Assembly of cellulases with synthetic protein scaffolds in vitro

Ting Yu,Xin Gao,Dongzhi Wei,Yuhong Ren

doi:10.1186/s40643-015-0046-8

Abstract

Abstract Background Enzymatic cascades in metabolic pathways are spatially organized in such a way as to facilitate the flow of substrates. The construction of artificial cellulase complexes that mimic natural multienzyme assemblies can potentially enhance the capacity for cellulose hydrolysis. In this study, an artificial cellulase complex was constructed by tethering three cellulases to a synthetic protein scaffold. Results Three pairs of interacting proteins were selected and characterized. The artificial protein scaffolds were constructed by fusing three interacting proteins. Cellulases were tethered to these synthetic scaffolds in different orders. The optimal assembly resulted in a 1.5-fold higher hydrolysis of cellulose than that achieved by unassembled cellulases. Conclusions A novel artificial protein scaffold was constructed and used to assemble three cellulases. The resultant increase in enzymatic activity suggests that this can be used as a strategy for enhancing the biocatalytic capacity of enzyme cascades.

Highlights

Enzymatic cascades in metabolic pathways are spatially organized in such a way as to facilitate the flow of substrates
To synthesize the protein scaffold, three pairs of interacting proteins from E. coli K12 were expressed in E. coli BL21 (DE3)
The response values (Figure 2B,C, D) confirmed their binding, and the KD calculated by Fortebio Data Analysis software version 7.0 for IPA-IPa (7.36 × 10−8 M), IPB-IPb (1.39 × 10−12 M), and IPC-IPc (5.27 × 10−8 M) demonstrated their high affinity, suggesting that they are suitable for constructing protein scaffolds

Summary

Introduction

Enzymatic cascades in metabolic pathways are spatially organized in such a way as to facilitate the flow of substrates. The construction of artificial cellulase complexes that mimic natural multienzyme assemblies can potentially enhance the capacity for cellulose hydrolysis. An artificial cellulase complex was constructed by tethering three cellulases to a synthetic protein scaffold. Multienzyme pathways in living systems comprise cascades in which enzymes are tethered together into assemblies that facilitate substrate flow between components, limit the diffusion of intermediate metabolic products, and increase the yield from sequential reactions [1,2]. There have been various attempts to produce multienzyme assemblies in vitro, including by gene fusion, protein or DNA scaffold construction, and chemical modification [3]. A selfassembled enzyme complex using cellulosome achieved a

Methods

Results

Conclusion