Peptide Synthetase Research Articles

Elucidation of the ferrichrome siderophore biosynthetic pathway in albomycin-producing Streptomyces sp. ATCC 700974

Sideromycins are a unique subset of siderophores comprising of a siderophore conjugated to an antimicrobial agent. The "Trojan horse" antibiotic albomycins are unique sideromycins consisting of a ferrichrome-type siderophore conjugated to a peptidyl nucleoside antibiotic. They exhibit potent antibacterial activities against many model bacteria and a number of clinical pathogens. Earlier studies have provided significant insight into the biosynthetic pathway of the peptidyl nucleoside moiety. We herein decipher the biosynthetic pathway of the ferrichrome-type siderophore in Streptomyces sp. ATCC 700974. Our genetic studies suggested that abmA, abmB, and abmQ are involved in the formation of the ferrichrome-type siderophore. Additionally, we performed biochemical studies to demonstrate that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential modifications of L-ornithine to generate N5-acetyl-N5-hydroxyornithine. Three molecules of N5-acetyl-N5-hydroxyornithine are then assembled to generate the tripeptide ferrichrome through the action of a nonribosomal peptide synthetase AbmQ. Of special note, we found out that orf05026 and orf03299, two genes scattered elsewhere in the chromosome of Streptomyces sp. ATCC 700974, have functional redundancy for abmA and abmB, respectively. Interestingly, both orf05026 and orf03299 are situated within gene clusters encoding putative siderophores. In summary, this study provided new insight into the siderophore moiety of albomycin biosynthesis and shed light on the contingency of multiple siderophores in albomycin-producing Streptomyces sp. ATCC700974.

Molecular Mechanisms to Optimize Gene Translation Elongation Differ Significantly in Bacteria with and without Non-Ribosomal Peptides

Non-ribosomal peptides play an important role in the vital activity of bacteria and have an extremely broad field of biological activity. In particular, they act as antibiotics, toxins, surfactants, siderophores, and also perform a number of other specific functions. Biosynthesis of these molecules does not occur on ribosomes but by special enzymes that form gene clusters in bacterial genomes. We hypothesized that the presence of non-ribosomal peptide synthesis pathways is a specific feature of bacterial metabolism, which may affect other vital processes of the cell, including translational ones. This work was the first to show the relationship between the translation regulation mechanism of protein-coding genes in bacteria, which is largely determined by the efficiency of translation elongation, and the presence of gene clusters in the genomes for the biosynthesis of non-ribosomal peptides. Bioinformatic analysis of the translation elongation efficiency of protein-coding genes was performed in 11679 bacterial genomes, some of which contained gene clusters of non-ribosomal peptide biosynthesis and some of which did not. The analysis showed that bacteria whose genomes contained clusters of non-ribosomal peptide biosynthetic genes and those without such gene clusters differ significantly in the molecular mechanisms that ensure translation efficiency. Thus, among microorganisms whose genomes contain gene clusters of non-ribosomal peptide synthetases, a significantly smaller part of them is characterized by optimized regulation of the number of local inverted repeats, while most of them have genomes optimized by the averaged energy of inverted repeats studs in mRNA and additionally by codon composition. Our results suggest that the presence of non-ribosomal peptide biosynthetic pathways in bacteria may influence the structure of the overall bacterial metabolism, which is also expressed in the specific mechanisms of ribosomal protein biosynthesis.

Antibacterial Activity of Freshwater Sponge Oncosclera asiatica Against Escherichia coli and Staphylococcus aureus

Freshwater sponges are animals from the Porifera phylum that live in freshwater. The sponge used is Oncosclera asiatica was taken from Kali Porong, East Java. Seventeen isolates of bacteria have been obtained from isolation. Antibacterial potential testing was performed by paper disc inhibition assay using Escherichia coli and Staphylococcus aureus as pathogenic bacteria and amoxicillin as a positive control. The antibacterial activity test showed that four isolates have the potential activity. The isolates with the highest inhibition zones were identified using a 16S rRNA.The results of BLAST showed isolate number 2 was Pseudomonas moraviensis with 99.51% similarity. The phylogenetic tree analysis was build using the MEGA X program. The results of the phylogenetic tree analysis showed that P.moraviensis had a bootstrap value of 100% with a genetic distance value of 0.001. P. moraviensis isolates screened for the presence of Non-Ribosomal Peptide Synthetase (NRPS) gene by A2gamF and A3gamR primers. The amplification result from NRPS gene showed positive meaning that P.moraviensis genome contained NRPS gene.

Investigation on Metabolites in Structure and Biosynthesis from the Deep-Sea Sediment-Derived Actinomycete Janibacter sp. SCSIO 52865

For exploring structurally diverse metabolites and uniquely metabolic mechanisms, we systematically investigated the chemical constituents and putative biosynthesis of Janibacter sp. SCSIO 52865 derived from the deep-sea sediment based on the OSMAC strategy, molecular networking tool, in combination with bioinformatic analysis. As a result, one new diketopiperazine (1), along with seven known cyclodipeptides (2-8), trans-cinnamic acid (9), N-phenethylacetamide (10) and five fatty acids (11-15), was isolated from the ethyl acetate extract of SCSIO 52865. Their structures were elucidated by a combination of comprehensive spectroscopic analyses, Marfey's method and GC-MS analysis. Furthermore, the analysis of molecular networking revealed the presence of cyclodipeptides, and compound 1 was produced only under mBHI fermentation condition. Moreover, bioinformatic analysis suggested that compound 1 was closely related to four genes, namely jatA-D, encoding core non-ribosomal peptide synthetase and acetyltransferase.

Enzymatic α-Ketothioester Decarbonylation Occurs in the Assembly Line of Barbamide for Skeleton Editing.

The decarbonylation reaction has been developed significantly in organic chemistry as an effective approach to various synthetic applications, but enzymatic precedents for this reaction are rare. Based on investigations into the hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line of barbamide, we report an on-line α-ketothioester decarbonylation reaction that leads to one-carbon truncation of the elongating skeleton. This enzymatic editing reaction occurs in the first round of lipopeptide extension and modification involving the multienzymes BarE and BarF, which successively house an NRPS module to initiate the biosynthesis and a PKS module to catalyze the first round of chain extension. Starting with processing a leucine-derived α-ketoacyl starter, the ketosynthase domain in BarE displays an unusual dual activity that results in net one-carbon chain elongation. It extrudes carbon monoxide from α-keto-isocaproyl thioester and then mediates decarboxylative condenses of the resultant isovaleryl thioester with malonyl thioester to form a diketide intermediate, followed by BarF-based O-methylation to stabilize the enol form of the β-carbonyl and afford an unusual E-double bond. Biochemical characterization, chemical synthesis, computational analysis, and the experimental outcome of site-directed mutagenesis illustrate the extraordinary catalytic capability of this ketosynthase domain. This work furthers the appreciation of assembly line chemistry and opens the door to new approaches for skeleton editing/engineering of related molecules using synthetic biology approaches.

Discovery of Cyclic Lipopeptides Ralstopeptins A and B from Ralstonia solanacearum Species Complex and Analysis of Biosynthetic Gene Evolution.

Ralstonia solanacearum species complex (RSSC) strains are plant pathogens that produce lipopeptides (ralstonins and ralstoamides) by the polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) enzyme hybrid. Recently, ralstonins were found to be key molecules in the parasitism of RSSC to other hosts, Aspergillus and Fusarium fungi. The PKS-NRPS genes of RSSC strains in the GenBank database suggest the production of additional lipopeptides, although it has not been confirmed to date. Here, we report the genome-driven and mass-spectrometry-guided discovery, isolation, and structural elucidation of ralstopeptins A and B from strain MAFF 211519. Ralstopeptins were found to be cyclic lipopeptides with two amino acid residues less than ralstonins. The partial deletion of the gene encoding PKS-NRPS obliterated the production of ralstopeptins in MAFF 211519. Bioinformatic analyses suggested possible evolutionary events of the biosynthetic genes of RSSC lipopeptides, where intragenomic recombination may have occurred within the PKS-NRPS genes, reducing the gene size. The chlamydospore-inducing activities of ralstopeptins A and B, ralstonins A and B, and ralstoamide A in the fungus Fusarium oxysporum indicated a structural preference for ralstonins. Altogether, we propose a model for the evolutionary processes that contribute to the chemical diversity of RSSC lipopeptides and its relation to the endoparasitism of RSSC in fungi.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

A putative terpene cyclase gene (CcPtc1) is required for fungal development and virulence in Cytospora chrysosperma.

Cytospora chrysosperma is a destructive plant pathogenic fungus, which causes canker disease on numerous woody plants. However, knowledge concerning the interaction between C. chrysosperma and its host remains limited. Secondary metabolites produced by phytopathogens often play important roles in their virulence. Terpene cyclases (TC), polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) are the key components for the synthesis of secondary metabolites. Here, we characterized the functions of a putative terpene type secondary metabolite biosynthetic core gene CcPtc1 in C. chrysosperma, which was significantly up-regulated in the early stages of infection. Importantly, deletion of CcPtc1 greatly reduced fungal virulence to the poplar twigs and they also showed significantly reduced fungal growth and conidiation compared with the wild-type (WT) strain. Furthermore, toxicity test of the crude extraction from each strain showed that the toxicity of crude extraction secreted by ΔCcPtc1 were strongly compromised in comparison with the WT strain. Subsequently, the untargeted metabolomics analyses between ΔCcPtc1 mutant and WT strain were conducted, which revealed 193 significantly different abundant metabolites (DAMs) inΔCcPtc1 mutant compared to the WT strain, including 90 significantly downregulated metabolites and 103 significantly up-regulated metabolites, respectively. Among them, four key metabolic pathways that reported to be important for fungal virulence were enriched, including pantothenate and coenzyme A (CoA) biosynthesis. Moreover, we also detected significant alterations in a series of terpenoids, among which (+)-ar-turmerone, pulegone, ethyl chrysanthemumate, and genipin were significantly down-regulated, while cuminaldehyde and (±)-abscisic acid were significantly up-regulated. In conclusion, our results demonstrated that CcPtc1 acts as a virulence-related secondary metabolism factor and provides new insights into the pathogenesis of C. chrysosperma.

Micrococcus spp. as a promising source for drug discovery: A review.

Historically, bacteria of the phylum Actinobacteria have been a very prominent source of bioactive compounds for drug discovery. Among the actinobacterial genera, Micrococcus has not generally been prioritized in the search for novel drugs. The bacteria in this genus are known to have very small genomes (generally<3 Mb). Actinobacteria with small genomes seldom contain the well-characterized biosynthetic gene clusters such as those encoding polyketide synthases and nonribosomal peptide synthetases that current genome mining algorithms are optimized to detect. Nevertheless, there are many reports of substantial pharmaceutically-relevant bioactivity of Micrococcus extracts. On the other hand, there are remarkably few descriptions of fully characterized and structurally-elucidated bioactive compounds from Micrococcus spp. This review provides a comprehensive summary of the bioactivity of Micrococcus spp., which encompasses antibacterial, antifungal, cytotoxic, antioxidant and anti-inflammatory activities. This review uncovers the considerable biosynthetic potential of this genus and highlights the need for a re-examination of these bioactive strains, with a particular emphasis on marine isolates, because of their potent bioactivity and high potential for encoding unique molecular scaffolds.

Streptomyces lividans 66 produces a protease inhibitor via a tRNA-utilizing enzyme interacting with a C-minus NRPS.

Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by nonribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-Utilizing Enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA-dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize. Significance The discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, e.g. of their evolution, activity and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.

Taxonomic Positions and Secondary Metabolite-Biosynthetic Gene Clusters of Akazaoxime- and Levantilide-Producers

Micromonospora sp. AKA109 is a producer of akazaoxime and A-76356, whereas Micromonospora sp. AKA38 is that of levantilide C. We aimed to clarify their taxonomic positions and identify biosynthetic gene clusters (BGCs) of these compounds. In 16S rRNA gene and DNA gyrase subunit B gene (gyrB) sequence analyses, strains AKA109 and AKA38 were the most closely related to Micromonospora humidisoli MMS20-R2-29T and Micromonospora schwarzwaldensis HKI0641T, respectively. Although Micromonospora sp. AKA109 was identified as M. humidisoli by the gyrB sequence similarity and DNA-DNA relatedness based on whole genome sequences, Micromonospora sp. AKA38 was classified to a new genomospecies. M. humidisoli AKA109 harbored six type-I polyketide synthase (PKS), one type-II PKS, one type-III PKS, three non-ribosomal peptide synthetase (NRPS) and three hybrid PKS/NRPS gene clusters, among which the BGC of akazaoxime and A-76356 was identified. These gene clusters are conserved in M. humidisoli MMS20-R2-29T. Micromonospora sp. AKA38 harbored two type-I PKS, one of which was responsible for levantilide C, one type-II PKS, one type-III PKS, two NRPS and five hybrid PKS/NRPS gene clusters. We predicted products derived from these gene clusters through bioinformatic analyses. Consequently, these two strains are revealed to be promising sources for diverse non-ribosomal peptide and polyketide compounds.

Characterization of Bacillus Strains from Natural Honeybee Products with High Keratinolytic Activity and Antimicrobial Potential

Two efficient feather-degrading bacteria were isolated from honeybee samples and identified as Bacillus sonorensis and Bacillus licheniformis based on 16S rRNA and genome sequencing. The strains were able to grow on chicken feathers as the sole carbon and nitrogen sources and degraded the feathers in a few days. The highest keratinase activity was detected by the B. licheniformis CG1 strain (3800 U × mL-1), followed by B. sonorensis AB7 (1450 U × mL-1). Keratinase from B. licheniformis CG1 was shown to be active across a wide range of pH, potentially making this strain advantageous for further industrial applications. All isolates displayed antimicrobial activity against Micrococcus luteus; however, only B. licheniformis CG1 was able to inhibit the growth of Mycobacterium smegmatis. In silico analysis using BAGEL and antiSMASH identified gene clusters associated with the synthesis of non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKSs) and/or ribosomally synthesized and post-translationally modified peptides (RiPPs) in most of the Bacillus isolates. B. licheniformis CG1, the only strain that inhibited the growth of the mycobacterial strain, contained sequences with 100% similarity to lichenysin (also present in the other isolates) and lichenicidin (only present in the CG1 strain). Both compounds have been described to display antimicrobial activity against distinct bacteria. In summary, in this work, we have isolated a strain (B. licheniformis CG1) with promising potential for use in different industrial applications, including animal nutrition, leather processing, detergent formulation and feather degradation.

Marine flatworm Acanthozoon sp.-associated bacteria with antibiotic property from the Java Sea

Neglected invertebrates, marine flatworms, have attracted global research interest due to their biological and chemical potential properties. The marine flatworms (Turbellaria), Phylum Platyhelminthes, belong to the Polycladida group. There are about 3000 species of free-living flatworms that make a living by hunting and eating other animals. A marine flatworm Acanthozoon sp. was used in this study due to its abundant presence in the site location. Staphylococcus epidermidis is an opportunistic pathogenic bacterium that was previously considered a harmless skin disease bacterium. This species is now considered to be in the first rank among the causative agents of nosocomial infection, specifically in the form of infections of the urinary tract, respiratory tract, and surgical site wounds. The aims of this study were to explore the biological diversity of marine flatworm-associated bacteria with antipathogenic properties and to detect the presence of polyketide synthase (PKS) and nonribosomal peptide synthetase NRPS gene clusters through a molecular approach. Recent studies have shown that S. epidermidis undergoes functional changes in the pro-inflammatory peptide family so that it has functions in immune evasion and biofilm development. Therefore, the search for new antimicrobial compounds is urgently needed due to the limited choice of antibiotic use. In the preliminary screening by overlay test, 7 out of 17 (41.2%) isolates showed antibacterial activities. These isolates were reselected and their activity confirmed by using plug agar and disk-diffusion methods. The FA02, FA03, FA05, FA07, FA13, FA16, and FA17 isolates demonstrated their inhibitory activities consistently against the causative agent of nosocomial infection S. epidermidis. Based on the morphological and 16S rRNA partial sequencing analysis, these isolates were closely related to the genus Virgibacillus, Brevibacterium, Alcanivorax, and Vibrio. None of these seven antibacterial strains possesses PKS-I and PKS-II, except NRPS genes for Virgibacillus salarius strain FA02, V. salarius strain FA16, and V. salarius strain FA17. The results of this study showed that bacteria associated with marine flatworms have future potential as a source of promising natural products for the development of antibiotics.

Genome Sequencing of Streptomyces griseus SCSIO PteL053, the Producer of 2,2′-Bipyridine and Actinomycin Analogs, and Associated Biosynthetic Gene Cluster Analysis

Marine symbiotic actinomycetes play a key role in drug development and their ecological niches can influence a variety of natural product biosynthesis, providing potential defensive benefits. In this study, we report the whole-genome sequence analysis of marine gastropod mollusk Planaxis sp.-associated Streptomyces griseus SCSIO PteL053, which harbors 28 putative biosynthetic gene clusters (BGCs). Among them, two BGCs encoded by a hybrid non-ribosomal peptide (NRPS)/polyketide (PKS) synthetase and non-ribosomal peptide synthetase (NRPS) are responsible for the synthesis of the known therapeutic metabolites 2,2′-bipyridine and actinomycin analogs, respectively. Detailed bioinformatics analysis revealed the putative BGCs and the functions of the involved genes in the biosynthesis of the known compounds SF2738D (1), SF2738F (2), actinomycin D (3), and Actinomycin Xoβ (4). In the present study, complete-genome sequencing allowed us to rediscover known, clinically useful secondary metabolites in the newly isolated Streptomyces griseus SCSIO PteL053.

Role of the nonribosomal peptide synthetase siderophore enzyme (Rfs) of Mucor lusitanicus in controlling the growth of fungal phytopathogens.

Dimorphic species of Mucor, which are cosmopolitan fungi belonging to subphylum Mucoromycotina, are metabolically versatile. Some species of Mucor are sources of biotechnological products, such as biodiesel from Mucor circinelloides and expression of heterologous proteins from Mucor lusitanicus. Furthermore, Mucor lusitanicus has been described as a model for understanding mucormycosis infections. However, little is known regarding the relationship between Mucor lusitanicus and other soil inhabitants. In this study, we investigated the potential use of Mucor lusitanicus as a biocontrol agent against fungal phytopathogens, namely Fusarium oxysporum f. sp. lycopersici, Fusarium solani, and Alternaria solani, which destroy economically important crops. Results showed that aerobic cell-free supernatants of the culture broth (SS) from Mucor lusitanicus inhibited the growth of the fungal phytopathogens in culture, soil, and tomato fruits. The SS obtained from a strain of Mucor lusitanicus carrying the deletion of rfs gene, which encodes an enzyme involved in the synthesis of siderophore rhizoferrin, had a decreased inhibitory effect against the growth of the phytopathogens. Contrarily, this inhibitory effect was more evident with the SS from an rfs-overexpressing strain compared to the wild-type. This study provides a framework for the potential biotechnological use of the molecules secreted from Mucor lusitanicus in the biocontrol of fungal phytopathogens.

Transcriptomic analysis of polyketide synthesis in dinoflagellate, Prorocentrum lima

The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. P. lima can produce polyketide compounds, such as okadaic acid (OA), dinophysistoxin (DTX) and their analogues, which are responsible for diarrhetic shellfish poisoning (DSP). Studying the molecular mechanism of DSP toxin biosynthesis is crucial for understanding the environmental driver influencing toxin biosynthesis as well as for better monitoring of marine ecosystems. Commonly, polyketides are produced by polyketide synthases (PKS). However, no gene has been confirmatively assigned to DSP toxin production. Here, we assembled a transcriptome from 94,730,858 Illumina RNAseq reads using Trinity, resulting in 147,527 unigenes with average sequence length of 1035 nt. Using bioinformatics analysis methods, we found 210 unigenes encoding single-domain PKS with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 15 transcripts encoding multi-domain PKS (forming typical type I PKSs modules) and 5 transcripts encoding hybrid nonribosomal peptide synthetase (NRPS)/PKS were found. Using comparative transcriptome and differential expression analysis, a total of 16 PKS genes were identified to be up-regulated in phosphorus-limited cultures, which was related to the up regulation of toxin expression. In concert with other recent transcriptome analyses, this study contributes to the building consensus that dinoflagellates may utilize a combination of Type I multi-domain and single-domain PKS proteins, in an as yet undefined manner, to synthesize polyketides. Our study provides valuable genomic resource for future research in order to understand the complex mechanism of toxin production in this dinoflagellate.

New approaches to track chemoenzymatic loading reactions of NRPS carrier protein domains using NMR.

The atomic-level detail provided by nuclear magnetic resonance (NMR) allows for the monitoring of biochemical reactions as substrates and intermediates are converted by enzymes. NMR signals are sensitive to chemical environments and this feature allows for scrutiny of the integrity of reactions. Routinely, time-consuming diversions are necessary with traditional approaches to monitor biochemical transformations as many require additional sample manipulations or their readouts are limited. Here, we highlight the advantages of NMR to both monitor and rescue a complex chemoenzymatic reaction that modifies an isotopically labeled nonribosomal peptide synthetase (NRPS) carrier protein with an unlabeled phosphopantetheine moiety through an enzymatic cascade. We present protocols to monitor this reaction that implement various 1D NMR experiments tailored to the detection of specific steps in the enzymatic cascade and can be easily applied even to reactions using unlabeled domains. Additionally, to highlight the signals of the unlabeled modification once attached to the labeled biomolecule, we present a modified diffusion and isotope-filtered pulse sequence. Importantly, when chemoenzymatic reactions are slow or are stalled, the technique provides an atomic-level readout of reaction byproducts and limiting or damaged reagents, and thus allows for quick interventions to rescue them. The advantages of using NMR highlighted in this study more generally serve as a dogma to monitor and restore complex chemoenzymatic reactions.

A genetic tool to express long fungal biosynthetic genes

BackgroundSecondary metabolites (SMs) from mushroom-forming fungi (Basidiomycota) and early diverging fungi (EDF) such as Mucoromycota are scarcely investigated. In many cases, production of SMs is induced by unknown stress factors or is accompanied by seasonable developmental changes on fungal morphology. Moreover, many of these fungi are considered as non-culturable under laboratory conditions which impedes investigation into SM. In the post-genomic era, numerous novel SM genes have been identified especially from EDF. As most of them encode multi-module enzymes, these genes are usually long which limits cloning and heterologous expression in traditional hosts.ResultsAn expression system in Aspergillus niger is presented that is suitable for the production of SMs from both Basidiomycota and EDF. The akuB gene was deleted in the expression host A. niger ATNT∆pyrG, resulting in a deficient nonhomologous end-joining repair mechanism which in turn facilitates the targeted gene deletion via homologous recombination. The ∆akuB mutant tLK01 served as a platform to integrate overlapping DNA fragments of long SM genes into the fwnA locus required for the black pigmentation of conidia. This enables an easy discrimination of correct transformants by screening the transformation plates for fawn-colored colonies. Expression of the gene of interest (GOI) is induced dose-dependently by addition of doxycycline and is enhanced by the dual TetON/terrein synthase promoter system (ATNT) from Aspergillus terreus. We show that the 8 kb polyketide synthase gene lpaA from the basidiomycete Laetiporus sulphureus is correctly assembled from five overlapping DNA fragments and laetiporic acids are produced. In a second approach, we expressed the yet uncharacterized > 20 kb nonribosomal peptide synthetase gene calA from the EDF Mortierella alpina. Gene expression and subsequent LC–MS/MS analysis of mycelial extracts revealed the production of the antimycobacterial compound calpinactam. This is the first report on the heterologous production of a full-length SM multidomain enzyme from EDF.ConclusionsThe system allows the assembly, targeted integration and expression of genes of > 20 kb size in A. niger in one single step. The system is suitable for evolutionary distantly related SM genes from both Basidiomycota and EDF. This uncovers new SM resources including genetically intractable or non-culturable fungi.

Classification and Secondary Metabolite-Biosynthetic Gene Clusters of Marine Streptomyces Strains Including a Lobophorin- and Divergolide-Producer

Two Streptomyces strains, named N11-26 and DC10-5, were isolated from deep-sea and non-photosynthetic stony coral, respectively. Strain N11-26 produces lobophorin C and divergolides, which are antimicrobial substances. This study aimed to classify these strains and reveal their cryptic potential to synthesize other secondary metabolites, such as polyketides and nonribosomal peptides. Strains N11-26 and DC10-5 showed 16S rRNA gene sequence similarities of 100% and 99.9% to Streptomyces olivaceus NRRL B-3009T, respectively. By digital DNA–DNA hybridization using whole-genome sequences, these strains were classified as Streptomyces olivaceus. Strain N11-26 was closer to the type strain of S. olivaceus than strain DC10-5 and possessed 17 clusters of polyketide synthase (PKS) and/or nonribosomal peptide synthetases (NRPS) genes, whereas strain DC10-5 harbored 19 clusters. Putative products by these gene clusters were predicted by bioinformatic analyses. Although 15 clusters were conserved between the two strains, two and four clusters were specific in strains N11-26 and DC10-5, respectively. This represents a diversity of potential polyketide and nonribosomal peptide compounds between strains of S. olivaceus. To the best of our knowledge, this is the first report annotating all the PKS and NRPS gene clusters in S. olivaceus strains with their putative products to provide useful information for genome mining.

Molecular Mechanisms to Optimize Gene Translation Elongation Differ Significantly in Bacteria with and without Nonribosomal Peptides

Nonribosomal peptides play an important role in the vital activity of bacteria and have an extremely broad field of biological activity. In particular, they act as antibiotics, toxins, surfactants, siderophores, and also perform a number of other specific functions. Biosynthesis of these molecules does not occur on ribosomes but by special enzymes that form gene clusters in bacterial genomes. We hypothesized that the presence of nonribosomal peptide synthesis pathways is a specific feature of bacterial metabolism, which may affect other vital processes of the cell, including translational ones. This work was the first to show the relationship between the translation regulation mechanism of protein-coding genes in bacteria, which is largely determined by the efficiency of translation elongation, and the presence of gene clusters in the genomes for the biosynthesis of nonribosomal peptides. Bioinformatic analysis of the translation elongation efficiency of protein-coding genes was performed in 11679 bacterial genomes, some of which contained gene clusters of nonribosomal peptide biosynthesis and some of which did not. The analysis showed that bacteria whose genomes contained clusters of nonribosomal peptide biosynthetic genes and those without such gene clusters differ significantly in the molecular mechanisms that ensure translation efficiency. Thus, among microorganisms whose genomes contain gene clusters of nonribosomal peptide synthetases, a significantly smaller part of them is characterized by optimized regulation of the number of local inverted repeats, while most of them have genomes optimized by the averaged energy of inverted repeats studs in mRNA and additionally by codon composition. Our results suggest that the presence of nonribosomal peptide biosynthetic pathways in bacteria may influence the structure of the overall bacterial metabolism, which is also expressed in the specific mechanisms of ribosomal protein biosynthesis.