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 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. 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 XlnRA871V mutation on amylase production was also highlighted.

Aims: Improvement of cellulase production of Penicillium decumbens by genome shuffling of an industrial catabolite‐repression‐resistant strain JU‐A10 with its mutants. Methods and Results: After two rounds of genome shuffling, three fusants, GS2‐15, GS2‐21 and GS2‐22, were obtained, showing 100%, 109% and 94% increase in FPase activity than JU‐A10 respectively. The cellulase production of the fusants on various substrates, such as corn stover, wheat straw, bagasse and the corncob residue, was studied. The maximum productivities of GS2‐15, GS2‐21 and GS2‐22 were 92·15, 102·63 and 92·35 FPU l⁻¹ h⁻¹ on the corncob residue at 44 h respectively, which were 117%, 142% and 118% higher than that of JU‐A10 (42·44 FPU l⁻¹ h⁻¹, at 90 h). The improvements of the fusants were possibly because of their enhanced growth rates, earlier cellulase synthesis and higher secretion of extracellular proteins. Conclusions: The fusants obtained after genome shuffling could produce abundant cellulase much earlier, and they could be potential candidates for bioconversion process. Significance and Impact of the Study: This is the first report on the improvement of cellulase production in fungi by genome shuffling, and this is a good technique to improve other important phenotypes in fungi.

Soil contaminated with Cd and Pb has caused sharp decrease of cultivatable soil and has been attracting increasing attention. Biosurfactants are efficient in solving the problem. However, little information is available about the influence of sophorolipids (SLs) on the remediation of Cd- or Pb-contaminated soil. The sophorolipids produced by Starmerella bombicola CGMCC 1576 were used to study the effects of Cd and Pb removal in batch soil washing from artificially contaminated soil. The removal efficiency of crude total SLs was better than both distilled water and synthetic surfactants. Furthermore, 83.6% of Cd and 44.8% of Pb were removed by 8% crude acidic SLs. Acidic SLs with high water solubility were more effective than lactonic SLs in enhancing remediation of heavy metal-contaminated soils. The complexation of Cd with the free carboxyl group of the acidic SLs was observed by Fourier-transform infrared spectroscopy study, and this complexation was effective in heavy metal removal from the soil. The fermentation broth of S. bombicola, without further preparation, removed 95% of Cd and 52% of Pb. These results suggested that SLs produced by S. bombicola could function as potential bioremediation agents for heavy metal-contaminated soil.