Regulators controlled by the sporulation gene flbA of Aspergillus niger

Abstract

Aspergillus is fungal genus that includes species used to produce compounds of industrial interest. A. niger is for example used for production of citric acid and carbohydrate-active enzymes such as glucoamylase. These enzymes are secreted in high amounts into the culture medium. However, protein secretion in A. niger is inhibited in sporulating zones of colonies. Gene flbA that encodes a Regulator of G-protein Signaling is one of the regulators involved in sporulation. Inhibition of protein secretion by sporulation can be overcome by inactivating flbA, which abolishes sporulation and allows secretion to occur in regions that normally would sporulate. Deletion of flbA however also changes secretome composition, and results in thinner cell walls and cell lysis, which hampers use of an flbA deletion strain as a cell factory. To disentangle these effects, transcription factor (TF) genes were studied that are differentially expressed in ΔflbA. Moreover, the influence of micro-colony size on the expression of secreted enzymes was investigated. Deletion of TF genes msnB and rpnR – that are both transcriptionally repressed by FlbA - affected protein secretion. MsnB represses protein secretion and genes that encode proteins catalyzing oxidation/reduction reactions. Deletion of rpnR decreased proteotoxic stress resistance, which was counteracted by reduced ribosomal and translation initiation factor eIF3 subunit expression, eventually causing a reduction in protein secretion. This reduction is in accordance with the role of FlbA in sporulation-inhibited protein secretion, where sporulating areas secrete less protein. Deletion of the FlbA-activated TF gene fum21 on the other hand abolished production of the mycotoxin fumonisin and reduced production of the antioxidant pyranonigrin A. This was associated with a reduction in expression of 10 out of 12 genes from the fumonisin cluster. These results show for the first time that FlbA controls secondary metabolism in A. niger. Next to this, the relationship between micro-colony size and protein synthesis was investigated for the first time within a single liquid shaken culture, as another way to increase protein secretion. To this end, GFP fluorescence per micro-colony volume was examined with this reporter gene under the control of the promoters of aamA, glaA, and aguA that encode the secreted enzymes acid amylase, glucoamlyase, and α-glucuronidase. Expression of aguA was near proportional to micro-colony volume, consistent with equal expression throughout the micro-colony. On the other hand, aamA and glaA expression per volume was inversely related to micro-colony volume, consistent with expression occurring in a concentric zone on the outside of the micro-colony. Taken together, this Thesis elucidates the effects on protein secretion of the FlbA-regulated TFs msnB and rpnR on the one hand, and establishes a relationship between micro-colony size and synthesis of secreted proteins on the other hand. These results can be used to improve A. niger as a cell factory.