1 Department of Systems Biology, Technical University of Denmark2 Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark3 Department of Chemical and Biochemical Engineering, Technical University of Denmark
Filamentous fungi are extensively used in the fermentation industry for synthesis of numerous products. One of the most important, is the fungus Aspergillus niger, used industrially for production of organic acids, and homologous as well as heterologous enzymes. This fungus has numerous of advantages, including tolerance for low pH, which is important for acid production. Furthermore, it has the capability of metabolizing a wide variety of carbon sources, possesses an exceptional efficient protein secretion capacity, and three genome sequences are publicly available. However, A. niger have some disadvantages as well, those are byproduct formation, secretion of proteolytic enzymes and formation of mycotoxins. The aim of this project was to reduce these disadvantages, though investigating the regulatory processes. The first objective was to study the regulatory events leading to A. niger’s citric acid overflow metabolism. This was done with analysis of both transcriptome and proteome profiles, from cultivations in manganese limitation and manganese excess conditions. Beside already described responses, that were used to verify the experimental setup, identification of novel events was done. The most interesting was the strong down regulation of phosphoenolpyruvate carboxykinase (PEPCK) at manganese limited conditions that could be one of the main initiators for the citrate overflow metabolism. To gain further insight into A. niger’s metabolism, a new metabolic engineering tool, termed transcription factor modulation was developed. Using this approach, two novel mutants were isolated and formed the basis for the following studies. Through knock out studies of putative trans-acting pH responding transcription factors, a mutant exhibiting an oxalate overproducing phenotype was identified and entitled Oxalic Acid repression Factor, OafA. This mutant was physiological characterized in details, using continues cultivation (chemostats), followed by transcriptional analysis. Two phosphoketolases were found to be down-regulated in the ΔoafA mutant and it was argued, that this was the main cause, for the increase oxalate formation. From similar knock out studies, another mutant was identified and this strain was shown to be a protease mutant and the responsible transcription factor was entitled Protease Regulator B, PrtB. This was compared against the already described, protease deficient strain, ΔprtT. The physiological batch characterization showed that the ΔprtT strain had the lowest protease activity (fivefold reduced), but also featured excessive CO2 yield, reduced growth rate and lower biomass yields. The ΔprtB strain had a close to twofold reduced levels of secreted proteases but with additional beneficial characteristics, as a lower oxalic ii acid formation and wild type growth performance; it was therefore argued that this strain could be an attractive alternative to ΔprtT. Finally, in order to characterize the formation of the carcinogenic mycotoxin fumonisin, a reporter strain of A. niger was constructed, where the promoter from the fumonisin synthase was fused to the green fluorescent protein. This strain was used together with the commercial large-scale nutrient profiling platform, Biolog Phenotype MicroArrays. Out of the 476 conditions tested, six compounds significantly induce fumonisin production, identified. These formed the basis for the subsequent examinations, which resulted in the identification of azelaic acid, a plant hormone and a very potent fumonisin inducer.