基于in vivo动力学分析的波动环境下黑曲霉产酶得率调控机制研究

项目名称： 基于in vivo动力学分析的波动环境下黑曲霉产酶得率调控机制研究

项目编号： No.21506052

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 有机化学

项目作者： 夏建业

作者单位： 华东理工大学

项目金额： 21万元

中文摘要： 黑曲霉因其高效的蛋白分泌特性而被作为重要的工业酶制剂生产菌种。但黑曲霉产酶发酵过程放大往往面临产酶得率降低的难题，成为限制其产业化的重要瓶颈。近年来发展起来的in vivo动力学研究，正逐渐成为揭示放大过程胞内调节机制的研究热点。本项目拟以黑曲霉产糖化酶为模式体系，通过“刺激-响应”实验，结合快速取样淬灭技术、高效胞内代谢物抽提技术及基于同位素稀释质谱法的高精度胞内代谢物定量技术，研究黑曲霉中心代谢途径在经历外界底物浓度刺激下的动态响应特性。分别从蛋白合成的关键氨基酸前体供应、ATP与NADPH供应，多元醇与有机酸等副产物生成等角度，结合形成的黑曲霉中心代谢in vivo动力学模型，通过实验及数值模拟研究，探讨波动环境下黑曲霉中心代谢的动力学调控对产酶得率的影响机制。揭示导致黑曲霉产酶放大困难的关键瓶颈，为黑曲霉菌种改造和反应器设计与优化提供理论预测工具，指导黑曲酶产酶的高效放大。

中文关键词： In；vivo；动力学；发酵过程放大；恒化培养；动力学建模；丝状真菌

英文摘要： Aspergillus niger is used as an important industrial working host for enzyme production as its highly efficient protein excretion capacity. But the scale up processes of enzyme production by A. niger always encounter reduced yields, which is the bottleneck in its industrialization process. The recently developed in vivo kinetics study of microbe is boosting, which is used for discovering the in vivo regulation mechanism of microorganism during industrial scale-up. In this proposal, the glucoamylase production by A. niger will be chosen as a model system, and the dynamic response characteristics of A. niger central metabolism under extracellular substrate stimulus will be studied, through “stimulus-response” experiment design, combining fast sampling and quenching technique, thoroughly intracellular metabolites extraction technique and high accuracy mass spectrum measurement based on IDMS. From aspects of supply of key amino acids, ATP and NADPH, and formation of byproducts - polyols and organic acids, combining with constructed in vivo kinetic model, the regulation mechanism of central metabolism on enzyme yield of A. niger will be investigated through experiment and simulation. The aim is trying to discover the bottleneck that leads to decreased enzyme yield during scale-up. The constructed model will provide theoretical prediction tool for strain improvement and bioreactor design and optimization, and also help for developing rational scale-up strategy of enzyme production by A. niger.

英文关键词： In vivo kinetics;Fermentation process scale-up;Chemostat culture;Kinetic modeling ;Filamentus fungi