Recombineering for Genetic Engineering of Natural Product Biosynthetic Pathways

Abstract

Microbial genomes encode many cryptic and uncharacterized biosynthetic gene clusters (BGCs). Exploiting this unexplored genetic wealth to discover microbial novel natural products (NPs) remains a challenging issue. We review homologous recombination (HR)-based recombineering, mediated by the recombinases RecE/RecT from Rac prophage and Redα/Redβ from lambda phage, which has developed into a highly inclusive tool for direct cloning of large DNA up to 100 kb, seamless mutation, multifragment assembly, and heterologous expression of microbial NP BGCs. Its utilization in the refactoring, engineering, and functional expression of long BGCs for NP biosynthesis makes it easy to elucidate NP-producing potential in microbes. This review also highlights various applications of recombineering in NP-derived drug discovery.

Keywords: Recombineering; biosynthetic gene cluster; direct cloning; homologous recombination; natural product.