Enhancement or Activation of Agricultural Antibiotic Production in Streptomyces by an Identified Strong Promoter P30S

We first screened for strong constitutive promoters by analyzing transcriptomics data under various fermentation conditions, focusing on genes with sustained high expression levels. Then, we validated the expression strength of 21 candidate promoters using β-glucuronidase enzymatic activity and qRT-PCR assays and observed that promoter P30S demonstrated consistently strong expression and efficacy in Streptomyces rimosus M527. The higher expression level and utility of P30S were further confirmed in three different host strains, including the industrial strain Streptomyces diastatochromogenes 1628. In this strain, overexpression of the toyA gene, a positive regulator, under the control of P30S significantly increased toyocamycin (TM) production, outperforming permE* in improving the TM yield. Similarly, in S. rimosus M527, P30S was superior to permE* in increasing rimocidin production through the overexpression of the accsr gene, which encodes acetyl-CoA carboxylase. Moreover, P30S successfully activated a cryptic gene cluster responsible for TM biosynthesis in S. rimosus M527 via CRISPR/Cas9-mediated knock-in.