Establishing an efficient salinomycin biosynthetic pathway in three heterologous Streptomyces hosts by constructing a 106‐kb multioperon artificial gene cluster

Abstract

Salinomycin is a promising anticancer drug for chemotherapy. A highly productive biosynthetic gene cluster will facilitate the creation of analogs with improved therapeutic activity and reduced side effects. In this study, we engineered an artificial 106-kb salinomycin gene cluster and achieved efficient heterologous expression in three hosts: Streptomyces coelicolor CH999, S. lividans K4-114, and S. albus J1074. The six-operon artificial gene cluster consists of 25 genes from the native gene cluster organized into five operons and five fatty acid β-oxidation genes into one operon. All operons are driven by strong constitutive promoters. For K4-114 and J1074 harboring the artificial gene cluster, salinomycin production in shake flask cultures was 14.3 mg L −1 and 19.3 mg L −1 , respectively. The production was 1.3-fold and 1.7-fold higher, respectively, than that of the native producer S. albus DSM41398. K4-114 and J1074 harboring the native gene cluster produced an undetectable amount of salinomycin and 0.5 mg L −1 , respectively. CH999 harboring the artificial gene cluster produced 10.3 mg L −1 of salinomycin, which was 92% of the production by DSM41398. The efficient heterologous expression system based on the 106-kb multioperon artificial gene cluster established in this study will facilitate structural diversification of salinomycin, which is valuable for drug development and structure-activity studies.

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity

“…With the development of synthetic biology, promoters have been widely used for modulating gene expression to optimize biosynthetic pathways for the synthesis of important compounds. Twenty-three genes for biosynthesis of the insecticide spinosad and twenty-five genes for biosynthesis of the anticancer drug salinomycin had been refactored using multiple endogenous strong constitutive promoters to obtain artificial gene clusters with higher transcription level and higher compound productions [ 45 , 46 ]. Gene cluster refactoring with strong constitutive promoters was also used to activate silent gene clusters for discovery and characterization of new natural products [ 47 – 49 ].…”

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacetrium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity

“…With the development of synthetic biology, gene cluster refactoring has been widely used to optimize biosynthetic pathways for the synthesis of important compounds. Twenty-three genes for biosynthesis of the insecticide spinosad and twenty-ve genes for biosynthesis of the anticancer drug salinomycin had been refactored using multiple endogenous strong constitutive promoters to obtain arti cial gene clusters with higher transcription level and higher compound productions [31,32]. Gene cluster refactoring with strong constitutive promoters was also used to activate silent gene clusters for discovery and characterization of new natural products [33][34][35].…”