Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering of Ministry of Education, SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Microb Cell Fact. 2020 May 12;19(1):102. doi: 10.1186/s12934-020-01363-8.
Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce (3R)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses.
In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting (3R)-acetoin surpassed 95%.
To the best of our knowledge, this is the highest titer of highly enantiomerically enriched (3R)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of (3R)-acetoin via microbial fermentation in the near future.
乙酰丙酮，尤其是光学纯的（3S）-或（3R）-对映异构体，是一种高附加值的生物基平台化学品，也是一种重要的潜在药物中间体。在过去的几十年中，人们投入了大量精力通过绿色生物技术生产乙酰丙酮。然而，高效且经济的方法来生产光学纯的乙酰丙酮对映异构体却很少有报道。此前，我们系统地对 GRAS 微生物谷氨酸棒杆菌进行工程改造，使其能够从葡萄糖高效生产（3R）-乙酰丙酮。然而，其产量和平均生产力仍不适合工业生物工艺。
在本研究中，通过几种代谢工程策略进一步将产乙酰丙酮菌 CGR6 中的细胞碳通量重新定向到乙酰丙酮合成，包括阻断氨甲酰磷酸合成途径、减弱 TCA 循环的关键基因的活性以及将额外的 alsSD 操纵子拷贝整合到基因组中。其中，柠檬酸合酶的减弱和磷酸烯醇丙酮酸羧激酶的失活组合对乙酰丙酮的生产表现出显著的协同作用。最终，优化后的工程菌株 CGS11 在 5 L 发酵罐中以 1.86 g/L/h 的速度生产了 102.45 g/L 的乙酰丙酮，得率为 0.419 g/g 葡萄糖，产物光学纯度超过 95%。
据我们所知，这是在简单、绿色的工艺中，不使用昂贵的添加剂或底物，以最高的（3R）-乙酰丙酮对映体富集的浓度、具有竞争力的产物得率和生产率实现的。该工艺为未来通过微生物发酵实现（3R）-乙酰丙酮的生产提供了一种简单、高效、经济和环保的可能性。
