Heterologous expression in well-studied model strains is a routinely applied method to investigate biosynthetic pathways. Here, we pursue a comparative approach of large-scale DNA-affinity-capturing assays (DACAs) coupled with semi-quantitative mass spectrometry (MS) to identify putative regulatory proteins from Streptomyces coelicolor M512, which bind to the heterologously expressed biosynthetic gene clusters (BGCs) of the liponucleoside antibiotics caprazamycin and liposidomycin. Both gene clusters share an almost identical genetic arrangement, including the location of promoter regions, as detected by RNA sequencing. A total of 2,214 proteins were trapped at the predicted promoter regions, with only three binding to corresponding promoters in both gene clusters. Among these, the overexpression of a yet uncharacterized TetR-family regulator (TFR), Sco4385, increased caprazamycin but not liposidomycin production. Protein-DNA interaction experiments using biolayer interferometry confirmed the binding of Sco4385 to Pcpz10 and PlpmH at different locations within both promoter regions, which might explain its functional variance. Sequence alignment allowed the determination of a consensus sequence present in both promoter regions, to which Sco4385 was experimentally shown to bind. Furthermore, we found that the overexpression of the Crp regulator, Sco3571, leads to a threefold increase in caprazamycin and liposidomycin production yields, possibly due to an increased expression of a precursor pathway.IMPORTANCEStreptomycetes are well-studied model organisms for the biosynthesis of pharmaceutically, industrially, and biotechnologically valuable metabolites. Their naturally broad repertoire of natural products can be further exploited by heterologous expression of biosynthetic gene clusters (BGCs) in non-native host strains. This approach forces the host to adapt to a new regulatory and metabolic environment. In our study, we demonstrate that a host regulator not only interacts with newly incorporated gene clusters but also regulates precursor supply for the produced compounds. We present a comprehensive study of regulatory proteins that interact with two genetically similar gene clusters for the biosynthesis of liponucleoside antibiotics. Thereby, we identified regulators of the heterologous host that influence the production of the corresponding antibiotic. Surprisingly, the regulatory interaction is highly specific for each biosynthetic gene cluster, even though they encode largely structurally similar metabolites.

The Arabidopsis thaliana Receptor-Like Protein RLP30 contributes to immunity against the fungal pathogen Sclerotinia sclerotiorum . Here we identify the RLP30-ligand as a small cysteine-rich protein (SCP) that occurs in many fungi and oomycetes and is also recognized by the Nicotiana benthamiana RLP RE02. However, RLP30 and RE02 share little sequence similarity and respond to different parts of the native/folded protein. Moreover, some Brassicaceae other than Arabidopsis also respond to a linear SCP peptide instead of the folded protein, suggesting that SCP is an eminent immune target that led to the convergent evolution of distinct immune receptors in plants. Surprisingly, RLP30 shows a second ligand specificity for a SCP-nonhomologous protein secreted by bacterial Pseudomonads. RLP30 expression in N. tabacum results in quantitatively lower susceptibility to bacterial, fungal and oomycete pathogens, thus demonstrating that detection of immunogenic patterns by Arabidopsis RLP30 is involved in defense against pathogens from three microbial kingdoms.

Nine dehydratases involved in the biosynthesis of secondary metabolites in addition to FabZ from fatty acid biosynthesis were investigated for their substrate scope using a panel of N-acetylcysteamine (SNAC) thioesters. The best performing enzyme BorDH2 was applied in kinetic resolutions.

Background Caprazamycins are liponucleoside antibiotics showing bioactivity against Gram-positive bacteria including clinically relevant Mycobacterium tuberculosis by targeting the bacterial MraY-translocase. Their chemical structure contains a unique 3-methylglutaryl moiety which they only share with the closely related liposidomycins. Although the biosynthesis of caprazamycin is understood to some extent, the origin of 3-methylglutaryl-CoA for caprazamycin biosynthesis remains elusive. Results In this work, we demonstrate two pathways of the heterologous producer Streptomyces coelicolor M1154 capable of supplying 3-methylglutaryl-CoA: One is encoded by the caprazamycin gene cluster itself including the 3-hydroxy-3-methylglutaryl-CoA synthase Cpz5. The second pathway is part of primary metabolism of the host cell and encodes for the leucine/isovalerate utilization pathway (Liu-pathway). We could identify the liu cluster in S. coelicolor M1154 and gene deletions showed that the intermediate 3-methylglutaconyl-CoA is used for 3-methylglutaryl-CoA biosynthesis. This is the first report of this intermediate being hijacked for secondary metabolite biosynthesis. Furthermore, Cpz20 and Cpz25 from the caprazamycin gene cluster were found to be part of a common route after both individual pathways are merged together. Conclusions The unique 3-methylglutaryl moiety in caprazamycin originates both from the caprazamycin gene cluster and the leucine/isovalerate utilization pathway of the heterologous host. Our study enhanced the knowledge on the caprazamycin biosynthesis and points out the importance of primary metabolism of the host cell for biosynthesis of natural products.

Antibiotic-producing microorganisms usually require one or more self-resistance determinants to survive antibiotic production. The effectors of these mechanisms are proteins that inactivate the antibiotic, facilitate its transport, or modify the target to render it insensitive to the molecule. Streptomyces bacteria biosynthesize various bioactive natural products and possess resistance systems for most metabolites, which are coregulated with antibiotic biosynthesis genes. Streptomyces olindensis strain DAUFPE 5622 produces the antitumor antibiotic cosmomycin D (COSD), a member of the anthracycline family. In this study, we propose three self-resistance mechanisms, anchored or based in the COSD biosynthetic gene cluster. These include cosIJ (an ABC transporter), cosU (a UvrA class IIa protein), and a new self-resistance mechanism encoded by cosP, which shows response against peroxides by the enzyme mycothiol peroxidase (MPx). Activity-based investigations of MPx and its mutant enzyme confirmed peroxidation during the production of COSD. Overexpression of the ABC transporter, the UvrA class IIa protein, and the MPx led to an effective response against toxic anthracyclines, such as cosmomycins. Our findings help to understand how thiol peroxidases play an antioxidant role in the anthracycline producer S. olindensis DAUFPE 5622, a mechanism which has been reported for neoplastic cells that are resistant to doxorubicin (DOX). IMPORTANCE Anthracycline compounds are DNA intercalating agents widely used in cancer chemotherapeutic protocols. This work focused on the self-resistance mechanisms developed by the cosmomycin-producing bacterium Streptomyces olindensis. Our findings showed that cysteine peroxidases, such as mycothiol peroxidase, encoded by the gene cosP, protected S. olindensis against peroxidation during cosmomycin production. This observation can contribute to much better understanding of resistance both in the producers, eventually enhancing production, and in some tumoral cell lines.

Brasilicardin A ( 1 ) consists of an unusual anti / syn / anti -perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone ( 5 ) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A ( 1 a ) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

Covering: up to September 2020Hundreds of nucleoside-based natural products have been isolated from various microorganisms, several of which have been utilized in agriculture as pesticides and herbicides, in medicine as therapeutics for cancer and infectious disease, and as molecular probes to study biological processes. Natural products consisting of structural modifications of each of the canonical nucleosides have been discovered, ranging from simple modifications such as single-step alkylations or acylations to highly elaborate modifications that dramatically alter the nucleoside scaffold and require multiple enzyme-catalyzed reactions. A vast amount of genomic information has been uncovered the past two decades, which has subsequently allowed the first opportunity to interrogate the chemically intriguing enzymatic transformations for the latter type of modifications. This review highlights (i) the discovery and potential applications of structurally complex pyrimidine nucleoside antibiotics for which genetic information is known, (ii) the established reactions that convert the canonical pyrimidine into a new nucleoside scaffold, and (iii) the important tailoring reactions that impart further structural complexity to these molecules.

A nonenzymatic Pictet-Spengler reaction has been postulated to give rise to a subset of naturally occurring uridyl peptide antibiotics (UPAs). Here, using a combination of strain engineering and synthetic chemistry, we demonstrate that Pictet-Spengler chemistry may be employed to generate even greater diversity in the UPAs. We use an engineered strain to afford access to meta-tyrosine containing pacidamycin 4. Pictet-Spengler diversification of this compound using a small series of aryl-aldehydes was achieved with some derivatives affording remarkable diastereomeric control.

Pyrones comprise a structurally diverse class of compounds. Although they are widespread in nature, their specific physiological functions remain unknown in most cases. We recently described that triketide pyrones mediate the sulfotransfer in caprazamycin biosynthesis. Herein, we report the identification of conexipyrones A-C, three previously unrecognized tetra-substituted α-pyrones, from the soil actinobacterium Conexibacter woesei. Insights into their biosynthesis via a type III polyketide synthase were obtained by feeding studies using isotope-enriched precursors. In vitro assays employing the genetically associated 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase CwoeST revealed conexipyrones as the enzymes' genuine sulfate acceptor substrates. Furthermore, conexipyrones were determined to function as sulfate shuttles in a two-enzyme assay, because their sulfated derivatives were accepted as donor molecules by the PAPS-independent arylsulfate sulfotransferase (ASST) Cpz4 to yield sulfated caprazamycin intermediates.

Cell wall biosynthesis represents a valid target for antibacterial action but only a limited number of chemical structure classes selectively interact with specific enzymes or protein structures like transporters of the cell envelope. The integral membrane protein MraY translocase is essential for peptidoglycan biosynthesis catalysing the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate, thereby generating the cell wall intermediate lipid I. Not present in eukaryotic cells, MraY is a member of the superfamily of yet not well-understood integral membrane enzymes which involve proteins for bacterial lipopolysaccharide and teichoic acid or eukaryotic N-linked saccharides biosynthesis. Different natural nucleoside antibiotics as inhibitors of MraY translocase have been discovered comprising a glycosylated heterocyclic pyrimidin base among other potential lipid-, peptidic- or sugar moieties. Caprazamycins are liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2. They possess activity in vitro against Gram-positive bacteria, in particular against the genus Mycobacterium including M. intracellulare, M. avium and M. tuberculosis. Structural elucidation revealed the (+)-caprazol core skeleton as a unique moiety, the caprazamycins share with other MraY inhibitors such as the liposidomycins, A-90289 and the muraminomicins. They also share structural features such as uridyl-, aminoribosyl- and fatty acyl-moieties with other MraY translocase inhibitors like FR-900493 and the muraymycins. Intensive studies on their biosynthesis during the last decade identified not only common initial biosynthetic steps, but also revealed possible branching points towards individual biosynthesis of the respective compound. Structural diversity of caprazamycins was generated by feeding experiments, genetic engineering of the biosynthetic gene clusters and chemical synthesis for structure activity relationship studies with its target, MraY translocase.

The generation of novel secondary metabolites by reengineering or refactoring biochemical pathways is a rewarding but also challenging goal of synthetic biology. For this, the development of tools for the reconstruction of secondary metabolite gene clusters as well as the challenge of understanding the obstacles in this process is of great interest. The artificial gene operon assembly system (AGOS) is a plug-and-play method developed as a tool to consecutively assemble artificial gene operons into a destination vector and subsequently express them under the control of a de-repressed promoter in a Streptomyces host strain. AGOS was designed as a set of entry plasmids for the construction of artificial gene operons and a SuperCos1 based destination vector, into which the constructed operons can be assembled by Red/ET-mediated recombination. To provide a proof-of-concept of this method, we disassembled the well-known novobiocin biosynthetic gene cluster into four gene operons, encoding for the different moieties of novobiocin. We then genetically reorganized these gene operons with the help of AGOS to finally obtain the complete novobiocin gene cluster again. The production of novobiocin precursors and of novobiocin could successfully be detected by LC-MS and LC-MS/MS. Furthermore, we demonstrated that the omission of terminator sequences only had a minor impact on product formation in our system.

Understanding the regulation of a heterologously expressed gene cluster in a host organism is crucial for activation of silent gene clusters or overproduction of the corresponding natural product. In this study, Streptomyces coelicolor M512(nov-BG1) containing the novobiocin biosynthetic gene cluster from Streptomyces niveus NCIMB 11891 was chosen as a model. An improved DNA affinity capturing assay (DACA), combined with semi-quantitative mass spectrometry, was used to identify proteins binding to the promoter regions of the novobiocin gene cluster. Altogether, 2475 proteins were identified in DACA studies with the promoter regions of the pathway-specific regulators novE (PnovE) and novG (PnovG), of the biosynthetic genes novH-W (PnovH) and of the vegetative σ-factor hrdB (PhrdB) as a negative control. A restrictive classification for specific binding reduced this number to 17 proteins. Twelve of them were captured by PnovH, among them, NovG, two were captured by PnovE, and three by PnovG. Unexpectedly some well-known regulatory proteins, such as the global regulators NdgR, AdpA, SlbR, and WhiA were captured in similar intensities by all four tested promoter regions. Of the 17 promoter-specific proteins, three were studied in more detail by deletion mutagenesis and by overexpression. Two of them, BxlR Sc and BxlR2 Sc , could be identified as positive regulators of novobiocin production in S. coelicolor M512. Deletion of a third gene, sco0460 , resulted in reduced novobiocin production, while overexpression had no effect. Furthermore, binding of BxlR Sc to PnovH and to its own promoter region was confirmed via surface plasmon resonance spectroscopy.

The shoot endophytic biocontrol strain Pseudomonas chlororaphis subsp. aurantiaca PB-St2 produces a wide range of exoproducts, including enzymes and antibiotics. The production of exoproducts is commonly tightly regulated. In order to get a deeper insight into the regulatory network of PB-St2, the strain was systematically investigated regarding its quorum sensing systems, both on the genetic and metabolic level. The genome analysis of PB-St2 revealed the presence of four putative acyl homoserine lactone (AHL) biosynthesis genes: phzI, csaI, aurI, and hdtS. LC-MS/MS analyses of the crude supernatant extracts demonstrated that PB-St2 produces eight AHLs. In addition, the concentration of all AHL derivatives was quantified time-resolved in parallel over a period of 42 h during the growth of P. aurantiaca PB-St2, resulting in production curves, which showed differences regarding the maximum levels of the AHLs (14.6 nM- 1.75 μM) and the production period. Cloning and heterologous overexpression of all identified AHL synthase genes in Escherichia coli proved the functionality of the resulting synthases PhzI, CsaI, and AurI. A clear AHL production pattern was assigned to each of these three AHL synthases, while the HdtS synthase did not lead to any AHL production. Furthermore, the heterologous expression study demonstrated unequivocally and for the first time that AurI directs the synthesis of two 3-oxo-AHLs.