Development of a salt-enhanced promoter strategy for activating silent biosynthetic gene clusters from streptomycetes

Activating silent biosynthetic gene clusters (BGCs) within various microorganisms is an important approach to uncover valuable natural products. In this study, we reported the capture and activation of two large silent BGCs from a marine-derived Streptomyces sp. SCSGAA 0027 in the heterologous host Streptomyces albus J1074 by inserting a widely used constitutive promoter kasOp∗ upstream of the core biosynthetic genes, which led to the production of coprisamides (COPs) and padanamides (PADs), respectively. Interestingly, the yields of COPs and PADs were significantly enhanced when potassium or sodium salts were supplemented in the fermentation media, especially 1 % KCl. The promoting strength of kasOp∗ was found to be obviously increased upon KCl addition by using the eGFP (enhanced green fluorescent protein) as an indicator. These findings revealed for the first time that the exogenous promoter kasOp∗ performed unexpectedly as a salt-enhanced element in S. albus J1074. Consequently, a "kasOp∗-KCl" strategy was developed to achieve the highest production of COPs A/B at 97.9 mg/L in fermentation with shaking flasks, along with the coproduction of a pair of new analogues, COPs E/F at 151.8 mg/L, leading to a maximum isolation titer of COPs at 171.7 mg/L, about 170-fold improvement comparing to previous reports. Similarly, the strategy increased the titers of the antimalarial agent PAD A to 76.7 mg/L and the diisonitrile copper chelator SF2768 to 72.8 mg/L in S. albus J1074, representing the highest yields reported to date for both compounds. Moreover, a small library of kasOp∗ variants were generated and validated to also be KCl-responsive, expanding the promoter toolkits for metabolic engineering and genome mining. These findings provide new insights into the salt-enhancing property of the widely used promoter kasOp∗, and offer a simple "kasOp∗-KCl" approach to efficiently activate silent BGCs and improve the production of the encoding natural products in multiple commonly used Streptomyces hosts.