Engineering Salinispora tropica for heterologous expression of natural product biosynthetic gene clusters

Abstract

The marine actinomycete genus Salinispora is a remarkably prolific source of structurally diverse and biologically active secondary metabolites. Herein, we select the model organism Salinispora tropica CNB-440 for development as a heterologous host for the expression of biosynthetic gene clusters (BGCs) to complement well-established Streptomyces host strains. In order to create an integratable host with a clean background of secondary metabolism, we replaced three genes (salA-C) essential for salinosporamide biosynthesis with a cassette containing the Streptomyces coelicolor ΦC31 phage attachment site attB to generate the mutant S. tropica CNB-4401 via double-crossover recombination. This mutagenesis not only knocks-in the attachment site attB in the genome of S. tropica CNB-440 but also abolishes production of the salinosporamides, thereby simplifying the strain's chemical background. We validated this new heterologous host with the successful integration and expression of the thiolactomycin BGC that we recently identified in several S. pacifica strains. When compared to the extensively engineered superhost S. coelicolor M1152, the production of thiolactomycins from S. tropica CNB-4401 was approximately 3-fold higher. To the best of our knowledge, this is the first example of using a marine actinomycete as a heterologous host for natural product BGC expression. The established heterologous host may provide a useful platform to accelerate the discovery of novel natural products and engineer biosynthetic pathways.

Keywords: Genetic engineering; Heterologous expression; Natural products; Salinispora.

Figure 1.

Schematic diagram depicting (A) the replacement of salA-C by Red/ET-mediated recombination with the attB-containing cassette; (B) subsequent removal of the resistance marker by FLP-mediated excision; and (C) replacement of the fosmid backbone chloramphenicol resistant gene cat with acc(3)IV (Apra^R) and oriT of RK2 to generate fosmid pMXT11 to simultaneously knock-in the attB attachment site and knock-out salA-C in S. tropica CNB-440.

Figure 2.

Verification of S. tropica CNB-4401 by (A) PCR amplification and (B) loss of production of salinosporamide A observed by LC-MS.

Figure 3.

Detection of thiolactomycin and its analogues in various producing strains by HPLC. (A) Schematic illustrating the thiolactomycin (tlm) biosynthetic gene cluster. HPLC chromatograms of ethyl acetate extracts from (B) S. tropica CNB-4401/tlm, (C) S. tropica CNB-4401, (D) S. pacifica CNS-863, and (E) S. coelicolor M1152/tlm. Detection at 239 nm. (F) Structures of thiolactomycin (1), 10-methyl thiolactomycin (2), 11-methyl thiolactomycin (3), and thiotetromycin (4).