Ligation independent cloning (LIC) as a rapid route to families of recombinant biocatalysts from sequenced prokaryotic genomes
Abstract
A technique is presented for the high throughput generation of families of recombinant biocatalysts sourced from prokaryotic genomes, providing rapid access to the naturally evolved diversity of enzyme specificity for biocatalyst discovery. The method exploits a novel ligation independent cloning strategy, based on the locally engineered vector pET-YSBLIC and has been used for the rapid generation of a suite of expression plasmids containing genes encoding a family of six Baeyer–Villiger monooxygenases (BVMOs) from Mycobacterium tuberculosis H37Rv (MTb). The six resultant recombinant strains of E. coli B834 (DE3) expressing the genes were assayed for oxygenating activity in respect of the target reaction; the resolution of