Unravelling the genetic complexity and redundancy of Aspergillus niger sugar metabolism

Abstract

Filamentous fungi are organisms with high industrial potential due to their inherent ability to directly convert biomass substrates to valuable metabolites. Additionally, their primary metabolism is a significant source of industrially important compounds, as well as of monomeric building blocks for the production of secondary metabolites and extracellular enzymes. Hence, large efforts have been made towards the development of suitable fungal strains for the industrial scale production of primary metabolites. However, to design better and more versatile industrial fungal cell factories, a thorough and holistic understanding of filamentous fungal metabolism is essential. While the primary metabolism of filamentous fungi has been a topic of study for many decades, the gaps in our knowledge regarding the metabolic enzymes involved in individual steps and the regulation of the pathways could be considered as actual bottlenecks in the development of more effective metabolic engineering strategies. The reconstruction of genome-scale metabolic models (GEMs) is currently one of the most promising approaches to generate an in silico prediction of cellular function in terms of physiology, providing a multi-level depiction of the metabolism and its regulation. However, while these novel models significantly improve our understanding of metabolism, their experimental validation is a prerequisite before being able to use their full potential as accurate predictive tools in metabolic engineering. Recently, a dynamic GEM of Aspergillus niger primary metabolism has been published, in which the genes involved in the various steps were verified using transcriptome data. In this GEM project, a network of reactions of primary metabolism based on the A. niger NRRL 3 gold-standard genome was generated and used to find orthologous genes and pathways involved in sugar catabolism in a set of closely related fungal species. The main objective of this thesis was to improve our understanding of A. niger primary carbon metabolism, by experimental validation of this recently published manually curated GEM of A. niger. Several pathways of A. niger sugar metabolism were dissected, while growth on plant biomass and re-routing of sugar catabolism was also evaluated to gain a better understanding of the flexibility of A. niger in using plant biomass-derived monomers as substrates. This did not only generate novel biological insights but also enabled refinement of the existing model. Taken together, the results obtained in this thesis have enriched the available knowledge on the primary carbon metabolism of A. niger by identifying new genes involved in the pentose, the L-rhamnose and the D-galactose catabolic pathways, as well as novel regulatory connections between the pathways. The difference between growth on pure monomeric sugars and plant biomass substrates was also highlighted. Through such an in-depth study of the A. niger metabolic network we have provided a detailed blueprint for fungal metabolic engineering to improve productivity of a broad range of metabolites.