Enhanced glycosyl hydrolase production in Aspergillus nidulans using transcription factor engineering approaches

Background: Engineered fungi are attractive platforms for the production of plant cell wall hydrolytic enzymes which, among other biotechnological applications, are required for the efficient conversion of biomass to glucose and other fermentable sugars. As a fungal model system, Aspergillus nidulans provides genetic tools that are of relevance in this context and potentially applicable to industrially important filamentous fungi. The goal of this study is to assess the utility of A. nidulans as a host for recombinant protein production. Results: We have successfully applied a transcription factor engineering approach to improve the efficiency of the A. nidulans xylanolytic XlnR-xlnp expression system. Specifically, endo-1,4-β-xylanases and an α-L-rhamnosidase were chosen as representatives of endogenous and heterologous glycosyl hydrolases involved in plant cell wall deconstruction. By deregulating the expression of the xylanolytic transcriptional activator XlnR and modulating the activity of the pH regulator PacC we improved protein production and reduced production times. Xylanase activity was about 200-fold greater in gpdAp::xlnR strains compared to controls 4 hours after transfer to inducing conditions, and 10-fold greater after 24 hours. Remarkably, 75% of the xylanase activity was present in the engineered strains within 4 hours. Engineering XlnR expression also had a considerable impact on foreign protein production, especially when the promoter of the 'acidic' xlnB gene was used to express the transgene. α-L-rhamnosidase activity in xlnBp::rhaA, gpdAp::xlnR strains was about 19-fold greater than that of controls 72 hours after transfer to xylan (about 85% of the total activity produced), and 10-fold greater at later times (120 hours). The performance of these strains was further enhanced by impeding the proteolytic activation of PacC; introduction of the palA1 allele in xlnBp::rhaA, gpdAp::xlnR strains resulted in an additional 2.7-fold increase in α-L-rhamnosidase activity by 48 hours (about 87% of the total activity produced) and a 1.7-fold increase at later times. Conclusions: Our results show that the XlnR-xlnp expression system is a valuable tool for manipulating the production of plant cell wall degrading enzymes in A. nidulans and establish the biotechnological potential of the transcription factors XlnR and PacC to boost and control the strength of xylanolytic promoters.