Department of Microbiology, University of Georgia, Athens, Georgia, USA.
School of Chemical, Materials and Biomedical Engineering, University of Georgia, Athens, Georgia, USA.
Appl Environ Microbiol. 2021 Jun 11;87(13):e0048721. doi: 10.1128/AEM.00487-21.
Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase () and pyruvate oxidase (). The effect of deleting phosphoenolpyruvate synthase () on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli. This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes.
改变代谢途径中的关键分支点的代谢通量通常通过基因敲除或调节基因表达来实现。另一种直接将代谢通量优先导向产物的方法是通过蛋白质工程降低关键酶的活性。在大肠杆菌中，当丙酮酸脱氢酶复合物中的碳通量受到抑制时，葡萄糖可以积累丙酮酸。基于这一原理，在大肠杆菌 C 中构建了 16 种染色体表达的 AceE 变体，并使用葡萄糖作为唯一碳源比较了它们的生长速率和丙酮酸积累。为了防止丙酮酸转化为其他产物，这些菌株还缺失了两条非必需途径：乳酸脱氢酶（）和丙酮酸氧化酶（）。还研究了删除磷酸烯醇丙酮酸合酶（）对丙酮酸同化的影响。在控制批处理和连续过程中检查了最佳的丙酮酸积累菌株。在氮限制恒化器过程中，在 0.15 至 0.28 h 的稳定生长速率下，表达 AceE[H106V]变体的工程菌株以 0.59 至 0.66 g 丙酮酸/g 葡萄糖的产率积累丙酮酸，比生产率为 0.78 至 0.92 g 丙酮酸/g 细胞·h。这些结果提供了概念验证，即丙酮酸脱氢酶复合物变体可以有效地将碳通量从中心碳代谢转移开，从而允许丙酮酸积累。这种方法可以潜在地应用于代谢中的其他关键酶，以将碳引导到生化产物上。 从可再生资源中生产生物化学物质已成为替代传统化学合成方法的高效且具有成本效益的方法。代谢工程工具对于优化在经济上可行的水平下运行的过程非常重要。本研究描述了一种修改中心代谢并将代谢通量导向产物的附加工具。我们已经表明，丙酮酸脱氢酶复合物的变体可以将代谢通量从细胞生长中转移出来，从而增加大肠杆菌中丙酮酸的产量。这种方法可以与现有的代谢优化策略结合使用，以创建具有工业相关性和经济可行性的过程。
