凝结芽孢杆菌中木糖代谢关键基因挖掘及XylA的克隆表达与酶学特性

Mining of key genes for xylose metabolism and cloning, expression, and enzymatic characterization of XylA in Bacillus coagulans

摘要:

凝结芽孢杆菌可以利用秸秆等农业废弃物水解碳源同型发酵生产乳酸，然而该菌株利用混合糖(葡萄糖和木糖)发酵时存在明显的碳源代谢阻遏效应，制约了乳酸产率。本研究通过改变培养基中葡萄糖/木糖比例，获得了凝结芽孢杆菌同步利用混合糖的发酵条件，并借助转录组测序筛选到多个木糖代谢关键基因。通过实时荧光定量PCR (quantitative real-time polymerase chain reaction, qRT-PCR)验证，发现木糖异构酶(xylose isomerase, XylA, EC 5.3.1.5)是影响凝结芽孢杆菌同步利用葡萄糖/木糖的关键酶，进而对该酶编码基因XylA进行了异源表达和特性分析。结果显示，XylA基因编码440个氨基酸的蛋白，编码蛋白质的二级结构以α螺旋和无规则卷曲为主，高级结构为同源四聚体。进一步将XylA基因在大肠杆菌BL21(DE3)中克隆表达，获得分子量约50 kDa的重组蛋白Bc-XlyA。酶学性质研究发现，Bc-XylA最适pH为8.0，最适温度为60 ℃，Mn²⁺、Mg²⁺和Co²⁺对其催化活性十分重要。本研究可为凝结芽孢杆菌的分子改造提供数据参考，为该菌种的木糖高效利用提供理论支撑。

Abstract:

Bacillus coagulans can utilize the hydrolyzed carbon source of agricultural waste to produce lactic acid via a homofermentative pathway. However, a significant carbon source metabolic repression effect was observed when the strain metabolized mixed sugars (glucose and xylose), reducing the productivity of lactic acid. In this study, we obtained the fermentation conditions for the simultaneous utilization of the mixed sugars by B. coagulans by changing the ratio of glucose to xylose in the medium. Through transcriptome sequencing, several key genes responsible for xylose utilization were identified. The critical role of xylose isomerase (XylA, EC 5.3.1.5) in the synchronous utilization of glucose/xylose in B. coagulans was investigated via qRT-PCR (quantitative real-time polymerase chain reaction). Subsequently, the heterologous expression and characterization of the XylA-encoding gene (XylA) were conducted. It was determined that the gene encoded a protein composed of 440 amino acid residues. The secondary structure of the encoded protein was predominantly composed of α-helixes and random coils, while the higher structure of the protein was identified as a homotetramer. Then, XylA was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant protein Bc-XlyA was obtained with a molecular weight of approximately 50 kDa. The optimal pH and temperature of Bc-XylA were 8.0 and 60 ℃, respectively, and Mn²⁺, Mg²⁺, and Co²⁺ had positive effects on the activity of Bc-XlyA. The present study provides scientific data on the molecular modification of B. coagulans, offering theoretical support for the efficient utilization of xylose in the strain.