DeepRES: Deep learning enables reaction-based comprehensive enzyme screening

The modern sequencing technologies are generating large volumes of information at the transcriptome and genome level. Translation of this information into a biological meaning is far behind the race due to which a significant portion of proteins discovered remain as proteins of unknown function (PUFs). Attempts to uncover the functional significance of PUFs are limited due to lack of easy and high throughput functional annotation tools. Here, we report an approach to assign putative functions to PUFs, identified in the transcriptome of mulberry, a perennial tree commonly cultivated as host of silkworm. We utilized the mulberry PUFs generated from leaf tissues exposed to drought stress at whole plant level. A sequence and structure based computational analysis predicted the probable function of the PUFs. For rapid and easy annotation of PUFs, we developed an automated pipeline by integrating diverse bioinformatics tools, designated as PUFs Annotation Server (PUFAS), which also provides a web service API (Application Programming Interface) for a large-scale analysis up to a genome. The expression analysis of three selected PUFs annotated by the pipeline revealed abiotic stress responsiveness of the genes, and hence their potential role in stress acclimation pathways. The automated pipeline developed here could be extended to assign functions to PUFs from any organism in general. PUFAS web server is available at http://caps.ncbs.res.in/pufas/ and the web service is accessible at http://capservices.ncbs.res.in/help/pufas.

Despite the current wealth of sequencing data, one-third of all biochemically characterized metabolic enzymes lack a corresponding gene or protein sequence, and as such can be considered orphan enzymes. They represent a major gap between our molecular and biochemical knowledge, and consequently are not amenable to modern systemic analyses. As 555 of these orphan enzymes have metabolic pathway neighbours, we developed a global framework that utilizes the pathway and (meta)genomic neighbour information to assign candidate sequences to orphan enzymes. For 131 orphan enzymes (37% of those for which (meta)genomic neighbours are available), we associate sequences to them using scoring parameters with an estimated accuracy of 70%, implying functional annotation of 16,345 gene sequences in numerous (meta)genomes. As a case in point, two of these candidate sequences were experimentally validated to encode the predicted activity. In addition, we augmented the currently available genome-scale metabolic models with these new sequence-function associations and were able to expand the models by on average 8%, with a considerable change in the flux connectivity patterns and improved essentiality prediction.

Bacillus amyloliquefaciens is an important agricultural microbial resource. This study focuses on the whole genome analysis and functional characterization of B. amyloliquefaciens SH-53, isolated from the Wuliang Mountain National Nature Reserve in Dali, Yunnan. The genomic feature analysis revealed that the genome of SH-53 contains 27 ribosomal RNA operons, 4078 protein-coding genes, and 250 prophage-related genes. Additionally, 12 biosynthetic gene clusters (BGCs) for secondary metabolites were predicted, of which 7 are novel gene clusters with unknown functions, showing significant differences compared to the known BGCs of conventional biocontrol strains. Functional potential analysis indicates that SH-53 possesses potential antagonistic activity against plant pathogenic bacteria and can colonize the plant rhizosphere through various mechanisms to exert growth-promoting effects. It is capable of synthesizing multiple antibacterial secondary metabolites, indole-3-acetic acid (IAA), iron carriers, secreting amylase, and efficiently utilizing sulfur sources. The genome also harbors a complete core gene network related to the induced systemic resistance (ISR) and supporting genes that maintain secondary metabolism homeostasis. In conclusion, B. amyloliquefaciens SH-53 exhibits rich biocontrol-related characteristics and unique secondary metabolic potential, indicating promising prospects for its development as an excellent biocontrol agent.

Bacillus amyloliquefaciens is an important agricultural microbial resource. This study focuses on the whole genome analysis and functional characterization of B. amyloliquefaciens SH-53, isolated from the Wuliang Mountain National Nature Reserve in Dali, Yunnan. The genomic feature analysis revealed that the genome of SH-53 contains 27 ribosomal RNA operons, 4,078 protein-coding genes, and 250 prophage-related genes. Additionally, 12 biosynthetic gene clusters (BGCs) for secondary metabolites were predicted, of which 7 are novel gene clusters with unknown functions, showing significant differences compared to the known BGCs of conventional biocontrol strains.Functional potential analysis indicates that SH-53 possesses potential antagonistic activity against plant pathogenic bacteria and can colonize the plant rhizosphere through various mechanisms to exert growth-promoting effects. It is capable of synthesizing multiple antibacterial secondary metabolites, indole-3-acetic acid (IAA), iron carriers, secreting amylase, and efficiently utilizing sulfur sources. The genome also harbors a complete core gene network related to the induced systemic resistance (ISR) and supporting genes that maintain secondary metabolism homeostasis.In conclusion, B. amyloliquefaciens SH-53 exhibits rich biocontrol-related characteristics and unique secondary metabolic potential, indicating promising prospects for its development as an excellent biocontrol agent.

Sinorhizobium sp. enhances plant vitality and stress resilience and improves soil structure. Underscoring their significance as agriculturally important bioagents for increased agricultural productivity requires understanding their taxonomic and functional relationships and the genetic foundations and pathways that drive plant growth-promoting traits. Genome sequencing, comparative genomics, functional annotation, and hybrid genome assemblies can achieve these. Comparative genomic analyses revealed a close relationship between the studied strains and Sinorhizobium meliloti and Sinorhizobium kummerowiae, which was further supported by phylogenomic, ANI, AAI, and dDDH analyses. Gene family cluster analysis identified 5999 gene families in the FRNB45 strain, 6116 in the FRNB101 strain, and 5996 in the FRNB126 strain. Functional genomic analysis identified several biosynthetic gene clusters (BGCs) related to secondary metabolite production, including polyketides, non-ribosomal peptides (NRPs), and siderophores, highlighting the metabolic versatility of these strains. KEGG pathway analysis confirmed the presence of nitrogen fixation and phosphate solubilization pathways, genes associated with the synthesis of indole-3-acetic acid (IAA), and siderophores. These findings support the potential application of FRNB45, FRNB101, and FRNB126 as plant growth-promoting rhizobacteria (PGPR), particularly suited for diverse climatic conditions and high-altitude ecosystems. However, further experimental validation is required to confirm their efficacy and consistency under field conditions.

Carnobacterium maltaromaticum is a non-starter lactic acid bacterium (LAB) of interest in the dairy industry for biopreservation. This study investigated the interference competition network and the specialized metabolites biosynthetic gene clusters (BGCs) content in this LAB in order to explore the relationship between the antimicrobial properties and the genome content. Network analysis revealed that the potency of inhibition tended to increase when the inhibition spectrum broadened, but also that several strains exhibited a high potency and narrow spectrum of inhibition. The C. maltaromaticum strains with potent anti-L. monocytogenes were characterized by high potency and a wide intraspecific spectrum. Genome mining of 29 strains revealed the presence of 12 bacteriocin BGCs: four of class I and eight of class II, among which seven belong to class IIa and one to class IIc. Overall, eight bacteriocins and one nonribosomal peptide synthetase and polyketide synthase (NRPS-PKS) BGCs were newly described. The comparison of the antimicrobial properties resulting from the analysis of the network and the BGC genome content allowed us to delineate candidate BGCs responsible for anti-L. monocytogenes and anti-C. maltaromaticum activity. However, it also highlighted that genome analysis is not suitable in the current state of the databases for the prediction of genes involved in the antimicrobial activity of strains with a narrow anti-C. maltaromaticum activity.

MotivationNatural products are often produced by a set of biosynthetic enzymes that are encoded by genes clustered together in the producer’s genome, referred to as a biosynthetic gene cluster (BGC). The ability to compare and cluster BGCs is essential for several applications, including predicting which bacteria will make a known product and assessing the potential diversity of natural products produced by a set of bacteria. There are multiple methods for comparing and clustering BGCs based on their similarity, but there has been a lack of investigation into how strongly BGC similarity relates to product structural similarity and how these methods perform relative to each other.ResultsUsing publicly available databases, we developed a benchmark dataset to assess how well different BGC similarity metrics correlate with the structural similarity of their products and how well these methods cluster BGCs. We found that all methods showed moderate correlation between BGC and structural similarity, with correlations improving for more similar BGCs and varying significantly by BGC biosynthetic class. Analysis of outliers revealed some outliers were due to mistakes or omissions in public datasets, while others represented deviation between BGC similarity and product structural similarity. All methods generally performed better on clustering metrics, with BiG-SCAPE performing the best after errors in the public datasets had been corrected.Availability and implementationScripts and data required to reproduce the results are available at https://github.com/aswalker-lab/BGC-clustering-benchmark and processed similarity, clusters, and scaffolds are also available at https://huggingface.co/datasets/allie-walker/BGC-clustering-benchmark. Code is also available at Zenodo: 10.5281/zenodo.17373546

Secondary metabolites are compounds not essential for an organism’s development, but provide significant ecological and physiological benefits. These compounds have applications in medicine, biotechnology and agriculture. Their production is encoded in biosynthetic gene clusters (BGCs), groups of genes collectively directing their biosynthesis. The advent of metagenomics has allowed researchers to study BGCs directly from environmental samples, identifying numerous previously unknown BGCs encoding unprecedented chemistry. Here, we present the BGC Atlas (https://bgc-atlas.cs.uni-tuebingen.de), a web resource that facilitates the exploration and analysis of BGC diversity in metagenomes. The BGC Atlas identifies and clusters BGCs from publicly available datasets, offering a centralized database and a web interface for metadata-aware exploration of BGCs and gene cluster families (GCFs). We analyzed over 35 000 datasets from MGnify, identifying nearly 1.8 million BGCs, which were clustered into GCFs. The analysis showed that ribosomally synthesized and post-translationally modified peptides are the most abundant compound class, with most GCFs exhibiting high environmental specificity. We believe that our tool will enable researchers to easily explore and analyze the BGC diversity in environmental samples, significantly enhancing our understanding of bacterial secondary metabolites, and promote the identification of ecological and evolutionary factors shaping the biosynthetic potential of microbial communities.

Natural products play important roles in the chemical interactions between bacteria and their host. Thousands of biosynthetic gene clusters for natural products have been identified through genomic analyses, but most of them are uncharacterized (“cryptic” gene clusters). Our goal is to identify the natural products encoded by these cryptic gene clusters and investigate their functions in bacteria‐host interaction. Using transcriptomics, we identified a cryptic gene cluster that is upregulated in Pseudomonas syringe during plant infection. This gene cluster is widely distributed in plant pathogens that cause worldwide crop damage. To identify the natural product encoded by this gene cluster, we develop an approach that combines heterologous expression and metabolomics with in vitro reconstitution of enzyme activity. We identify a novel natural product, phevamine, which harbors a distinct chemical structure that partly resembles spider and wasp toxins. We discover that phevamine is a virulence factor that suppresses plant immune response. The biosynthesis of phevamine involves ATP‐Grasp type amide ligases. Our work identifies a new virulence factor that pathogens use to invade plant defense. Our work also highlights amide ligases as an important and growing class of biosynthetic enzymes for natural products.Support or Funding InformationThis work was supported by Rita Allen Foundation (B.L.), National Institutes of Health (R00 GM099904, B.L.), NSF‐IOS‐1343020 INSPIRE Track 2 (J.D.), and DOE‐BER‐0000217779 (J.D.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deep-sea cold seeps harbor unique microbial communities that play crucial roles in biogeochemical cycles and possess potential biotechnological applications. Herein, we report the isolation, characterization, and genomic analysis of a novel Bacillus species, Bacillus haimaensis sp. nov. (type strain CSS-39T, CCTCC M20241382), obtained from sediments collected at a depth of 1,350 m in the Haima cold seep, South China Sea. Phylogenomic analysis, revealing an average nucleotide identity of 87.78% and a digital DNA-DNA hybridization value of 34.0% with its closest relative B. tianshenii DSM 25879T, confirms the taxonomic novelty of the genus Bacillus. The complete 4.54 Mb genome of B. haimaensis reveals adaptations to the cold seep environment, including enhanced nutrient acquisition capabilities and stress response mechanisms. Comparative genomic analysis identifies 27 unique gene clusters related to spore germination and sulfate assimilation, suggesting specialized metabolic strategies for this extreme habitat. Furthermore, six biosynthetic gene clusters, including a novel lassopeptide cluster, indicate a potential for secondary metabolite production. Phenotypic characterization demonstrates the strain's ability to utilize diverse carbon sources and tolerate a wide range of environmental conditions. Our findings provide insights into microbial adaptations to deep-sea cold seeps and highlight the potential of B. haimaensis for biotechnological applications in bioremediation and natural product discovery. This study expands our understanding of microbial diversity in extreme marine environments and offers a new model bacterium for investigating bacterial adaptations to deep-sea ecosystems.IMPORTANCEThe discovery of Bacillus haimaensis sp. nov. in the Haima cold seep of the South China Sea represents a significant advancement in our understanding of microbial adaptations to extreme marine environments. This novel species exhibits remarkable metabolic versatility and unique genomic features, providing insights into bacterial survival strategies in nutrient-variable, high-pressure deep-sea ecosystems. Comprehensive genomic analysis reveals distinctive biosynthetic gene clusters, suggesting untapped potential for discovering novel natural product. Furthermore, B. haimaensis exhibits promising capabilities for aromatic compound degradation, indicating potential applications in marine bioremediation. This work not only expands our knowledge of microbial diversity in understudied deep-sea habitats but also highlights the biotechnological promise of extremophiles. The adaptive mechanisms elucidated in B. haimaensis, particularly those related to sporulation and sulfate assimilation, contribute to our broader understanding of microbial ecology in cold seeps and may inform future research on climate change impacts on deep-sea ecosystems.

Streptomyces sp. BRA-346 is an Actinobacteria isolated from the Brazilian endemic tunicate Euherdmania sp. We have reported that this strain produces epoxyketone peptides, as dihydroeponemycin (DHE) and structurally related analogs. This cocktail of epoxyketone peptides inhibits the proteasome chymotrypsin-like activity and shows high cytotoxicity to glioma cells. However, low yields and poor reproducibility of epoxyketone peptides production by BRA-346 under laboratory cultivation have limited the isolation of epoxyketone peptides for additional studies. Here, we evaluated several cultivation methods using different culture media and chemical elicitors to increase the repertoire of peptide epoxyketone production by this bacterium. Furthermore, BRA-346 genome was sequenced, revealing its broad genetic potential, which is mostly hidden under laboratory conditions. By using specific growth conditions, we were able to evidence different classes of secondary metabolites produced by BRA-346. In addition, by combining genome mining with untargeted metabolomics, we could link the metabolites produced by BRA-346 to its genetic capacity and potential regulators. A single biosynthetic gene cluster (BGC) was related to the production of the target epoxyketone peptides by BRA-346. The candidate BGC displays conserved biosynthetic enzymes with the reported eponemycin (EPN) and TMC-86A (TMC) BGCs. The core of the putative epoxyketone peptide BGC (ORFs A-L), in which ORF A is a LuxR-like transcription factor, was cloned into a heterologous host. The recombinant organism was capable to produce TMC and EPN natural products, along with the biosynthetic intermediates DH-TMC and DHE, and additional congeners. A phylogenetic analysis of the epn/tmc BGC revealed related BGCs in public databases. Most of them carry a proteasome beta-subunit, however, lacking an assigned specialized metabolite. The retrieved BGCs also display a diversity of regulatory genes and TTA codons, indicating tight regulation of this BGC at the transcription and translational levels. These results demonstrate the plasticity of the epn/tmc BGC of BRA-346 in producing epoxyketone peptides and the feasibility of their production in a heterologous host. This work also highlights the capacity of BRA-346 to tightly regulate its secondary metabolism and shed light on how to awake silent gene clusters of Streptomyces sp. BRA-346 to allow the production of pharmacologically important biosynthetic products.

Natural microbial communities produce a diverse array of secondary metabolites with ecologically and biotechnologically relevant activities. Some of them have been used clinically as drugs, and their production pathways have been identified in a few culturable microorganisms. However, since the vast majority of microorganisms in nature have not been cultured, identifying the synthetic pathways of these metabolites and tracking their hosts remain a challenge. The microbial biosynthetic potential of mangrove swamps remains largely unknown. Here, we examined the diversity and novelty of biosynthetic gene clusters in dominant microbial populations in mangrove wetlands by mining 809 newly reconstructed draft genomes and probing the activities and products of these clusters by using metatranscriptomic and metabolomic techniques. A total of 3,740 biosynthetic gene clusters were identified from these genomes, including 1,065 polyketide and nonribosomal peptide gene clusters, 86% of which showed no similarity to known clusters in the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) repository. Of these gene clusters, 59% were harbored by new species or lineages of Desulfobacterota-related phyla and Chloroflexota, whose members are highly abundant in mangrove wetlands and for which few synthetic natural products have been reported. Metatranscriptomics revealed that most of the identified gene clusters were active in field and microcosm samples. Untargeted metabolomics was also used to identify metabolites from the sediment enrichments, and 98% of the mass spectra generated were unrecognizable, further supporting the novelty of these biosynthetic gene clusters. Our study taps into a corner of the microbial metabolite reservoir in mangrove swamps, providing clues for the discovery of new compounds with valuable activities. IMPORTANCE At present, the majority of known clinical drugs originated from cultivated species of a few bacterial lineages. It is vital for the development of new pharmaceuticals to explore the biosynthetic potential of naturally uncultivable microorganisms using new techniques. Based on the large numbers of genomes reconstructed from mangrove wetlands, we identified abundant and diverse biosynthetic gene clusters in previously unsuspected phylogenetic groups. These gene clusters exhibited a variety of organizational architectures, especially for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), implying the presence of new compounds with valuable activities in the mangrove swamp microbiome.

BackgroundEfforts to construct the Streptomyces host strain with enhanced yields of heterologous product have focussed mostly on engineering of primary metabolism and/or the deletion of endogenous biosynthetic gene clusters. However, other factors, such as chromosome compactization, have been shown to have a significant influence on gene expression levels in bacteria and fungi. The expression of genes and biosynthetic gene clusters may vary significantly depending on their location within the chromosome. Little is known about the position effect in actinomycetes, which are important producers of various industrially relevant bioactive molecules.ResultsTo demonstrate an impact of the chromosomal position effect on the heterologous expression of genes and gene clusters in Streptomyces albus J1074, a transposon mutant library with randomly distributed transposon that includes a β-glucuronidase reporter gene was generated. Reporter gene expression levels have been shown to depend on the position on the chromosome. Using a combination of the transposon system and a φC31-based vector, the aranciamycin biosynthetic cluster was introduced randomly into the S. albus genome. The production levels of aranciamycin varied up to eightfold depending on the location of the gene cluster within the chromosome of S. albus J1074. One of the isolated mutant strains with an artificially introduced attachment site produced approximately 50% more aranciamycin than strains with endogenous attBs.ConclusionsIn this study, we demonstrate that expression of the reporter gene and aranciamycin biosynthetic cluster in Streptomyces albus J1074 varies up to eightfold depending on its position on the chromosome. The integration of the heterologous cluster into different locations on the chromosome may significantly influence the titre of the produced substance. This knowledge can be used for the more efficient engineering of Actinobacteria via the relocation of the biosynthetic gene clusters and insertion of additional copies of heterologous constructs in a suitable chromosomal position.

Antarctic microbes are important agents for evolutionary adaptation and natural resource of bioactive compounds, harboring the particular metabolic pathways to biosynthesize natural products. However, not much is known on symbiotic microbiomes of fish in the Antarctic zone. In the present study, the culture method and whole-genome sequencing were performed. Natural product analyses were carried out to determine the biosynthetic potential. We report the isolation and identification of a symbiotic bacterium Serratia myotis L7-1, that is highly adaptive and resides within Antarctic fish, Trematomus bernacchii. As revealed by genomic analyses, Antarctic strain S. myotis L7-1 possesses carbohydrate-active enzymes (CAZymes), biosynthetic gene clusters (BGCs), stress response genes, antibiotic resistant genes (ARGs), and a complete type IV secretion system which could facilitate competition and colonization in the extreme Antarctic environment. The identification of microbiome gene clusters indicates the biosynthetic potential of bioactive compounds. Based on bioactivity-guided fractionation, serranticin was purified and identified as the bioactive compound, showing significant antibacterial and antitumor activity. The serranticin gene cluster was identified and located on the chrome. Furthermore, the multidrug resistance and strong bacterial antagonism contribute competitive advantages in ecological niches. Our results highlight the existence of a symbiotic bacterium in Antarctic fish largely represented by bioactive natural products and the adaptability to survive in the fish living in Antarctic oceans.

Biosynthetic gene clusters (BGCs) bridging genotype and phenotype continuously evolve through gene mutations and recombinations to generate chemical diversity. Phenazine BGCs are widespread in bacteria, and the biosynthetic mechanisms of the formation of the phenazine structural core have been illuminated in the last decade. However, little is known about the complex phenazine core-modification machinery. Here, we report the diversity-oriented modifications of the phenazine core through two distinct BGCs in the entomopathogenic bacterium Xenorhabdus szentirmaii, which lives in symbiosis with nematodes. A previously unidentified aldehyde intermediate, which can be modified by multiple enzymatic and non-enzymatic reactions, is a common intermediate bridging the pathways encoded by these BGCs. Evaluation of the antibiotic activity of the resulting phenazine derivatives suggests a highly effective strategy to convert Gram-positive specific phenazines into broad-spectrum antibiotics, which might help the bacteria-nematode complex to maintain its special environmental niche.