磷酸盐对耐热凝结芽孢杆菌L-乳酸脱氢酶基因的转录激活机制研究

耐热凝结芽孢杆菌具有营养要求简单，发酵浓度高，高温不易染菌等优势，成为乳酸发酵的主流菌种。磷酸盐成本低，并且成分单一有利于减轻下游处理压力等优势而广泛应用于工业发酵过程。在前期研究过程中，我们发现磷酸盐可以显著激活凝结芽孢杆菌L-乳酸脱氢酶基因的转录，提高乳酸脱氢酶的比酶活，从而提高乳酸产量。磷酸盐如何激活乳酸脱氢酶的基因表达，目前还不清楚，也未有类似的研究报道。当前磷酸盐的代谢调控研究均集中在Pho调节子，探讨细菌如何精确调控从环境中获取适量无机磷满足自身生长需求，还未涉及细菌在其他重要细胞代谢方面的研究。本项目拟对凝结芽孢杆菌响应磷酸盐的调控机制进行深入研究，对凝结芽孢杆菌乳酸脱氢酶基因上游的未知功能蛋白进行鉴定，解析凝结芽孢杆菌对磷酸盐代谢的调控网络，期望揭示其对磷酸盐的调控应答机制，有望获得一种新的响应磷酸盐的调控元件，为提高其他生物化学品的合成效率改造提供新的理论基础。

Thermophilic Bacillus coagulans species have several traits, such as low nutritional requirements, capacity to high production titer and growth with high temperature, which is expected to minimize contamination in industrial-scale fermentations under non-sterile fermentation conditions. Therefore, in recent years, there has been an interest in studies on this species. Compared to organic nitrogen sources, inorganic nitrogen sources for lactic acid fermentation have advantages in facilitating downstream decolorization, purification, and waste treatment processes. Thus, diammonium phosphate has been widely used in industrial fermentation process. In our previous study, we have demonstrated that phosphate stimulates the gene expression of L-lactate dehydrogenase in B. coagulans 2-6, providing higher specific enzyme activity in vivo and increasing L-lactic acid production. While the stimulating mechanism of phosphate on L-Lactate dehydrogenase transcription in thermotolerant B. coagulans strain remains unknown, and even there is no any related reports. To date, the main known mechanism responding to phosphate starvation is the Pho regulon, which constitutes a very efficient and sensible regulatory mechanism in bacteria to secure this nutrient from environments. Indeed, the Pho regulon is also found to be involved in regulation of nutritional cross-talk, secondary metabolite production and pathogenesis, among other important cell processes, but the information is still very limited. In this proposal, we will pioneer to investigate the stimulating mechanism of phosphate on lactate dehydrogenase gene transcription in B. coagulans strains. Based on characterization of the role of unknown functional protein in the upstream of L-lactate dehydrogenase and response network to phosphate in B. coagulans strain 2-6, we aim to reveal its regulatory response mechanism and expect to obtain a new phosphate-responsive regulatory element, which will also provide a new theoretical basis for improving the bio-synthesis efficiency of other chemicals.