Mining the cryptic specialized metabolome of Streptomyces cyanogenus S136

Abstract

Streptomyces cyanogenus S136 is known to produce landomycin family antibiotics, particularly its largest congener, landomycin A. Except for landomycins and polyene lucensomycin, no other specialized metabolites were sourced from S136. Nevertheless, S136 genome sequencing revealed over 40 biosynthetic gene clusters (BGCs), implying underappreciated potential of this strain for the production of novel bioactive natural compounds. We set out to gain deeper insight into the specialized metabolome of this strain. First, trans­criptomic analysis of S136 grown under landomycin production conditions has been carried out, revealing that most of them are expressed at a basal level. This, likely, leads to a phenotypic silence of most of the BGCs. Nevertheless, several notable exceptions have been spotted. First of all, landomycin BGC is expressed at high level (at least 100 Transcripts Per Million mapped reads (TPM); and around 1000 TPM for minimal polyketide synthase genes lanFABC). Similarly, high levels of expression showed BGCs # 2, 4, 7 and 33, of which #2, encoding unknown saccharide, is the most dissimilar to the described precedents. RNAseq data also allowed us to delineate better the borders of several presumed BGCs. In the next phase of the work we singled out a few BGCs within S136 that appeared to be promising. First, these BGCs exhibited low similarity to the other gene clusters directing the production of known natural products. Second, the BGCs harbored cluster-situated regulatory genes that can be employed in the attempts to activate the expression of cryptic pathways. For one such BGC we constructed two plasmids for expression of several such regulatory genes and introduced them into S136 and its derivative deficient in production of landomycin A. Bioassays showed no differences in bioactivity of the recombinant strains as compared to the initial strains. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of several S. cyanogenus samples revealed the effects of genotype, growth conditions and extraction on specialized metabolome of this species, setting reference point for further stu­dies.