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Abstract：
Identifying and overcoming the limitations preventing efficient high-yield production of chemicals remain important tasks in metabolic engineering. In an attempt to rewire Corynebacterium glutamicum to produce ethanol, we attained a low yield (63% of the theoretical) when using resting cells on glucose, and large amounts of succinate and acetate were formed. To prevent the by-product formation, we knocked out the malate dehydrogenase and replaced the native E3 subunit of the pyruvate dehydrogenase complex (PDHc) with that from Escherichia coli, which is active only under aerobic conditions. However, this tampering resulted in a 10-times-reduced glycolytic flux as well as a greatly increased NADH/NAD+ ratio. When we replaced glucose with fructose, we found that the glycolytic flux was greatly enhanced, which led us to speculate whether the source of reducing power could be the pentose phosphate pathway (PPP) that is bypassed when fructose is metabolized. Indeed, after shutting down the PPP by deleting the zwf gene, encoding glucose-6-phosphate dehydrogenase, the ethanol yield on glucose increased significantly, to 92% of the theoretical. Based on that, we managed to rechannel the metabolism of C. glutamicum into d-lactate with high yield, 98%, which is the highest that has been reported. It is further demonstrated that the PPP-inactivated platform strain can offer high-yield production of valuable chemicals using lactose contained in dairy waste as feedstock, which paves a promising way for potentially turning dairy waste into a valuable product.IMPORTANCE The widely used industrial workhorse C. glutamicum possesses a complex anaerobic metabolism under nongrowing conditions, and we demonstrate here that the PPP in resting C. glutamicum is a source of reducing power that can interfere with otherwise redox-balanced metabolic pathways and reduce yields of desired products. By harnessing this physiological insight, we employed the PPP-inactivated platform strains to produce ethanol, d-lactate, and alanine using the dairy waste whey permeate as the feedstock. The production yield was high, and our results show that inactivation of the PPP flux in resting cells is a promising strategy when the aim is to use nongrowing C. glutamicum cells for producing valuable compounds. Overall, we describe the benefits of disrupting the oxidative PPP in nongrowing C. glutamicum and provide a feasible approach toward waste valorization.
摘要：
识别和克服阻碍化学品高效高产生产的限制仍然是代谢工程中的重要任务。为了重新连接谷氨酸棒杆菌以生产乙醇，在葡萄糖上使用静息细胞时，我们获得了低产量（理论值的63％），生成大量的琥珀酸酯和乙酸酯。为了防止副产物的形成，我们敲除苹果酸脱氢酶，用大肠杆菌取代丙酮酸脱氢酶复合物（PDHc）的天然E3亚基，只有在有氧条件下才有活性。然而,这种篡改导致糖酵解通量降低了10倍，NADH/NAD比率大大提高。当我们用果糖代替葡萄糖时，我们发现糖酵解通量大大增强，这使我们推测还原力的来源是否可能是果糖代谢时绕过的磷酸戊糖途径（PPP）。的确,在通过删除zwf基因关闭PPP之后，编码葡萄糖-6-磷酸脱氢酶，葡萄糖的乙醇产量显著增加，92％的理论。基于此,我们设法将谷氨酸棒杆菌的代谢以高产率重新转化为d-乳酸，98%,这是报告中最高的。它进一步证明，PPP灭活的平台菌株可以提供高产量生产的有价值的化学品，使用包含在乳制品废物作为原料的乳糖，这为可能将乳制品废物转化为有价值的产品铺平了一条有希望的道路。重要性广泛使用的工业主力谷氨酸棒杆菌在非生长条件下具有复杂的无氧代谢，我们在此证明了静息谷氨酸棒杆菌中的PPP是还原力的来源，可以干扰其他氧化还原平衡的代谢途径并降低所需产物的产量。通过利用这种生理洞察力，我们使用PPP灭活的平台菌株生产乙醇，d-乳酸，和丙氨酸使用乳品废物乳清渗透物作为原料。产量高，我们的结果表明，当目的是使用非生长的谷氨酸棒杆菌细胞生产有价值的化合物时，静息细胞中PPP通量的失活是一种有前途的策略。总的来说,我们描述了在非生长的谷氨酸棒杆菌中破坏氧化PPP的好处，并提供了一种可行的废物增值方法。
