The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan Universitygrid.258151.a, Wuxi, People's Republic of China.
Appl Environ Microbiol. 2022 Sep 13;88(17):e0097622. doi: 10.1128/aem.00976-22. Epub 2022 Aug 18.
As an important metabolic intermediate, 2-ketoisovalerate has significant potential in the pharmaceutical and biofuel industries. However, a low output through microbial fermentation inhibits its industrial application. The microbial production of 2-ketoisovalerate is representative whereby redox imbalance is generated with two molecules of NADH accumulated and an extra NADPH required to produce one 2-ketoisovalerate from glucose. To achieve efficient 2-ketoisovalerate production, metabolic engineering strategies were evaluated in Escherichia coli. After deleting the competing routes, overexpressing the key enzymes for 2-ketoisovalerate production, tuning the supply of NADPH, and recycling the excess NADH through enhancing aerobic respiration, a 2-ketoisovalerate titer and yield of 46.4 g/L and 0.644 mol/mol glucose, respectively, were achieved. To reduce the main by-product of isobutanol, the activity and expression of acetolactate synthase were modified. Additionally, a protein degradation tag was fused to pyruvate dehydrogenase (PDH) to curtail the conversion of pyruvate precursor into acetyl-CoA and the generation of NADH. The resulting strain, 050TY/pCTSDTQ487S-RBS55, was initially incubated under aerobic conditions to attain sufficient cell mass and then transferred to a microaerobic condition to degrade PDH and inhibit the remaining activity of PDH. Intracellular redox imbalance was relieved with titer, productivity and yield of 2-ketoisovalerate improved to 55.8 g/L, 2.14 g/L h and 0.852 mol/mol glucose. These results revealed metabolic engineering strategies for the production of a redox-imbalanced fermentative metabolite with high titer, productivity, and yield. An efficient microbial strain was constructed for 2-ketoisovalerate synthesis. The positive effect of the deletion on 2-ketoisovalerate production was found. An optimal combination of overexpressing the target genes was obtained by adjusting the positions of the multiple enzymes on the plasmid frame and the presence of terminators, which could also be useful for the production of downstream products such as isobutanol and l-valine. Reducing the isobutanol by-product by engineering the acetolactate synthase called for special attention to decreasing the promiscuous activity of the enzymes involved. Redox-balancing strategies such as tuning the expression of the chromosomal pyridine nucleotide transhydrogenase, recycling NADH under aerobic cultivation, switching off PDH by degradation, and inhibiting the expression and activity under microaerobic conditions were proven effective for improving 2-ketoisovalerate production. The degradation of PDH and inhibiting this enzyme's expression would serve as a means to generate a wide range of products from pyruvate.
作为一种重要的代谢中间产物，2-酮异戊酸在制药和生物燃料行业具有巨大的潜力。然而，微生物发酵产量低，限制了其工业应用。微生物生产 2-酮异戊酸就是一个很好的例子，在此过程中会产生氧化还原失衡，需要两分子 NADH 才能将一分子葡萄糖转化为 2-酮异戊酸，同时还需要额外的 NADPH。为了实现高效的 2-酮异戊酸生产，对大肠杆菌中的代谢工程策略进行了评估。在删除竞争途径后，通过过表达 2-酮异戊酸生成的关键酶、调节 NADPH 的供应以及通过增强有氧呼吸回收多余的 NADH，将 2-酮异戊酸的产量和得率分别提高到 46.4 g/L 和 0.644 mol/mol 葡萄糖。为了减少异丁醇的主要副产物，对乙酰乳酸合酶的活性和表达进行了修饰。此外，在丙酮酸脱氢酶（PDH）上融合了一个蛋白降解标签，以抑制丙酮酸前体转化为乙酰辅酶 A 和 NADH 的生成。初始条件下，将得到的菌株 050TY/pCTSDTQ487S-RBS55 在有氧条件下培养，以获得足够的细胞量，然后转移到微氧条件下，降解 PDH 并抑制剩余的 PDH 活性。通过提高 2-酮异戊酸的产量、生产力和得率，缓解了细胞内氧化还原失衡，使 2-酮异戊酸的产量、生产力和得率分别提高到 55.8 g/L、2.14 g/L·h 和 0.852 mol/mol 葡萄糖。这些结果揭示了用于生产高浓度、高生产力和高得率的氧化还原失衡发酵代谢物的代谢工程策略。构建了一种高效的微生物菌株，用于 2-酮异戊酸的合成。发现删除对 2-酮异戊酸生产有积极影响。通过调整质粒框架上多个酶的位置和终止子的存在，获得了最佳的目标基因过表达组合，这对于生产下游产品如异丁醇和 L-缬氨酸也可能很有用。通过工程改造乙酰乳酸合酶来减少异丁醇副产物，需要特别注意降低涉及的酶的混杂活性。事实证明，调节染色体吡啶核苷酸转氢酶的表达、在有氧培养条件下回收 NADH、通过降解关闭 PDH 以及在微氧条件下抑制表达和活性等氧化还原平衡策略，对于提高 2-酮异戊酸的生产非常有效。PDH 的降解和抑制该酶的表达将成为从丙酮酸生成广泛产品的一种手段。
