Systematic regulation of mycelial extracellular proteolysis, endoplasmic reticulum homeostasis, morphology, and bioprocess boosts recombinant laccase production in Coprinopsis cinerea

Laccase is an important green catalyst, particularly basidiomycete laccases, which have significant industrial applications in fields, such as food and biomass energy. However, the natural secretion of laccase by basidiomycetes is low. Heterologous expression of basidiomycete laccase in yeast or Aspergillus often changes the enzyme's properties due to inappropriate post-translational modifications. Therefore, using basidiomycetes for homologous recombination expression of basidiomycete laccase has great industrial prospects and economic benefits. This study used the native host for the homologous recombination expression of Laccase Lcc5 from Coprinopsis cinerea and developed a novel strategy to increase its extracellular activity. Silencing the protease Ccmpr , overexpressing the endoplasmic reticulum stress response gene Anhac 1, and silencing the morphological regulation gene rac 1 all increased the recombinant Lcc5 activity. The extracellular laccase activity of the rac 1-silenced Cc lcc5 -anti rac 1-2 was 1.84-fold higher than that of the original Cc lcc5 -13. In particular, the Cc lcc5 -anti ChiE 2& mpr -3, obtained by simultaneously silencing the chitinase ChiE2 and the protease Ccmpr , exhibited the highest extracellular laccase activity, 67.71 U/mL, which was 2.56-fold higher than that of Cc lcc5 -13. Optimizing the fermentation process of Cc lcc5 -anti ChiE 2& mpr -3 increased laccase activity in shake flasks to 76.03 U/mL when using 20 g/L yeast extract as the nitrogen source. At a stirring speed of 200 rpm, the laccase activity in a 1.5-L fermenter reached 109.95 U/mL. To our knowledge, this is the highest extracellular laccase Lcc5 activity in basidiomycetes reported to date. This is the first report of a multidimensional integrated strategy to enhance recombinant protein expression in C. cinerea by modulating protease gene expression, optimizing post-translational folding and secretion, and modifying mycelial morphology. This study will pave the way for laccase industrial production and accelerate the development of the C. cinerea high-expression system.