This technology is ready for efficient production of cellulase-free recombinant xylanase in industrial scale. Xylanase is an enzyme that is widely applied in various industrial processes, such as cellulosic ethanol production, feedstock additives, and biobleaching, etc. T. reesei is regarded as an important producer of xylanase. Induced by arabinose-rich plant hydrolysates and lactose in fed-batch cultures, the mutant strain T. reesei Rut C-30 produces up to 1350 IU/ml of xylanase. However, under induced conditions, T. reesei produces a large amount of cellulase aside from xylanase, and the cellulase side product is problematic for the application of xylanase in some industrial processes, such as biobleaching. To resolve this problem, we screened strong constitutive promoters of T. reesei through q RT-PCR, and developed a novel approach to produce cellulase-free xylanase by using the promoters of the pdc gene. The T. reesei pxyn2 recombinant strain, in which the pdc promoter were used to conduct the xyn2 gene expression, produced 9266 IU/ml of activity in a modified Mandels medium. Moreover, under constitutive production with high glucose concentrations, the recombinant strains produce little non-objective proteins as revealed by the SDS-PAGE image, and only a trace of cellulase activity is detected, with 0.29 IU/ml of CMCase activity and 0.03 FPIU/ml of filter paper activity. As xyanlases are a group of industrial enzymes with applications in feed and food, pulp and paper, textiles, etc., a number of production systems for these enzymes have been developed, including native production of xylanases in T. reesei, and heterologous production of xylanase in bacteria or fungi. To date, the highest native production of obtained in T. reesei is 1800 IU/ml with a fed-batch cultivation process, and the highest heterologous production of xylanase obtained is 3676 IU/ml with a fed-batch cultivation of Pchia pastoris expressing A. niger xylB . We used strong constitutive promoters in the xylanase production in T. reesei, and obtained recombinant productivity of 9266 IU/ml for the pdc promoter and 8866 IU/ml for the eno promoter in batch cultivation, which are much higher than the best native and heterologous xylanase production levels to date.
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