Co-expression Of Cellulase And Xylanase In Saccharomyces Cerevisiae And Its Application

Lignocellulose is an abundant renewable resource that can be used to produce renewable energy sources such as bioethanol,which is considered a substitute for fossil fuel resources.Lignocellulosic biomass is mainly composed of cellulose,hemicellulose and lignin,forming a strong network structure.Cellulose and hemicellulose account for 40-50%and 25-35%of the lignocellulosic material,respectively.Due to the strong combination of lignocellulosic network structure and components,hydrolysis of lignocellulose has become the most important technology and economic limiting factor for its large-scale application,and is also a major problem in the current use of lignocellulose as a raw material for the production of bioenergy.Enzymatic hydrolysis is a good method because of its mild and environmental friendly reaction conditions.Cellulase hydrolyzed cellulose has many limitations,and it has been reported that the enzymatic hydrolysis of xylan components in hemicellulose by xylanase favors the hydrolysis of cellulase.The previous study also showed that the cellulase and xylanase had significant synergistic effects during the enzymatic hydrolysis of lignocellulose.Therefore,it is of great significance to construct a synergistic S.cerevisiae for enzymatic hydrolysis and transformation of lignocellulose.Two Saccharomyces cerevisiae engineering bacteria co-expressing cellulase and xylanase were constructed in this experiment.One source of cellulase genes and two different sources of xylanase genes were combined in the same strain of Saccharomyces cerevisiae.Expression in order to increase the efficiency of transformation and utilization of lignocellulose by Saccharomyces cerevisiae engineering bacteria.First,the cellulase geneCBH(GenBank:KC751534.1)was cloned into the pHBM368?PGK vector to obtain pHBM368-PGK-CBH,which was transformed into Saccharomyces cerevisiae by electroporation for expression.The enzyme activity was verified by CMC-Na.The laboratory xylanase gene TS(GenBangk:AY156910.1)and the xylan gene CA(GenBank:XX12596.1)were cloned into the vector pHBM368-PGK to obtain pHBM368-PGK-TS and pHBM368-PGK-CA,respectively.It was transformed into Saccharomyces cerevisiae by electroporation for expression,and its enzyme activity was verified by indica xylan.Reconstruct pHBM368-PGK-CBH-Ura,pHBM368-PGK-TS-Ura,pHBM368-PGK-CA-ura,respectively,and transform the uracil screening marker of the vector into a leucine screening marker,which will connect the cellulase gene and wood The vector of the glycanase gene was mixed and electrotransformed into Saccharomyces cerevisiae and screened using SC double auxotrophy(Ura-.Lue-)to obtain a wine strain in which the cellulase gene and the xylan gene were co-expressed.After functional verification,they were named:S.cerevisiae INVSc-CBH-TS,S.cerevisiae INVSc-CBH-CA.Determination of xylanase activity and cellulase activity of co-expressing S.cerevisiae strains INVSc-CBH-TS and INVSc-CBH-CA,cellulase activity of recombinant Saccharomyces cerevisiae strain INVSc-CBH-TS At 931.27/mL,the xylanase activity reached 413.70 U/mL.The cellulase activity of the recombinant Saccharomyces cerevisiae strain IEVSc-CBH-CA reached 716.43 U/mL,and the xylanase activity reached 205.13 U/mL.Co-expressed using the pretreated corn stover as the sole carbon source.The strains,experimental results show that the engineered bacteria can use the pretreated corn stover as a carbon source for growth.Recombinant Saccharomyces cerevisiae strain pHBM368-PGK-CBH-TS used pretreated corn stover as the sole carbon source for anaerobic fermentation to produce ethanol at a rate of 1.9 g/L,an increase of 342%over the type of INVSc Saccharomyces cerevisiae ethanol production.