Abstract 1662 Initial Success in Functional Expression of a GILT-Like Reductase from Attagenus unicolor in the Heterologous Host Trichoderma reesei

Trichoderma reesei is widely used as an industrial producer of enzymes due to its high protein expression yield and nontoxic nature. However, there are no successful expression of heterologous disulfide-reducing enzymes reported in this system. Our objective was to express the gene coding for a gamma interferon inducible lysosomal thiol (GILT)-like reductase from the black carpet beetle A. unicolor in the filamentous fungus T. reesei and to determine the disulfide-reducing and keratinolytic potential of the enzyme. The gene's full sequence is 594bp with a 60bp signal sequence. Without the signal peptide, the mature protein's predicted molecular mass is 20 kDa. The gene exhibits a GILT signature sequence and CXXC active site that is also found in thioredoxins. The plasmid used is named pSLH, which is 11kb in length and contains a CBHI promoter region inducible by lactose and a hygromycin B resistance gene. The gene was fused with the T. reesei endoglucanase II signal peptide, carbohydrate-binding module and linker region on the N-terminus and a 6x histag on the C-terminus. A T. reesei Rut C-30 derived low-protein background strain TSL126 was used as the expression host. The plasmid was transformed into the host through PEG-mediated transformation and colonies selected using hygromycin B at 100ug/mL concentration. The transformants were passaged twice under hygromycin B selection to ensure mitotic stability. With shake flask cultures, the enzyme was expressed using 2% lactose. Partial purification was done through Nickel bead chromatography. Successful gene integration and expression was confirmed through genomic PCR and RT-qPCR. Western blot results showed the expression of the GILT-like reductase as a dimer, with partial cleavage into monomeric form with the addition of 8M urea in 2x Laemmli buffer. The molecular mass of the enzyme was estimated to be 42kDa as a dimer and 21kDa as a monomer, including the 6x histag. The enzyme demonstrated disulfide bond reduction of Ellman's reagent (DTNB) and feather powder, which presented optimal activity at a pH of 6.0 and temperature range of 30-40C. Co-incubation of this enzyme with proteinase K revealed enhanced hydrolysis of feather powder compared with proteinase K alone. In conclusion, we were able to express a heterologous disulfide reductase in T. reesei and the enzyme showed a potential to enhance hydrolysis of keratinous substrates by proteinase K. This project is supported in part by a USDA grant (2019-69012-29905).