产丁醇梭菌属微生物木糖与葡萄糖同步利用调控机制研究

Carbon catabolite repression is a common phenomenon during the fermentation of mixed carbon sources. The simultaneous utilization of pentoses and hexoses in the production of butanol from lignocellulosic biomass by Clostridium species has been an important task to accomplish. Favoring certain types of monosaccharides during fermentation results in problems such as prolonged fermentation period, lowered efficiency of substrate utilization and high costs for wastewater treatment. Developing strains that can simultaneously utilize pentoses and hexoses is of paramount importance for highly efficient lignocellulose bioconversion. Investigations carried out in this project focuses on the glucose-xylose simultaneously utilizing C. beijerinckii SE-2 that the applicant previously isolated and identified, as well as C. acetobutylicum ATCC824 for which the ‘glucose repression effect’ is apparent. Using glucose and xylose as the respective model monosaccharide for hexose and pentose, the research on the properties and differences of monosaccharide transportation will be carried out for butanol-producing Clostridium species. The mechanisms of glucose and xylose transporting pathway will be further elucidated, which will reveal the mechanisms of the key proteins in this pathway in Clostridium species. These investigations will set the basis for improving the efficiency of lignocellulose hydrolysate utilization by constructing engineer strains that have the capability of pentose-hexose simultaneous utilization.

微生物混合碳源发酵过程普遍存在“代谢物阻遏效应”，产丁醇梭菌属微生物转化木质纤维素原料生产丁醇过程中，水解液中五碳糖与六碳糖的同步同等利用成为亟待解决的难题。单糖利用的偏好性导致其在利用水解液发酵时，出现发酵周期长、底物利用率低、废水处理成本高等一系列问题。开发能够同步利用五碳糖与六碳糖的菌株，对于木质纤维素原料生物转化至关重要。以申请者筛选得到的一株能够同步利用葡萄糖与木糖的拜氏梭菌C. beijerinckii SE-2及存在“葡萄糖效应”的丙酮丁醇梭菌C. acetobutylicum ATCC 824为研究对象，以木糖与葡萄糖分别作为五六碳糖模式物，研究产丁醇梭菌属微生物五六碳糖的转运特性及差异，阐明葡萄糖与木糖转运途径，揭示产丁醇梭菌属微生物葡萄糖与木糖转运过程关键蛋白基因的作用机制，为构建五六碳糖同步利用基因工程菌提高木质纤维素水解液发酵过程底物利用率奠定理论基础。

以混合糖发酵过程中可同步利用葡萄糖与木糖菌株C. beijerinckii SE-2及木糖利用受葡萄糖明显抑制的C. acetobutylicum824菌株为基础，对其木糖转运过程进行研究。木糖转运及代谢相关基因RT-qPCR研究显示， C. beijerinckii SE-2在混合糖发酵过程中“葡萄糖阻遏效应”依然存在，木糖转运及代谢相关基因同样受葡萄糖的抑制，区别在于木糖转运虽受葡萄糖抑制，但转运至胞内木糖能够满足代谢过程需要，保证了木糖代谢途径的顺利进行，该结论在进一步2-DOG与木糖胞内积累研究中得到证。两菌株中木糖与2-DOG的转运途径类似，都是共用相同的转运蛋白xylT，但是两菌转运蛋白（Cbei_0109，来自C. beijerinckii SE-2；CAC1345，来自C. acetobutylicum824）的编码基因序列差别很大，同源性只有39%。敲除C. beijerinckii SE-2中xylT（Cbei_0109）基因，导致其葡萄糖与木糖利用率分别下降36.1%与58.8%；而在C. acetobutylicum824中过表达xylT（Cbei_0109）基因，木糖的利用率提高47.5%，总糖的利用率提高21.4%，进一步说明xylT（Cbei_0109）在木糖同步转运中发挥了重要作用。因此，基于xylT基因的深入研究，对于产丁醇梭菌微生物混合糖的同步利用具有重要意义。