Mining And Functional Study On The Regulator Of Cellulase Expression In Penicillium Oxalicum

Since the beginning of the 21 st century,the petrochemical energy crisis has become increasingly prominent.At the same time,the use of oil has also brought a series of environmental problems.It is urgent to find new sustainable green energy.Biomass is the largest renewable resource in nature.Using lignocellulosic biomass to produce degradable products to replace part of oil is an important guarantee for human sustainable development.Lignocellulose is degraded into fermentable sugars by enzymes,which plays a key role in the process of biotransformation.Filamentous fungi are important sources of lignocellulose degrading enzymes,and can secrete cellulase efficiently.Therefore,improving the efficiency of cellulase production by filamentous fungi is conducive to the industrial application of cellulase producing strains.At present,the rational transformation of strains by genetic engineering is of great significance to improve the enzyme production efficiency of filamentous fungi.Penicillium oxalicum114-2 is an important strain to study the regulation mechanism of cellulase expression.P oxalicum is rich in lignocellulose and its proportion is reasonable.The determination of genomic sequence of P oxalicum provides abundant targets for the multi-directional transformation of the strain.In addition to the known genes related to the expression of lignocellulose,it is of great significance to explore the genes involved in the regulation of lignocellulose expression in the genome of P oxalicum,and to study their new functions in the regulation of lignocellulose expression.Based on the transcriptome data of P oxalicum lignocellulose key transcription factor mutants under different carbon source culture conditions,we first identified 59 genes with significant expression changes,and further constructed a single gene knockout strain with 59 genes.According to the enzyme producing activity and growth phenotype of gene knockout mutants,new functional genes with significant effect on the expression of lignocellulose were screened.In addition,based on the annotated signal transduction proteins of P oxalicum genome,we constructed a single gene knockout library of signal transduction proteins,screened the key genes of signal transduction proteins that affect the expression of cellulase in P oxalicum,and preliminarily studied their regulatory functions.The main results are as follows:1.Effect of dpp-Ⅴ gene on the hydrolase system of P oxalicumAccording to the transcriptome data of P oxalicum strains cultured in different carbon sources,59 genes with significant differences in gene expression were screened,59 genes were single gene knockout,and the enzyme activity and phenotype of mutant strains were tested.Compared with the original strain 114-2,dpp V gene knockout strain was screened.The results showed that the activity of cellulase and amylase of Δdpp V strain decreased significantly,and there was no obvious starch hydrolysis circle on the plate with starch as the sole carbon source.The results showed that dpp V gene might be involved in the regulation of cellulase and amylase synthesis and secretion of P oxalicum.2.Study on the function of calcium signal transduction protein from P oxalicumBased on the signal transduction protein information annotated by P oxalicum 114-2 transcriptional group,the enzyme activity and phenotype of the knockout four single gene knockout strains of calcium ion signal transduction protein were tested.The results showed that the cellulase activity of the ΔPDE＿04965 strain was lower than that of the original strain Δku42.On the cellulose phenotype plate,the phenotype of the knockout strain ΔPDE＿04965 was affected.The decreased expression of the major transcriptional regulator and cellulase gene in the knockout strain indicated that the gene might affect the cellulase synthesis of P oxalicum.3.Study on the function of cAMP signal transduction protein in P oxalicumIn this study,three single gene knockout strains of cAMP signal protein were constructed according to the cAMP signal transduction protein information annotated by the transcriptome of P oxalicum.The enzyme activity and phenotype of the knockout strain were tested.The results showed that the enzyme activity of filter paper was increased and the amylase activity was decreased after the cAMP signal protein gene was knocked out,which indicated that the cAMP signal affected the expression of cellulase and amylase in P oxalicum.