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

Abstract

Heterologous expression of natural product biosynthetic 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 TAR cloning large NP BGCs, detailing the process of choosing TAR plasmids, designing pathway-specific TAR vectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressing BGCs 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.

Keywords: Biosynthesis; Heterologous expression; Natural products; Yeast homologous recombination.

Fig. 1

Scheme for TAR cloning and heterologous expression of biosynthetic gene clusters using pCAP vectors.

Fig. 2

Vector maps for (A) pCAP01, (B) pCAP03, (C) pCAPB02, and (D) pCAP05.

Fig. 3

Two methods for generating pathway-specific capture vectors involving (A) PCR amplification and assembly of two 1kb homology arms or (B) assembly of synthetic ds-DNA fragment containing two 50bp homology arms.