Cellulase Expression Analysis Of Kinase Gene Stk-12 Of Neurospora Crassa

Protein phosphorylation plays an important role in many important cellular processes,such as the extracellular cellulase induction signal sensing and intracellular signal transduction processes in filamentous fungi. Protein phosphorylation is controlled by protein kinases and phosphatases. In order to dissect the roles of protein kinases in cellulase induction signaling pathway, the 62 kinase mutants were cultured on crystalline cellulose and a comparison of levels of secreted protein and cellulase activity relative to the wild type strain resulted in the identification of five hyper-production (?NCU07378, ?NCU00914,?NCU06638, ?NCU01187 and ?NCU01940) and three hypo-production (?NCU06685,?NCU04426 and ?NCU08346) strains. Intriguingly, the ?NCU07378 (?stk-12) mutant exhibited the highest protein secretion among tested strains. Thus, we focused on its functional characterization. Reintroduction of the wild type stk-12 gene complemented the Astk-12 mutant phenotypes, suggesting that STK-12 negatively regulates cellulase production. The deletion of stk-12 resulted in growth defect when grown on different carbon sources, such as glucose, glycerol, xylose or cellobiose. STK-12 also was involved in high osmolarity resistance, oxidative stress response and cell wall integrity maintenance.Furthermore, the ?stk-12Acre-1 strain exhibited stronger protein production capability than either the Acre-1 strain or the ?stk-12 strain. It indicated that deletion both stk-12 and cre-1 had a significant synergistic effect on lignocellulase production. Thus, the enhanced cellulase gene expression in the ?stk-12 is not the result of relief from catabolite repression mediated by cre-1. To test the effect of stk-12 on cellulase gene expression under cellobiose condition, a ?stk-12?3?G quardruple mutant was constructed by crossing. The consumption assay indicated that the quadruple mutant had significantly increased cellobiose consumption compared with its parental strain. On the other hand, a real-time PCR analysis also indicated that the expression level of cellobiose transporters in?stk-12?3?G were dramatically up-regulated. These data suggested that stk-12 is involved in cellulase induction pathway. In addition, stk-12 also probablely affected cellulase production via PKA pathway. To determine the function of stk-12 is conserved in the filamentous fungi, we constructed the strain carrying deletion of stk-12 in the Myceliophthora thermophile. The ?Mt-stk-12 secreted significantly higher quantities of enzymatically active cellulases, suggesting that the role of stk-12 in cellulase production is highly conserved. In summary, these observations could be helpful for a better understanding of the roles of protein kinases in cellulase induction pathway.