Genome Mining of actinomycete bacteria for new natural products
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Here, the genome data of seven different strains of Streptomyces and a strain from the Actinoalloteichus genus were analysed for the presence of biosynthetic gene clusters (BGC) encoding for potential new natural products (NP). Altogether, using genome mining approach, six new compounds were identified (three lasso peptides, one thiopeptide, and two polyketides), two of them with antimicrobial activities. Identified lasso peptide BGCs were heterologously expressed with a novel orthogonal expression system developed in this work based on a newly-identified regulatory circuit from Actinoalloteichus fjordicus. Six lasso peptide gene clusters, mainly originating from marine Actinobacteria, were chosen for proofof- concept studies. By varying the Streptomyces expression hosts and cultivation conditions, three new lasso peptides were successfully produced and characterized by tandem mass spectrometry (MS). A newly isolated Streptomyces sp. YIM 130001 from the tropical rainforest in Xishuangbanna (Yunnan, China) displayed antibacterial activity against Bacillus subtilis. To investigate which compound is responsible for the bioactivity, genome sequence of this bacterium was examined, revealing 18 putative BGCs for secondary metabolites (SM) biosynthesis. Inactivation of a lantibiotic dehydrogenase-encoding gene from the BGC presumed to govern biosynthesis of a thiopeptide resulted in the loss of bioactivity. The compound, designated geninthiocin B, was purified, and its structure elucidated, confirming its novelty and similarity to several 35-membered macrocyclic thiopeptides. Bioinformatics analysis of the geninthiocin B BGC revealed its possible biosynthetic steps, which appears to be similar to that of berninamycins. Genome analyses of another strain isolated in Yunnan province (China), Streptomyces sp. YIM 121038 led to identification of a remarkable SM biosynthesis potential. More than 60 secondary metabolite BGCs were predicted, 17 of which were localized to a giant liner plasmid of ca 1 Mb. A genome mining study was attempted aimed at identification of a product specified by a unique trans-AT polyketide synthase gene (PKS) cluster in this strain via gene disruption and overexpression of the pathway-specific regulators (LysR and SARP). Overexpression of SARP regulator led detection of a putative compound specified by this cluster, but the production level was too low for purification and structure elucidation. Production of the two other compounds was detected in the recombinant strain overexpressing lysR regulator-gene. Subsequent purification and structure elucidation revealed them being macrolides featuring glycosylated 42-membered ring with aminoacyl and aminoacyl-succinyl side chains, apparently originating from another PKS type I gene cluster. Based on the bioinformatics analysis, biosynthetic pathway for the new compounds named Gratumycins A and B could be suggested, which involves unusual starter unit most likely originating from the polyamine pathway. Gratumycin B demonstrated activity against Grampositive bacteria, while Gratumycin A, which differed by a succinate moiety blocking free amino group of Gratumycin B, was inactive. Finally, the first insights into the biosynthesis and unusually high accumulation of free fatty acids (FFAs) by environmental isolate Streptomyces sp. NP10 was gained after its genome sequencing and bioinformatics-based genome mining. The latter led to identification of involved putative BGC that may be involved in free fatty acids biosynthesis. Mutational analysis of the identified BGC confirmed it being responsible for unusually high FFAs production in Streptomyces sp. NP10.