Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China.
Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China.
J Biotechnol. 2021 May 20;332:20-28. doi: 10.1016/j.jbiotec.2021.03.014. Epub 2021 Mar 23.
To explore the suitability of Corynebacterium glutamicum as a chassis for diacetyl production from glucose, diacetyl metabolic pathway and the respiratory chain were linked to achieve redox balance. The carbon flux was redirected from pyruvate to diacetyl by overexpressing the α-acetolactate synthase, in combination with disruption the biosynthetic pathways of lactate, acetoin, 2,3-butanediol and acetate in C. glutamicum ATCC 13032. These modifications resulted in a sharp increase of the NADH/NAD ratio from 0.53 to 1.10, and produced 0.58 g/L diacetyl under aerobic conditions, representing a 58-fold increase over the wild type. Although the modification of the by-product pathways is an effective strategy, these disruption led to intracellular cofactor imbalance. NADH re-oxidization was further successfully solved by overexpressing of cytochrome bd oxidase. We constructed an efficient respiration-dependent cell factory by modification of the respiratory chain, improving diacetyl titer to 1.29 g/L in CGC11, decreased NADH/NAD ratio to 0.45, increased the ATP concentration from 8.51 to 10.64 μM/gDCW. To our best knowledge, this is the first report of diacetyl synthesis in C. glutamicum. Intracellular cofactor imbalance can be reduced by modification of the respiratory chain for production of diacetyl as well as other bio-based products with cofactor imbalance in C. glutamicum.
为了探索谷氨酸棒杆菌作为从葡萄糖生产双乙酰的底盘的适宜性，通过连接双乙酰代谢途径和呼吸链来实现氧化还原平衡。通过过表达α-乙酰乳酸合酶，将碳通量从丙酮酸重新定向到双乙酰，同时敲除谷氨酸棒杆菌 ATCC 13032 中的乳酸、乙酰醇、2,3-丁二醇和乙酸的生物合成途径。这些修饰使 NADH/NAD 比从 0.53 增加到 1.10，在有氧条件下产生 0.58 g/L 的双乙酰，比野生型增加了 58 倍。虽然修饰副产物途径是一种有效的策略，但这些修饰导致细胞内辅因子失衡。通过过表达细胞色素 bd 氧化酶，进一步成功解决了 NADH 的再氧化问题。我们通过修饰呼吸链构建了一个有效的依赖呼吸的细胞工厂，在 CGC11 中使双乙酰产量提高到 1.29 g/L，将 NADH/NAD 比降低到 0.45，将 ATP 浓度从 8.51 提高到 10.64 μM/gDCW。据我们所知，这是首次在谷氨酸棒杆菌中报道双乙酰的合成。通过修饰呼吸链可以减少细胞内辅因子失衡，从而在谷氨酸棒杆菌中生产双乙酰和其他具有辅因子失衡的生物基产品。
