A closed-loop strategy for on-site production of saccharolytic enzymes for lignocellulose biorefinery using internal lignocellulosic hydrolysates

The cost-effective production of lignocellulose-saccharolytic enzymes is crucial for the bioconversion of plant biomass into fuels and chemicals. The integrated mode for producing these enzymes using lignocellulosic feedstocks as natural inducers has a cost advantage; however, enzyme production level is limited because of the solid-state and complex composition of the raw materials. This study aimed to establish an innovative process for the integrated production of lignocellulolytic enzymes using lignocellulosic hydrolysate as the carbon source. The cellulase-producing workhorse Trichoderma reesei was reprogrammed by combinatorial engineering of transcription factors and the introduction of heterologous beta-glucosidases. This enabled inducer-independent production of a complete lignocellulose-saccharolytic enzyme cocktail using lignocellulose-derived monosaccharides as sole carbon source. Via continuous feeding of the enzymatic hydrolysate of pretreated corn stover, crude enzymes with filter paper enzyme activity of 60.4 U/ml and beta-glucosidase activity of 503 U/ml were produced. Crude fermentation broth can be used directly to prepare hydrolysates, enabling nex-round enzyme production by recycling 3.1% of the hydrolysate. These results demonstrate a closed-loop strategy for integrated enzyme production using internally produced lignocellulosic sugars from biorefinery plants.