Pyruvate Production by Escherichia coli by Use of Pyruvate Dehydrogenase Variants
Pyruvate Production by Escherichia coli by Use of Pyruvate Dehydrogenase Variants
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大肠杆菌利用丙酮酸脱氢酶变体生产丙酮酸
DOI：
10.1128/aem.00487-21
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发表时间：
2021-07-01
影响因子：
4.4
通讯作者：
Eiteman, Mark A.
中科院分区：
文献类型：
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作者：
Moxley, W. Chris;Eiteman, Mark A.
在代谢的一个关键分支点改变代谢通量通常是通过基因敲除或通过调节基因表达来完成的。另一种将代谢通量优先导向产品的方法是通过蛋白质工程降低关键酶的活性。在埃希氏螺旋体中，当丙酮酸脱氢酶复合物的碳通量受到抑制时，丙酮酸可以从葡萄糖中积累。基于这一原理，在E - coil C中构建了16个染色体表达的AceE变异，并以葡萄糖为唯一碳源，比较了其生长速率和丙酮酸积累。为了防止丙酮酸转化为其他产物，菌株还含有两个非必需途径的缺失：乳酸脱氢酶（IdhA）和丙酮酸氧化酶（poxB）。研究了去除磷酸烯醇丙酮酸合成酶（ppsA）对丙酮酸同化的影响。在控制间歇和连续工艺条件下考察了最佳的丙酮酸积累菌株。在限氮化过程中，在0.15至028 h（-1）的稳态生长速率下，表达AceE[-1106V]变体的工程菌株以059至0.66 g丙酮酸/g葡萄糖的产量积累丙酮酸，比生产率为0.78至0.92 g丙酮酸/g细胞。这些结果证明了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到允许丙酮酸积累。这种方法可以潜在地应用于新陈代谢中的其他关键酶，以将碳导向生化产物。微生物利用可再生资源生产生物化学品已成为传统化学合成方法的一种高效、经济的替代方法。代谢工程工具对于在经济上可行的水平上优化过程是很重要的。本研究描述了一个额外的工具，以改变中心代谢和直接代谢通量的产品。我们已经证明，丙酮酸脱氢酶复合物的变体可以引导代谢通量远离细胞生长，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策略配对，以优化新陈代谢，并创建工业相关和经济上可行的过程。
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 coil, 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 coil 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 (IdhA) and pyruvate oxidase (poxB). The effect of deleting phosphoenolpyruvate synthase (ppsA) 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 028 h(-1), an engineered strain expressing the AceE[-1106V] variant accumulated pyruvate at a yield of 059 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.IMPORTANCE 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.