Titer improvement of milbemycins via coordinating metabolic competition and transcriptional co‐activation controlled by Streptomyces antibiotic regulatory protein family regulator in Streptomyces bingchenggensis

Abstract

Streptomyces bingchenggensis is a promising producer of milbemycins (MILs), the macrolide pesticide used widely in agriculture. The relationship between different biosynthetic gene clusters (BGCs) and the MIL BGC remains unclear, which hinders the precise metabolic engineering of S. bingchenggensis for titer improvement. To address this issue, this study discovered the regulatory function of a previously unidentified regulator KelR on a type‐II polyketide BGC, MIL BGC, and two other BGCs, and caused titer improvement. First, a type II polyketide synthase (PKS) gene cluster kel with a bidirectional effect on MIL biosynthesis was found using transcriptome analysis. A Streptomyces antibiotic regulatory protein (SARP) family regulator KelR from the kel cluster was then characterized as an activator of several BGCs including mil and kel clusters. Metabolic competition between mil and kel clusters at the late fermentation stage was confirmed. Finally, KelR and those BGCs were manipulated in S. bingchenggensis, which led to a 71.7% titer improvement of MIL A3/A4 to 4058.2 ± 71.0 mg/L. This study deciphered the regulatory function of a previously unidentified regulatory protein KelR on several BGCs including mil in S. bingchenggensis and provided an example of coordinating metabolic competition and coregulation for titer improvement of secondary metabolites.

Omics-assisted systematic exploration of the intricate regulatory network of guvermectin biosynthesis centered by the cluster-situated regulator GvmR inStreptomyces

“…For example, the overexpression of the red cluster activator gene, redZ from S. coelicolor could transiently increase transcription of cda, red and act clusters (Huang et al, 2005). Inactivation of kelR, a positive regulatory gene within a type II PKS cluster in S. bingchenggensis, not only leads to the absence of cognate compound but also results in nearly negligible expression of several other BGCs, including the milbemycin BGC (Wang et al, 2022).…”

“…For example, the overexpression of the red cluster activator gene, redZ from S. coelicolor could transiently increase transcription of cda , red and act clusters(Huang et al, 2005). Inactivation of kelR , a positive regulatory gene within a type II PKS cluster in S. bingchenggensis , not only leads to the absence of cognate compound but also results in nearly negligible expression of several other BGCs, including the milbemycin BGC(Wang et al, 2022). The key CSR of the nemadectin cluster, nemR , in S. cyaneogriseus , also directly controls four non-nemadectin BGC genes involved in precursor supply, morphological development and chemical secretion(Li et al, 2019).…”

Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V–F CRISPR/Cas Nuclease AsCas12f1

“…SIPI-054 is an industrial high-yield milbemycin-producing strain, which is a genetically intractable microorganism. 11 Here, four different sizes of DNA fragments were selected as deletion targets (Figure 6A), including milR2 (390 bp, encoding a TetR family regulator involved in regulation of milbemycin biosynthesis), 35 milF (603 bp, encoding a C5-ketoreductase responsible for transforming 5-oxomilbemycins to milbemycins), 36 kelBCD (3.3 kb) from the kel BGC responsible for the biosynthesis of the yellow compound KEL, 37 and a 62-kb functionally unknown DNA region, which divides the milbemycin BGC into two parts, including the large part (from milA2 to milR) and the small part (milA1and milD). 38 First, milR2 and milF were tested for AsCas12f1-based editing.…”

The roles of SARP family regulators involved in secondary metabolism in Streptomyces

“…In Streptomyces binchenggensis , milR3 / kelR is located in a type II PKS BGC that is responsible for producing the yellow compound. The milbemycin BGC is far from the type II PKS BGC in the genome of S. bingchenggensis , but MilR3/KelR has been shown to coactivate the biosynthesis of milbemycin and the yellow compound ( Wang et al, 2022 ; Yan et al, 2022 ). In addition, S. bingchenggensis can also produce nanchangmycin.…”