Abstract
In the past decade, the number of available genomes from filamentous fungi has increased exponentially. In many ascomycete fungi, especially Aspergillus niger and Aspergillus nidulans, this has led to the identification of many genes and enzymes involved in carbohydrate metabolism. Despite the genome era, the identification of genes encoding aromatic
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compound converting enzymes in fungi is still lacking and only a handful of genes have been identified. The aim of this thesis was to study the aromatic metabolic pathways from A. niger using genetic and biochemical approaches.
In chapter 2, aromatic metabolic pathways from bacteria and fungi were reviewed. This led to an update overview of the aromatic pathways observed in microorganisms and the enzymes involved.
Chapter 3 demonstrates the power of adaptive evolution in A. niger and was grown on ferulic acid, incrementally increasing the concentration gradually over six months. The obtained adaptive evolution strain showed improved tolerance against ferulic acid and consume ferulic acid faster than the parental strain.
Chapter 4 focuses on the transcriptional regulation of cdcA and padA, which encode for the enzymes involved in the decarboxylation of cinnamic acid, by SdrA. The transcriptional response of ΔsdrA was studied and revealed that SdrA tightly regulates cdcA and padA and several neighbouring genes.
The benzoic acid metabolic pathway of A. niger has been studied for decades and led to the identification of BphA which catalyses the first step in this pathway. Chapter 5 describes the identification of p-hydroxybenzoate-m-hydroxylase and protocatechuate 3,4-dioxygenase (PrcA) of A. niger using whole-genome transcriptome data. The deletion of prcA revealed an alternative protocatechuic acid pathway that hydroxylases protocatechuic acid to hydroxyquinol.
In chapter 6, we continued with the alternative protocatechuic acid pathway and identified a novel protocatechuate hydroxylase (PhyA). The double deletion mutant ΔphyAΔprcA grown of p-hydroxyphenyl and related aromatic compounds resulted in the accumulation of protocatechuic acid and demonstrated new possibilities for A. niger as a cell factory.
Chapter 7 describes the salicylic acid metabolic pathway. Using literature and transcriptome data, salicylate hydroxylase and catechol 1,2-dioxygenase of A. niger were identified. Both enzymes were produced in E. coli and can be used to convert salicylic acid to catechol and cis,cis-muconic acid.
Chapter 8 focuses on the metabolism of vanillin and vanillic acid in A. niger. Using transcriptome data of A. niger, three genes encoding vanillin dehydrogenase, vanillate hydroxylase, and methoxyhydroquinone 1,2-dioxygenase were identified. Deletion mutants grown of guaiacyl and related aromatic compounds resulted in the accumulation of vanillic acid and methoxyhydroquinone.
In Chapter 9, we combined the transcriptome datasets from the previous chapters to identify the genes involved in the metabolism of ferulic acid, p-coumaric acid, and caffeic acid. The gene deletion mutants grown on these compounds revealed that they are part of the CoA-dependent β-oxidative decarboxylation.
The results described in this thesis provide a better understanding of the aromatic metabolic pathways and the identification of the involved genes and enzymes. Furthermore, the knowledge obtained in this thesis was applied to create fungal cell factories that can produce specific aromatic compounds.
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