Search

Globomycin is a cyclic lipodepsipeptide originally isolated from several Streptomyces species which displays strong and selective antibacterial activity against Gram-negative pathogens. Its mode of action is based on the competitive inhibition of the lipoprotein signal peptidase II (LspA), which is absent in eukaryotes and considered an attractive target for the development of new antibiotics. Despite its interesting biological properties, the gene cluster encoding its biosynthesis has not yet been identified. In this study we employed a genome-mining approach in the globomycin-producing Streptomyces sp. CA-278952 to identify a candidate gene cluster responsible for its biosynthesis. A null mutant was constructed using CRISPR base editing where production was abolished, strongly suggesting its involvement in the biosynthesis. The putative gene cluster was then cloned and heterologously expressed in Streptomyces albus J1074 and Streptomyces coelicolor M1146, therefore unambiguously linking globomycin and its biosynthetic gene cluster. Our work paves the way for the biosynthesis of new globomycin derivatives with improved pharmacological properties. [ABSTRACT FROM AUTHOR]

Many drugs are derived from small molecules produced by microorganisms and plants, so-called natural products. Natural products have diverse chemical structures, but the biosynthetic pathways producing those compounds are often organized as biosynthetic gene clusters (BGCs) and follow a highly conserved biosynthetic logic. This allows for the identification of core biosynthetic enzymes using genome mining strategies that are based on the sequence similarity of the involved enzymes/genes. However, mining for a variety of BGCs quickly approaches a complexity level where manual analyses are no longer possible and require the use of automated genome mining pipelines, such as the antiSMASH software. In this review, we discuss the principles underlying the predictions of antiSMASH and other tools and provide practical advice for their application. Furthermore, we discuss important caveats such as rule-based BGC detection, sequence and annotation quality and cluster boundary prediction, which all have to be considered while planning for, performing and analyzing the results of genome mining studies. [ABSTRACT FROM AUTHOR]

Actinomycetes are excellent sources for novel bioactive compounds, which serve as potential drug candidates for antibiotics development. While industrial efforts to find and develop novel antimicrobials have been severely reduced during the past two decades, the increasing threat of multidrug-resistant pathogens and the development of new technologies to find and produce such compounds have again attracted interest in this field. Based on improvements in whole-genome sequencing, novel methods have been developed to identify the secondary metabolite biosynthetic gene clusters by genome mining, to clone them, and to express them in heterologous hosts in much higher throughput than before. These technologies now enable metabolic engineering approaches to optimize production yields and to directly manipulate the pathways to generate modified products. [ABSTRACT FROM AUTHOR]

Abstract: Natural products of bacteria and fungi are the most important source for antimicrobial drug leads. For decades, such compounds were exclusively found by chemical/bioactivity-guided screening approaches. The rapid progress in sequencing technologies only recently allowed the development of novel screening methods based on the genome sequences of potential producing organisms. The basic principle of such genome mining approaches is to identify genes, which are involved in the biosynthesis of such molecules, and to predict the products of the identified pathways. Thus, bioinformatics methods and tools are crucial for genome mining. In this review, a comprehensive overview is given on programs and databases for the identification and analysis of antibiotic biosynthesis gene clusters in genomic data. [Copyright &y& Elsevier]

The main steps in the biosynthesis of complex secondary metabolites such as the antibiotic kirromycin are catalyzed by modular polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS). During antibiotic assembly, the biosynthetic intermediates are attached to carrier protein domains of these megaenzymes via a phosphopantetheinyl arm. This functional group of the carrier proteins is attached post-translationally by a phosphopantetheinyl transferase (PPTase). No experimental evidence exists about how such an activation of the carrier proteins of the kirromycin PKS/NRPS is accomplished. Here we report on the characterization of the PPTase KirP, which is encoded by a gene located in the kirromycin biosynthetic gene cluster. An inactivation of the kirP gene resulted in a 90% decrease in kirromycin production, indicating a substantial role for KirP in the biosynthesis of the antibiotic. In enzymatic assays, KirP was able to activate both acyl carrier protein and petidyl carrier domains of the kirromycin PKS/NRPS. In addition to coenzyme A (CoA), which is the natural substrate of KirP, the enzyme was able to transfer acyl-phosphopantetheinyl groups to the apo forms of the carrier proteins. Thus, KirP is very flexible in terms of both CoA substrate and carrier protein specificity. Our results indicate that KirP is the main PPTases that activates the carrier proteins in kirromycin biosynthesis. [ABSTRACT FROM AUTHOR]

Summary Pristinamycin, produced by Streptomyces pristinaespiralis Pr11, is a streptogramin antibiotic consisting of two chemically unrelated compounds, pristinamycin I and pristinamycin II. The semi-synthetic derivatives of these compounds are used in human medicine as therapeutic agents against methicillin-resistant Staphylococcus aureus strains. Only the partial sequence of the pristinamycin biosynthetic gene cluster has been previously reported. To complete the sequence, overlapping cosmids were isolated from a S. pristinaespiralis Pr11 gene library and sequenced. The boundaries of the cluster were deduced, limiting the cluster size to approximately 210 kb. In the central region of the cluster, previously unknown pristinamycin biosynthetic genes were identified. Combining the current and previously identified sequence information, we propose that all essential pristinamycin biosynthetic genes are included in the 210 kb region. A pristinamycin biosynthetic pathway was established. Furthermore, the pristinamycin gene cluster was found to be interspersed by a cryptic secondary metabolite cluster, which probably codes for a glycosylated aromatic polyketide. Gene inactivation experiments revealed that this cluster has no influence on pristinamycin production. Overall, this work provides new insights into pristinamycin biosynthesis and the unique genetic organization of the pristinamycin gene region, which is the largest antibiotic 'supercluster' known so far. [ABSTRACT FROM AUTHOR]

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel "semi-targeted" approach focusing on activating "silent" BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10. [ABSTRACT FROM AUTHOR]

Understanding a complex working mechanism of biosynthetic gene clusters (BGCs) encoding secondary metabolites is a key to discovery of new antibiotics. Computational resources continue to be developed in order to better process increasing volumes of genome and chemistry data, and thereby better understand BGCs. In this context, this review highlights recent advances in computational resources for secondary metabolites with emphasis on genome mining, compound identification and dereplication as well as databases. We also introduce an updated version of Secondary Metabolite Bioinformatics Portal (SMBP; http://www.secondarymetabolites.org ), which we previously released as a curated gateway to all the computational tools and databases useful for discovery and engineering of secondary metabolites. [ABSTRACT FROM AUTHOR]

Summary: In the biosynthesis of complex polyketides, acyltransferase domains (ATs) are key determinants of structural diversity. Their specificity and position in polyketide synthases (PKSs) usually controls the location and structure of building blocks in polyketides. Many bioactive polyketides, however, are generated by trans-AT PKSs lacking internal AT domains. They were previously believed to use mainly malonyl-specific free-standing ATs. Here, we report a mechanism of structural diversification, in which the trans-AT KirCII regiospecifically incorporates the unusual extender unit ethylmalonyl-CoA in kirromycin polyketide biosynthesis. [Copyright &y& Elsevier]

Abstract: Amycolatopsis balhimycina produces the vancomycin-analogue balhimycin. The strain therefore serves as a model strain for glycopeptide antibiotic production. Previous characterisation of the balhimycin biosynthetic cluster had shown that the border sequences contained both, a putative 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dahp), and a prephenate dehydrogenase (pdh) gene. In a metabolic engineering approach for increasing the precursor supply for balhimycin production, the dahp and pdh genes from the biosynthetic cluster were overexpressed both individually and together and the resulting strains were subjected to quantitative physiological characterisation. The constructed strains expressing an additional copy of the dahp gene and the strain carrying an extra copy of both dahp and pdh showed improved specific glycopeptide productivities by approximately a factor three, whereas the pdh overexpression strain showed a production profile similar to the wild type strain. In addition to the overexpression strains, corresponding deletion mutants, Δdahp and Δpdh, were constructed and characterised. Deletion of dahp resulted in significant reduction in balhimycin production whereas the Δpdh strain had production levels similar to the parent strain. Based on these results the relation between primary and secondary metabolism with regards to Dahp and Pdh is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Streptomyces tendae Tü 4042 produces the aromatic polyketide antibiotic lysolipin. Lysolipin has strong antibacterial activity against a variety of multidrug-resistant pathogens. The complete lysolipin biosynthetic gene cluster was isolated and fully sequenced. Within a 42-kb genomic region, 42 genes were identified that code for a type II polyketide synthase (llpF, E, and D), cyclases (llpCI–CIII), methyltransferases (llpMI–MVI), a halogenase (llpH), an amidotransferase (llpA), a ferredoxin (llpK), a transporter (llpN) and regulatory proteins (llpRI–RV). In addition, 15 genes encoding enzymes involved in redox modifications of the polyketide precursor molecule (llpOI–OVIII, ZI–ZIV, U, L, and S) were present in the lysolipin biosynthetic gene cluster. With this high number of oxidoreductases, lysolipin is among the most highly modified aromatic polyketides known to date. The heterologous expression of the cluster in Streptomyces albus led to lysolipin production with a yield comparable to that of wild-type, indicating that all biosynthetic genes were successfully cloned. [Copyright &y& Elsevier]

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications. [ABSTRACT FROM AUTHOR]

Highlights: [•] The kirromycin producer Streptomyces collinus Tü 365 was completely sequenced. [•] The S. collinus Tü 365 genome consists of an 8.27MB chromosome and two linear plasmids of 85 and 19.3kb. [•] An antiSMASH analysis revealed 32 gene clusters encoding the biosynthesis of secondary metabolites. [ABSTRACT FROM AUTHOR]