Jojima Toru, Igari Takafumi, Noburyu Ryoji, Watanabe Akira, Suda Masako, Inui Masayuki
Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto, 619-0292, Japan.
Faculty of Agriculture, Department of Environmental Management, Kindai University, 3327-204 Nakamachi, Nara, 631-8505, Japan.
Biotechnol Biofuels. 2021 Feb 16;14(1):45. doi: 10.1186/s13068-021-01876-3.
It is interesting to modify sugar metabolic pathways to improve the productivity of biocatalysts that convert sugars to value-added products. However, this attempt often fails due to the tight control of the sugar metabolic pathways. Recently, activation of the Entner-Doudoroff (ED) pathway in Escherichia coli has been shown to enhance glucose consumption, though the mechanism underlying this phenomenon is poorly understood. In the present study, we investigated the effect of a functional ED pathway in metabolically engineered Corynebacterium glutamicum that metabolizes glucose via the Embden-Meyerhof-Parnas (EMP) pathway to produce ethanol under oxygen deprivation. This study aims to provide further information on metabolic engineering strategies that allow the Entner-Doudoroff and Embden-Meyerhof-Parnas pathways to coexist.
Three genes (zwf, edd, and eda) encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis were expressed in a genetically modified strain, C. glutamicum CRZ2e, which produces pyruvate decarboxylase and alcohol dehydrogenase from Z. mobilis. A C-labeling experiment using [1-C] glucose indicated a distinctive C distribution of ethanol between the parental and the ED-introduced strains, which suggested an alteration of carbon flux as a consequence of ED pathway introduction. The ED-introduced strain, CRZ2e-ED, consumed glucose 1.5-fold faster than the parental strain. A pfkA deletion mutant of CRZ2e-ED (CRZ2e-EDΔpfkA) was also constructed to evaluate the effects of EMP pathway inactivation, which showed an almost identical rate of glucose consumption compared to that of the parental CRZ2e strain. The introduction of the ED pathway did not alter the intracellular NADH/NAD ratio, whereas it resulted in a slight increase in the ATP/ADP ratio. The recombinant strains with simultaneous overexpression of the genes for the EMP and ED pathways exhibited the highest ethanol productivity among all C. glutamicum strains ever constructed.
The increased sugar consumption observed in ED-introduced strains was not a consequence of cofactor balance alterations, but rather the crucial coexistence of two active glycolytic pathways for enhanced glucose consumption. Coexistence of the ED and EMP pathways is a good strategy for improving biocatalyst productivity even when NADPH supply is not a limiting factor for fermentation.
改造糖代谢途径以提高将糖转化为增值产品的生物催化剂的生产力是一件有趣的事情。然而，由于糖代谢途径受到严格调控，这种尝试往往会失败。最近，已证明在大肠杆菌中激活Entner-Doudoroff（ED）途径可增强葡萄糖消耗，尽管对这一现象背后的机制了解甚少。在本研究中，我们研究了功能性ED途径在代谢工程改造的谷氨酸棒杆菌中的作用，该菌株在缺氧条件下通过Embden-Meyerhof-Parnas（EMP）途径代谢葡萄糖以生产乙醇。本研究旨在提供更多关于使Entner-Doudoroff途径和Embden-Meyerhof-Parnas途径共存的代谢工程策略的信息。
编码葡萄糖-6-磷酸脱氢酶、6-磷酸葡萄糖酸脱水酶和2-酮-3-脱氧-6-磷酸葡萄糖酸醛缩酶的三个基因（zwf、edd和eda）来自运动发酵单胞菌，在基因改造菌株谷氨酸棒杆菌CRZ2e中表达，该菌株产生来自运动发酵单胞菌的丙酮酸脱羧酶和乙醇脱氢酶。使用[1-C]葡萄糖进行的碳标记实验表明，亲本菌株和引入ED途径的菌株之间乙醇的碳分布不同，这表明引入ED途径导致了碳通量的改变。引入ED途径的菌株CRZ2e-ED消耗葡萄糖的速度比亲本菌株快1.5倍。还构建了CRZ2e-ED的pfkA缺失突变体（CRZ2e-EDΔpfkA）以评估EMP途径失活的影响，其葡萄糖消耗速率与亲本CRZ2e菌株几乎相同。ED途径的引入并未改变细胞内NADH/NAD比率，然而却导致ATP/ADP比率略有增加。在所有构建的谷氨酸棒杆菌菌株中，同时过表达EMP和ED途径基因的重组菌株表现出最高的乙醇生产力。
在引入ED途径的菌株中观察到的糖消耗增加不是辅因子平衡改变的结果，而是两条活跃糖酵解途径共存以增强葡萄糖消耗的关键。即使NADPH供应不是发酵的限制因素，ED和EMP途径的共存也是提高生物催化剂生产力的良好策略。
