Yuan Panhong, Xu Mengtao, Mao Chengyao, Zheng Han, Sun Dongchang
College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China.
ACS Synth Biol. 2023 Oct 20;12(10):2983-2995. doi: 10.1021/acssynbio.3c00315. Epub 2023 Sep 4.
In response to a high concentration of glucose, , a microbial chassis for producing many industrial metabolites, rapidly takes up glucose using the phosphotransferase system (PTS), leading to overflow metabolism, a common phenomenon observed in many bacteria. Although overflow metabolism affects cell growth and reduces the production of many metabolites, effective strategies that reduce overflow metabolism while maintaining normal cell growth remain to be developed. Here, we used a quorum sensing (QS)-mediated circuit to tune the glucose uptake rate and thereby relieve overflow metabolism in an engineered for producing d-pantothenic acid (DPA). A low-efficiency non-PTS system was used for glucose uptake at the early growth stages to avoid a rapid glycolytic flux, while an efficient PTS system, which was activated by a QS circuit, was automatically activated at the late growth stages after surpassing a threshold cell density. This strategy was successfully applied as a modular metabolic engineering process for the high production of DPA. By enhancing the translation levels of key enzymes (3-methyl-2-oxobutanoate hydroxymethytransferase, pantothenate synthetase, aspartate 1-decarboxylase proenzyme, 2-dehydropantoate 2-reductase, dihydroxy-acid dehydratase, and acetolactate synthase) with engineered 5'-untranslated regions (UTRs) of mRNAs, the metabolic flux was promoted in the direction of DPA production, elevating the yield of DPA to 5.11 g/L in shake flasks. Finally, the engineered produced 21.52 g/L of DPA in fed-batch fermentations. Our work not only revealed a new strategy for reducing overflow metabolism by adjusting the glucose uptake rate in combination with promoting the translation of key metabolic enzymes through engineering the 5'-UTR of mRNAs but also showed its power in promoting the bioproduction of DPA in , exhibiting promising application prospects.
作为一种用于生产多种工业代谢产物的微生物底盘，在高浓度葡萄糖的刺激下，会通过磷酸转移酶系统（PTS）迅速摄取葡萄糖，从而导致溢流代谢，这是在许多细菌中都观察到的常见现象。尽管溢流代谢会影响细胞生长并降低许多代谢产物的产量，但在维持细胞正常生长的同时减少溢流代谢的有效策略仍有待开发。在此，我们使用了一种群体感应（QS）介导的回路来调节葡萄糖摄取速率，从而缓解工程化生产d -泛酸（DPA）过程中的溢流代谢。在生长早期阶段，使用低效的非PTS系统进行葡萄糖摄取，以避免糖酵解通量过快，而由QS回路激活的高效PTS系统在细胞密度超过阈值后的生长后期阶段会自动被激活。该策略作为一种模块化代谢工程过程成功应用于DPA的高产生产。通过用工程化的mRNA 5'非翻译区（UTR）提高关键酶（3 -甲基 - 2 -氧代丁酸羟甲基转移酶、泛酸合成酶、天冬氨酸1 -脱羧酶原酶、2 -脱氢泛酸2 -还原酶、二羟基酸脱水酶和乙酰乳酸合酶）的翻译水平，代谢通量朝着DPA生产的方向得到促进，在摇瓶中DPA产量提高到5.11 g/L。最后，工程菌在补料分批发酵中生产了21.52 g/L的DPA。我们的工作不仅揭示了一种通过调节葡萄糖摄取速率并结合工程化mRNA的5'UTR促进关键代谢酶翻译来减少溢流代谢的新策略，还展示了其在促进工程菌中DPA生物合成方面的能力，具有广阔的应用前景。
