Natural Product Biosynthetic Gene Clusters Research Articles

Discovery, characterization, and engineering of an advantageous Streptomyces host for heterologous expression of natural product biosynthetic gene clusters

BackgroundStreptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential.ResultsWe engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host.ConclusionOur engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces.

Nucleobase-Coupled Xanthones with Anti-ROS Effects from Marine-Derived Fungus Aspergillus sydowii.

A MS/MS-based molecular networking approach compared to the Global Natural Product Social Molecular Networking library, in association with genomic annotation of natural product biosynthetic gene clusters within a marine-derived fungus, Aspergillus sydowii, identified a suite of xanthone metabolites. Chromatographic techniques applied to the cultured fungus led to the isolation of 11 xanthone-based alkaloids, dubbed sydoxanthones F-M. The structures of these alkaloids were elucidated using extensive spectroscopic data, including electronic circular dichroism and single-crystal X-ray diffraction data for configurational assignments. Among these analogues, sydoxanthones F-K exhibit structure features typical of nucleobase-coupled xanthones, with sydoxanthone H being an N-bonded xanthone dimer. Notably, (±)sydoxanthones F (1a/1b), (±)sydoxanthones H (3b/3a), and (±)sydoxanthones J (5b/5a) are enantiomeric pairs, while sydoxanthones G (2), I (4), and K (6) are stereoisomers of 1, 3, and 5, respectively. Furthermore, (+)sydoxanthone H (3a) demonstrated significant rescue of cell viability in H2O2-injuried SH-SY5Y cells by inhibiting reactive oxygen species production, suggesting its potential for neuroprotection.

Elucidation of genes enhancing natural product biosynthesis through co-evolution analysis.

Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we proposed that a set of genes co-evolved with BGCs to support biosynthetic proficiency must exist in those strains, and that their identification may provide universal strategies to improve the productivity of other strains. We show here that genes co-evolved with natural product BGCs in Streptomyces can be identified by phylogenomics analysis. Among the 597 genes that co-evolved with polyketide BGCs, 11 genes in the 'coenzyme' category have been examined, including a gene cluster encoding for the cofactor pyrroloquinoline quinone. When the pqq gene cluster was engineered into 11 Streptomyces strains, it enhanced production of 16,385 metabolites, including 36 known natural products with up to 40-fold improvement and several activated silent gene clusters. This study provides an innovative engineering strategy for improving polyketide production and finding previously unidentified BGCs.

Promoter engineering of natural product biosynthetic gene clusters in actinomycetes: concepts and applications.

Covering 2011 to 2022Low titers of natural products in laboratory culture or fermentation conditions have been one of the challenging issues in natural products research. Many natural product biosynthetic gene clusters (BGCs) are also transcriptionally silent in laboratory culture conditions, making it challenging to characterize the structures and activities of their metabolites. Promoter engineering offers a potential solution to this problem by providing tools for transcriptional activation or optimization of biosynthetic genes. In this review, we summarize the 10 years of progress in promoter engineering approaches in natural products research focusing on the most metabolically talented group of bacteria actinomycetes.

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Streptomyces has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering. The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick, Golden Gate, CATCH, yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly. This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters. To demonstrate the utility of this toolkit, we quantified a library of modular regulatory parts, and engineered a gene cluster (act) using characterized promoters that led to increased production. Overall, this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.

Phylogenetic classification of natural product biosynthetic gene clusters based on regulatory mechanisms.

The natural products (NPs) biosynthetic gene clusters (BGCs) represent the adapting biochemical toolkit for microorganisms to thrive different microenvironments. Despite their high diversity, particularly at the genomic level, detecting them in a shake-flask is challenging and remains the primary obstacle limiting our access to valuable chemicals. Studying the molecular mechanisms that regulate BGC expression is crucial to design of artificial conditions that derive on their expression. Here, we propose a phylogenetic analysis of regulatory elements linked to biosynthesis gene clusters, to classify BGCs to regulatory mechanisms based on protein domain information. We utilized Hidden Markov Models from the Pfam database to retrieve regulatory elements, such as histidine kinases and transcription factors, from BGCs in the MIBiG database, focusing on actinobacterial strains from three distinct environments: oligotrophic basins, rainforests, and marine environments. Despite the environmental variations, our isolated microorganisms share similar regulatory mechanisms, suggesting the potential to activate new BGCs using activators known to affect previously characterized BGCs.

Identification of the lipodepsipeptide selethramide encoded in a giant nonribosomal peptide synthetase from a Burkholderia bacterium

Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.

Assessing the Biosynthetic Inventory of the Biocontrol Fungus Trichoderma afroharzianum T22.

Natural products biosynthesized from biocontrol fungi in the rhizosphere can have both beneficial and deleterious effects on plants. Herein, we performed a comprehensive analysis of natural product biosynthetic gene clusters (BGCs) from the widely used biocontrol fungus Trichoderma afroharzianum T22 (ThT22). This fungus encodes at least 64 BGCs, yet only seven compounds and four BGCs were previously characterized or mined. We correlated 21 BGCs of ThT22 with known primary and secondary metabolites through homologous BGC comparison and characterized one unknown BGC involved in the biosynthesis of eujavanicol A using heterologous expression. In addition, we performed untargeted transcriptomics and metabolic analysis to demonstrate the activation of silent ThT22 BGCs via the "one strain many compound" (OSMAC) approach. Collectively, our analysis showcases the biosynthetic capacity of ThT22 and paves the way for fully exploring the roles of natural products of ThT22.

EasyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from Proteobacteria.

The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.

Deciphering chemical logic of fungal natural product biosynthesis through heterologous expression and genome mining.

Covering: 2010 to 2022Heterologous expression of natural product biosynthetic gene clusters (BGCs) has become a widely used tool for genome mining of cryptic pathways, bottom-up investigation of biosynthetic enzymes, and engineered biosynthesis of new natural product variants. In the field of fungal natural products, heterologous expression of a complete pathway was first demonstrated in the biosynthesis of tenellin in Aspergillus oryzae in 2010. Since then, advances in genome sequencing, DNA synthesis, synthetic biology, etc. have led to mining, assignment, and characterization of many fungal BGCs using various heterologous hosts. In this review, we will highlight key examples in the last decade in integrating heterologous expression into genome mining and biosynthetic investigations. The review will cover the choice of heterologous hosts, prioritization of BGCs for structural novelty, and how shunt products from heterologous expression can reveal important insights into the chemical logic of biosynthesis. The review is not meant to be exhaustive but is rather a collection of examples from researchers in the field, including ours, that demonstrates the usefulness and pitfalls of heterologous biosynthesis in fungal natural product discovery.

Isolation, complete genome sequencing and in silico genome mining of Burkholderia for secondary metabolites

Recent years, Burkholderia species have emerged as a new source of natural products (NPs) with increasing attractions. Genome mining suggests the Burkholderia genomes include many natural product biosynthetic gene clusters (BGCs) which are new targets for drug discovery. In order to collect more Burkholderia, here, a strain S-53 was isolated from the soil samples on a mountain area in Changde, P.R. China and verified by comparative genetic analysis to belong to Burkholderia. The complete genome of Burkholderia strain S-53 is 8.2 Mbps in size with an average G + C content of 66.35%. Its taxonomy was both characterized by 16S rRNA- and whole genome-based phylogenetic trees. Bioinformatic prediction in silico revealed it has a total of 15 NP BGCs, some of which may encode unknown products. It is expectable that availability of these BGCs will speed up the identification of new secondary metabolites from Burkholderia and help us understand how sophisticated BGC regulation works.

Navigating and expanding the roadmap of natural product genome mining tools.

Natural products are structurally highly diverse and exhibit a wide array of biological activities. As a result, they serve as an important source of new drug leads. Traditionally, natural products have been discovered by bioactivity-guided fractionation. The advent of genome sequencing technology has resulted in the introduction of an alternative approach towards novel natural product scaffolds: Genome mining. Genome mining is an in-silico natural product discovery strategy in which sequenced genomes are analyzed for the potential of the associated organism to produce natural products. Seemingly universal biosynthetic principles have been deciphered for most natural product classes that are used to detect natural product biosynthetic gene clusters using pathway-encoded conserved key enzymes, domains, or motifs as bait. Several generations of highly sophisticated tools have been developed for the biosynthetic rule-based identification of natural product gene clusters. Apart from these hard-coded algorithms, multiple tools that use machine learning-based approaches have been designed to complement the existing genome mining tool set and focus on natural product gene clusters that lack genes with conserved signature sequences. In this perspective, we take a closer look at state-of-the-art genome mining tools that are based on either hard-coded rules or machine learning algorithms, with an emphasis on the confidence of their predictions and potential to identify non-canonical natural product biosynthetic gene clusters. We highlight the genome mining pipelines' current strengths and limitations by contrasting their advantages and disadvantages. Moreover, we introduce two indirect biosynthetic gene cluster identification strategies that complement current workflows. The combination of all genome mining approaches will pave the way towards a more comprehensive understanding of the full biosynthetic repertoire encoded in microbial genome sequences.

Amino acid (acyl carrier protein) ligase-associated biosynthetic gene clusters reveal unexplored biosynthetic potential.

This study aimed to evaluate the postulated cellular function of a novel family of amino acid (acyl carrier protein) ligases (AALs) in natural product biosynthesis. Here, we analyzed the manually curated, putative, aal-associated natural product biosynthetic gene clusters (NP BGCs) using two computational platforms for NP prediction, antiSMASH-BiG-SCAPE-CORASON and DeepBGC. The detected BGCs included a diversity of type I polyketide/nonribosomal peptide (PKS/NRPS) hybrid BGCs, exemplified by the guadinomine BGC, which suggested a dedicated function of AALs in the biosynthesis of rare (2S)-aminomalonyl-ACP extension units. Besides modular PKS/NRPSs and NRPSs, AAL-associated BGCs were predicted to assemble arylpolyenes, ladderane lipids, phosphonates, aminoglycosides, β-lactones, and thioamides of both nonribosomal and ribosomal origins. Additionally, we revealed a frequent association of AALs with putative, seldom observed transglutaminase-like and BtrH-like transferases of the cysteine protease superfamily, which may form larger families of ACP-dependent amide bond catalysts used in NP synthesis. Our results disclosed an exceptional chemical novelty and biosynthetic potential of the AAL-associated BGCs in NP biosynthesis. The presented in silico evidence supports the initial hypothesis and provides an important foundation for future experimental studies aimed at discovering novel pharmaceutically relevant active compounds.

Complete genome sequencing and in silico genome mining reveal the promising metabolic potential in Streptomyces strain CS-7.

Gram-positive Streptomyces bacteria can produce valuable secondary metabolites. Streptomyces genomes include huge unknown silent natural product (NP) biosynthetic gene clusters (BGCs), making them a potential drug discovery repository. To collect antibiotic-producing bacteria from unexplored areas, we identified Streptomyces sp. CS-7 from mountain soil samples in Changsha, P.R. China, which showed strong antibacterial activity. Complete genome sequencing and prediction in silico revealed that its 8.4 Mbp genome contains a total of 36 BGCs for NPs. We purified two important antibiotics from this strain, which were structurally elucidated to be mayamycin and mayamycin B active against Staphylococcus aureus. We identified functionally a BGC for the biosynthesis of these two compounds by BGC direct cloning and heterologous expression in Streptomyces albus. The data here supported this Streptomyces species, especially from unexplored habitats, having a high potential for new NPs.

Metabolomic Profiling and Molecular Networking of Nudibranch-Associated Streptomyces sp. SCSIO 001680

Antibiotic-resistant bacteria are the primary source of one of the growing public health problems that requires global attention, indicating an urgent need for new antibiotics. Marine ecosystems are characterized by high biodiversity and are considered one of the essential sources of bioactive chemical compounds. Bacterial associates of marine invertebrates are commonly a source of active medicinal and natural products and are important sources for drug discovery. Hence, marine invertebrate-associated microbiomes are a fruitful resource for excavating novel genes and bioactive compounds. In a previous study, we isolated Streptomyces sp. SCSIO 001680, coded as strain 63, from the Red Sea nudibranch Chromodoris quadricolor, which exhibited antimicrobial and antitumor activity. In addition, this isolate harbors several natural product biosynthetic gene clusters, suggesting it has the potential to produce bioactive natural products. The present study aimed to investigate the metabolic profile of the isolated Streptomyces sp. SCSIO 001680 (strain 63) and to predict their potential role in the host’s survival. The crude metabolic extracts of strain 63 cultivated in two different media were characterized by ultra-high-performance liquid chromatography and high-resolution mass spectrometry. The metabolomics approach provided us with characteristic chemical fingerprints of the cellular processes and the relative abundance of specific compounds. The Global Products Social Molecular Networking database was used to identify the metabolites. While 434 metabolites were detected in the extracts, only a few compounds were identified based on the standards and the public spectral libraries, including desferrioxamines, marineosin A, and bisucaberin, halichoblelide, alternarin A, pachastrelloside A, streptodepsipeptide P1 1B, didemnaketal F, and alexandrolide. This finding suggests that this strain harbors several novel compounds. In addition, the metabolism of the microbiome of marine invertebrates remains poorly represented. Thus, our data constitute a valuable complement to the study of metabolism in the host microbiome.

Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor.

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

Discovery and Activation of the Cryptic Cluster from Aspergillus sp. CPCC 400735 for Asperphenalenone Biosynthesis.

Postgenomic analysis manifested that filamentous fungi contain numerous natural product biosynthetic gene clusters in their genome, yet most clusters remain cryptic or down-regulated. Herein, we report the successful manipulation of strain Aspergillus sp. CPCC 400735 that enables its genetic engineering via targeted overexpression of pathway-specific transcriptional regulator AspE. The down-regulated metabolic pathway encoded by the biosynthetic gene cluster asp was successfully up-activated. Analyses of mutant Ai-OE::aspE extracts led to isolation and characterization of 13 asperphenalenone derivatives, of which 11 of them are new compounds. All of the asperphenalenones exhibited conspicuous anti-influenza A virus effects with IC50 values of 0.45-2.22 μM. Additionally, their identification provided insight into biosynthesis of asperphenalenones and might benefit studies of downstream combinatorial biosynthesis. Our study further demonstrates the effective application of targeted overexpressing pathway-specific activator and novel metabolite discovery in microorganisms. These will accelerate the exploitation of the untapped resources and biosynthetic capability in filamentous fungi.

Genome-based classification of Streptomyces pinistramenti sp. nov., a novel actinomycete isolated from a pine forest soil in Poland with a focus on its biotechnological and ecological properties.

A genomic-based polyphasic study was undertaken to establish the taxonomic status and biotechnological and ecological potential of a Streptomyces strain, isolate SF28T, that was recovered from the litter layer in a Polish Pinus sylvestris forest. The isolate had morphological characteristics and chemotaxonomic properties consistent with its classification in the genus Streptomyces. It formed long straight chains of spores with smooth surfaces, contained LL-diaminopimelic acid, glucose and ribose in whole-organism hydrolysates, produced major proportions of straight, iso- and anteiso- fatty acids, hexa- and octa-hydrogenated menaquinones with nine isoprene units and had a polar lipid pattern composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, glycophospholipids and three uncharacterized components. Phylogenetic trees prepared using 16S rRNA gene and multilocus gene sequences of conserved housekeeping genes showed that the isolate formed a branch that was loosely associated with the type strains of several validly published Streptomyces species. A draft genome generated for the isolate was rich in natural product-biosynthetic gene clusters with the potential to produce new specialised metabolites, notably antibiotics, and stress related genes which provide an insight into how it may have become adapted to the harsh conditions that prevail in acidic forest soils. A phylogenomic tree based on the genomes of the isolate and its phylogenetic neighbours confirmed that it formed a distinct lineage well separated from its closest evolutionary relatives. The isolate shared low average nucleotide identity and digital DNA:DNA hybridization values with its phylogenomic neighbours and was also distinguished from them using a combination of cultural and micromorphological properties. Given this wealth of taxonomic data it is proposed that isolate SF28T (= DSM 113360T = PCM 3163T) be classified in the genus Streptomyces as Streptomyces pinistramenti sp. nov. The isolate showed pronounced antimicrobial activity, especially against fungal plant pathogens.

Microbe Profile: Salinispora tropica: natural products and the evolution of a unique marine bacterium.

Salinispora tropica was originally cultured from tropical marine sediments and described as the first obligate marine actinomycete genus. Soon after its discovery, it yielded the potent proteasome inhibitor salinosporamide A, a structurally novel natural product that is currently in phase III clinical trials for the treatment of cancer. If approved, it will be the first natural product derived from a cultured marine microbe to achieve clinical relevance. S. tropica produces many other biologically active natural products, including some linked to chemical defence, thus providing ecological context for their production. However, genomic analyses reveal that most natural product biosynthetic gene clusters remain orphan, suggesting that more compounds await discovery. The abundance of biosynthetic gene clusters in S. tropica supports the concept that the small molecules they encode serve important ecological functions, while their evolutionary histories suggest a potential role in promoting diversification. Better insights into the ecological functions of microbial natural products will help inform future discovery efforts.

Genome insights into the pharmaceutical and plant growth promoting features of the novel species Nocardia alni sp. nov

BackgroundRecent studies highlighted the biosynthetic potential of nocardiae to produce diverse novel natural products comparable to that of Streptomyces, thereby making them an attractive source of new drug leads. Many of the 119 Nocardia validly named species were isolated from natural habitats but little is known about the diversity and the potential of the endophytic nocardiae of root nodule of actinorhizal plants.ResultsThe taxonomic status of an actinobacterium strain, designated ncl2T, was established in a genome-based polyphasic study. The strain was Gram-stain-positive, produced substrate and aerial hyphae that fragmented into coccoid and rod-like elements and showed chemotaxonomic properties that were also typical of the genus Nocardia. It formed a distinct branch in the Nocardia 16S rRNA gene tree and was most closely related to the type strains of Nocardia nova (98.6%), Nocardia jiangxiensis (98.4%), Nocardia miyuensis (97.8%) and Nocardia vaccinii (97.7%). A comparison of the draft genome sequence generated for the isolate with the whole genome sequences of its closest phylogenetic neighbours showed that it was most closely related to the N. jiangxiensis, N. miyuensis and N. vaccinii strains, a result underpinned by average nucleotide identity and digital DNA-DNA hybridization data. Corresponding taxogenomic data, including those from a pan-genome sequence analysis showed that strain ncl2T was most closely related to N. vaccinii DSM 43285T. A combination of genomic, genotypic and phenotypic data distinguished these strains from one another. Consequently, it is proposed that strain ncl2T (= DSM 110931T = CECT 30122T) represents a new species within the genus Nocardia, namely Nocardia alni sp. nov. The genomes of the N. alni and N. vaccinii strains contained 36 and 29 natural product-biosynthetic gene clusters, respectively, many of which were predicted to encode for a broad range of novel specialised products, notably antibiotics. Genome mining of the N. alni strain and the type strains of its closest phylogenetic neighbours revealed the presence of genes associated with direct and indirect mechanisms that promote plant growth. The core genomes of these strains mainly consisted of genes involved in amino acid transport and metabolism, energy production and conversion and transcription.ConclusionsOur genome-based taxonomic study showed that isolate ncl2T formed a new centre of evolutionary variation within the genus Nocardia. This novel endophytic strain contained natural product biosynthetic gene clusters predicted to synthesize novel specialised products, notably antibiotics and genes associated with the expression of plant growth promoting compounds.