Search

Metabolic profiling of Streptomyces sp. IB2014/016‐6 led to the identification of three new tetrahydroisoquinoline natural products, perquinolines A–C (1–3). Labelled precursor feeding studies and the cloning of the pqr biosynthetic gene cluster revealed that 1–3 are assembled by the action of several unusual enzymes. The biosynthesis starts with the condensation of succinyl‐CoA and l‐phenylalanine catalyzed by the amino‐7‐oxononanoate synthase‐like enzyme PqrA, representing rare chemistry in natural product assembly. The second condensation and cyclization events are conducted by PqrG, an enzyme resembling an acyl‐CoA ligase. Last, ATP‐grasp RimK‐type ligase PqrI completes the biosynthesis by transferring a γ‐aminobutyric acid or β‐alanine moiety. The discovered pathway represents a new route for assembling the tetrahydroisoquinoline cores of natural products. [ABSTRACT FROM AUTHOR]

Pyrrolopyrimidines are an important class of natural products with a broad spectrum of biological activities, including antibacterial, antifungal, antiviral, anticancer or anti-inflammatory. Here, we present the identification of a biosynthetic gene cluster from the rare actinomycete strain Kutzneria albida DSM 43870, which leads to the production of huimycin, a new member of the pyrrolopyrimidine family of compounds. The huimycin gene cluster was successfully expressed in the heterologous host strain Streptomyces albus Del14. The compound was purified, and its structure was elucidated by means of nuclear magnetic resonance spectroscopy. The minimal huimycin gene cluster was identified through sequence analysis and a series of gene deletion experiments. A model for huimycin biosynthesis is also proposed in this paper. [ABSTRACT FROM AUTHOR]

Abstract Natural products are a rich source of potential drugs for many applications. Discovery of natural products through the activation of cryptic gene clusters encoding their biosynthetic pathways, engineering of those biosynthetic pathways and optimization of production yields often rely on the expression of these gene clusters in suitable heterologous host strains. Streptomyces albus J1074 provides high success rates of heterologous cluster expression with high levels of metabolite production, rapid growth and amenability to genetic manipulations. Here, we report the construction of S. albus chassis strains optimized for the discovery of natural products through heterologous expression of secondary metabolite clusters. 15 clusters encoding secondary metabolite biosynthetic pathways were deleted in the chromosome of S. albus Del14. This strain provides a substantially improved compound detection limit, owing to the lack of native secondary metabolites. Furthermore, the production yield of natural products heterologously expressed in S. albus Del14 was higher than in commonly used S. albus J1074 and S. coelicolor hosts. S. albus strains B2P1 and B4 were generated by introduction of additional phage phi C31 attB sites into the chromosome of S. albus Del14, allowing integration of up to four copies of a heterologous gene cluster. Amplification of gene clusters in the chromosome of the constructed strains further improved production yields of the encoded compounds. One cryptic cluster from Streptomyces spp. and two clusters from distantly related Frankia spp. strains were successfully activated in these new chassis strains, leading to the isolation of a new compound fralnimycin. Highlights • A novel genome engineering platform based on the ordered BAC library is presented. • A panel of advanced Streptomyces albus -based chassis strains lacking biosynthetic gene clusters is constructed. • Yield of the target compounds achieved by expression in the constructed chassis strains is superior to that in the initial strain. • Several new compounds have been discovered through the heterologous expression of uncharacterised gene clusters. [ABSTRACT FROM AUTHOR]

Abstract We present a random rational approach enabling the construction of overproducing strains in two steps. The approach first involves creating a library of clusters of interest, in which native promoters are substituted with randomly generated constitutive synthetic promoters, and then expressing this library in an appropriate host strain. This strategy is fast, easy to use, accounts for the architecture of a cluster and completely decouples the expression of a gene cluster from complex native regulatory networks. The strategy was applied to improve the production of a macrocyclic peptide, bottromycin, which possesses antibacterial activity against multidrug-resistant bacteria and is a blueprint for a new class of antibacterials. We successfully optimized the expression of genes in operons and created several variants of the bottromycin gene cluster that provide 5–50 fold higher titres of bottromycin than the natural one, thus resulting in the identification of several new bottromycin derivatives not previously described. Moreover, due to the higher bottromycin yield, bottromycin derivatization was performed via the biosynthetic engineering of the gene cluster. The abovementioned features make this generic strategy a promising tool for the overproduction of known secondary metabolites and the activation of silent secondary metabolites in Actinobacteria. Highlights • A random rational approach enabling the construction of overproducing strains in two steps is presented. • The strategy completely decouples the expression of a gene cluster from complex native regulatory networks. • Several variants of the bottromycin gene cluster that provide 5–50 folds higher titres of bottromycin were created. • Several new bottromycin derivatives not previously described were identified. [ABSTRACT FROM AUTHOR]