Abstract：
Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel \"PEACES\" system by (1) expressing the NAD+ kinase gene ppnk from Clostridium glutamicum and the NADP+-dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.
摘要：
维生素B5[D-泛酸（D-PA）]是一种必需的水溶性维生素，广泛用于食品和饲料工业。目前,相对较低的发酵效率限制了D-PA的工业应用。这里,使用系统的代谢工程策略构建了无质粒的D-PA超生产者。首先,丙酮酸通过删除非磷酸转移酶系统来富集，抑制丙酮酸竞争性分支，并动态地控制TCA循环。接下来,通过筛选限速酶PanBC并逐个调节该途径的其他酶，可以增强（R）-泛解酸途径。然后，为了增强NADPH的可持续性，通过新的“PEACES”系统实现NADPH再生，方法是（1）表达谷氨酸梭菌的NAD激酶基因ppnk和乙酰丁酸梭菌的NADP依赖性gapCcae，（2）敲除内源性sthA基因，在D-PA生物合成途径中与ilvC和panE相互作用。结合转录组分析，发现膜蛋白OmpC和TolR通过增加膜流动性促进D-PA外排。菌株PA132通过两阶段补料分批发酵产生的D-PA滴度为83.26g/L,这是迄今为止报道的最高D-PA滴度。这项工作为D-PA的工业生产建立了有竞争力的生产者，并为相关产品的生产提供了有效的策略。
