Aspergillus nidulans as a platform for discovery and characterization of complex biosynthetic pathways

Abstract

Filamentous fungi produce a wide range of bioactive compounds, classified as secondary metabolites,which have the potential to be used as pharmaceuticals, insecticides, fungicides and food additives.Secondary metabolites also include mycotoxins, which are produced by fungi that contaminate food andfeed. Secondary metabolites therefore both have a positive and deleterious impact on the human health.The increase in available genome sequences of fungi has revealed that there is a large number of putativesecondary metabolite biosynthetic gene clusters to be discovered and potentially exploited aspharmaceuticals. Access to this unexploited reservoir is hampered as many of the clusters are silent orbarely expressed under laboratory conditions. Methods for activating these pathways are thereforeessential for pathway discovery and elucidation.  Filamentous fungi and Aspergillus species in particular are used in industrial applications for the productionof these bioactive compounds and other chemicals as well as for enzyme production. Especially Aspergillusniger and Aspergillus oryzae are used as industrial workhorses for the production of various enzymes. Manyof the secreted proteins are glycosylated, indicating that glycosylation plays an important role in thesecretory pathway. Thus, understanding the role and process of glycosylation will enable directedglycoengineering in Aspergilli to improve protein production and expand the repertoire of proteins, whichcan be produced by these fungi. Aspergillus nidulans has been used as a model organism for a range of research disciplines and manygenetic engineering tools are available for working in this organism. This PhD study therefore employed A.nidulans as a model system to address the following on two aspects: 1) Developing A. nidulans as aplatform for pathway discovery of secondary metabolites and 2) Developing A. nidulans as a model systemfor protein production with human-like glycan structure.  The first part of this study resulted in the development of a method for the transfer and expression ofintact biosynthetic gene clusters to A. nidulans to facilitate pathway and product discovery. As proof ofconcept the biosynthetic gene cluster for production of the polyketide geodin was identified andtransferred from A. terreus to A. nidulans. The cluster was integrated in a well characterized locus in A.nidulans. Reconstitution of the cluster resulted in the production of geodin. Expression of the enzymes inthe pathway was validated by transcription analysis and the functions of specific genes were investigatedby gene deletions. This proved that this method is a fast and easy way to transfer biosynthetic gene clustersregardless of size and characterize them. Furthermore, a different approach to activate silent clusters wasdemonstrated, as the heterologous expression of a putative transcription factor from A. niger in A. nidulansinduced the synthesis of insect juvenile hormones in A. nidulans, which had previously not been reportedas fungal metabolites.  The second part of the study focused on understanding the glycosylation pathway in A. nidulans andengineering a strain capable of producing precursors for the further modification of the glycan structuretowards a more human-like pattern. Previous studies have shown that the deletion of the firstmannosyltransferase in the ER (alg3) resulted in the accumulation of sizes from Man3GlcNAc2 toMan7GlcNAc2. This study shows that the remaining mannosyltransferases in the ER do not use thetruncated structure generated from the alg3 deletion as a substrate, thus the remainingmannosyltransferase activity must take place in the Golgi. Furthermore, there is an indication that theMan5GlcNAc2 structure generated from the alg3 deletion is trimmed to Man3GlcNAc2 and extended toMan5GlcNAc2, which has a different structure that the first generated structure by the alg3 deletion. Thework done during this study gives more insight into the glycosylation pathway of A. nidulans, which can beused as a basis for further engineering of the pathway to produce humanized glycans in Aspergilli.