Abstract
BackgroundLignocellulolytic 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.ResultsHere, we verified that a point mutation XlnRA871V in transcription factor XlnR enhanced the expression of lignocellulolytic enzymes, particularly hemicellulases, in P. oxalicum. Then, overexpression of XlnRA871V 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, XlnRA871V led to decreased production of β-glucosidase and amylase, which could be attributed to the reduced transcription of corresponding enzyme-encoding genes.ConclusionsThe 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 XlnRA871V mutation on amylase production was also highlighted.
Highlights
Introduction ofXlnRA871V resulted in dramatically reduced amylase production, which could be attributed to the decreased expression of amy15A and amyR
In P. oxalicum, we found that XlnR was indispensible for hemicellulase induction and to a less extent for cellulase expression [10]
Enhanced expression of lignocellulolytic enzyme genes due to the point mutation X lnRA871V In our previous study, we identified that overexpression of xlnR resulted in significantly improved xylanase production and a slight increase in cellulase production in P. oxalicum [10]
Summary
Introduction ofXlnRA871V resulted in dramatically reduced amylase production, which could be attributed to the decreased expression of amy15A and amyR. The decreased amylase activity in xlnRA871V should release less glucose from starch than wild-type strain in the wheat bran medium, and reduced the carbon catabolite repression on lignocellulolytic enzyme expression. 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. Three main cellobiohydrolases (CBHs), fifteen endoglucanases (EGs), eleven β-glucosidases (BGLs), and fifty-one hemicellulases were predicted to be encoded in its genome [9, 10] Owing to these enzymes, the lignocellulolytic enzyme system of P. oxalicum is more diverse. A transcription factor gene deletion library was constructed in P. oxalicum and several regulators including ClrB, CreA, XlnR, and AmyR were proved to regulate cellulase expression [10]. The potential of this strategy has not been fully explored regarding the manipulation of transcription factors at the activity level
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