ralstonia eutropha菌株。在乙酰乙酰-辅酶a还原步骤进行进一步修饰，确定共聚物组成的通量分布。当主要的乙酰乙酰辅酶a还原酶（PhaB1）被低活性的类似酶（PhaB2）或反向β氧化酶取代时，葡萄糖可以有效地合成具有高3HHx组成的共聚酯，这可能是由于乙酰乙酰辅酶a通过(S)-3HB-CoA增强了丁基辅酶a的形成。P（3HB-co-3HHx）由7.0 mol%和12.1 mol%的3HHx组分组成，分别以71.4 wt%和75.3 wt%的细胞含量生产，足以用于实际应用。PhaB1低亲和力突变体的替代对PHA在葡萄糖上的生物合成影响不大，但对果糖的生物合成影响不大，这表明依赖于糖转运机制的代谢调节发生了改变。当细胞生长在甘油上时，PhaB1主要作用于乙酰乙酰辅酶a的转化，因为缺乏PhaB1会严重损害共聚酯的生物合成。结论本研究结果表明，真菌菌乙酰乙酰辅酶a节点的通量分布对于结构不相关的化合物合成具有调节组成的PHA共聚酯具有重要意义。
BackgroundPoly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is a bacterial polyester with high biodegradability, even in marine environments. Ralstonia eutropha has been engineered for the biosynthesis of P(3HB-co-3HHx) from vegetable oils, but its production from structurally unrelated carbon sources remains unsatisfactory.ResultsRalstonia eutropha strains capable of synthesizing P(3HB-co-3HHx) from not only fructose but also glucose and glycerol were constructed by integrating previously established engineering strategies. Further modifications were made at the acetoacetyl-CoA reduction step determining flux distribution responsible for the copolymer composition. When the major acetoacetyl-CoA reductase (PhaB1) was replaced by a low-activity paralog (PhaB2) or enzymes for reverse β-oxidation, copolyesters with high 3HHx composition were efficiently synthesized from glucose, possibly due to enhanced formation of butyryl-CoA from acetoacetyl-CoA via (S)-3HB-CoA. P(3HB-co-3HHx) composed of 7.0 mol% and 12.1 mol% 3HHx fractions, adequate for practical applications, were produced at cellular contents of 71.4 wt% and 75.3 wt%, respectively. The replacement by low-affinity mutants of PhaB1 had little impact on the PHA biosynthesis on glucose, but slightly affected those on fructose, suggesting altered metabolic regulation depending on the sugar-transport machinery. PhaB1 mostly acted in the conversion of acetoacetyl-CoA when the cells were grown on glycerol, as copolyester biosynthesis was severely impaired by the lack of phaB1.ConclusionsThe present results indicate the importance of flux distribution at the acetoacetyl-CoA node in R. eutropha for the biosynthesis of the PHA copolyesters with regulated composition from structurally unrelated compounds.
影响因子：
5.7
作者：
Price, AC;Zhang, YM;White, SW
通讯作者：
White, SW