Engineered citrate synthase alters Acetate Accumulation in Escherichia coli
Engineered citrate synthase alters Acetate Accumulation in Escherichia coli
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工程柠檬酸合酶改变大肠杆菌中的乙酸积累
DOI：
10.1016/j.ymben.2020.06.006
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发表时间：
2020
影响因子：
8.4
通讯作者：
Eiteman, Mark A.
中科院分区：
文献类型：
--
作者：
Tovilla-Coutiño, D. Brisbane;Momany, Cory;Eiteman, Mark A.
代谢工程用于提高宿主微生物（如大肠杆菌）中生化产物的滴度、产量和生成速率。许多生化物质都来源于中心碳代谢物乙酰辅酶A，它是大多数微生物中最大的天然乙酰辅酶A排泄物，包括E。大肠杆菌通过柠檬酸合酶（由gltA基因编码）进入三羧酸（TCA）循环。由于从乙酰辅酶A衍生的任何生物化学的途径最终必须与柠檬酸合酶竞争，柠檬酸合酶活性的降低应促进从乙酰辅酶A衍生的产物的形成增加。为了验证这一假设，我们整合了E。coliC Δ poxB 28种柠檬酸合酶变体，其具有预期降低柠檬酸合酶活性的特定点突变。在摇瓶中评估这些变体的生长和乙酸盐的产生，乙酸盐是源自乙酰辅酶A的模型产物。与天然柠檬酸合酶（0.05 g/g）相比，柠檬酸合酶在残基W260、A267和V361处的突变导致最大的乙酸盐产率（约0.24 g/g葡萄糖）。在受控的分批和连续工艺中进一步检查了这些变体。这些结果为改善乙酰辅酶A衍生化合物的生产提供了重要的见解。
Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such asEscherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes includingE. coliis entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by thegltAgene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated intoE. coliC ΔpoxBtwenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.