Direct cloning and heterologous expression of natural product biosynthetic gene clusters by transformation-associated recombination

Abstract Heterologous expressionof natural productbiosynthetic gene clusters(BGCs) is a robust approach not only to decipher biosynthetic logic behind natural product(NP) biosynthesis, but also to discover new chemicals from uncharacterized BGCs. This approach largely relies on techniques used for cloning large BGCs into suitable expression vectors. Recently, several whole-pathway direct cloning approaches, including full-length RecE-mediated recombination in Escherichia coli, Cas9-assisted in vitro assembly, and transformation-associated recombination ( TAR) in Saccharomyces cerevisiae, have been developed to accelerate BGC isolation. In this chapter, we summarize a protocol for TARcloning large NP BGCs, detailing the process of choosing TARplasmids, designing pathway-specific TARvectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressingBGCs in various host strains. We believe that the established platforms can accelerate the process of discovering new NPs, understanding NP biosynthetic logic, and engineering biosynthetic pathways.