Functional Analysis Of The Filamentous Fungus Podospora Anserina Protein Kinase SNF1 And Response Regulator SSK1

Lignocellulose is the most abundant renewable resource on the planet and has great application prospects in the production of biofuels and other value-added chemicals.Podospora anserine,a filamentous fungus,contains a large number of proteases related to the degradation of lignocellulose,and has broad prospects for bioenergy conversion.The biosynthesis of cellulase in P.anserina involves many physiological processes,including external signal sensing,signal transduction,transcription and translation of cellulase genes,and their secretion.Among them,external signal sensing is the first step in cellulase production.To investigate the potential mechanism of Surose non-fermenting protein kinase?SNF1?and response regulator SSK1 for external signal-induced conduction and cellulose utilization in P.anserina,we conducted this experiment.In this study,homologs of SNF1 and SSK1 were identified in the genome of P.anserina,and the PaSNF1 and PaSSK1 genes were knocked out by the Split-Marker method.Positive transformants were verified by resistance screening,PCR verification and Southern Blot.Mutant strains?PaSNF1 and?PaSSK1 were obtained,and at the same time,mutant replenishing strains CPPaSNF1 and CPPaSSK1 were constructed.Subsequently,wild type WT,mutant strains?PaSNF1 and?PaSSK1,supplemental strains CPPaSNF1 and CPPaSSK1 were analyzed for growth phenotype,alternative carbon source utilization,osmotic stress tolerance reaction,cellulase activity determination and secondary metabolite synthesis identification.The results showed that?1??PaSNF1 showed a significant decrease in colony growth rate compared with the wild type strain,while the growth rate of?PaSSK1 was the same as that of wild type.The microscopic observation showed that the diameter of?PaSNF1 and?PaSSK1 was significantly reduced compared with the wild type.?2?The number of?PaSNF1 fruiting bodies was significantly reduced,only 52% of wild-type strains,and the developmental maturity of?PaSNF1 fruiting body was significantly delayed,while the number and developmental maturity of?PaSSK1 fruiting bodies were similar to wild type.?3?The growth rate of?PaSNF1on the medium with different carbon sources was significantly reduced,especially in the lignin,and the inhibition rate was as high as 52%,and the growth inhibition was also observed on the glucose carbon source,while?PaSSK1grows under different carbon sources,it is no different from wild type.?4??PaSNF1 was significantly inhibited on osmotic stress medium such as 0.5M NaCl,0.5M KCl,0.5M Sorbitol and 0.75M Glycerol.?PaSSK1 also showed growth inhibition under 0.2M NaCl,0.3M KCl and 0.5M Sorbitol high osmotic pressure treatment,but showed strong tolerance to0.75M Glycerol.?5?The oxidant 50?M menadione also inhibited the growth of?PaSNF1,but?PaSNF1 was not sensitive to 0.1%H₂O₂.?PaSSK1 is not sensitive to 0.1%H₂O₂and 50?M menaquinone.?6??PaSNF1 showed sensitivity to heat shock reaction,its growth slowed down after heat shock treatment,and the inhibition rate increased significantly.However,?PaSSK1 was not sensitive to heat shock reaction.?7??PaSNF1 showed resistance to the cell membrane inhibitor fluconazole,and the growth of?PaSNF1 was significantly improved compared with the wild type.?PaSSK1 is not sensitive to this condition.?8?The enzyme activity assay showed that the FPA of?PaSNF1 was reduced to17.2%of the wild type strain,the CMCA was only 27.4%of the wild type,and the?-glucanase enzyme activity was equivalent to 50.3%of the wild type.The exoglucanase activity of?PaSSK1 was significantly higher than that of the wild type strain,and the?-glucanase enzyme activity was lower than that of the wild type strain.?9?It was found by HPLC and NMR analysis that the sterigmatocystin content of the?PaSNF1 mutant was significantly increased as compared with the wild type.In contrast,the sterigmatocystin content of the?PaSSK1 mutant was significantly reduced.This indicates that PaSNF1negatively regulates sterigmatocystin biosynthesis,and PaSSK1 positively regulates sterigmatocystin biosynthesis.In addition,the?PaSSK1 mutant produced a new peak at 7.5min.According to the results above,we can conculude that:?1?SNF1 plays a key role in stress response,utilization of alternative carbon sources,and regulation of heat tolerance.Importantly,SNF1 is a key regulator of the growth,development,and biosynthesis of secondary metabolites of filamentous fungi.?2?SSK1 plays an important role in osmotic stress and is essential for the synthesis of secondary metabolites of filamentous fungi.