钾离子调控聚γ-谷氨酸发酵过程中粘度变化的规律及机理研究

Poly-γ-glutamic acid (PGA) is a biodegradable polymer from microorganisms with widely applications in the field of food, medicine, agriculture, environmental protection and cosmetics, etc. This project aims to solve the key problem of PGA fermentation process -- "high viscosity of fermentation broth", to carry out basic research for application. Previous experiments found that the viscosity of fermentation broth was reduced and the yield of PGA was increased significantly, but the molecular weight of PGA was not decreased after adding a certain concentration of K+ to the PGA fermentation medium. The goal of this research is to study the regularity of rheological properties, mixing and mass transfer characteristics in response to the viscosity change, to ascertain the effect of cell physiological structure changes and PGA secreted protein on products secretion and cell aggregation, to analyze the relationship between the molecular conformation of PGA and the viscosity of fermentation broth under potassium regulation. Then, revealing the regularity and mechanism of viscosity change in the PGA fermentation process by potassium regulation, from a relevance perspective of cell physiological structure, PGA molecular conformation and fermentation broth rheology. These results will provide more experimental evidence for further improving PGA yield and substrate conversion efficiency from a new perspective, and provide theoretical guidance for developing fermentation control strategy, so as to promote the development of the PGA industry. It can also provide a reference for the study of fermentative production of other similar biopolymers.

聚γ-谷氨酸（PGA）是一种微生物源可降解高分子聚合物，在食品、医药、农业、环保和化妆品等领域具有广泛用途。本项目针对PGA发酵过程中的关键问题——“发酵液高粘度”，开展应用基础研究。前期实验发现，添加一定浓度K+至发酵培养基中，能够显著降低发酵液粘度，提高PGA产量且不降低PGA分子量。本项目拟在K+调控下，深入研究PGA发酵液流变特性、混合特性和传质特性响应粘度变化的规律，探明菌体细胞生理结构变化以及PGA分泌相关的酶蛋白对产物分泌和菌体聚集的影响，解析PGA分子构象与发酵液粘度的关系，进而从菌体生理结构-PGA分子构象-发酵液流体学的关联角度阐释K+调控PGA发酵过程中粘度变化的规律及机理。研究结果有望从新的视角为进一步提高PGA产量和底物转化率提供实验依据，并为发酵控制策略的开发提供理论指导，从而促进PGA产业的发展，同时也可为其它类似生物高分子的发酵生产研究提供参考。

项目针对聚γ-谷氨酸（PGA）发酵过程中的关键问题——“发酵液高粘度”，围绕KCl能够显著降低PGA发酵液粘度这一现象开展研究。在KCl调控下，研究PGA发酵体系的流变特性、混合特性和传质特性响应粘度变化的规律；在此基础上探究菌体细胞生理结构变化对产物分泌和菌体聚集的影响，以及KCl调控发酵液粘度过程中菌体细胞内基因转录的响应情况；解析PGA分子结构变化对发酵液粘度的作用。主要研究结果如下：（1）在B. subtilis GXA-28发酵生产PGA过程中，培养基中添加高浓度（201.2 mM）KCl时，PGA发酵液粘度降低58.98%，PGA产量提高30.75%；发酵液稠度系数和屈服应力相应降低，但流变指数、体积氧传递系数和有效混合体积比例升高。（2）添加KCl使菌体和PGA之间的交联程度减弱、菌体聚集现象减少；菌体表面zeta电位降低使得菌体与PGA之间的排斥力增强；细胞膜渗透性提高、完整性降低；上述菌体生理结构变化是PGA发酵液粘度降低的重要原因。（3）通过RNA-seq技术筛选获得了多个响应发酵液粘度变化的差异表达基因，有16个基因被高、低浓度KCl共同调控，其中2个上调、14个下调。这些差异基因主要涉及膜转运和碳水化合物代谢途径，可能参与KCl调控PGA合成及发酵液粘度。（4）添加KCl未显著改变PGA的重均分子量、数均分子量和多聚分散性。但是，PGA分子中的D-谷氨酸比例提高；且在水溶液中易聚集，形成了更加紧密的结构；构象由β-折叠转向β-折叠和α-螺旋并存。PGA分子结构的上述变化对其发酵液粘度的降低起着重要作用。本研究从菌体生理结构-PGA分子结构-发酵液流体学的关联角度阐释KCl调控PGA发酵过程中粘度变化的规律及机理，为PGA发酵控制策略的开发提供了理论指导。

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