Construction Of A Multifunctional Cellulase And Expression In Saccharomyces Cerevisiae

Lignocelluloses have received great attention owning to their abundance,renewability,and non-competition with human demand.Cellulose is the most important component of lignocellulose and its complete degradation requires synergy between three types of enzymes with different cleavage sites,including endoglucanase,exoglucanase and ?-glucosidase.With extensive research on cellulases,these biocatalysts are widely used in many industrial fields.Considering the demand for a synergistic action of multiple enzymes,the expression of a variety of enzymes in a single cell is likely to improve the efficiency of cellulose degradation and reduce the production process.There are many ways to express multiple genes in a single cell,but most rely on rmultiple screening methods and are complicated to operate.There have been reports of single gene duality,but no gene has been described to have more than two functions at present.Under this condition,we were the first to use a fusion strategy to construct a single gene with three different cellulase functions to reduce the complexity of the manipulation in the expression of multifunctional cellulases.The gene was expressed in Saccharomyces cerevisiae and screened for strains having three cellulase functions in order to demonstrate the feasibility and potential value of constructing a single-gene multifunctional enzymeIn this study,we designed primers to amplify the catalytic domains of the three cellulase genes.The reverse coding sequence of the flexible linker(G4S)3 and the restriction enzyme sites were added to the reverse primer ends of the first two genes.The PCR product was digested with the corresponding restriction enzymes and ligated with T4 DNA ligase after purification The 3.0 kb DNA fragment obtained by PCR using the enzyme-linked mixture as a template is the trifunctional cellulase single gene designated bee.It was cloned into the S.cerevisiae expression vector pHBM368-pgk,and the recombinant vector pHBM368-pgk-bce was obtained after resistance screening and enzyme digestion verification.The vector was used to electroporate S.cerevisiae INVSc1,followed by uracil auxotrophic screening and chromogenic substrate plate screening.After the determination of total cellulase activity and colony PCR validation,a recombinant strain with high cellulase activity was selected and named INVScl-BCE.We performed specific substrate activity analysis and comparison of single-gene trifunctional cellulase BCE and monofunctional cellulase.At 45? and pH 6.0,the ?-glucosidase activity and endoglucanase activity of BCE reached 2.24 U/mL and 2.76 U/mL,which were higher than BG and EG by 30.93%and 12.34%,respectively.However,the BCE exoglucanase activity was 8.63%lower than CBH,reaching 0.28 U/mL.The results showed that BCE has three different cellulase activities.In addition,the total cellulase activity of BCE and monofunctional cellulase was determined.At 45? and pH 6.0,the FPase activity of BCE reached 272.5 U/L,which was higher than that of BG and EG,which was comparable to that of CBH.The results demonstrated that the single-gene trifunctional cellulase has the ability to degrade complex substrate(filter paper).Comparing the differences in specific substrate activities,it could be speculated that this enzyme exhibited a synergistic effect during degradation.Finally,the optimum reaction conditions for the FPase activity of BCE were determined to be 55?,pH 6.5,and the activity reached 443.0 U/L under this condition.In summary,it was simple to operate,effective,feasible and in line with the three cellulase synergistic degradation mode to construct a single-gene trifunctional cellulase using a fusion strategy.Besides,it had significant reference value for the process of degradation of lignocellulose by many other types of enzymes.