Mevalonate is a biochemical precursor to a wide range of isoprenoids. The mevalonate pathway uses three moles of acetyl‐CoA, and therefore native pathways which metabolize acetyl‐CoA compete with mevalonate synthesis. Moreover, the final step in mevalonate formation, mediated by hydroxymethylglutaryl‐CoA reductase, requires NADPH as a co‐substrate. This study focuses on chromosomal modification of citrate synthase (GltA) involved in acetyl‐CoA utilization and phosphofructokinase (PfkA) involved in NADPH formation to increase the yield and productivity of mevalonate in Escherichia coli overexpressing the three genes of the heterologous mevalonate pathway. Nine GltA variants were compared for mevalonate production with the ΔgltA knockout and the wild‐type GltA strain in shake flasks in the absence and presence of casamino acids. In the presence of casamino acids, all variants generated mevalonate at a greater yield than the wild‐type control, but less than the GltA knockout. In the absence of casamino acids, the strain expressing wild‐type GltA generated the greatest yield of mevalonate, while most variants instead accumulated primarily acetate. Using the wild‐type strain and two citrate synthase variants, four phosphofructokinase variants were also compared with the ΔpfkA knockout and the wild‐type strain, but PfkA variants generated less mevalonate than the corresponding wild‐type PfkA strain. Controlled processes at the 1‐liter scale comparing five strains demonstrated the inverse relationship between yield and productivity, with the GltA[K167A] variant showing the best balance for the yield (0.20 g/g) and productivity (0.87 g/L h). A nitrogen‐limited process using the GltA[K167A] variant generated 36.9 g/L mevalonate in 31 h at a yield of 0.31 g/g. This study demonstrates that GltA variants offer a means to affect intracellular acetyl‐CoA pools for the generation of acetyl‐CoA derived products and that the acetyl‐CoA pool rather than NADPH availability is the important limiting factor for mevalonate production.

中文翻译：

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使用工程化柠檬酸盐合酶和大肠杆菌的磷酸果糖激酶变体增加甲羟戊酸的产生


甲羟戊酸酯是多种类异戊二烯的生化前体。甲羟戊酸途径使用三摩尔乙酰辅酶 A，因此代谢乙酰辅酶 A 的天然途径与甲羟戊酸合成竞争。此外，由羟甲基戊二酰辅酶 A 还原酶介导的甲羟戊酸形成的最后一步需要 NADPH 作为共底物。本研究的重点是参与乙酰辅酶 A 利用的柠檬酸合酶 （GltA） 和参与 NADPH 形成的磷酸果糖激酶 （PfkA） 的染色体修饰，以提高过表达异源甲羟戊酸通路三个基因的大肠杆菌中甲羟戊酸的产量和生产力。在不存在和存在酪蛋白氨基酸的情况下，比较了 9 个 GltA 变体在摇瓶中用 ΔgltA 敲除和野生型 GltA 菌株产生甲羟戊酸的情况。在酪蛋白氨基酸存在下，所有变体生成甲羟戊酸的产量都高于野生型对照，但低于 GltA 敲除。在不存在酪蛋白氨基酸的情况下，表达野生型 GltA 的菌株产生了最大的甲羟戊酸产量，而大多数变体反而主要积累乙酸盐。使用野生型菌株和两种柠檬酸盐合酶变体，还将四种磷酸果糖激酶变体与 ΔpfkA 敲除和野生型菌株进行了比较，但 PfkA 变体产生的甲羟戊酸比相应的野生型 PfkA 菌株少。比较 5 种菌株的 1 升尺度的受控过程表明，产量和生产率之间存在反比关系，其中 GltA[K167A] 变体显示出产量 （0.20 g/g） 和生产率 （0.87 g/L h） 的最佳平衡。使用 GltA[K167A] 变体的限氮工艺在 31 小时内以 0.31 g/g 的产量生成 36.9 g/L 甲羟戊酸酯。 本研究表明，GltA 变体提供了一种影响细胞内乙酰辅酶 A 库以产生乙酰辅酶 A 衍生产物的方法，并且乙酰辅酶 A 库而不是 NADPH 可用性是甲羟戊酸生产的重要限制因素。