Antisense inhibition of xylitol dehydrogenase gene, xdh1 from Trichoderma reesei

Abstract

To inhibit xylitol dehydrogenase (XDH) in Trichoderma reesei by antisense inhibition strategy and construct novel strains capable of accumulating xylitol. The xdh1 antisense expression plasmid pGTA-xdh was constructed by inserting xdh1 DNA fragment inversely between the gpdA promoter and the trpC terminator from Aspergillus nidulans into a pUC19 plasmid backbone. Trichoderma reesei protoplasts were co-transformated with pGTA-xdh and hygromycin B resistance plasmid pAN7-1. Of 20 transformants screened from the selective medium, one transformant with the highest xylitol accumulation, designated ZY15, showed a distinct reduction (c. 52%) in XDH activity compared with the original strain Rut-C30. The results of Southern hybridization and PCR assay showed that the antisense expression cassette of xdh1 was integrated into the genome of T. reesei. The RT-PCR analysis proved that antisense RNA effectively inhibited XDH expression (c. 65%). Xylitol accumulation (2.37 mg ml(-1)) of ZY15 was five times higher than that (0.46 mg ml(-1)) of the original strain Rut-C30. Strain ZY15 successfully downregulated XDH production and exhibited xylitol accumulation in xylose liquid medium. This study contributed to the budding field of fungal genetics in two points. First, it confirmed that antisense RNA strategy could be used as a means of reducing gene expression in the filamentous fungus T. reesei. Secondly, it verified that the strategy appears most promising for creating novel filamentous fungi strains capable of accumulating intermediary metabolites.