Abstract
BackgroundThe generation of focused mutant libraries at hotspot residues is an important strategy in directed protein evolution. Existing methods, such as combinatorial active site testing and residual coupling analysis, depend primarily on the evolutionary conserved information to find the hotspot residues. Hardly any attention has been paid to another important functional and structural determinants, the functionally correlated variation information--coevolution.ResultsIn this paper, we suggest a new method, named combinatorial coevolving-site saturation mutagenesis (CCSM), in which the functionally correlated variation sites of proteins are chosen as the hotspot sites to construct focused mutant libraries. The CCSM approach was used to improve the thermal stability of α-amylase from Bacillus subtilis CN7 (Amy7C). The results indicate that the CCSM can identify novel beneficial mutation sites, and enhance the thermal stability of wild-type Amy7C by 8°C ( ), which could not be achieved with the ordinarily rational introduction of single or a double point mutation.ConclusionsOur method is able to produce more thermostable mutant α-amylases with novel beneficial mutations at new sites. It is also verified that the coevolving sites can be used as the hotspots to construct focused mutant libraries in protein engineering. This study throws new light on the active researches of the molecular coevolution.
Highlights
The generation of focused mutant libraries at hotspot residues is an important strategy in directed protein evolution
While the aforementioned methods depend primarily on the evolutionary conservation information to find out the hotspot residues, there are some other important functional and structural determinants desirable to be taken into consideration, such as the functionally correlated variation information–coevolution
We propose a method, combinatorial coevolving-site saturation mutagenesis (CCSM), which chooses the coevolving sites of proteins as hotspot residues to construct focused mutant libraries
Summary
The generation of focused mutant libraries at hotspot residues is an important strategy in directed protein evolution Existing methods, such as combinatorial active site testing and residual coupling analysis, depend primarily on the evolutionary conserved information to find the hotspot residues. Directed protein evolution is invaluable in engineering biocatalysts for better properties, such as enhancements in activity, stability, and enzyme selectivity [1,2] It is usually limited by its inability to generate highquality mutant libraries containing more beneficial variants. All existing focused mutant library methods can be essentially classified into two categories: structure-based approaches and sequence-based approaches The former includes combinatorial active site testing (CAST), B-factors, and knowledge-based potential analysis [6,7,8,9,10]. While the aforementioned methods depend primarily on the evolutionary conservation information to find out the hotspot residues, there are some other important functional and structural determinants desirable to be taken into consideration, such as the functionally correlated variation information–coevolution
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