BAC cloning and heterologous expression of a giant biosynthetic gene cluster encoding antifungal neotetrafibricin in streptomyces rubrisoli

Polyene natural products including nystatin A1, amphotericin B, ECO-02301, and mediomycin belong to a large family of valuable antifungal polyketide compounds typically produced by soil actinomycetes. A previous study (Park et al., Front. Bioeng. Biotechnol., 2021, 9, 692340) isolated Streptomyces rubrisoli Inha501 with strong antifungal activity and analyzed a large-sized biosynthetic gene cluster (BGC) of a linear polyene compound named Inha-neotetrafibricin (I-NTF) using whole genome sequencing and bioinformatics. In the present study, an entire I-NTF BGC (& SIM;167 kb) was isolated through construction and screening of Streptomyces BAC library. Overexpression of the cloned I-NTF BGC in the wild-type S. rubrisoli Inha501 and its heterologous expression in S. lividans led to 2.6-fold and 2.8-fold increase in I-NTF yields, respectively. The qRT-PCR confirmed that the transcription levels of I-NTF BGC were significantly increased in both homologous and heterologous hosts containing the BAC integration of I-NTF BGC. In addition, the I-NTF aglycone-producing strains were constructed by a target-specific deletion of glycosyltransferase gene present in I-NTF BGC. A comparison of the in vitro biological activities of I-NTF and I-NTF aglycone confirmed that the rhamnose sugar motif of I-NTF plays a critical role in both antifungal and antibacterial activities. These results suggest that the Streptomyces BAC cloning of a large-sized natural product BGC is a valuable approach for natural product titer improvement and biological activity screening of natural product in actinomycetes.

Automated summary

This study isolated a Streptomyces strain with strong antifungal activity and identified a large biosynthetic gene cluster (BGC) for a polyene compound. Overexpression of the BGC in the strain and heterologous expression in another strain led to increased production of the compound. The researchers also created strains lacking the sugar moiety of the compound and found that the sugar played a critical role in its biological activity. BAC cloning of large natural product BGCs in Streptomyces is a valuable approach for improving production and screening for biological activity.