Diversity and distribution of biosynthetic gene clusters in agricultural soil microbiomes.

Bacterial secondary metabolites serve as an important source of molecules for drug discovery. They also play an important function in mediating the interactions of microbial producers with their living environment and surrounding organisms. However, little is known about the genetic novelty, distribution, and community-level impacts of soil bacterial biosynthetic potential on a large geographic scale. Here, we constructed the first catalog of 11,149 biosynthetic gene clusters (BGCs) from agricultural soils across China and unearthed hidden biosynthetic potential for new natural product discovery from the not-yet-cultivated soil bacteria. Notably, we revealed soil pH as the strongest environmental driver of BGC biogeography and predicted that soil acidification and global climate change could damage the biosynthetic potential of the soil microbiome. The co-occurrence network of bacterial genomes revealed two BGC-rich species, i.e., Nocardia niigatensis from Actinobacteriota and PSRF01 from Acidobacteriota, as the module hub and connector, respectively, indicating their keystone positions in the soil microbial communities. We also uncovered a dominant role of BGC-inferred biotic interactions over environmental drivers in structuring the soil microbiome. Overall, this study achieved novel insights into the BGC landscape in agricultural soils of China, substantially expanding our understanding of the diversity and novelty of bacterial secondary metabolism and the potential role of secondary metabolites in microbiota assembly.IMPORTANCEBacterial secondary metabolites not only serve as the foundation for numerous therapeutics (e.g., antibiotics and anticancer drugs), but they also play critical ecological roles in mediating microbial interactions (e.g., competition and communication). However, our knowledge of bacterial secondary metabolism is limited to only a small fraction of cultured strains, thus restricting our comprehensive understanding of their diversity, novelty, and potential ecological roles in soil ecosystems. Here, we used culture-independent metagenomics to explore biosynthetic potentials in agricultural soils of China. Our analyses revealed a high degree of genetic diversity and novelty within biosynthetic gene clusters in agricultural soil environments, offering valuable insights for biochemists seeking to synthesize novel bioactive products. Furthermore, we uncovered the pivotal role of BGC-rich species in microbial communities and the significant relationship between BGC richness and microbial phylogenetic turnover. This information emphasizes the importance of biosynthetic potential in the assembly of microbial communities.

Heterologous production of a new lanthipeptide boletupeptin using a cryptic biosynthetic gene cluster of the myxobacterium Melittangium boletus.

Myxobacteria have comparatively large genomes that contain many biosynthetic genes with the potential to produce secondary metabolites. Based on genome mining, we discovered a new biosynthetic gene cluster of class III lanthipeptide in the genome of the myxobacterium Melittangium boletus. The biosynthetic gene cluster contained a precursor peptide-coding gene bolA, and a class III lanthipeptide synthetase-coding gene bolKC. The expression vector containing bolA and bolKC was constructed using synthetic DNA with codon-optimized sequences based on the commercially available vector pET29b. Co-expression of the two genes in the host Escherichia coli BL21(DE3) yielded a new class III lanthipeptide named boletupeptin. The structure of boletupeptin was proposed to have one unit of labionin, as determined by mass spectrometry experiments after reductive cleavage. This is the first report of a class III lanthipeptide from a myxobacterial origin.

Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster.

Psychoactive mushrooms in the genus Psilocybe have immense cultural value and have been used for centuries in Mesoamerica. Despite the recent surge of interest in these mushrooms due to the psychotherapeutic potential of their natural alkaloid psilocybin, their phylogeny and taxonomy remain substantially incomplete. Moreover, the recent elucidation of the psilocybin biosynthetic gene cluster is known for only five of ~165 species of Psilocybe, four of which belong to only one of two major clades. We set out to improve the phylogeny of Psilocybe using shotgun sequencing of fungarium specimens, from which we obtained 71 metagenomes including from 23 types, and conducting phylogenomic analysis of 2,983 single-copy gene families to generate a fully supported phylogeny. Molecular clock analysis suggests the stem lineage of Psilocybe arose ~67 mya and diversified ~56 mya. We also show that psilocybin biosynthesis first arose in Psilocybe, with 4 to 5 possible horizontal transfers to other mushrooms between 40 and 9 mya. Moreover, predicted orthologs of the psilocybin biosynthetic genes revealed two distinct gene orders within the biosynthetic gene cluster that corresponds to a deep split within the genus, possibly a signature of two independent acquisitions of the cluster within Psilocybe.

The selection landscape and genetic legacy of ancient Eurasians.

The Holocene (beginning around 12,000 years ago) encompassed some of the most significant changes in human evolution, with far-reaching consequences for the dietary, physical and mental health of present-day populations. Using a dataset of more than 1,600 imputed ancient genomes1, we modelled the selection landscape during the transition from hunting and gathering, to farming and pastoralism across West Eurasia. We identify key selection signals related to metabolism, including that selection at the FADS cluster began earlier than previously reported and that selection near the LCT locus predates the emergence of the lactase persistence allele by thousands of years. We also find strong selection in the HLA region, possibly due to increased exposure to pathogens during the Bronze Age. Using ancient individuals to infer local ancestry tracts in over 400,000 samples from the UK Biobank, we identify widespread differences in the distribution of Mesolithic, Neolithic and Bronze Age ancestries across Eurasia. By calculating ancestry-specific polygenic risk scores, we show that height differences between Northern and Southern Europe are associated with differential Steppe ancestry, rather than selection, and that risk alleles for mood-related phenotypes are enriched for Neolithic farmer ancestry, whereas risk alleles for diabetes and Alzheimer's disease are enriched for Western hunter-gatherer ancestry. Our results indicate that ancient selection and migration were large contributors to the distribution of phenotypic diversity in present-day Europeans.

Receptor tyrosine kinase gene expression profiling of orbital rhabdomyosarcoma unveils MET as a potential biomarker and therapeutic target.

Receptor tyrosine kinases (RTKs) serve as molecular targets for the development of novel personalized therapies in many malignancies. In the present study, expression pattern of receptor tyrosine kinases and its clinical significance in orbital RMS has been explored. Eighteen patients with histopathologically confirmed orbital RMS formed part of this study. Comprehensive q-PCR gene expression profiles of 19 RTKs were generated in the cases and controls. The patients were followed up for 59.53 ± 20.93 years. Clustering and statistical analysis tools were applied to identify the significant combination of RTKs associated with orbital rhabdomyosarcoma patients. mRNA overexpression of RTKs which included MET, AXL, EGFR was seen in 60-80% of cases; EGFR3, IGFR2, FGFR1, RET, PDGFR1, VEGFR2, PDGFR2 in 30-60% of cases; and EGFR4, FGFR3,VEGFR3 and ROS,IGFR1, EGFR1, FGFR2, VEGFR1 in 10-30% of cases. Immunoexpression of MET was seen in 89% of cases. A significant association was seen between MET mRNA and its protein expression. In all the cases MET gene expression was associated with worst overall survival (P = 0.03).There was a significant correlation of MET mRNA expression with RET, ROS, AXL, FGFR1, FGFR3, PDGFR1, IGFR1, VEGFR2, and EGFR3 genes. Association between MET gene and collective expression of RTKs was further evaluated by semi-supervised gene cluster analysis and Principal component analysis, which showed well-separated tumor clusters. MET gene overexpression could be a useful biomarker for identifying high risk orbital rhabdomyosarcoma patients. Well-separated tumor clusters confirmed the association between MET gene and collective expression of RTK genes. Therefore, the therapeutic potential of multi-kinase inhibitors targeting MET and the 9 other significant RTKs needs to be explored.

ABC-HuMi: the Atlas of Biosynthetic Gene Clusters in the Human Microbiome.

The human microbiome has emerged as a rich source of diverse and bioactive natural products, harboring immense potential for therapeutic applications. To facilitate systematic exploration and analysis of its biosynthetic landscape, we present ABC-HuMi: the Atlas of Biosynthetic Gene Clusters (BGCs) in the Human Microbiome. ABC-HuMi integrates data from major human microbiome sequence databases and provides an expansive repository of BGCs compared to the limited coverage offered by existing resources. Employing state-of-the-art BGC prediction and analysis tools, our database ensures accurate annotation and enhanced prediction capabilities. ABC-HuMi empowers researchers with advanced browsing, filtering, and search functionality, enabling efficient exploration of the resource. At present, ABC-HuMi boasts a catalog of 19 218 representative BGCs derived from the human gut, oral, skin, respiratory and urogenital systems. By capturing the intricate biosynthetic potential across diverse human body sites, our database fosters profound insights into the molecular repertoire encoded within the human microbiome and offers a comprehensive resource for the discovery and characterization of novel bioactive compounds. The database is freely accessible at https://www.ccb.uni-saarland.de/abc_humi/.

The gene cluster associated with strong biofilm-formation capacity by histamine-producing Lentilactobacillus parabuchneri encodes a sortase-mediated pilus and is located on a plasmid.

Histamine is a biogenic amine synthesized through the enzymatic decarboxylation of the amino acid histidine. It can accumulate at high concentrations in foods through the metabolism of certain bacteria, sometimes leading to adverse reactions in consumers. In cheese, histamine can accumulate at toxic levels; Lentilactobacillus parabuchneri has been identified the major cause of this problem. Previous studies have shown some L. parabuchneri strains to form biofilms on different surfaces, posing a contamination risk during cheese production, particularly for cheeses that are processed post-ripening (e.g., grating or slicing). The food contamination they cause can result in economic losses and even foodborne illness if histamine accumulates in the final product. The aim of the present work was to identify the genes of L. parabuchneri involved in biofilm formation, and to determine their function. The genomes of six strains with different biofilm-production capacities (strong, moderate and weak) were sequenced and analysed. A cluster of four genes, similar to those involved in sortase-mediated pilus formation, was identified in the strong biofilm-producers, suggesting it to have a role in surface adhesion. Cloning and heterologous expression in Lactococcus cremoris NZ9000 confirmed its functionality and involvement in adhesion and, therefore, in biofilm formation. PacBio sequencing showed this cluster to be located on a 33.4 kb plasmid, which might increase its chances of horizontal transmission. These findings provide insight into the genetic factors associated with biofilm formation in histamine-producing L. parabuchneri, and into the risks associated with this bacterium in cheese production.

Hox, homology, and parsimony: An organismal perspective.

Hox genes are important regulators in animal development. They often show a mosaic of conserved (e.g., longitudinal axis patterning) and lineage-specific novel functions (e.g., development of skeletal, sensory, or locomotory systems). Despite extensive research over the past decades, it remains controversial at which node in the animal tree of life the Hox cluster evolved. Its presence already in the last common metazoan ancestor has been proposed, although the genomes of both putative earliest extant metazoan offshoots, the ctenophores and the poriferans, are devoid of Hox sequences. The lack of Hox genes in the supposedly "simple"-built poriferans and their low number in cnidarians and the basally branching bilaterians, the xenacoelomorphs, seems to support the classical notion that the number of Hox genes is correlated with the degree of animal complexity. However, the 4-fold increase of the Hox cluster in xiphosurans, a basally branching chelicerate clade, as well as the situation in some teleost fishes that show a multitude of Hox genes compared to, e.g., human, demonstrates, that there is no per se direct correlation between organismal complexity and Hox number. Traditional approaches have tried to base homology on the morphological level on shared expression profiles of individual genes, but recent data have shown that, in particular with respect to Hox and other regulatory genes, complex gene-gene interactions rather than expression signatures of individual genes alone are responsible for shaping morphological traits during ontogeny. Accordingly, for sound homology assessments and reconstructions of character evolution on organ system level, additional independent datasets (e.g., morphological, developmental) need to be included in any such analyses. If supported by solid data, proposed structural homology should be regarded as valid and not be rejected solely on the grounds of non-parsimonious distribution of the character over a given phylogenetic topology.

Endophyte genomes support greater metabolic gene cluster diversity compared with non-endophytes in Trichoderma.

Trichoderma is a cosmopolitan genus with diverse lifestyles and nutritional modes, including mycotrophy, saprophytism, and endophytism. Previous research has reported greater metabolic gene repertoires in endophytic fungal species compared to closely-related non-endophytes. However, the extent of this ecological trend and its underlying mechanisms are unclear. Some endophytic fungi may also be mycotrophs and have one or more mycoparasitism mechanisms. Mycotrophic endophytes are prominent in certain genera like Trichoderma, therefore, the mechanisms that enable these fungi to colonize both living plants and fungi may be the result of expanded metabolic gene repertoires. Our objective was to determine what, if any, genomic features are overrepresented in endophytic fungi genomes in order to undercover the genomic underpinning of the fungal endophytic lifestyle. Here we compared metabolic gene cluster and mycoparasitism gene diversity across a dataset of thirty-eight Trichoderma genomes representing the full breadth of environmental Trichoderma's diverse lifestyles and nutritional modes. We generated four new Trichoderma endophyticum genomes to improve the sampling of endophytic isolates from this genus. As predicted, endophytic Trichoderma genomes contained, on average, more total biosynthetic and degradative gene clusters than non-endophytic isolates, suggesting that the ability to create/modify a diversity of metabolites potential is beneficial or necessary to the endophytic fungi. Still, once the phylogenetic signal was taken in consideration, no particular class of metabolic gene cluster was independently associated with the Trichoderma endophytic lifestyle. Several mycoparasitism genes, but no chitinase genes, were associated with endophytic Trichoderma genomes. Most genomic differences between Trichoderma lifestyles and nutritional modes are difficult to disentangle from phylogenetic divergences among species, suggesting that Trichoderma genomes maybe particularly well-equipped for lifestyle plasticity. We also consider the role of endophytism in diversifying secondary metabolism after identifying the horizontal transfer of the ergot alkaloid gene cluster to Trichoderma.

Genome-wide identification and expression analysis of GA20ox and GA3ox genes during pod development in peanut.

Background: Gibberellins (GAs) play important roles in regulating peanut growth and development. GA20ox and GA3ox are key enzymes involved in GA biosynthesis. These enzymes encoded by a multigene family belong to the 2OG-Fe (II) oxygenase superfamily. To date, no genome-wide comparative analysis of peanut AhGA20ox and AhGA3ox-encoding genes has been performed, and the roles of these genes in peanut pod development are not clear. Methods: A whole-genome analysis of AhGA20ox and AhGA3ox gene families in peanut was carried out using bioinformatic tools. The expression of these genes at different stage of pod development was analyzed using qRT-PCR. Results: In this study, a total of 15 AhGA20ox and five AhGA3ox genes were identified in peanut genome, which were distributed on 14 chromosomes. Phylogenetic analysis divided the GA20oxs and GA3oxs into three groups, but AhGA20oxs and AhGA3oxs in two groups. The conserved pattern of gene structure, cis-elements, and protein motifs further confirmed their evolutionary relationship in peanut. AhGA20ox and AhGA3ox genes were differential expressed at different stages of pod development. The strong expression of AhGA20ox1/AhGA20ox4, AhGA20ox12/AhGA20ox15, AhGA3ox1 and AhGA3ox4/AhGA3ox5 in S1-stage indicated that these genes could have a key role in controlling peg elongation. Furthermore, AhGA20ox and AhGA3ox also showed diverse expression patterns in different peanut tissues including leaves, main stems, flowers and inflorescences. Noticeably, AhGA20ox9/AhGA20ox11 and AhGA3o4/AhGA3ox5 were highly expressed in the main stem, whereas the AhGA3ox1 and AhGA20ox10 were strongly expressed in the inflorescence. The expression levels of AhGA20ox2/AhGA20ox3, AhGA20ox5/AhGA20ox6, AhGA20ox7/AhGA20ox8, AhGA20ox13/AhGA20ox14 and AhGA3ox2/AhGA3ox3 were high in the flowers, suggesting their involvement in flower development. These results provide a basis for deciphering the roles of AhGA20ox and AhGA3ox in peanut growth and development, especially in pod development.

Genome wide identification, structural characterization and phylogenetic analysis of High-Affinity potassium (HAK) ion transporters in common bean (Phaseolus vulgaris L.).

BACKGROUND: High-Affinity Potassium ions represent one of the most important and large group of potassium transporters. Although HAK genes have been studied in a variety of plant species, yet, remain unexplored in common bean. RESULTS: In the current study, 20 HAK genes were identified in common bean genome. Super-family "K_trans" domain was found in all PvHAK genes. Signals for localization of PvHAK proteins were detected in cell membrane. Fifty three HAKs genes, across diverse plant species, were divided into 5 groups based on sequential homology. Twelve pairs of orthologs genes were found in various plant species. PvHAKs genes were distributed unequally on 7 chromosomes with maximum number (7) mapped on chromosome 2 while only 1 PvHAK found on each chromosome 1, 4, and 6. Tandem gene duplication was witnessed in 2 paralog pairs while 1 pair exhibited segmental gene duplication. Five groups were made in PvHAK gene family based on Phylogeny. Maximum PvHAKs (10) were detected in Group-V while group-II composed of only 1 PvHAK gene. Variation was witnessed in number and size of motifs, and structure of PvHAKs associated with different groups. Light and hormone responsive elements contributed 57 and 24% share, respectively, to cis regulatory elements. qRT-PCR based results revealed significant increase in expression of all 4 PvHAK genes under low-potassium stress. CONCLUSION: The current study provides valuable information for further functional characterization and uncovering the molecular mechanism associated with Potassium transportation in plants.

The Hypervariable Tpr Multigene Family of Theileria Parasites, Defined by a Conserved, Membrane-Associated, C-Terminal Domain, Includes Several Copies with Defined Orthology Between Species.

Multigene families often play an important role in host-parasite interactions. One of the largest multigene families in Theileria parva, the causative agent of East Coast fever, is the T. parva repeat (Tpr) gene family. The function of the putative Tpr proteins remains unknown. The initial publication of the T. parva reference genome identified 39 Tpr family open reading frames (ORFs) sharing a conserved C-terminal domain. Twenty-eight of these are clustered in a central region of chromosome 3, termed the "Tpr locus", while others are dispersed throughout all four nuclear chromosomes. The Tpr locus contains three of the four assembly gaps remaining in the genome, suggesting the presence of additional, as yet uncharacterized, Tpr gene copies. Here, we describe the use of long-read sequencing to attempt to close the gaps in the reference assembly of T. parva (located among multigene families clusters), characterize the full complement of Tpr family ORFs in the T. parva reference genome, and evaluate their evolutionary relationship with Tpr homologs in other Theileria species. We identify three new Tpr family genes in the T. parva reference genome and show that sequence similarity among paralogs in the Tpr locus is significantly higher than between genes outside the Tpr locus. We also identify sequences homologous to the conserved C-terminal domain in five additional Theileria species. Using these sequences, we show that the evolution of this gene family involves conservation of a few orthologs across species, combined with gene gains/losses, and species-specific expansions.

Efficient Protein Expression and Biosynthetic Gene Cluster Regulation in Bacillus subtilis Driven by a T7-BOOST System.

Bacillus subtilis is a generally recognized as safe microorganism that is widely used for protein expression and chemical production, but has a limited number of genetic regulatory components compared with the Gram-negative model microorganism Escherichia coli. In this study, a two-module plug-and-play T7-based optimized output strategy for transcription (T7-BOOST) systems with low leakage expression and a wide dynamic range was constructed based on the inducible promoters Phy-spank and PxylA. The first T7 RNA polymerase-driven module was seamlessly integrated into the genome based on the CRISPR/Cpf1 system, while the second expression control module was introduced into low, medium, and high copy plasmids for characterization. As a proof of concept, the T7-BOOST systems were successfully employed for whole-cell catalysis production of γ-aminobutyric acid (109.8 g/L with a 98.0% conversion rate), expression of human αS1 casein and human lactoferrin, and regulation of exogenous lycopene biosynthetic gene cluster and endogenous riboflavin biosynthetic gene cluster. Overall, the T7-BOOST system serves as a stringent, controllable, and effective tool for regulating gene expression in B. subtilis.

Yeast-based heterologous production of the Colletochlorin family of fungal secondary metabolites.

Transcriptomic studies have revealed that fungal pathogens of plants activate the expression of numerous biosynthetic gene clusters (BGC) exclusively when in presence of a living host plant. The identification and structural elucidation of the corresponding secondary metabolites remain challenging. The aim was to develop a polycistronic system for heterologous expression of fungal BGCs in Saccharomyces cerevisiae. Here we adapted a polycistronic vector for efficient, seamless and cost-effective cloning of biosynthetic genes using in vivo assembly (also called transformation-assisted recombination) directly in Escherichia coli followed by heterologous expression in S. cerevisiae. Two vectors were generated with different auto-inducible yeast promoters and selection markers. The effectiveness of these vectors was validated with fluorescent proteins. As a proof-of-principle, we applied our approach to the Colletochlorin family of molecules. These polyketide secondary metabolites were known from the phytopathogenic fungus Colletotrichum higginsianum but had never been linked to their biosynthetic genes. Considering the requirement for a halogenase, and by applying comparative genomics, we identified a BGC putatively involved in the biosynthesis of Colletochlorins in C. higginsianum. Following the expression of those genes in S. cerevisiae, we could identify the presence of the precursor Orsellinic acid, Colletochlorins and their non-chlorinated counterparts, the Colletorins. In conclusion, the polycistronic vectors described herein were adapted for the host S. cerevisiae and allowed to link the Colletochlorin compound family to their corresponding biosynthetic genes. This system will now enable the production and purification of infection-specific secondary metabolites of fungal phytopathogens. More widely, this system could be applied to any fungal BGC of interest.

Genome-wide identification, comparative analysis and functional roles in flavonoid biosynthesis of cytochrome P450 superfamily in pear (Pyrus spp.).

BACKGROUND: The cytochrome P450 (CYP) superfamily is the largest enzyme metabolism family in plants identified to date, and it is involved in many biological processes, including secondary metabolite biosynthesis, hormone metabolism and stress resistance. However, the P450 gene superfamily has not been well studied in pear (Pyrus spp.). RESULTS: Here, the comprehensive identification and a comparative analysis of P450 superfamily members were conducted in cultivated and wild pear genomes. In total, 338, 299 and 419 P450 genes were identified in Chinese white pear, European pear and the wild pear, respectively. Based on the phylogenetic analyses, pear P450 genes were divided into ten clans, comprising 48 families. The motif and gene structure analyses further supported this classification. The expansion of the pear P450 gene family was attributed to whole-genome and single-gene duplication events. Several P450 gene clusters were detected, which have resulted from tandem and proximal duplications. Purifying selection was the major force imposed on the long-term evolution of P450 genes. Gene dosage balance, subfunctionalization and neofunctionalization jointly drove the retention and functional diversification of P450 gene pairs. Based on the association analysis between transcriptome expression profiles and flavonoid content during fruit development, three candidate genes were identified as being closely associated with the flavonoid biosynthesis, and the expression of one gene was further verified using qRT-PCR and its function was validated through transient transformation in pear fruit. CONCLUSIONS: The study results provide insights into the evolution and biological functions of P450 genes in pear.

The complete sequence of a human Y chromosome.

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure that includes long palindromes, tandem repeats and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4,5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, showing the complete ampliconic structures of gene families TSPY, DAZ and RBMY; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a previous assembly of the CHM13 genome4 and mapped available population variation, clinical variants and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

Identification and characterization of a novel bacteriocin gene cluster in Lysinibacillus boronitolerans.

Although the antibiotics inhibit or kill pathogens, the abuse leads to the resistance formation and even "Super Bacteria." Therefore, it is urgent to explore the natural and safe alternatives such as bacteriocin. In this study, an uncharacterized bacteriocin gene cluster for Lysinibacillus boronitolerans was first predicted by genome sequencing and bioinformatics analysis, of which including two biosynthetic genes, a regulatory gene, a transport-related gene, and six other genes. Subsequently, the 10.24-kb gene cluster was expressed in Escherichia coli BL21, and the lysate effectively inhibited the growths of pathogenic bacteria containing Bacillus pumilus, Bacillus velezensis, Pseudomonas syringae pv. tomato DC3000, and Xanthomonas axonopodis pv. manihotis. The antibacterial substance was purified by 70% ammonium sulfate precipitation and further identified by liquid chromatography-tandem mass spectrometry. The results showed that the antibacterial substance consisted of 44 amino acids and had 24.1% sequence identity with the cyanobacterin Piricyclamide 7005 E4 PirE4, a bacteriocin analogue. The minimal set of genes required for the biosynthesis of the antibacterial substance was determined by site-directed mutagenesis, suggesting both a transcriptional repressor and a phosphohydroxythreonine transaminase were essential. Subsequently, the evolution and conservation of the two proteins were analyzed among 22 Lysinibacillus species. Among them, the residues responsible for functions were identified. Collectively, our results set a solid foundation for investigation of the biosynthesis and application of bacteriocin.

Global analyses of biosynthetic gene clusters in phytobiomes reveal strong phylogenetic conservation of terpenes and aryl polyenes.

There are gaps in our understandings on how did the evolutionary relationships among members of the phytobiomes shape their ability to produce tremendously complex specialized metabolites under the influence of plant host. To determine these relationships, we investigated the phylogenetic conservation of biosynthetic gene clusters (BGCs) on a global collection of 4,519 high-quality and nonredundant (out of 12,181) bacterial isolates and metagenome-assembled genomes from 47 different plant hosts and soil, by adopting three independent phylogenomic approaches (D-test, Pagel's λ, and consenTRAIT). We report that the BGCs are phylogenetically conserved to varying strengths and depths in their different classes. We show that the ability to produce specialized metabolites qualifies as a complex trait, and the depth of conservation is equivalent to ecologically relevant complex microbial traits. Interestingly, terpene and aryl polyene BGCs had the strongest phylogenetic conservation in the phytobiomes, but not in the soil microbiomes. Furthermore, we showed that terpenes are largely uncharacterized in phytobiomes and pinpointed specific clades that harbor potentially novel terpenes. Taken together, this study sheds light on the evolution of specialized metabolites' biosynthesis potential in phytobiomes under the influence of plant hosts and presents strategies to rationally guide the discovery of potentially novel classes of metabolites. IMPORTANCE This study expands our understandings of the biosynthetic potential of phytobiomes by using such worldwide and extensive collection of microbiomes from plants and soil. Apart from providing such vital resource for the plant microbiome researchers, this study provides fundamental insights into the evolution of biosynthetic gene clusters (BGCs) in phytobiomes under the influence of plant host. Specifically, we report that the strength of phylogenetic conservation in microbiomes varies for different classes of BGCs and is influenced as a result of plant host association. Furthermore, our results indicate that biosynthetic potential of specialized metabolites is deeply conserved equivalent to other complex and ecologically relevant microbial traits. Finally, for the most conserved class of specialized metabolites (terpenes), we identified clades harboring potentially novel class of molecules. Future studies could focus on plant-microbe coevolution and interactions through specialized metabolites building upon these findings.