Resistance gene-guided genome mining reveals the roseopurpurins as inhibitors of cyclin-dependent kinases.

With the significant increase in the availability of microbial genome sequences in recent years, resistance gene-guided genome mining has emerged as a powerful approach for identifying natural products with specific bioactivities. Here, we present the use of this approach to reveal the roseopurpurins as potent inhibitors of cyclin-dependent kinases (CDKs), a class of cell cycle regulators implicated in multiple cancers. We identified a biosynthetic gene cluster (BGC) with a putative resistance gene with homology to human CDK2. Using targeted gene disruption and transcription factor overexpression in Aspergillus uvarum, and heterologous expression of the BGC in Aspergillus nidulans, we demonstrated that roseopurpurin C (1) is produced by this cluster and characterized its biosynthesis. We determined the potency, specificity, and mechanism of action of 1 as well as multiple intermediates and shunt products produced from the BGC. We show that 1 inhibits human CDK2 with a Kiapp of 44 nM, demonstrates selectivity for clinically relevant members of the CDK family, and induces G1 cell cycle arrest in HCT116 cells. Structural analysis of 1 complexed with CDK2 revealed the molecular basis of ATP-competitive inhibition.

A systematic comparison of natural product potential, with an emphasis on RiPPs, by mining of bacteria of three large ecosystems.

The implementation of several global microbiome studies has yielded extensive insights into the biosynthetic potential of natural microbial communities. However, studies on the distribution of several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs), non-ribosomal peptides (NRPs) and polyketides (PKs) in different large microbial ecosystems have been very limited. Here, we collected a large set of metagenome-assembled bacterial genomes from marine, freshwater and terrestrial ecosystems to investigate the biosynthetic potential of these bacteria. We demonstrate the utility of public dataset collections for revealing the different secondary metabolite biosynthetic potentials among these different living environments. We show that there is a higher occurrence of RiPPs in terrestrial systems, while in marine systems, we found relatively more terpene-, NRP-, and PK encoding gene clusters. Among the many new biosynthetic gene clusters (BGCs) identified, we analyzed various Nif-11-like and nitrile hydratase leader peptide (NHLP) containing gene clusters that would merit further study, including promising products, such as mersacidin-, LAP- and proteusin analogs. This research highlights the significance of public datasets in elucidating the biosynthetic potential of microbes in different living environments and underscores the wide bioengineering opportunities within the RiPP family.

Unearthing New ccr Genes and Staphylococcal Cassette Chromosome Elements in Staphylococci Through Genome Mining.

Staphylococcal cassette chromosome mec (SCCmec) typing is crucial for investigating methicillin-resistant Staphylococcus aureus (MRSA), relying primarily on the combination of ccr and mec gene complexes. To date, 19 ccr genes and 10 ccr gene complexes have been identified, forming 15 SCCmec types. With the vast release of bacterial genome sequences, mining the database for novel ccr gene complexes and SCC/SCCmec elements could enhance MRSA epidemiological studies. In this study, we identified 12 novel ccr genes (6 ccrA, 3 ccrB, and 3 ccrC) through mining of the National Center for Biotechnology Information (NCBI) database, forming 12 novel ccr gene complexes and 10 novel SCC elements. Overexpression of 5 groups of novel Ccr recombinases (CcrA9B3, CcrA10B1, CcrC3, CcrC4, and CcrC5) in a mutant MRSA strain lacking the ccr gene and extrachromosomal circular intermediate (ciSCC) production significantly promoted ciSCC production, demonstrating their biological activity. This discovery provides an opportunity to advance MRSA epidemiological research and develop database-based bacterial typing methods.

Are there conserved biosynthetic genes in lichens? Genome-wide assessment of terpene biosynthetic genes suggests ubiquitous distribution of the squalene synthase cluster.

Lichen-forming fungi (LFF) are prolific producers of functionally and structurally diverse secondary metabolites, most of which are taxonomically exclusive and play lineage-specific roles. To date, widely distributed, evolutionarily conserved biosynthetic pathways in LFF are not known. However, this idea stems from polyketide derivatives, since most biochemical research on lichens has concentrated on polyketide synthases (PKSs). Here, we present the first systematic identification and comparison of terpene biosynthetic genes of LFF using all the available Lecanoromycete reference genomes and 22 de novo sequenced ones (111 in total, representing 60 genera and 23 families). We implemented genome mining and gene networking approaches to identify and group the biosynthetic gene clusters (BGCs) into networks of similar BGCs. Our large-scale analysis led to the identification of 724 terpene BGCs with varying degrees of pairwise similarity. Most BGCs in the dataset were unique with no similarity to a previously known fungal or bacterial BGC or among each other. Remarkably, we found two BGCs that were widely distributed in LFF. Interestingly, both conserved BGCs contain the same core gene, i.e., putatively a squalene/phytoene synthase (SQS), involved in sterol biosynthesis. This indicates that early gene duplications, followed by gene losses/gains and gene rearrangement are the major evolutionary factors shaping the composition of these widely distributed SQS BGCs across LFF. We provide an in-depth overview of these BGCs, including the transmembrane, conserved, variable and LFF-specific regions. Our study revealed that lichenized fungi do have a highly conserved BGC, providing the first evidence that a biosynthetic gene may constitute essential genes in lichens.

Genome mining of Lactiplantibacillus plantarum PA21: insights into its antimicrobial potential.

BACKGROUND: The dramatic increase of antimicrobial resistance in the healthcare realm has become inexorably linked to the abuse of antibiotics over the years. Therefore, this study seeks to identify potential postbiotic metabolites derived from lactic acid bacteria such as Lactiplantibacillus plantarum that could exhibit antimicrobial properties against multi-drug resistant pathogens. RESULTS: In the present work, the genome sequence of Lactiplantibacillus plantarum PA21 consisting of three contigs was assembled to a size of 3,218,706 bp. Phylogenomic analysis and average nucleotide identity (ANI) revealed L. plantarum PA21 is closely related to genomes isolated from diverse niches such as dairy products, food, and animals. Genome mining through the BAGEL4 and antiSMASH database revealed four bacteriocins in a single cluster and four regions of biosynthetic gene clusters responsible for the production of bioactive compounds. The potential probiotic genes indirectly responsible for postbiotic metabolites production were also identified. Additionally, in vitro studies showed that the L. plantarum PA21 cell-free supernatant exhibited antimicrobial activity against all nine methicillin-resistant Staphylococcus aureus (MRSA) and three out of 13 Klebsiella pneumoniae clinical isolates tested. CONCLUSION: Results in this study demonstrates that L. plantarum PA21 postbiotic metabolites is a prolific source of antimicrobials against multi-drug resistant pathogens with potential antimicrobial properties.

Functional analysis and comparative genomics of Rahnella perminowiae S11P1 and Variovorax sp. S12S4, two plant growth-promoting rhizobacteria isolated from Crocus sativus L. (saffron) rhizosphere.

BACKGROUND: Rahnella perminowiae S11P1 and Variovorax sp. S12S4 are two plant growth-promoting rhizobacteria that were previously isolated from the rhizosphere of Crocus sativus L. (saffron), and have demonstrated interesting PGP activities and promising results when used as inoculants in field trials. To further elucidate the molecular mechanisms underlying their beneficial effects on plant growth, comprehensive genome mining of S11P1 and S12S4 and comparative genomic analysis with closely related strains were conducted. RESULTS: Functional annotation of the two strains predicted a large number of genes involved in auxin and siderophore production, nitrogen fixation, sulfur metabolism, organic acid biosynthesis, pyrroloquinoline quinone production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, volatile organic compounds production, and polyamine biosynthesis. In addition, numerous genes implicated in plant-bacteria interactions, such as those involved in chemotaxis and quorum sensing, were predicted. Moreover, the two strains carried genes involved in bacterial fitness under abiotic stress conditions. Comparative genomic analysis revealed an open pan-genomic structure for the two strains. COG annotation showed that higher fractions of core and accessory genes were involved in the metabolism and transport of carbohydrates and amino acids, suggesting the metabolic versatility of the two strains as effective rhizosphere colonizers. Furthermore, this study reports the first comparison of Multilocus sequence analysis (MLSA) and core-based phylogenies of the Rahnella and Variovorax genera. CONCLUSIONS: The present study unveils the molecular mechanisms underlying plant growth promotion and biocontrol activity of S11P1 and S12S4, and provides a basis for their further biotechnological application in agriculture.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Genome mining leads to the identification of a stable and promiscuous Baeyer-Villiger monooxygenase from a thermophilic microorganism.

Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 ºC), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95% conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub-stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH-L1-ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer-Villiger oxidations.

Chemical Diversity of Aspergillus alliaceus Phenotypes: Discovery of Brominated Bianthrones with Activity against Triple-Negative Breast Cancer Cell Lines.

Marine-derived fungi have emerged as a source for novel metabolites with a broad range of bioactivities. However, accessing the full potential of fungi under standard laboratory conditions remains challenging. LC-MS-based metabolomics in combination with varied culture conditions is a fast and powerful tool to detect new metabolites. Here, three developmental forms of the marine-derived fungus Aspergillus alliaceus were analyzed and 14 fungal metabolites, including new brominated polyketides (11-14) were isolated. Structure elucidation relied mainly on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry and DFT-based computations. We sequenced the A. alliaceus genome, identified the bianthrone-producing biosynthetic gene cluster, and conducted expression analysis on genes involved in sexual development and biosynthesis. The NCI-60 cell line panel revealed selective in vitro activity against triple-negative breast cancer (TNBC) for the halogenated allianthrones and their full antiproliferative and cytotoxic effects were evaluated in five TNBC cell lines.

Bacterial Hydrazine Biosynthetic Pathways Featuring Cupin/Methionyl tRNA Synthetase-like Enzymes.

Nitrogen-Nitrogen (N-N) bond-containing functional groups in natural products and synthetic drugs play significant roles in exerting biological activities. The mechanisms of N-N bond formation in natural organic molecules have garnered increasing attention over the decades. Recent advances have illuminated various enzymatic and nonenzymatic strategies, and our understanding of natural N-N bond construction is rapidly expanding. A group of didomain proteins with zinc-binding cupin/methionyl-tRNA synthetase (MetRS)-like domains, also known as hydrazine synthetases, generates amino acid-based hydrazines, which serve as key biosynthetic precursors of diverse N-N bond-containing functionalities such as hydrazone, diazo, triazene, pyrazole, and pyridazinone groups. In this review, we summarize the current knowledge on hydrazine synthetase mechanisms and the various pathways employing this unique bond-forming machinery.

Phylogeny-guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase-like Enzymes.

Cupin/methionyl-tRNA synthetase (MetRS)-like didomain enzymes catalyze nitrogen-nitrogen (N-N) bond formation between Nω-hydroxylamines and amino acids to generate hydrazines, key biosynthetic intermediates of various natural products containing N-N bonds. While the combination of these two building blocks leads to the creation of diverse hydrazine products, the full extent of their structural diversity remains largely unknown. To explore this, we herein conducted phylogeny-guided genome-mining of related hydrazine biosynthetic pathways consisting of two enzymes: flavin-dependent Nω-hydroxylating monooxygenases (NMOs) that produce Nω-hydroxylamine precursors and cupin/MetRS-like enzymes that couple the Nω-hydroxylamines with amino acids via N-N bonds. A phylogenetic analysis identified the largely unexplored sequence spaces of these enzyme families. The biochemical characterization of NMOs demonstrated their capabilities to produce various Nω-hydroxylamines, including those previously not known as precursors of N-N bonds. Furthermore, the characterization of cupin/MetRS-like enzymes identified five new hydrazine products with novel combinations of building blocks, including one containing non-amino acid building blocks: 1,3-diaminopropane and putrescine. This study substantially expanded the variety of N-N bond forming pathways mediated by cupin/MetRS-like enzymes.

Mining biosynthetic gene clusters in Paenibacillus genomes to discover novel antibiotics.

BACKGROUND: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied. RESULTS: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin. CONCLUSIONS: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.

Recent advances in genome mining and synthetic biology for discovery and biosynthesis of natural products.

Natural products have long served as critical raw materials in chemical and pharmaceutical manufacturing, primarily which can provide superior scaffolds or intermediates for drug discovery and development. Over the last century, natural products have contributed to more than a third of therapeutic drug production. However, traditional methods of producing drugs from natural products have become less efficient and more expensive over the past few decades. The combined utilization of genome mining and synthetic biology based on genome sequencing, bioinformatics tools, big data analytics, genetic engineering, metabolic engineering, and systems biology promises to counter this trend. Here, we reviewed recent (2020-2023) examples of genome mining and synthetic biology used to resolve challenges in the production of natural products, such as less variety, poor efficiency, and low yield. Additionally, the emerging efficient tools, design principles, and building strategies of synthetic biology and its application prospects in NPs synthesis have also been discussed.

Genome mining of Bacillus licheniformis MCC2514 for the identification of lasso peptide biosynthetic gene cluster and its characterization.

The probiotic strain Bacillus licheniformis MCC2514 has been shown to produce a strong antibacterial peptide and the whole genome sequence of this strain is also reported in our previous study. The present study is focused on the genome level investigation of this peptide antibiotic and its characterization. Genome mining of the culture revealed the presence of three putative bacteriocin clusters, viz. lichenicidin, sonorensin and lasso peptide. Hence, the mode of action of the peptide was investigated by reporter assay, scanning electron microscopy, and Fourier Transform Infrared spectroscopy. Additionally, the peptide treated groups of Kocuria rhizophila showed a reduction in the fold expression for transcription-related genes. The gene expression studies, quantitative ß-galactosidase induction assay using the RNA stress reporter strain, yvgS along with the homology studies concluded that lasso peptide is responsible for the antibacterial activity of the peptide which acts as an inhibitor of RNA biosynthesis. Gene expression analysis showed a considerable increase in fold expression of lasso peptide genes at various fermentation hours. Also, the peptide was isolated, and its time-kill kinetics and minimum inhibitory concentration against the indicator pathogen K. rhizophila were examined. The peptide was also purified and the molecular weight was determined to be ~ 2 kDa. Our study suggests that this bacteriocin can function as an effective antibacterial agent in food products as well as in therapeutics as it contains lasso peptide, which inhibits the RNA biosynthesis.

Endophytic Streptomyces: an underexplored source with potential for novel natural drug discovery and development.

Streptomyces has long been considered as key sources for natural compounds discovery in medicine and agriculture. These compounds have been demonstrated to possess different biological activities, including antibiotic, antifungal, anticancer, and antiviral effects. As a result, new pharmaceuticals and antibiotics have been developed. Nevertheless, there have been only a few novel discoveries of bioactive compounds in the past decades from Streptomyces in natural habitats. There is, therefore, now a renewed search for new Streptomyces species having the potential to produce many compounds from one strain in lesser explored natural habitats that may be helpful in fighting diseases. Consequently, modern genome mining approaches are imperative for discovering structurally novel natural compounds with therapeutic applications from untapped sources. In light of these facts, endophytic Streptomyces from plants may offer new avenues for the discovery of bioactive compounds with distinctive chemical properties and activities. In the present review, we present the progress made in isolating natural compounds from endophytic Streptomyces originating from plants which have remarkable antimicrobial, cytotoxic, and antifungal properties. A different of distinct structural classes of compounds were reported from endophytic Streptomyces, such as indolosequiterpene, macrolides, flavones, peptides, naphthoquinones, and terpenoids. Further, we discussed modern genomics progress in finding biosynthetic gene clusters (BGCs) encoding compounds. Overall, this review might provide valuable insights into the potential for novel drug discovery from untapped endophytic Streptomyces in the future.

Genome mining and physiological analyses uncover adaptation strategies and biotechnological potential of Virgibacillus dokdonensis T4.6 isolated from high-salt shrimp paste.

Virgibacillus spp. stand out as a potent starter culture for accelerating the fermention of fish sauces and shrimp pastes. However, the underlying molecular mechanisms responsible for their adaptation and biotechnological potential remain elusive. Therefore, the present study focuses on phenotypic and genomic analyses of a halophilic bacterium Virgibacillus dokdonensis T4.6, derived from Vietnamese high-salt fermented shrimp paste. The draft genome contained 4,096,868 bp with 3780 predicted coding sequences. Genome mining revealed the presence of 143 genes involved in osmotic adaptation explaining its resistant phenotype to 24% (w/v) NaCl. Among them, 37 genes making up the complete ectoine metabolism pathway, confirmed its ability to produce 4.38 ± 0.29 wt% ectoine under 12.5% NaCl stress. A significant finding was the identification of 39 genes responsible for an entire degradation pathway of the toxic biogenic amine histamine, which was in agreement with its histamine degradation rate of 42.7 ± 2.1% in the HA medium containing 5 mM histamine within 10 days at 37 °C. Furthermore, 114 proteolytic and 19 lipolytic genes were detected which might contribute to its survival as well as the nutrient quality and flavor of shrimp paste. Of note, a putative gene vdo2592 was found as a possible novel lipase/esterase due to its unique Glycine-Aspartate-Serine-Leucine (GDSL) sequence motif. This is the first report to reveal the adaptative strategies and related biotechnological potential of Virgibacillus associated with femented foods. Our findings indicated that V. dokdonensis T4.6 is a promising starter culture for the production of fermented shrimp paste products.

Aquincola agrisoli sp. nov., isolated from rhizospheric soil of eggplant and in silico genome mining for the prediction of biosynthetic gene clusters.

A Gram-stain negative, aerobic, rod-shaped, motile and flagellated novel bacterial strain, designated MAHUQ-54T, was isolated from the rhizospheric soil of eggplant. The colonies were observed to be light pink coloured, smooth, spherical and 0.2-0.6 mm in diameter when grown on R2A agar medium for 2 days. MAHUQ-54T was able to grow at 15-40 °C, at pH 5.5-9.0 and in the presence of 0-0.5 % NaCl (w/v). The strain gave positive results for both catalase and oxidase tests. The strain was positive for hydrolysis of l-tyrosine, urea, Tween 20 and Tween 80. On the basis of the results of 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Aquincola and is closely related to Aquincola tertiaricarbonis L10T (98.8 % sequence similarity) and Leptothrix mobilis Feox-1T (98.2 %). MAHUQ-54T has a draft genome size of 5 994 516 bp (60 contigs), annotated with 5348 protein-coding genes, 45 tRNA and 5 rRNA genes. The average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values between MAHUQ-54T and its closest phylogenetic neighbours were 75.8-83.3 and 20.8-25.3 %, respectively. In silico genome mining revealed that MAHUQ-54T has a significant potential for the production of novel natural products in the future. The genomic DNA G+C content was determined to be 70.4 %. The predominant isoprenoid quinone was ubiquinone-8. The major fatty acids were identified as C16  :  0, summed feature 3 (comprising C16  :  1ω7c and/or C16  :  1ω6c) and summed feature 8 (comprising C18  :  1ω7c and/or C18  :  1ω6c). On the basis of dDDH, ANI value, genotypic analysis, chemotaxonomic and physiological data, strain MAHUQ-54T represents a novel species within the genus Aquincola, for which the name Aquincola agrisoli sp. nov. is proposed, with MAHUQ-54T (=KACC 22001T = CGMCC 1.18515T) as the type strain.

Rhodococcus indonesiensis sp. nov. a new member of the Rhodococcus ruber lineage isolated from sediment of a neutral hot spring and reclassification of Rhodococcus electrodiphilus (Ramaprasad et al. 2018) as a later heterotypic synonym of Rhodococcus ruber (Kruse 1896) Goodfellow and Alderson 1977 (Approved Lists 1980).

A polyphasic study was designed to determine the taxonomic status of isolate CSLK01-03T, which was recovered from an Indonesian neutral hot spring and provisionally assigned to the genus Rhodococcus. The isolate was found to have chemotaxonomic, cultural and morphological properties typical of rhodococci. It has a rod-coccus lifecycle and grows from 10 to 39 °C, from pH 6.5 to 8.0 and in the presence of 0-10 % (w/v) sodium chloride. Whole-organism hydrolysates contain meso-diaminopimelic acid, arabinose and galactose, the predominant menaquinone is MK-8 (H2), the polar lipid pattern consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylmethylethanolamine and two unidentified components, it produces mycolic acids, and C16:0 is the major fatty acid. Whole-genome analyses show that the isolate and Rhodococcus electrodiphilus LMG 29881T (GenBank accession: JAULCK000000000) have genome sizes of 5.5 and 5.1 Mbp, respectively. These strains and Rhodococcus aetherivorans DSM 44752T and Rhodococcus ruber DSM 43338T form well-supported lineages in 16S rRNA and whole-genome trees that are close to sister lineages composed of the type strains of Rhodococcus rhodochrous and related Rhodococcus species. The isolate can be distinguished from its closest evolutionary neighbours using combinations of cultural and phenotypic features, and by low DNA-DNA hybridization values. Based on these data it is proposed that isolate CSLK01-03T (=CCMM B1310T=ICEBB-06T=NCIMB 15214T) be classified in the genus Rhodococcus as Rhodococcus indonesiensis sp. nov. The genomes of the isolate and its closest phylogenomic relatives are rich in biosynthetic gene clusters with the potential to synthesize new natural products, notably antibiotics. In addition, whole-genome-based taxonomy revealed that Rhodococcus electrodiphilus LMG 29881T and Rhodococcus ruber DSM 43338T belong to a single species. It is, therefore, proposed that R. electrodiphilus be recognized as a heterotypic synonym of R. ruber.

Genome-guided purification of high amounts of the siderophore ornibactin and detection of potentially novel burkholdine derivatives produced by Burkholderia catarinensis 89T.

AIM: The increased availability of genome sequences has enabled the development of valuable tools for the prediction and identification of bacterial natural products. Burkholderia catarinensis 89T produces siderophores and an unknown potent antifungal metabolite. The aim of this work was to identify and purify natural products of B. catarinensis 89T through a genome-guided approach. MATERIALS AND METHODS: The analysis of B. catarinensis 89T genome revealed 16 clusters putatively related to secondary metabolism and antibiotics production. Of particular note was the identification of a nonribosomal peptide synthetase (NRPS) cluster related to the production of the siderophore ornibactin, a hybrid NRPS-polyketide synthase Type 1 cluster for the production of the antifungal glycolipopeptide burkholdine, and a gene cluster encoding homoserine lactones (HSL), probably involved in the regulation of both metabolites. We were able to purify high amounts of the ornibactin derivatives D/C6 and F/C8, while also detecting the derivative B/C4 in mass spectrometry investigations. A group of metabolites with molecular masses ranging from 1188 to 1272 Da could be detected in MS experiments, which we postulate to be new burkholdine analogs produced by B. catarinensis. The comparison of B. catarinensis BGCs with other Bcc members corroborates the hypothesis that this bacterium could produce new derivatives of these metabolites. Moreover, the quorum sensing metabolites C6-HSL, C8-HSL, and 3OH-C8-HSL were observed in LC-MS/MS analysis. CONCLUSION: The new species B. catarinensis is a potential source of new bioactive secondary metabolites. Our results highlight the importance of genome-guided purification and identification of metabolites of biotechnological importance.

Identification of three novel P450 enzymes involved in the oxidative modification of a newly discovered fusicoccane diterpene.

Fusicoccane (FC)-type diterpenoids are a class of diterpenoids characterized by a unique 5-8-5 ring system and exhibit diverse biological activities. Recently, we identified a novel FC-type diterpene synthase MgMS, which produces a myrothec-15(17)-en-7-ol (1) hydrocarbon skeleton, however, its tailoring congeners have not been elucidated. Here, we discovered two additional gene clusters Bn and Np, each encoding a highly homologous terpene synthase to MgMS but distinct tailoring enzymes. Heterologous expression of the terpene synthases BnMS and NpMS yielded the same product as MgMS. Subsequent introduction of three P450 enzymes MgP450, BnP450 and NpP450 from individual gene clusters resulted in four new FC-type diterpenoids 2-5. Notably, MgP450 serves as the first enzyme responsible for hydroxylation of the C19 methyl group, whereas NpP450 functions as a multifunctional P450 enzyme involved in the oxidations at C5, C6, and C19 positions of the 5-8-5 tricyclic skeleton. C5 oxidation of the hydrocarbon skeleton 1 led to broadening of the NMR signals and incomplete spectra, which was resolved by high-temperature NMR spectral analysis.