State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Rd., P.O. box 329#, Shanghai, 200237, People's Republic of China.
Shanghai Acebright Pharmaceuticals Group Co., Ltd, Shanghai, 201203, People's Republic of China.
Microb Cell Fact. 2017 Sep 12;16(1):150. doi: 10.1186/s12934-017-0764-z.
Riboflavin, an intermediate of primary metabolism, is one kind of important food additive with high economic value. The microbial cell factory Bacillus subtilis has already been proven to possess significant importance for the food industry and have become one of the most widely used riboflavin-producing strains. In the practical fermentation processes, a sharp decrease in riboflavin production is encountered along with a decrease in the dissolved oxygen (DO) tension. Influence of this oxygen availability on riboflavin biosynthesis through carbon central metabolic pathways in B. subtilis is unknown so far. Therefore the unveiled effective metabolic pathways were still an unaccomplished task till present research work.
In this paper, the microscopic regulation mechanisms of B. subtilis grown under different dissolved oxygen tensions were studied by integrating C metabolic flux analysis, metabolomics and transcriptomics. It was revealed that the glucose metabolic flux through pentose phosphate (PP) pathway was lower as being confirmed by smaller pool sizes of metabolites in PP pathway and lower expression amount of ykgB at transcriptional level. The latter encodes 6-phosphogluconolactonase (6-PGL) under low DO tension. In response to low DO tension in broth, the glucose metabolic flux through Embden-Meyerhof-Parnas (EMP) pathway was higher and the gene, alsS, encoding for acetolactate synthase was significantly activated that may result due to lower ATP concentration and higher NADH/NAD ratio. Moreover, ResE, a membrane-anchored protein that is capable of oxygen regulated phosphorylase activity, and ResD, a regulatory protein that can be phosphorylated and dephosphorylated by ResE, were considered as DO tension sensor and transcriptional regulator respectively.
This study shows that integration of transcriptomics, C metabolic flux analysis and metabolomics analysis provides a comprehensive understanding of biosynthesized riboflavin's regulatory mechanisms in B. subtilis grown under different dissolved oxygen tension conditions. The two-component system, ResD-ResE, was considered as the signal receiver of DO tension and gene regulator that led to differences between biomass and riboflavin production after triggering the shifts in gene expression, metabolic flux distributions and metabolite pool sizes.
核黄素是初级代谢的中间产物，是一种具有高经济价值的重要食品添加剂。枯草芽孢杆菌微生物细胞工厂已被证明对食品工业具有重要意义，已成为最广泛使用的生产核黄素的菌株之一。在实际发酵过程中，随着溶解氧（DO）张力的降低，核黄素的产量会急剧下降。到目前为止，这种氧可用性对枯草芽孢杆菌碳中心代谢途径中核黄素生物合成的影响尚不清楚。因此，到目前为止，揭示有效的代谢途径仍然是一项未完成的任务。
本文通过整合 C 代谢通量分析、代谢组学和转录组学，研究了不同溶解氧张力下枯草芽孢杆菌生长的微观调控机制。结果表明，通过戊糖磷酸（PP）途径的葡萄糖代谢通量较低，这可以通过 PP 途径中代谢物池的大小较小和转录水平上 ykgB 的表达量较低来证实。后者在低 DO 张力下编码 6-磷酸葡萄糖酸内酯酶（6-PGL）。在发酵液中 DO 张力较低时，通过 EMP 途径的葡萄糖代谢通量较高，并且编码乙酰乳酸合酶的基因 alsS 被显著激活，这可能是由于 ATP 浓度降低和 NADH/NAD 比升高所致。此外，ResE 是一种能够氧调节磷酸化酶活性的膜锚定蛋白，ResD 是一种可以被 ResE 磷酸化和去磷酸化的调节蛋白，它们分别被认为是 DO 张力传感器和转录调节剂。
本研究表明，转录组学、C 代谢通量分析和代谢组学分析的整合为不同溶解氧张力条件下枯草芽孢杆菌生物合成核黄素的调控机制提供了全面的了解。ResD-ResE 二组分系统被认为是 DO 张力的信号接收器和基因调节剂，它导致了 DO 张力触发基因表达、代谢通量分布和代谢物池大小的变化后生物量和核黄素产量的差异。
