凝结芽孢杆菌乳酸转运蛋白LutP的生理功能及其分子调控机制

L-Lactic acid is a building block widely used in food, pharmaceutical, and chemical industries. Use of L-lactic acid in the synthesis of poly-lactic-acid has grown over the years. This carboxylic acid is currently mainly produced using lactic-acid bacteria. The use of microorganisms that are more tolerant to low pH values could strongly decrease the amount of neutralizing agents required and lower the cost of down-stream processing, and metabolic pathway manipulation for improving low-pH-tolerance of microorganisms is becoming a well-established concept. Lactate transporter LutP is responsible for the permeation of lactate. It plays a vital role for the maintenance of cells in low-pH environment. Bacillus coagulans is an efficient polymer-grade L-lactic acid producer. Inspection of the genomic sequence of B. coagulans revealed that lutP and lutR (encoding a transcriptional regulator) are adjacently arranged. In this study, LutP, together with its regulator, will be purified from B. coagulans, and its biochemical and kinetic characterization will be studied. Molecular biological methods, such as gene knockout in vivo, mutant strain complementation and site-directed mutagenesis, will also be used to verify the functions of LutP. Furthermore, the mechanisms for molecular regulation of LutP will also be investigated. This study is expected to obtain a new lactate transporter and provide the fundamental information for the construction of excellence performance of lactic-acid-producing strains.

光学纯L-乳酸是迅速发展起来的生物塑料─聚乳酸的主要前体。由于乳酸对微生物生长有抑制作用，因此构建耐酸L-乳酸生产菌株一直是研究热点。目前研究主要集中在酵母工程菌的构建，发现乳酸转运蛋白对于酵母菌耐酸性具有重要作用。乳酸通透酶（LutP）具有协同转运乳酸功能。作为乳酸代谢操纵子中的关键组分，LutP对于维持细胞在低酸环境中生长起着至关重要的作用。凝结芽孢杆菌是L-乳酸发酵的主要菌种。通过基因组分析，发现了LutP基因，但未发现乳酸利用酶基因，以上结果暗示凝结芽孢杆菌中LutP的表达调控机制具有独特之处。本申请通过LutP与调节蛋白的酶学特性分析，结合基因敲除手段，揭示LutP在乳酸发酵过程中所起的具体作用；采用生物化学与分子生物学相结合手段，阐明LutP的表达调控机制。通过该项目的研究，有望获得一种新的乳酸转运元件，研究结果可为构建耐酸高产乳酸发酵菌株和细胞工厂提供有价值的理论参考。

凝结芽孢杆菌是同型L-乳酸发酵菌种。乳酸通透酶（LutP）具有协同转运乳酸功能。作为乳酸代谢操纵子中的关键组分，LutP对于维持细胞在低酸环境中生长起着至关重要的作用。通过基因组分析，发现了lutP基因及其调节基因lutR位于一个基因簇中，但未发现乳酸利用酶基因，以上结果暗示凝结芽孢杆菌中LutP的表达调控机制具有独特之处。本申请采用RT-PCR技术，分析乳酸和葡萄糖对LutP转录水平的影响，发现LutP的转录受乳酸激活，受葡萄抑制。通过LutP编码基因的敲除与回补，发现LutP的缺失抑制了菌株的生长和葡萄糖的消耗。利用同位素标记实验证实了LutP具有向细胞内转运乳酸的能力。进一步采用分子互作手段（凝胶阻滞实验和生物膜干涉实验），确定了LutP转录调控结合位点，葡萄糖和乳酸正是通过改变调控蛋白与结合位点的亲和力来调控LutP的表达。通过该项目的研究，首次揭示凝结芽孢杆菌中LutP在乳酸发酵过程中的动态调控机制，研究结果可为构建耐酸高产乳酸发酵菌株和细胞工厂提供有价值的理论参考。