Construction And Characterization Of A Protein Scaffold System Based On Colicin Dnases And Their Inhibitors

Artificial protein scaffold system is a cutting-edge technology to achieve multienzyme co-domainization.It can use protein reaction pairs to assemble an efficient and stable multi-enzyme molecular machine and obtain better performance than free enzyme systems.To achieve the orderly self-assembly of enzyme molecules,most of the common artificial protein scaffold systems are currently constructed using Cohesin-Dockerin,which is derived from natural cellulosomes with species-specific and ultra-high affinity(Kd=10^-9?10^-12)characteristics.However,the limited variety of Cohesin-Dockerin protein reaction pairs and the difficulty of compatibility with thermophilic enzyme systems have limited the application of this system to some extent.Therefore,new orthologous protein reaction pairs need to be explored to construct artificial protein scaffold systems with a wider range of applications.The family of colicin DNases(CE2,CE7,CE8 and CE9)derived from Escherichia coli is a class of non-specific endonucleases with highly similar structures.They bind with their respective inhibitors(Im2,Im7,Im8 and Im9)with high affinity(Kd=10^-14?10^-17)and high specificity.The truncated mutant CL7 with inactivation of the CE7 DNase domain makes it possible for the four groups of CE-Im to become a new type of orthogonal protein reaction pair.In this study,we constructed a protein scaffold system using inhibitor Im as the scaffold protein backbone and successfully applied it to thermostable designer cellulosomes.The details of the study are as follows.(1)Truncated mutants of CE nuclease inactivation(CL2,CL7,CL8 and CL9)and their corresponding inhibitors(Im2,Im7,Im8 and Im9)were constructed and efficiently expressed by an E.coli expression system.(2)The good specificity and high temperature tolerance of the four homologous paired CL-Im proteins were verified.(3)Scaffold proteins with Im proteins as the backbone were constructed and the functional integrity of the tetravalent scaffold protein(Scaf-Im2789)was successfully verified by fluorescent protein labeling.(4)Application of Scaf-Im2789 in designer cellulosomesDesigner cellulosomes were successfully assembled by combining exoglucanase CL2-Cel48S_m3,endoglucanase Cel8A_4m-CL7 with GH5D-CL8,and?-glucosidase Co GH1A-CL9with Scaf-Im2789.Compared with the free enzyme systems,the designer cellulosomes yielded about 60%more reducing sugars after a 3 h reaction at 70?with phosphoric acid swollen cellulose(PASC)as the substrate,and about 80%more reducing sugars after a 3 h reaction at75?with microcrystalline cellulose(Avicel)as the substrate.In summary,the CL-Im protein scaffold system constructed in this study possessed high affinity,high specificity and high thermal stability,and was coupled with CL-tagged thermophilic cellulases to obtain designer cellulosomes by self-assembly.Compared with the free enzyme systems,the efficiency of hydrolyzing cellulose under high temperature conditions was significantly enhanced.This study provides a new tool for the construction of artificial multi-enzyme molecular machines,which has some application prospects.