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

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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.