Tracing the fungal carbon metabolic roadmap

Abstract

Plant biomass is one of the major sources of energy on Earth, used by living organisms and industry. However, its complete conversion remains a major industrial challenge. Fungi are the main degraders of plant biomass. They produce enzymes to degrade the polymers to mono- or short oligomers that can be taken up and metabolized by the cells. Aspergillus niger is considered an industrial workhorse due to its ability to produce different compounds and enzymes. The aim of this thesis was to reconstruct the metabolic network of A. niger focusing on carbon metabolism and predict metabolic capabilities in other species across the fungal kingdom. Genome-scale metabolic network reconstruction links genetic, metabolic and bibliomic information in a mathematical equation for a better estimation of the metabolism of an organism. We have updated and expanded the A. niger genome-scale metabolic network creating strain specific metabolic networks for three of the most common A. niger strains used in academia. By extending the model to other strains, we enable users to update and validate the model against the experimental data. During plant biomass degradation, activation of gene expression via specific inducers is balanced with gene repression via carbon catabolite repression (CCR) mediated by the CreA repressor protein. CCR prevents producing unnecessary proteins, when sufficient monosaccharides are present in the environment. We studied carbon utilization of A. niger WT and ΔcreA strain using a manually curated carbon catabolic network based on the A. niger NRRL 3 gold-standard genome. Starting with simple monosaccharide utilization, we showed pathway specific induction according to substrate and constitutive expression of the main carbon catabolic pathways (glycolysis, TCA and glyoxylic acid cycle), and identified CreA specific target genes. In nature, sugars do not occur as pure substrates, but in a heterogeneous mixture, after the crude substrates are depolymerized by enzymes. We explored carbon utilization by growing A. niger on a mixture of monosaccharides. This showed how in the presence of several carbon sources, the fungus utilized first high-energy content sugars and when they were finished, continued with less preferred sugars regardless of whether CreA was present or not. We went also further and studied gene expression during growth on different crude substrates and showed how substrate composition shapes gene expression over time. Moreover, our data showed that a ΔcreA strain needed more time to adapt to a new environment, after which gene expression became similar to the WT. A. niger is not the only fungus able to degrade plant biomass, and therefore we studied whether or not genome content can be used to predict metabolic abilities in other species. Our data showed that although genome content between closely related species was similar, regulatory systems and alternative pathways are important to understand their metabolic abilities. Taken together, this thesis depicts A. niger carbon metabolism and its further implementation in the fungal kingdom, focusing on plant biomass degradation and its industrial applications, using bioinformatics as driver.