基于多尺度分析的凝结芽孢杆菌木糖代谢机理研究

The efficient utilization of xylose is one of the key prombles for comprehensive use of agricultural and forestry wastes. At present, the research of xylose is mainly focused on fermentation of ethanol by yeast strains, and less attention is paid on other microorganisms. Bacillus coagulans is a new kind of industrial microbe that can ferment xylose to lactic acid with a high theoretical yield and a simple fermentation process. Recently, we found that, for B. coagulans, the use efficiency of xylose was lower than that of glucose, which would limit its application potential in wood fiber resources. In this study, an independent selected strain, B. coagulans NL01, was used for adaptive evolution to improve its ferment ability on xylose and the positive mutation was obtained through conditioned screening. The network of xylose to lactic acid of B. coagulans was constructed based on the combination of genome data and metabolic flux analysis The original strain and the mutantion were compared on gene sequences, transcription, translation, and enzyme activities of the key genes in xylose metabolic network by multi-scale analysis. The mechanism on improved metabolic efficiency of positive mutation was discussed, which will lay a theoretical foundation on modifying of B. coagulans by metabolic engineering.

木糖的高效利用是农林废弃物全面充分利用必须解决的关键问题之一。本申请中的凝结芽孢杆菌是近年来引起广泛关注的一类能代谢木糖产乳酸的菌株，相较于酵母菌等其他木糖代谢菌株，该菌株木糖代谢途径简单、理论转化率高、发酵过程易于调控。前期工作中发现凝结芽孢杆菌对木糖的利用效率低于其对葡萄糖的利用效率，鉴于此，本申请拟以自主筛选获得的凝结芽孢杆菌NL01为对象，利用适应性进化技术促进其木糖代谢进化，条件筛选获得木糖代谢产乳酸正向突变菌株；结合凝结芽孢杆菌基因组数据分析和代谢流分析构建其木糖代谢产乳酸网络；通过对比出发菌株和正向突变菌株木糖代谢产乳酸网络中的关键基因在基因序列、转录水平、翻译水平和酶活力水平的差异，对二者的木糖代谢途径进行多尺度全方位比较研究，探讨正向突变菌株木糖代谢效率提高机理，解析凝结芽孢杆菌木糖代谢产乳酸过程的本质，从而为进一步利用代谢工程手段构建木糖高效利用菌株提供理论依据。

Bacillus coagulans是一种近年来受到广泛关注的工业乳酸菌，绝大多数该种属的菌株都可以通过同型乳酸发酵途径转化葡萄糖和木糖生产高光学纯度的L-乳酸。相较于其对葡萄糖的利用情况，Bacillus coagulans利用木糖时存在发酵速度慢，产率较低，木糖耐受浓度低等问题。.本项目中在出发菌株B. coagulans NL01的基础上，利用适应性进化技术促进其木糖代谢进化后筛选获得到了B. coagulans CC17。经过培养基和培养条件的优化后，B. coagulans CC17的木糖乳酸转化率最高达到了94.4%，为木质纤维原料中己糖戊糖的全组份利用奠定了良好的基础。.对B. coagulans NL01进行了高通量基因组测序，在全基因组信息基础上，对NL01的木糖代谢和L-乳酸合成等相关特性进行了分析。菌株NL01中的木糖操纵子完整，包括木糖转运蛋白编码基因xylT，木糖异构酶编码基因xylA，木酮糖激酶编码基因xylB，以及调控蛋白编码基因xylR。首次对凝结芽孢杆菌来源的木糖异构酶和木酮糖激酶进行了外源克隆表达及酶学性质分析。重组木糖异构酶最适pH为7，最适温度为85oC，Ni2+对木糖异构酶有强烈的激活作用；重组木酮糖激酶最适pH为8，最适温度为80oC，Co2+对木酮糖激酶有最好的激活效果。.利用链特异性RNA-Seq测序技术，研究了B. coagulans NL01不同木糖浓度（60 g/L和140 g/L）条件下基因表达谱的差异和转录调控的变化，共筛选出593个差异基因。结合差异基因的KEGG以及GO数据，对细胞内磷酸戊糖途径，木糖的跨膜转运，乳酸及副产物的生成以及能量代谢进行了多层次的解析。实验结果表明B. coagulans NL01在高木糖浓度下发酵产L-乳酸的关键瓶颈在于细胞生长受到抑制，这可以通过提高与菌体产能相关基因的转录表达水平来克服。