Dodelin Jeffrey K, Rose Abigail E, Rajpurohit Hemshikha, Eiteman Mark A
Department of Microbiology, University of Georgia, Athens, Georgia, USA.
School of Chemical, Materials and Biomedical Engineering, University of Georgia, Athens, Georgia, USA.
Biotechnol Bioeng. 2025 Mar;122(3):548-560. doi: 10.1002/bit.28902. Epub 2024 Dec 9.
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.
甲羟戊酸是多种类异戊二烯的生化前体。甲羟戊酸途径消耗三摩尔乙酰辅酶A，因此代谢乙酰辅酶A的天然途径与甲羟戊酸合成相互竞争。此外，由甲羟戊酸单酰辅酶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。本研究聚焦于参与乙酰辅酶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，以提高在异源甲羟戊酸途径的三个基因过表达的大肠杆菌中甲羟戊酸的产量和生产率。在有无酪蛋白氨基酸的情况下，在摇瓶中比较了九个GltA变体与ΔgltA敲除菌株和野生型GltA菌株的甲羟戊酸产量。在有酪蛋白氨基酸的情况下，所有变体产生甲羟戊酸的产量均高于野生型对照，但低于GltA敲除菌株。在无酪蛋白氨基酸的情况下，表达野生型GltA的菌株产生甲羟戊酸的产量最高，而大多数变体主要积累乙酸盐。使用野生型菌株和两个柠檬酸合酶变体，还将四个磷酸果糖激酶变体与ΔpfkA敲除菌株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。在1升规模下比较五个菌株的受控过程表明产量和生产率之间呈反比关系，GltA[K167A]变体在产量（0.20 g/g）和生产率（0.87 g/L·h）方面表现出最佳平衡。使用GltA[K167A]变体的氮限制过程在31小时内产生了36.9 g/L甲羟戊酸，产量为0.31 g/g。本研究表明，GltA变体提供了一种影响细胞内乙酰辅酶A库以生成乙酰辅酶A衍生产品的方法，并且乙酰辅酶A库而非NADPH的可用性是甲羟戊酸生产的重要限制因素。
