沙柳水解物混糖共发酵过程CCR调控机理及产高光学纯L-乳酸研究

In this study, the hydrolysate of lignocellulosic material-Salix cheilophila is used as the raw material for L-lactic acid production. By enhancing cellobiose intracellular metabolism, improving the homofermentation of mixed sugars, and regulating the metabolic pathway and flux, this study is aimed at solving the problem of carbon catabolite repression (CCR) during fermentation of mixed sugars and improving the optical purity of L-lactic acid. The main content of this research is as follows: analyzing the effect of the concentration ratio of different sugars, intracellular enzyme activity, and C/N ratio on the growth of lactic acid bacteria to determine the mechanism of mitigating the CCR phenomenon during co-fermentation of mixed sugars; regulating the activity of key enzyme for direct metabolizing cellobiose to pyruvate to avoid glucose accumulation and establish a homofermentation process using mixed sugars of cellobiose and xylose without CCR; Using C13 isotope tracer method and whole-genome analysis to determine the metabolism flux distribution of different sugar metabolism pathways, optimize sugar metabolism distribution to produce L-lactic acid, and build a fermentation regulation mode for high optically pure L-lactic acid production from cofermentation of cellibiose and xylose. This study can help to understand the essence of Salix cheilophila hydralysate fermentation, enrich the theory of lactic acid fermentation, and provide scientific basis for high-efficient and stable homofermentation of mixes sugars to advance its industrialization.

本研究为解决混糖乳酸发酵过程中的碳代谢阻遏效应（CCR）和L-乳酸光学纯度问题，深入探讨使用纤维素类物质沙柳水解物为原料，强化纤维二糖胞内酶代谢，同型高效共发酵及混糖代谢途径和流量调控中的几个关键性技术及科学问题。分析多糖体系的浓度比例、乳酸菌胞内酶酶活、C/N比等参数的变化对乳酸菌生长的影响，研究混糖同型共发酵不同于单糖发酵的糖酸复杂转化特征，尤其是戊糖和己糖之间的CCR特征及抑制消除机理；调控二糖直接代谢生成丙酮酸的关键性酶活，避免葡萄糖积累，确定纤维二糖/木糖无CCR的同型共发酵工艺；利用C13同位素示踪和全基因组分析方法确定糖代谢各途径流量分布，优化L-乳酸生成的碳源代谢分布，构建高光学纯L-乳酸最大产量下的乳酸菌强化纤维二糖共发酵调控模式，由此掌握沙柳水解物混糖发酵本质，丰富乳酸发酵理论，为混糖同型共发酵模式的高效、稳定地进行、并向产业化推进提供可靠的依据。

本研究为解决混糖乳酸发酵过程中的碳代谢阻遏效应（CCR）和L-乳酸光学纯度问题，深入探讨使用纤维素类物质沙柳水解物为原料，强化纤维二糖胞内酶代谢，同型高效共发酵及混糖代谢途径和流量调控中的几个关键性技术及科学问题。分析多糖体系的浓度比例、乳酸菌胞内酶酶活、C/N比等参数的变化对乳酸菌生长的影响，研究结果显示在木糖/纤维二糖混糖体系下LDH活性最高，活性为232.59 ng/mL、葡萄糖/木糖体系下LDH活性最高，活性为155.08 ng/mL，该结果与木糖/纤维二糖体系下乳酸产量29.65 g/L远高于葡萄糖/木糖体系下的11.75 g/L，说明LDH酶活对乳酸产量呈现正向相关；研究混糖同型共发酵不同于单糖发酵的糖酸复杂转化特征，尤其是戊糖和己糖之间的CCR特征及抑制消除机理；调控二糖直接代谢生成丙酮酸的关键性酶活，避免葡萄糖积累，确定纤维二糖/木糖无CCR的同型共发酵工艺；利用B族维生素优化L-乳酸生成的碳源代谢分布，构建高光学纯L-乳酸最大产量下的乳酸菌强化纤维二糖共发酵调控模式，微生物B1、B2、B3、B5、B6、B7、B9、B12的添加均能促进乳酸生产， B6、B7、B9、B12这四种维生素的添加对木糖/纤维二糖体系的促进作用明显优于B1、B2、B3、B5，其中维生素B12促进效果最好，最佳添加量为0.5 mg/L，由此掌握沙柳水解物混糖发酵本质，丰富乳酸发酵理论，为混糖同型共发酵模式的高效、稳定地进行、并向产业化推进提供可靠的依据