Combining manipulation of transcription factors and overexpression of the target genes to enhance lignocellulolytic enzyme production in Penicillium oxalicum

Abstract

Lignocellulolytic enzymes are the main enzymes to saccharify lignocellulose from renewable plant biomass in the bio-based economy. The production of these enzymes is transcriptionally regulated by multiple transcription factors. We previously engineered Penicillium oxalicum for improved cellulase production via manipulation of three genes in the cellulase expression regulatory network. However, the potential of combinational engineering of multiple regulators and their targets at protein abundance and activity levels has not been fully explored. Here, we verified that a point mutation XlnR A871V in transcription factor XlnR enhanced the expression of lignocellulolytic enzymes, particularly hemicellulases, in P. oxalicum . Then, overexpression of XlnR A871V with a constitutive PDE_02864 promoter was combined with the overexpression of cellulase transcriptional activator ClrB and deletion of carbon catabolite repressor CreA. The resulted strain RE-7 showed 8.9- and 51.5-fold increased production of cellulase and xylanase relative to the starting strain M12, respectively. Further overexpression of two major cellulase genes cbh1 - 2 and eg1 enabled an additional 13.0% improvement of cellulase production. In addition, XlnR A871V led to decreased production of β-glucosidase and amylase, which could be attributed to the reduced transcription of corresponding enzyme-encoding genes. The results illustrated that combinational manipulation of the involved transcription factors and their target genes was a viable strategy for efficient production of lignocellulolytic enzymes in filamentous fungi. The striking negative effect of XlnR A871V mutation on amylase production was also highlighted.