凝结芽孢杆菌磷酸盐响应启动子的研究

Phosphate-responsive promoter region in Heyndrickxia coagulans

摘要:

耐热凝结芽孢杆菌因其对营养要求简单、发酵产物浓度高以及耐高温等特点，已成为乳酸发酵的主要菌种。在前期的研究中，我们发现磷酸盐可以激活凝结芽孢杆菌l-乳酸脱氢酶基因的转录，从而提高乳酸产量。然而，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。【目的】对凝结芽孢杆菌响应磷酸盐的调控机制进行研究。【方法】通过RT-PCR分析磷酸盐添加时凝结芽孢杆菌乳酸脱氢酶转录水平变化，确定响应磷酸盐的关键元件区域，进一步通过分子生物学手段，分析凝结芽孢杆菌响应磷酸盐的关键基因片段。【结果】确定了响应磷酸盐的关键元件位于乳酸脱氢酶基因上游启动子区，解析了响应磷酸盐的l-乳酸脱氢酶启动子核心区，利用该启动子及核心区能够有效驱动外源d-乳酸脱氢酶基因的表达，实现在凝结芽孢杆菌中d-乳酸的合成。【结论】本研究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参考。

Abstract:

Heyndrickxia coagulans characterized by low nutrient requirements, high titers of fermentation products, and thermal tolerance has become a major microbial species for lactic acid fermentation. We have demonstrated that phosphate stimulates the gene expression of l-lactate dehydrogenase in H. coagulans to increase l-lactic acid production, the mechanism of which, however, remains unknown. [Objective] To primarily investigated the mechanism of phosphate in stimulating the gene transcription of lactate dehydrogenase in H. coagulans. [Methods] RT-PCR was employed to analyze the transcriptional level changes of lactate dehydrogenase in H. coagulans after phosphate addition. The core promotor region responsive to phosphate stimulation was identified by conventional methods of molecular biology. [Results] The key element responsive to phosphate was located in the upstream promoter region of the l-lactate dehydrogenase gene. The core region of the promoter was responsible for phosphate stimulation. The identified promoter was employed to promote d-lactate production with diammonium phosphate addition. [Conclusion] This study reported a new phosphate-responsive gene element, providing a theoretical basis for improving the synthesis efficiency of other biochemicals.