“Top-down” overexpression optimization of butelase-1 in Escherichia coli and its application in anti-tumor peptides

Butelase 1, the fastest known Asn/Asp-specific peptide ligase capable of catalyzing peptide ligation and cyclization, holds promising application prospects in the fields of food and biology. However, limited research exists on its recombinant expression and potential applications in peptide drugs. In this study, the activity of recombinantly-produced butelase 1 was enhanced by co-expressing it with a molecular chaperone in the SHuffle T7 strain. By introducing single or multiple synonymous rare codons at the beginning of the coding regions of beta-strand or alpha-helix, in combination with ribosomal binding site engineering, the activity of butelase 1 could be further improved. Consequently, the butelase 1 with a specific activity of 386.93 U/mg and a catalytic efficiency of 11,048 M