Abstract：
Erythritol is a natural non-caloric sweetener, which is produced by fermentation and extensively applied in food, medicine and chemical industries. The final step of the erythritol synthesis pathway is involved in erythritol reductase, whose activity and NADPH-dependent become the limiting node of erythritol production efficiency. Herein, we implemented a strategy combining molecular docking and thermal stability screening to construct an ER mutant library. And we successfully obtained a double mutant ERK26N/V295M (ER*) whose catalytic activity was 1.48 times that of wild-type ER. Through structural analysis and MD analysis, we found that the catalytic pocket and the enzyme stability of ER* were both improved. We overexpressed ER* in the engineered strain ΔKU70 to obtain the strain YLE-1. YLE-1 can produce 39.47 g/L of erythritol within 144 h, representing a 35% increase compared to the unmodified strain, and a 10% increase compared to the strain overexpressing wild-type ER. Considering the essentiality of NADPH supply, we further co-expressed ER* with two genes from the oxidative phase of PPP, ZWF1 and GND1. This resulted in the construction of YLE-3, which exhibited a significant increase in production, producing 47.85 g/L of erythritol within 144 h, representing a 63.90% increase compared to the original chassis strain. The productivity and the yield of the engineered strain YLE-3 were 0.33 g/L/h and 0.48 g/g glycerol, respectively. This work provided an ER mutation with excellent performance, and also proved the importance of cofactors in the process of erythritol synthesis, which will promote the industrial production of erythritol by metabolic engineering of Y. lipolytica.
摘要：
赤藓糖醇是一种天然的无热量甜味剂，它是通过发酵生产的，广泛应用于食品中，医药和化学工业。赤藓糖醇合成途径的最后一步涉及赤藓糖醇还原酶，其活性和NADPH依赖性成为赤藓糖醇生产效率的限制节点。在这里，我们实施了结合分子对接和热稳定性筛选的策略构建ER突变体文库.并且我们成功获得了双突变体ERK26N/V295M（ER*），其催化活性是野生型ER的1.48倍。通过结构分析和MD分析,我们发现ER*的催化口袋和酶稳定性都得到了改善。我们在工程菌株ΔKU70中过表达ER*以获得菌株YLE-1。YLE-1可在144h内产生39.47g/L的赤藓糖醇，与未修饰的菌株相比，增加了35%，与过表达野生型ER的菌株相比增加10%。考虑到NADPH供应的必要性，我们进一步将ER*与PPP氧化期的两个基因共表达，ZWF1和GND1。这导致了YLE-3的建造，其产量显着增加，在144小时内生产47.85g/L赤藓糖醇，与原始底盘应变相比，增加了63.90%。工程菌株YLE-3的生产率和产量分别为0.33g/L/h和0.48g/g甘油，分别。这项工作提供了一个具有优异性能的ER突变，也证明了辅因子在赤藓糖醇合成过程中的重要性，这将促进Y的代谢工程赤藓糖醇的工业生产。
