丝状真菌液体深层发酵过程中菌丝聚集分子调控机制

Filamentous fungus is one of the expression systems for the microbial fermented products. The specific characteristic of filamentous fungus submerged fermentation process is that fermentation environment significantly affected mycelium aggregation, which leaded to the significant change of the rheology of fermentation broth. Then the transfer of mass, heat and momentum will be remarkably affected, which finally affect the biosynthesis of target products and the efficiency of fermentation process. While, the molecular regulatory mechanism about mycelium aggregation during filamentous fungi submerged fermentation process has not been studied yet. In this project, the special edible mushroom of Tuber melanosporum is taken as the typical example of filamentous fungus. Firstly, the method of automated image analysis for morphological quantification will be developed and applied to digitally describe the dynamics of mycelium aggregation. Secondly, suppression subtractive hybridization will be employed to screen the regulatory and structural genes which related to the mycelium aggregation. Thirdly，the expression of regulatory and structural genes is regulated through the fermentation environmental disturbance. Finally, a progressive control strategy referred as "fermentation environment affecting the expression of regulatory and structural genes, then affecting mycelium aggregation, and finally improving the biosynthesis of target products" will be developed. The expected results will be very useful to control the mycelium aggregation during the submerged fermentation process of filamentous fungi, to enhance the biosynthesis of target products, to improve the efficiency of the fermentation process, and then to promote the application of filamentous fungi as the expression system for the commercial products in the field of medicine, agriculture and industry.

丝状真菌是微生物发酵产品的重要表达体系，其液体深层发酵过程的典型特征是发酵环境显著影响菌丝聚集，菌丝聚集影响发酵体系流变特性，进而影响传质、传热、传动，最终影响目标产物生物合成和发酵过程效率。然而，发酵环境影响菌丝聚集的分子调控机制尚未明晰。本项目拟以名贵食用菌黑孢块菌为丝状真菌研究对象，采用计算机图像识别分析技术建立丝状真菌液体深层发酵过程菌体形态表征方法学，对菌丝聚集过程进行数字化表征；根据菌体形态变化，构建消减差异杂交文库，筛选出调控菌丝聚集的调节基因和结构基因；通过发酵环境扰动，调控调节基因和结构基因的表达，阐明其对菌丝聚集的影响；建立"发酵环境影响调节基因和结构基因表达水平、进而调控菌丝聚集，最终强化目标产物合成"的递进调控策略。研究结果预期将为丝状真菌发酵过程菌体形态控制，强化目标产物合成效率，提高发酵过程效率奠定理论基础，推动丝状真菌表达体系在医药、工业和农业的应用。

以黑孢块菌为典型丝状真菌为研究对象，建立了数字化表征菌丝聚集过程的菌体形态特征的方法，筛选了调控菌丝聚集的关键基因几丁质合成酶基因chs4和chs7，揭示其发挥调控作用的机制可通过钙信号传导产生应激，进而对菌体形态发挥调控作用。本项目研究至今已在本领域国际知名学术期刊如Metabolic Engineering (IF=8.258，1篇)、Scientific Reports (IF=5.578，6篇)、Applied Microbiology and Biotechnology (IF=3.337，3篇)等上发表SCI论文14篇, 较好的完成了计划任务书中规定的目标。