Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A

Abstract

Recent developments in next-generation sequencing technologies have brought recognition of microbial genomes as a rich resource for novel natural product discovery. However, owing to the scarcity of efficient procedures to connect genes to molecules, only a small fraction of secondary metabolomes have been investigated to date. Transformation-associated recombination (TAR) cloning takes advantage of the natural in vivo homologous recombination of Saccharomyces cerevisiae to directly capture large genomic loci. Here we report a TAR-based genetic platform that allows us to directly clone, refactor, and heterologously express a silent biosynthetic pathway to yield a new antibiotic. With this method, which involves regulatory gene remodeling, we successfully expressed a 67-kb nonribosomal peptide synthetase biosynthetic gene cluster from the marine actinomycete Saccharomonospora sp. CNQ-490 and produced the dichlorinated lipopeptide antibiotic taromycin A in the model expression host Streptomyces coelicolor. The taromycin gene cluster (tar) is highly similar to the clinically approved antibiotic daptomycin from Streptomyces roseosporus, but has notable structural differences in three amino acid residues and the lipid side chain. With the activation of the tar gene cluster and production of taromycin A, this study highlights a unique "plug-and-play" approach to efficiently gaining access to orphan pathways that may open avenues for novel natural product discoveries and drug development.

Keywords: biosynthesis; drug discovery; genome mining.

Fig. 1.

Design and strategy of TAR-based cloning and expression. (A) Physical map of the gene cluster capture vector pCAP01. The vector consists of three elements that allow direct cloning and manipulation of pathways in yeast (blue), maintenance and manipulation in E. coli (green), and chromosomal integration and expression of cloned pathways in heterologous actinobacteria (red). For the construction of a pathway-specific capture vector, homology arms corresponding to both ends of the pathway are introduced into the capture arm cloning sites. (B) The procedure for TAR-based natural product discovery involves three steps. In step 1, transformation-associated recombination in yeast involves homologous recombination between the linearized pathway specific capture vector and cointroduced genomic DNA fragments to yield a circular construct that can be grown on selective media. In step 2, the cloned pathway can be manipulated using either in vivo yeast recombination or E. coli-based genetic manipulations, such as λ-Red recombination. Finally, in step 3, through conjugal DNA transfer from E. coli, the cloned and manipulated pathway is integrated into the chromosome of an actinomycete host strain for small-molecule expression studies.

Fig. 2.

Gene organization of lipopeptide biosynthetic gene clusters. Examples include the daptomycin (dpt) gene cluster in Streptomyces roseosporus NRRL11379 (A), an orphan lipopeptide biosynthetic gene cluster in Saccharomonospora viridis DSM43017 (B), and the taromycin (tar) biosynthetic gene cluster in Sacharomonospora sp. CNQ-490 (C). (D) The modular architecture of the taromycin NRPS assembly line and the predicted amino acid substrate specificity of each module A domain. A, adenylation domain; C, condensation domain; E, epimerization domain; T, thiolation domain; TE, thioesterase domain.

Fig. 3.

Physical maps of the TAR-cloned tar gene cluster and the pKY01-based complementation vectors. (A) The 73-kb genomic region containing the tar gene cluster was directly cloned in yeast, yielding pCAP01-tar. The tar regulatory genes tar19/tar20 and unrelated genes beyond the cluster border on pCAP01-tar (dotted arrows) were replaced with the URA3 auxotrophic marker gene in yeast, generating regulatory gene-deficient tar gene cluster expression constructs pCAP01-tarM1 and pCAP01-tarM2. The three constructs were introduced into the φC31 attachment site on the chromosome of the heterologous host S. coelicolor M1146. (B) Both of the eliminated regulatory genes and the codon redressed tar20 luxR regulator gene were introduced separately into the expression mutants using the φBT1 integrative expression vector pKY01. Color codes for the genes are the same as in Fig. 2.

Fig. 4.

HPLC analysis of the taromycins produced heterologously by S. coelicolor mutants and structures of daptomycin and taromycin A. Extracts from S. coelicolor M1146/pCAP01-tarM2 (∆tar20 luxR) (A), S. coelicolor M1146/pCAP01-tarM1 (∆tar19 sarp, ∆tar20 luxR) (B), and S. coelicolor M1146/pCAP01-tar (intact) (C) were analyzed by C18 reversed-phase HPLC. UV absorption at 254 nm was monitored. *Taromycin A derivatives. The taromycin structure is elucidated in SI Appendix, Tables S3–S5 and Figs. S8–S11. Structural differences between taromycin A and daptomycin are highlighted in red.