Studies On Enzyme Production And Growth Characteristics Of Engineered Trichoderma Reesei Strains And Optimization Of The Enzyme System

Biomass is the most abundant renewable resource on earth,of which more than 80%is lignocellulose.Lignocellulose can be decomposed into small molecules that can be further converted under the action of various degrading enzymes,but the cost of using these enzymes is still high.The filamentous fungus Trichoderma reesei is one of the most important lignocellulose-degrading enzyme producers in industry.It is of great significance to study the enzyme-producing characteristics of T.reesei and engineer it for better performances.The main results of this research are as follows:1.Cellulase production capacity of XYR1 mutant overexpressed strainXYR1 is a major activator that regulates the expression of T.reesei lignocellulosedegrading enzyme genes.Previously,our group obtained OExyr1*strain by overexpressing the transcription factor mutant XYR1A824V in T.reesei QP4,in which the expression of cellulase can get rid of the dependence on inducing carbon sources such as cellulose.In order to reveal the enzyme production characteristics of OExyr1*strain,we studied the ability of OExyr1*strain to produce cellulase on different kinds of carbon sources,we found that its ability to produce cellulase on medium with glucose as the sole carbon source exceeded that on cellulose medium by 14%,and its xylanase activity on xylose medium is 3.2-fold higher than that on glucose medium.By analyzing the transcriptome data of OExyr1*and QM9414 under glucose and xylose conditions,it was found that the transcriptional abundance of cellulase and hemicellulase genes in OExyr1*strain were up-regulated under both glucose and xylose conditions.However,the expression levels of some degrading enzymes were still affected by the type of carbon source.In addition,the saccharification solution obtained from hydrolysis of corn pericarp by OExyr1*crude enzyme solution could be reused for enzyme production by OExyr1*and used as a carbon source for the growth of Candida utilis.2.Construction and test of biomass-degrading enzyme systems from Trichoderma reesei and Penicillium oxalicumThe composition and properties of biomass degrading enzymes produced by different strains are different.Using two or more fungi with complementary advantages of enzyme system or directly mixed fermentation,it is possible to obtain enzyme systems with diverse components and higher degradation efficiency.In order to obtain enzyme systems with diverse components and higher degradation efficiency,we used two fungi with complementary advantages,T.reesei OExyr1*and P.oxalicum MCAX,and investigated the enzyme-producing capacity of them under mono-or co-cultivation conditions.The crude enzymes were applied to the saccharification of corn pericarp and steam-exploded corn straw respectively.It was found that co-cultivation had more advantages than mono-cultivation in the synthesis of filter paper enzymes.Co-cultivation or mixture of enzymes from mono-cultivations can improve the degradation efficiency of substrates to a certain extent.In addition,by replacing the 5%protein content of T.reesei enzymes with crude amylase from P.oxalicum,the glucose release from corn pericarp can be accelerated by 170%at 5 h.3.Construction of growth control system in T.reesei in response to carbon source speciesThe dynamic metabolic regulation system can be used to regulate the allocation of cellular resources,which is conducive to the coordination between the synthesis of biomass and the formation of target products.In order to provide parts for the construction of growth regulation system in T.reesei,we controlled the expression of the auxotrophic complementary gene pyrG using the promoter Pglt1 that is responsive to carbon source species,we demonstrated that the strategy could indeed control the growth of T.reesei on media containing a single carbon source.However,the ability of Pgltl promoter to regulate gene expression under switched carbon source conditions still needs to be improved.Furthermore,we tested the feasibility of erg 10 and gscl as essential genes for growth control in T.reesei.The results provided parts for further construction and optimization of dynamic metabolic regulation system in T.reesei.