The Role Of CAMP-related Genes In The Regulation Of Cellulase Expression In Myceliophthora Thermophilum

Lignocellulose is one of the largest,most widely distributed and most abundant renewable resources.Cellulase produced by microorganisms degrades cellulose to produce glucose and further converts it into other energy sources such as ethanol.It can alleviate current energy stress.Myceliophthora thermophila produces thermostable cellulase.It is known as a potentially efficient medium-high-temperature enzyme library.Cyclic adenosine monophosphate(cAMP)is an important substance involved in regulating the metabolism and biological functions of cells.It is an important second messenger in organism.It can reduce the carbon metabolism repressing effect of fungi thereby increase the ability of fungi to produce cellulase.The adenylate cyclase and phosphodiesterase can jointly regulate intracellular cAMP levels.In this study,we mainly used RNAi technology to interfere with the ac and pde genes of Myceliophthora thermophilum,and exogenously added cAMP,in order to investigate the role of cAMP and related genes in the cellulase gene expression regulation of Myceliophthora thermophila.The main results of this paper are as follows:1)RNA interfering with the adenylate cyclase gene: adenylate cyclase is a key enzyme that regulates intracellular cAMP levels in response to environmental signals,and is the only enzyme that catalyzes the formation of cAMP by ATP.In the present study,interference sequences were designed for the ac of Myceliophthora thermophilum,and the recombinant interference expression vectors were constructed using the pUC19-MtPpdc-MtTpdc plasmid.The interference expression vectors and the pAN7-1 plasmid were cotransformed into Myceliophthora thermophila,and the interfering transformed strains AC1,AC2-1,AC2-3 and AC2 C were successfully obtained.RT-qPCR was used to screen out the interference efficiency of AC1 and AC2-3.The better one was used for enzyme activity experiments.After measuring by filter paper enzyme activity(FPA),endonuclease activity(CMC),and cellobiohydrolase(BG),it was found that when cultured on the 4th day under avicel induction conditions,the FPA,BG,and CMC activities of transformant AC 1 respectively decreased by43%,24%,and 20% compared with the enzyme activity of WT.However,the FPA,CMC andBG activities of transformant AC2-3 did not decrease significantly.The mRNA levels of cellulase-related gene in AC1 and WT were detected by RT-qPCR.The expression levels of bgl2 and egl1 in transformant AC1 decreased by 24%?53% compared with those in WT,respectively.The mRNA levels of other cellulase genes bgl1,bgl3,egl3,and regulatory factors cre1,ace1,xyr1 were not decreased.2)The exogenous addition of cAMP: in this study,Myceliophthora thermophila was introduced into avicel medium,corncob powder medium,and cellobiose medium with 2mM cAMP,Myceliophthora thermophila cultured in each medium without cAMP was as control.The FPA,BG,and CMC enzyme activities were measured.It was found that in the avicel medium,the FPA and CMC activity increased in the strains cultured with cAMP medium.At the 7th day and the 5th day of culture,it was 1.76-fold and 1.75-fold higher than those of WT,the change of BG enzyme activity is not obvious.However,in the cellobiose medium,the changes of FPA,BG and CMC activities were not obvious.In the corncob powder medium with cAMP,the FPA activity on the 4th day was 2.25-fold higher than that of WT,while the BG and CMC activities have not obvious changes.The results of RT-qPCR showed that the expression levels of bgl1,bgl3,egl3 and cre1 mRNA increased by 3.97-fold,3.35-fold,2.97-fold and 1.27-fold,respectively in avicel medium with cAMP.No significant changes were detected in expression level of other genes.The iTRAQ results showed that there were 58 different proteins between the cAMP-added strain and the cAMP-free strain.These proteins were involved in many kinds of metabolic pathways,such as carbon metabolism,secondary metabolism,metabolism,starch and sucrose metabolism,pentose phosphate pathway,and glycolysis,glyoxylic acid and dicarboxylic acid metabolism,pentose and glucuronic acid interconversion,amino sugar and ribose,galactose metabolism,tryptophan metabolism,antibiotic biosynthesis etc.3)RNA interfering with the pde gene: phosphodiesterase(PDE)specifically catalyzes the hydrolysis of second messenger cAMP and cyclic guanosine monophosphate,and regulates the level of intracellular cAMP together with adenylate cyclase.In the present study,interference sequences were designed for the pde gene in Myceliophthora thermophilum,and recombinant interference expression vectors were constructed.The interference expression vectors and the pAN7-1 plasmid were cotransformed into Myceliophthora thermophila,andthe transformants pde1-1,pde1-2,pde1-3,pde3-1,pde3-2 and pde3-5 were successfully obtained.The interference efficiency of pde 3-1 was the best during all the transformants detected by RT-qPCR.The FPA,BG and CMC activities in pde 3-1 cultured in avicel medium did not change significantly compared with the activities of wild type.However,in the cellobiose medium,the FPA,BG and CMC activities in pde 3-1 continued to decrease.On the4 th day of culture,the decrease was was 41%,40% and 56% compared with wild type,respectively.The results of RT-qPCR showed that the mRNA expression levels of four cellulase genes of bgl1,bgl2,bgl3 and egl1 were significantly decreased by 23%,75%,88%and 66%,respectively.The mRNA expression level of the carbon metabolism repressor cre1 in pde 3-1 strain was 2.54-fold compared with that of WT.The results of this study indicated that interference with the ac gene reduces cAMP formation from ATP synthesis,thereby partially inhibiting the expression of the cellulase gene in Myceliophthora thermophila;increasing extracellular cAMP levels can increase the expression of main cellulase genes of Myceliophthora thermophila;however,the enzyme activities in transformants interfered with the pde gene were not significantly changed.The xyr1 is one of the positive regulators of the cellulase gene,and cre1 is one of the negative regulator in Myceliophthora thermophila.Both of the expression levels of the two regulators were up-regulated.Their effects on the downstream genes regulated need to be synthesized by multiple factors,and then expressed as corresponding protein activity,which reflects the complexity on regulation of intracellular gene expression.