BACKGROUND 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. RESULTS 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%. CONCLUSION 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.

中文翻译：

---

谷氨酸棒杆菌工业级 (3R)-乙偶姻生物合成的工程中心途径。


背景技术乙偶姻，特别是光学纯的(3S)-或(3R)-对映体，是一种高附加值的生物基平台化学品和重要的潜在医药中间体。在过去的几十年里，人们致力于通过绿色生物技术生产乙偶姻。然而，用于生产光学纯乙偶姻对映体的有效且经济的方法却很少报道。此前，我们系统地改造了 GRAS 微生物谷氨酸棒杆菌，以从葡萄糖中高效生产 (3R)-乙偶姻。然而，其产量和平均生产率对于工业生物过程来说仍然不能令人满意。结果在这项研究中，使用多种代谢工程策略，乙偶姻生产者 CGR6 中的细胞碳通量进一步重定向至乙偶姻合成，包括阻断回补途径、减弱 TCA 循环的关键基因以及将 alsSD 操纵子的额外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化酶的失活相结合，对乙偶姻生产表现出显着的协同效应。最终，优化的工程菌株CGS11在5 L发酵罐中以1.86 g/L/h的速率生产乙偶姻，滴度为102.45 g/L，葡萄糖产量为0.419 g/g。所得(3R)-乙偶姻的光学纯度超过95%。结论 据我们所知，这是高度对映体富集的 (3R)-乙偶姻的最高滴度，以及具有竞争力的产品产率和生产率，通过简单、绿色的工艺实现，无需昂贵的添加剂或底物。 因此，该工艺为在不久的将来通过微生物发酵实现简单、高效、经济和环保的 (3R)-乙偶姻生产提供了可能性。