Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia 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 including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). 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 into E. coli C ΔpoxB twenty-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.

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、产量和生成率。广泛的生化物质来源于中心碳代谢物乙酰辅酶 A，大多数微生物（包括大肠杆菌）中乙酰辅酶 A 的最大天然排泄是通过柠檬酸合酶（由gltA编码）进入三羧酸 (TCA) 循环基因）。由于任何源自乙酰辅酶 A 的生化物质的途径最终都必须与柠檬酸合酶竞争，因此柠檬酸合酶活性的降低应促进源自乙酰辅酶 A 的产物的形成增加。为了验证这一假设，我们将大肠杆菌C Δ poxB整合到二十八种柠檬酸合酶变体具有预期会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酯的产生，乙酸酯是源自乙酰辅酶A的模型产物。与天然柠檬酸合酶 (0.05 g/g) 相比，残基 W260、A267 和 V361 处的柠檬酸合酶突变导致最大的醋酸盐产量（约 0.24 g/g 葡萄糖）。在受控的分批和连续过程中进一步检查了这些变体。结果为改进乙酰辅酶A衍生化合物的生产提供了重要的见解。