Abstract
Many bioactive natural products are glycosylated compounds in which the sugar components usually participate in interaction and molecular recognition of the cellular target. Therefore, the presence of sugar moieties is important, in some cases essential, for bioactivity. Searching for novel glycosylated bioactive compounds is an important aim in the field of the research for natural products from actinomycetes. A great majority of these sugar moieties belong to the 6-deoxyhexoses and share two common biosynthetic steps catalyzed by a NDP-D-glucose synthase (GS) and a NDP-D-glucose 4,6-dehydratase (DH). Based on this fact, seventy one Streptomyces strains isolated from the integument of ants of the Tribe Attini were screened for the presence of biosynthetic gene clusters (BGCs) for glycosylated compounds. Total DNAs were analyzed by PCR amplification using oligo primers for GSs and DHs and also for a NDP-D-glucose-2,3-dehydratases. Amplicons were used in gene disruption experiments to generate non-producing mutants in the corresponding clusters. Eleven mutants were obtained and comparative dereplication analyses between the wild type strains and the corresponding mutants allowed in some cases the identification of the compound coded by the corresponding cluster (lobophorins, vicenistatin, chromomycins and benzanthrins) and that of two novel macrolactams (sipanmycin A and B). Several strains did not show UPLC differential peaks between the wild type strain and mutant profiles. However, after genome sequencing of these strains, the activation of the expression of two clusters was achieved by using nutritional and genetic approaches leading to the identification of compounds of the cervimycins family and two novel members of the warkmycins family. Our work defines a useful strategy for the identification new glycosylated compounds by a combination of genome mining, gene inactivation experiments and the activation of silent biosynthetic clusters in Streptomyces strains.
Highlights
Natural products are an important source of clinical and pharmaceutical compounds and they contribute with compounds showing agricultural and veterinary applications
polymerase chain reaction (PCR) Analysis of a Collection of Actinomycetes Searching for Deoxy Sugar Biosynthesis Genes
The second pair of primers corresponded to internal and conserved regions for NDP-D-glucose-2,3-dehydratases, enzymes catalyzing the 2,3-dehydration step in the biosynthesis of 2,6-DOH
Summary
Natural products are an important source of clinical and pharmaceutical compounds and they contribute with compounds showing agricultural and veterinary applications. A large proportion of natural products are synthesized by microorganisms which constitute a prolific source of structurally diverse bioactive metabolites and have yielded some of the most important products of the pharmaceutical industry. With the bioinformatic analyses of these genomes became evident that actinomycetes carry the genetic potential to produce many more natural products than those detected under laboratory conditions. It is frequent to observe that the sequenced genomes of actinomycetes contain between 20 and 30 biosynthetic gene clusters (BGCs) for secondary metabolites production. Many of these BGCs are not expressed (or are very low expressed) under laboratory cultivation conditions and are designated as “silent” clusters. Several strategies have been applied to awake these clusters including nutritional (Bode et al, 2002) and genetic approaches (Challis, 2008; Laureti et al, 2011; Olano et al, 2014; Thanapipatsiri et al, 2016; Zhang et al, 2017b) and ribosomal engineering (Ochi et al, 2004; Ochi and Hosaka, 2013)
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