Control of mono-species and multi-species pellet heterogeneity and their implications on product formation in the cell factory Aspergillus niger

包括黑曲霉在内的丝状真菌是生物技术中的细胞工厂，用于生产有机酸、蛋白质和生物活性化合物。然而，真菌宏观形态的进化，如分散的菌丝和球团，仍然是一个不可预测的多因素过程，这强烈限制了生产力。该项目的总体目标是在搅拌槽生物反应器（STR）和摇摇生物反应器（RMB）培养中获得黑曲霉在个体和大形态种群内异质大形态特征的机理见解。尽管在种群内和生物反应器室内的异质性已经知道了很长时间，但到目前为止，丝状真菌异质性的遗传基础以及合理工艺设计的选择都没有以系统的方式进行研究，也没有全面了解丝状真菌的细胞-细胞和细胞-生物反应器相互作用。为了全面了解单种黑曲霉（A. niger）和多种黑曲霉（A. niger和S. coelicolor）颗粒形态在实验室规模和大型生物反应器中的进化，以及它们对黑曲霉已建立的产物组合（初级代谢物柠檬酸、葡萄糖淀粉酶、次生代谢物enniatin B）的影响，以及丝状细菌coelicolor的存在对新产物（次生代谢物）的诱导，我们将测试不同的STR和RMB设计和接种方案。这些因素对生长、生理、形态和产品形成的影响将使用在线和离线显微镜工具、活力分析、转录组学、代谢组学和x射线微计算机断层扫描进行研究，这些工具表征了颗粒的三维内部结构。最终目标是了解种群异质性发展背后的遗传和过程工程机制，平衡菌丝生长和产品形成。了解基因调控和代谢途径与变化的工艺环境之间的联系将最终确定黑曲霉中的基因开关，其靶向操作将合理地提高工业发酵过程中该细胞工厂的生产率-不仅在产品形成方面，而且在改善宏观形态方面。了解工艺设计对生物系统的影响将反过来为优化工艺控制提供线索。我们进一步建议建立一个多尺度建模平台，将丝状微生物的大形态异质性演化与氧气供应和产物形成结合起来。这种模型框架最终将支持基于模型的工艺设计，以改进生物工艺。因此，通过合理的遗传工程和工艺工程来预测和控制黑曲霉在无菌或混合生物过程中的宏观形态将成为可能。

Filamentous fungi including Aspergillus niger are cell factories in biotechnology for the production of organic acids, proteins and bioactive compounds. However, the evolution of the fungal macroscopic morphologies such as dispersed mycelia and pellets remains an unpredictable multifactorial process, which strongly limits productivities.The overall objective of the project is to gain mechanistic insights into the heterogeneous macromorphological characteristics of A. niger in stirred tank bioreactor (STR) and rocking-motion bioreactor (RMB) cultivations - within individual and among macromorphological populations. Although heterogeneities within a population and within bioreactor compartments are known for a long time, the genetic basis of filamentous heterogeneity as well as options for rational process design have neither been studied so far in a systematic manner nor are cell-cell and cell-bioreactor interactions comprehensively understood for filamentous fungi. To comprehensively understand the evolution of mono-species (A. niger) and multi-species (A. niger and S. coelicolor) pellet morphologies in lab-scale and large scale bioreactors, their impact on the established product portfolio of A. niger (primary metabolite citric acid, enzyme glucoamylase, secondary metabolite enniatin B) and the induction of new products (secondary metabolites) due to the presence of the filamentous bacterium S. coelicolor, we will test different STR and RMB designs and inoculation schemes. The impact of these factors on growth, physiology, morphology and product formation will be studied using on line and off line microscopic tools, viability analyses, transcriptomics, metabolomics, and X-ray µ-computed tomography, which characterize the 3-dimensional inner structure of pellets. The ultimate goal is in understanding both genetic and process engineering mechanisms behind the development of population heterogeneities that balance hyphal growth with product formation. Understanding the link between gene regulatory and metabolic pathways with changing process environments will ultimately identify gene switches in A. niger whose targeted manipulation will rationally improve productivity of this cell factory during industrial fermentation – not only with respect to product formation, but also with respect to improved macromorphologies. Understanding the effect of process design on the biological system will in turn generate leads for optimized process control. We further propose the establishment of a multiscale modelling platform which couples the evolution of macromorphological heterogeneities of filamentous microorganisms with oxygen supply and product formation. Such model framework will eventually support model-based process design for improved bioprocesses. Hence, rational genetic and process engineering to predict and control macroscopic morphologies of A. niger in axenic or mixed bioprocesses will become possible.