Construction And Application Of An Efficient Multi-point Combination Mutation Method

Compared with traditional chemical catalysts,biocatalysts with high specificity and high catalytic performance are widely used in the synthesis of complex pharmaceutical intermediates,fine chemicals and so on.Natural enzymes lack the properties required for commercial applications of enzyme preparation,and the evolving technology of directed evolution combined with high-quality mutagenesis methods and efficient screening methods can rapidly screen for better quality or new functions.Directed evolution is a powerful and widely used method of enzyme engineering.With the in-depth research of protein structure and functions and the continuous improvement of computational programs and computer capabilities,computer-aided design has often been applied to enzyme engineering.The structure and function of protein depends on the synergy between amino acid residues.The directed evolutionary mutagenesis method of existing multi-point combination mutations based on computer-aided design screening of amino acid targets cannot simultaneously achieve mutation diversity and a large number of clones.Therefore,it is necessary to develop a new directed evolution mutagenesis method to combine mutations at different key amino acid positions.In this research,a multi-point combinatorial mutagenesis(MCM)method was developed to construct plasmid libraries.A highly abundant mutation rate and a large number of clones can be achieved by this method simultaneously.Firstly,this method utilized DNA assembly to splice the mutant target gene;subsequently,two linear plasmid DNA fragments with gaps at different positions were obtained by fusion PCR between target genes and vector fragments;the last two types of linear plasmids were hybridized in vitro to obtain circular plasmid molecules,multi-point combination mutation was performed by transformation into Escherichia coli competent state.And then,the fusion PCR reaction conditions and linear plasmid hybridization conditions were optimized,the number of Colony-Forming Units(CFUs)obtained by electroporation to competent Escherichia.coli TreliefTM 5a exceeded 106 CFUs/?g DNA.The efficiency of MCM method was tested by using the optimized method to the directed evolution of benzoylformate decarboxylase(BFD).Colony PCR validation and DNA sequencing results showed that 90/100 single colonies were accurately assembled;the frequency of simultaneous mutations at 5 sites was over 80%;12 possible amino acid residues derived from the NDT degenerate codon were included.Finally,a 2-fold increase in enzyme catalytic activity were screened(BFD-F3:L109Y,L110D,H281G,Q282V and A460M).The kinetic parameters determined that the kcat of the mutant BFD-F3 was increased by about 6 times,and the final kcet/Km was 1.35.M-1 s-1,which was about 10 times that of wild type.Gas chromatography-mass spectrometry(GC-MS)results showed that BFD protein did catalyze the synthesis of hydroxyacetaldehyde.This research project successfully constructed a directed evolution method of multi-point combinatorial mutation,which introduced rich diversity of mutations at the targeted site,and at the same time a large number of clones were created to cover the possibility of mutation.Therefore,if combined with computer-aided design methods,this method can significantly improve the efficiency of enzyme engineering and can be expected to have a significant impact on the development of directed evolution of enzymes.