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1.
Genes (Basel) ; 14(1)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36672935

RESUMO

The APETALA2/Ethylene-Responsive Transcriptional Factors containing conservative AP2/ERF domains constituted a plant-specific transcription factor (TF) superfamily, called AP2/ERF. The configuration of the AP2/ERF superfamily in maize has remained unresolved. In this study, we identified the 229 AP2/ERF genes in the latest (B73 RefGen_v5) maize reference genome. Phylogenetic classification of the ZmAP2/ERF family members categorized it into five clades, including 27 AP2 (APETALA2), 5 RAV (Related to ABI3/VP), 89 DREB (dehydration responsive element binding), 105 ERF (ethylene responsive factors), and a soloist. The duplication events of the paralogous genes occurred from 1.724-25.855 MYA, a key route to maize evolution. Structural analysis reveals that they have more introns and few exons. The results showed that 32 ZmAP2/ERFs regulate biotic stresses, and 24 ZmAP2/ERFs are involved in responses towards abiotic stresses. Additionally, the expression analysis showed that DREB family members are involved in plant sex determination. The real-time quantitative expression profiling of ZmAP2/ERFs in the leaves of the maize inbred line B73 under ABA, JA, salt, drought, heat, and wounding stress revealed their specific expression patterns. Conclusively, this study unveiled the evolutionary pathway of ZmAP2/ERFs and its essential role in stress and developmental processes. The generated information will be useful for stress resilience maize breeding programs.


Assuntos
Família Multigênica , Zea mays , Zea mays/genética , Zea mays/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Melhoramento Vegetal , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Etilenos
2.
Philos Trans R Soc Lond B Biol Sci ; 378(1871): 20220026, 2023 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-36633280

RESUMO

Non-ribosomal peptide synthetases (NRPSs) biosynthesize many pharmaceuticals and virulence factors. The biosynthesis of these natural peptide products from biosynthetic gene clusters depends on complex regulations in bacteria. However, our current knowledge of NRPSs is based on enzymological studies using full NRPS systems and/or a single NRPS domain in heterologous hosts. Chemical and/or biochemical strategies to capture the endogenous activities of NRPSs facilitate studies on NRPS cell biology in bacterial cells. Here, we describe a chemical scaffold for the rapid and selective photoaffinity labelling of NRPSs in purified systems, crude biological samples and living bacterial cells. We synthesized photoaffinity labelling probes coupled with 5'-O-N-(phenylalanyl)sulfamoyladenosine with clickable alkyl diazirine or trifluoromethyl phenyl diazirine. We found that a trifluoromethyl phenyl diazirine-based probe cross-linked the Phe-activating domain of a GrsA-NRPS with high selectivity and sensitivity at shorter ultraviolet (UV) irradiation times (less than 5 min) relative to a prototypical benzophenone-based probe. Our results demonstrated that this quick labelling protocol can prevent damage to proteins and cells caused by long UV irradiation times, providing a mild photoaffinity labelling method for biological samples. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.


Assuntos
Bactérias , Diazometano , Diazometano/metabolismo , Bactérias/genética , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Família Multigênica
3.
Int J Mol Sci ; 24(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36674628

RESUMO

Streptomyces lunaelactis strains have been isolated from moonmilk deposits, which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of a Streptomyces species with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup of S. lunaelactis strains for which in silico analysis predicted the production of nonribosomal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts of S. lunaelactis strains either holding or not holding BGC 28a combined with MS/MS-guided peptidogenomics and 1H/13C NMR allowed us to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, with 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a significant bactericidal activity of lunaemycins against Gram-positive bacteria, including multi-drug resistant clinical isolates. Our work demonstrates how an accurate in silico analysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.


Assuntos
Anti-Infecciosos , Streptomyces , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Espectrometria de Massas em Tandem , Anti-Infecciosos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Família Multigênica
4.
Arch Microbiol ; 205(2): 75, 2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36708387

RESUMO

Fungi of the genus Penicillium section Sclerotiora have as their main characteristic the presence of orange-pigmented mycelium, which is associated with sclerotiorin, a chlorinated secondary metabolite of the azaphilone subclass of polyketides. Sclerotiorin presents anti-diabetes, antioxidant, anti-inflammatory, anti-Alzheimer, antiviral, and antimicrobial activities, which has always attracted the attention of researchers worldwide. During our ongoing search for azaphilone-producing Amazonian fungi, the strain of Penicillium MMSRG-058 was isolated as an endophyte from the roots of Duguetia stelechantha and showed great capacity for producing sclerotiorin-like metabolites. Using multilocus phylogeny, this strain was identified as Penicillium meliponae. Moreover, based on the genome mining of this strain through the reverse approach, a cluster of putative biosynthetic genes (BGC) responsible for the biosynthesis of sclerotiorin-like metabolites (scl cluster) was identified. The knockout of the sclA (highly reducing PKS) and sclI (non-reducing PKS) genes resulted in mutants with loss of mycelial pigmentation and terminated the biosynthesis of sclerotiorin-like metabolites: geumsanol B, chlorogeumsanol B, 7-deacetylisochromophilone VI, isochromophilone VI, ochrephilone, isorotiorin, and sclerotiorin. Based on these results, a biosynthetic pathway was proposed considering the homology of BGC scl genes with the azaphilone BGCs that have already been functionally characterized.


Assuntos
Penicillium , Técnicas de Inativação de Genes , Penicillium/genética , Penicillium/metabolismo , Fungos/genética , Família Multigênica
5.
J Am Chem Soc ; 145(4): 2342-2353, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36669196

RESUMO

Investigating the ecological context of microbial predator-prey interactions enables the identification of microorganisms, which produce multiple secondary metabolites to evade predation or to kill the predator. In addition, genome mining combined with molecular biology methods can be used to identify further biosynthetic gene clusters that yield new antimicrobials to fight the antimicrobial crisis. In contrast, classical screening-based approaches have limitations since they do not aim to unlock the entire biosynthetic potential of a given organism. Here, we describe the genomics-based identification of keanumycins A-C. These nonribosomal peptides enable bacteria of the genus Pseudomonas to evade amoebal predation. While being amoebicidal at a nanomolar level, these compounds also exhibit a strong antimycotic activity in particular against the devastating plant pathogen Botrytis cinerea and they drastically inhibit the infection of Hydrangea macrophylla leaves using only supernatants of Pseudomonas cultures. The structures of the keanumycins were fully elucidated through a combination of nuclear magnetic resonance, tandem mass spectrometry, and degradation experiments revealing an unprecedented terminal imine motif in keanumycin C extending the family of nonribosomal amino acids by a highly reactive building block. In addition, chemical synthesis unveiled the absolute configuration of the unusual dihydroxylated fatty acid of keanumycin A, which has not yet been reported for this lipodepsipeptide class. Finally, a detailed genome-wide microarray analysis of Candida albicans exposed to keanumycin A shed light on the mode-of-action of this potential natural product lead, which will aid the development of new pharmaceutical and agrochemical antifungals.


Assuntos
Anti-Infecciosos , Lipopeptídeos , Lipopeptídeos/farmacologia , Lipopeptídeos/química , Aminoácidos/genética , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Genômica , Família Multigênica
6.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614182

RESUMO

Auxin is a key regulator that virtually controls almost every aspect of plant growth and development throughout its life cycle. As the major components of auxin signaling, auxin response factors (ARFs) play crucial roles in various processes of plant growth and development. In this study, a total of 35 PtrARF genes were identified, and their phylogenetic relationships, chromosomal locations, synteny relationships, exon/intron structures, cis-elements, conserved motifs, and protein characteristics were systemically investigated. We also analyzed the expression patterns of these PtrARF genes and revealed that 16 of them, including PtrARF1, 3, 7, 11, 13-17, 21, 23, 26, 27, 29, 31, and 33, were preferentially expressed in primary stems, while 15 of them, including PtrARF2, 4, 6, 9, 10, 12, 18-20, 22, 24, 25, 28, 32, and 35, participated in different phases of wood formation. In addition, some PtrARF genes, with at least one cis-element related to indole-3-acetic acid (IAA) or abscisic acid (ABA) response, responded differently to exogenous IAA and ABA treatment, respectively. Three PtrARF proteins, namely PtrARF18, PtrARF23, and PtrARF29, selected from three classes, were characterized, and only PtrARF18 was a transcriptional self-activator localized in the nucleus. Moreover, Y2H and bimolecular fluorescence complementation (BiFC) assay demonstrated that PtrARF23 interacted with PtrIAA10 and PtrIAA28 in the nucleus, while PtrARF29 interacted with PtrIAA28 in the nucleus. Our results provided comprehensive information regarding the PtrARF gene family, which will lay some foundation for future research about PtrARF genes in tree development and growth, especially the wood formation, in response to cellular signaling and environmental cues.


Assuntos
Populus , Madeira , Madeira/metabolismo , Populus/metabolismo , Filogenia , Família Multigênica , Proteínas de Plantas/metabolismo , Perfilação da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ácidos Indolacéticos/farmacologia , Ácidos Indolacéticos/metabolismo , Hormônios , Regulação da Expressão Gênica de Plantas
7.
PeerJ ; 11: e14690, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36710860

RESUMO

FCS-like zinc finger (FLZ) is a plant-specific gene family that plays an important regulatory role in plant growth and development and its response to stress. However, studies on the characteristics and functions of cotton FLZ family genes are still lacking. This study systematically identified members of the cotton FLZ gene family based on cotton genome data. The cotton FLZ family genes were systematically analyzed by bioinformatics, and their expression patterns in different tissues and under low-temperature stress were analyzed by transcriptome and qRT-PCR. The G. hirsutum genome contains 56 FLZ genes distributed on 20 chromosomes, and most of them are located in the nucleus. According to the number and evolution analysis of FLZ family genes, FLZ family genes can be divided into five subgroups in cotton. The G. hirsutum FLZ gene has a wide range of tissue expression types, among which the expression is generally higher in roots, stems, leaves, receptacles and calyx. Through promoter analysis, it was found that it contained the most cis-acting elements related to methyl jasmonate (MeJA) and abscisic acid (ABA). Combined with the promoter and qRT-PCR results, it was speculated that GhFLZ11, GhFLZ25, GhFLZ44 and GhFLZ55 were involved in the response of cotton to low-temperature stress. Taken together, our findings suggest an important role for the FLZ gene family in the cotton response to cold stress. This study provides an important theoretical basis for further research on the function of the FLZ gene family and the molecular mechanism of the cotton response to low temperature.


Assuntos
Genoma de Planta , Gossypium , Gossypium/genética , Genoma de Planta/genética , Temperatura , Família Multigênica/genética , Dedos de Zinco/genética
8.
Int J Biol Macromol ; 229: 766-777, 2023 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-36610562

RESUMO

Invertases are ubiquitous enzymes that catalyze the unalterable cleavage of sucrose into glucose and fructose, and are crucially involved in plant growth, development and stress response. In this study, a total of 17 putative invertase genes, including 3 cell wall invertases, 3 vacuolar invertases, and 11 neutral invertases were identified in apple genome. Subcellular localization of MdNINV7 and MdNINV11 indicated that both invertases were located in the cytoplasm. Comprehensive analyses of physicochemical properties, chromosomal localization, genomic characterization, and gene evolution of MdINV family were conducted. Gene duplication revealed that whole-genome or segmental duplication and random duplication might have been the major driving force for MdINVs expansion. Selection index values, ω, showed strong evidence of positive selection signatures among the INV clusters. Gene expression analysis indicated that MdNINV1/3/6/7 members are crucially involved in fruit development and sugar accumulation. Similarly, expression profiles of MdCWINV1, MdVINV1, and MdNINV1/2/7/11 suggested their potential roles in response to cold stress. Furthermore, overexpression of MdNINV11 in apple calli at least in part promoted the expression of MdCBF1-5 and H2O2 detoxification in response to cold. Overall, our results will be useful for understanding the functions of MdINVs in the regulation of apple fruit development and cold stress response.


Assuntos
Malus , beta-Frutofuranosidase , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo , Malus/genética , Malus/metabolismo , Peróxido de Hidrogênio/metabolismo , Família Multigênica , Filogenia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
9.
Genome Res ; 33(1): 32-44, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36617663

RESUMO

Homeobox genes encode transcription factors with essential roles in patterning and cell fate in developing animal embryos. Many homeobox genes, including Hox and NK genes, are arranged in gene clusters, a feature likely related to transcriptional control. Sparse taxon sampling and fragmentary genome assemblies mean that little is known about the dynamics of homeobox gene evolution across Lepidoptera or about how changes in homeobox gene number and organization relate to diversity in this large order of insects. Here we analyze an extensive data set of high-quality genomes to characterize the number and organization of all homeobox genes in 123 species of Lepidoptera from 23 taxonomic families. We find most Lepidoptera have around 100 homeobox loci, including an unusual Hox gene cluster in which the lab gene is repositioned and the ro gene is next to pb A topologically associating domain spans much of the gene cluster, suggesting deep regulatory conservation of the Hox cluster arrangement in this insect order. Most Lepidoptera have four Shx genes, divergent zen-derived loci, but these loci underwent dramatic duplication in several lineages, with some moths having over 165 homeobox loci in the Hox gene cluster; this expansion is associated with local LINE element density. In contrast, the NK gene cluster content is more stable, although there are differences in organization compared with other insects, as well as major rearrangements within butterflies. Our analysis represents the first description of homeobox gene content across the order Lepidoptera, exemplifying the potential of newly generated genome assemblies for understanding genome and gene family evolution.


Assuntos
Borboletas , Genes Homeobox , Animais , Filogenia , Família Multigênica , Genômica , Evolução Molecular
10.
Nat Prod Rep ; 40(1): 7-8, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36622035

RESUMO

Filamentous fungi are highly diverse eukaryotes that inhabit all known ecosystems on earth. Estimates suggest that more than 2 × 106 species are likely to exist, and analyses of typical fungal genomes suggest they harbour around 50 biosynthetic gene clusters on average. The biosynthetic potential of these organisms is thus vast. Fungi produce all the main classes of secondary metabolites, and numerous hybrid compounds. Many are highly useful in medicine such as the 'classic' special metabolites penicillins, cephalosporins, statins and mycophenolic acid, and new antimicrobial agents such as the pleuromutilins and enfumafungins that overcome specific patterns of resistance. Fungi differentiated from bacteria more than a billion years ago, so there has been plenty of time for uniquely fungal biosynthetic systems to evolve.


Assuntos
Produtos Biológicos , Produtos Biológicos/metabolismo , Ecossistema , Fungos/genética , Fungos/metabolismo , Metabolismo Secundário/genética , Genoma Fúngico , Família Multigênica
11.
Sci Rep ; 13(1): 84, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36596810

RESUMO

The SQUAMOSA promoter binding-like protein (SPL) is a specific transcription factor that affects plant growth and development. The SPL gene family has been explored in various plants, but information about these genes in alfalfa is limited. This study, based on the whole genome data of alfalfa SPL, the fundamental physicochemical properties, phylogenetic evolution, gene structure, cis-acting elements, and gene expression of members of the MsSPL gene family were analyzed by bioinformatics methods. We identified 82 SPL sequences in the alfalfa, which were annotated into 23 genes, including 7 (30.43%) genes with four alleles, 10 (43.47%) with three, 3 (13.04%) with two, 3 (13.04%) with one allele. These SPL genes were divided into six groups, that are constructed from A. thaliana, M. truncatula and alfalfa. Chromosomal localization of the identified SPL genes showed arbitary distribution. The subcellular localization predictions showed that all MsSPL proteins were located in the nucleus. A total of 71 pairs of duplicated genes were identified, and segmental duplication mainly contributed to the expansion of the MsSPL gene family. Analysis of the Ka/Ks ratios indicated that paralogs of the MsSPL gene family principally underwent purifying selection. Protein-protein interaction analysis of MsSPL proteins were performed to predict their roles in potential regulatory networks. Twelve cis-acting elements including phytohormone and stress elements were detected in the regions of MsSPL genes. We further analyzed that the MsSPLs had apparent responses to abiotic stresses such as drought and salt and the biotic stress of methyl jasmonate. These results provide comprehensive information on the MsSPL gene family in alfalfa and lay a solid foundation for elucidating the biological functions of MsSPLs. This study also provides valuable on the regulation mechanism and function of MsSPLs in response to biotic and abiotic stresses.


Assuntos
Medicago sativa , Proteínas de Plantas , Medicago sativa/genética , Medicago sativa/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Estresse Fisiológico/genética , Regulação da Expressão Gênica de Plantas , Família Multigênica , Genoma de Planta
12.
BMC Genomics ; 24(1): 7, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624379

RESUMO

BACKGROUND: ORP (Oxysterol-binding protein-related proteins) genes play a role in lipid metabolism, vesicular transferring and signaling, and non-vesicular sterol transport. However, no systematic identification and analysis of ORP genes have been reported in cotton. RESULT: In this study, we identified 14, 14, 7, and 7 ORP genes in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. Phylogenetic analysis showed that all ORP genes could be classified into four groups. Gene structure and conserved motif analysis suggest that the function of this gene family was conserved. The Ka/Ks analysis showed that this gene family was exposed to purifying selection during evolution. Transcriptome data showed that four ORP genes, especially GhORP_A02, were induced by abiotic stress treatment. The cis-acting elements in the ORP promoters were responsive to phytohormones and various abiotic stresses. The silenced plants of GhORP_A02 were more sensitive to drought stress when compared to control. CONCLUSION: The major finding of this study shed light on the potential role of ORP genes in abiotic stress and provided a fundamental resource for further analysis in cotton.


Assuntos
Gossypium , Gossypium/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Família Multigênica , Estresse Fisiológico/genética
13.
PeerJ ; 11: e14637, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36655051

RESUMO

Background: The calmodulin-like (CML) protein is a crucial Ca2+-binding protein that can sense and conduct the Ca2+ signal in response to extracellular stimuli. The CML protein families have been identified and characterized in many species. Nevertheless, scarce information on cucumber CML is retrievable. Methods: In this study, bioinformatic analyses, including gene structure, conserved domain, phylogenetic relationship, chromosome distribution, and gene synteny, were comprehensively performed to identify and characterize CsCML gene members. Spatiotemporal expression analysis in different organs and environment conditions were assayed with real-time quantitative polymerase chain reaction (qRT-PCR). Results: Forty-four CsCMLs family members were well characterized, and the results showed that the 44 CsCML proteins contained one to four EF-hand domains without other functional domains. Most of the CsCML proteins were intron-less and unevenly distributed on seven chromosomes; two tandemly duplicated gene pairs and three segmentally duplicated gene pairs were identified in the cucumber genome. Cis-acting element analysis showed that the hormone, stress, and plant growth and development-related elements were in the promotor regions. In addition, spatiotemporal expression analysis revealed distinctive expression patterns for CsCML genes in different tissues and environmental conditions, and a putative protein interaction network also confirmed their potential role in responding to various stimuli. These results provide a foundation for understanding CsCMLs and provide a theoretical basis for further study of the physiological functions of CsCMLs.


Assuntos
Cucumis sativus , Cucumis sativus/genética , Genoma de Planta/genética , Calmodulina/genética , Filogenia , Família Multigênica/genética
14.
BMC Plant Biol ; 23(1): 43, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658501

RESUMO

BACKGROUND: Cyclin-dependent kinases (CDKs) are a predominant group of serine/threonine protein kinases that have multi-faceted functions in eukaryotes. The plant CDK members have well-known roles in cell cycle progression, transcriptional regulation, DNA repair, abiotic stress and defense responses, making them promising targets for developing stress adaptable high-yielding crops. There is relatively sparse information available on the CDK family genes of cultivated oilseed crop peanut and its diploid progenitors. RESULTS: We have identified 52 putative cyclin-dependent kinases (CDKs) and CDK-like (CDKLs) genes in Arachis hypogaea (cultivated peanut) and total 26 genes in each diploid parent of cultivated peanut (Arachis duranensis and Arachis ipaensis). Both CDK and CDKL genes were classified into eight groups based on their cyclin binding motifs and their phylogenetic relationship with Arabidopsis counterparts. Genes in the same subgroup displayed similar exon-intron structure and conserved motifs. Further, gene duplication analysis suggested that segmental duplication events played major roles in the expansion and evolution of CDK and CDKL genes in cultivated peanuts. Identification of diverse cis-acting response elements in CDK and CDKL genes promoter indicated their potential fundamental roles in multiple biological processes. Various gene expression patterns of CDKs and CDKLs in different peanut tissues suggested their involvement during growth and development. In addition, qRT-PCR analysis demonstrated that most representing CDK and CDKL gene family members were significantly down-regulated under ABA, PEG and mannitol treatments. CONCLUSIONS: Genome-wide analysis offers a comprehensive understanding of the classification, evolution, gene structure, and gene expression profiles of CDK and CDKL genes in cultivated peanut and their diploid progenitors. Additionally, it also provides cell cycle regulatory gene resources for further functional characterization to enhance growth, development and abiotic stress tolerance.


Assuntos
Arachis , Genoma de Planta , Arachis/genética , Arachis/metabolismo , Filogenia , Duplicação Gênica , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Regulação da Expressão Gênica de Plantas , Família Multigênica
15.
Microb Cell Fact ; 22(1): 15, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658647

RESUMO

BACKGROUND: Spinosad is a macrolide insecticide with the tetracyclic lactone backbone to which forosamine and tri-O-methylrhamnose are attached. Both the sugar moieties are essential for its insecticidal activity. In biosynthesis of spinosad, the amino group of forosamine is dimethylated by SpnS and then transferred onto the lactone backbone by SpnP. Because the spinosad native producer is difficult to genetically manipulate, we previously changed promoters, ribosome binding sites and start codons of 23 spinosad biosynthetic genes to construct an artificial gene cluster which resulted in a 328-fold yield improvement in the heterologous host Streptomyces albus J1074 compared with the native gene cluster. However, in fermentation of J1074 with the artificial gene cluster, the N-monodesmethyl spinosad with lower insecticidal activity was always produced with the same titer as spinosad. RESULTS: By tuning expression of SpnS with an inducible promotor, we found that the undesired less active byproduct N-monodesmethyl spinosad was produced when SpnS was expressed at low level. Although N-monodesmethyl spinosad can be almost fully eliminated with high SpnS expression level, the titer of desired product spinosad was only increased by less than 38%. When the forosaminyl transferase SpnP was further overexpressed together with SpnS, the titer of spinosad was improved by 5.3 folds and the content of N-desmethyl derivatives was decreased by ~ 90%. CONCLUSION: N-monodesmethyl spinosad was produced due to unbalanced expression of spnS and upstream biosynthetic genes in the refactored artificial gene cluster. The accumulated N-desmethyl forosamine was transferred onto the lactone backbone by SpnP. This study suggested that balanced expression of biosynthetic genes should be considered in the refactoring strategy to avoid accumulation of undesired intermediates or analogues which may affect optimal production of desired compounds.


Assuntos
Streptomyces griseus , Transferases , Transferases/genética , Metiltransferases/genética , Streptomyces griseus/metabolismo , Macrolídeos/metabolismo , Família Multigênica
16.
BMC Plant Biol ; 23(1): 48, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36683040

RESUMO

BACKGROUND: The AP2/ERF gene family is a superfamily of transcription factors that are important in the response of plants to abiotic stress and development. However, comprehensive research of the AP2/ERF genes in the Solanaceae family is lacking. RESULTS: Here, we updated the annotation of AP2/ERF genes in the genomes of eight Solanaceae species, as well as Arabidopsis thaliana and Oryza sativa. We identified 2,195 AP2/ERF genes, of which 368 (17%) were newly identified. Based on phylogenetic analyses, we observed expansion of the copy number of these genes, especially those belonging to specific Ethylene-Responsive Factor (ERF) subgroups of the Solanaceae. From the results of chromosomal location and synteny analyses, we identified that the AP2/ERF genes of the pepper (Capsicum annuum), the tomato (Solanum lycopersicum), and the potato (Solanum tuberosum) belonging to ERF subgroups form a tandem array and most of them are species-specific without orthologs in other species, which has led to differentiation of AP2/ERF gene repertory among Solanaceae. We suggest that these genes mainly emerged through recent gene duplication after the divergence of these species. Transcriptome analyses showed that the genes have a putative function in the response of the pepper and tomato to abiotic stress, especially those in ERF subgroups. CONCLUSIONS: Our findings will provide comprehensive information on AP2/ERF genes and insights into the structural, evolutionary, and functional understanding of the role of these genes in the Solanaceae.


Assuntos
Variações do Número de Cópias de DNA , Solanum tuberosum , Filogenia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Família Multigênica , Solanum tuberosum/genética , Etilenos , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas
17.
Microbiome ; 11(1): 13, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36691088

RESUMO

BACKGROUND: It is well-known that the microbiome produces a myriad of specialised metabolites with diverse functions. To better characterise their structures and identify their producers in complex samples, integrative genome and metabolome mining is becoming increasingly popular. Metabologenomic co-occurrence-based correlation scoring methods facilitate the linking of metabolite mass fragmentation spectra (MS/MS) to their cognate biosynthetic gene clusters (BGCs) based on shared absence/presence patterns of metabolites and BGCs in paired omics datasets of multiple strains. Recently, these methods have been made more readily accessible through the NPLinker platform. However, co-occurrence-based approaches usually result in too many candidate links to manually validate. To address this issue, we introduce a generic feature-based correlation method that matches chemical compound classes between BGCs and MS/MS spectra. RESULTS: To automatically reduce the long lists of potential BGC-MS/MS spectrum links, we match natural product (NP) ontologies previously independently developed for genomics and metabolomics and developed NPClassScore: an empirical class matching score that we also implemented in the NPLinker platform. By applying NPClassScore on three paired omics datasets totalling 189 bacterial strains, we show that the number of links is reduced by on average 63% as compared to using a co-occurrence-based strategy alone. We further demonstrate that 96% of experimentally validated links in these datasets are retained and prioritised when using NPClassScore. CONCLUSION: The matching genome-metabolome class ontologies provide a starting point for selecting plausible candidates for BGCs and MS/MS spectra based on matching chemical compound class ontologies. NPClassScore expedites genome/metabolome data integration, as relevant BGC-metabolite links are prioritised, and researchers are faced with substantially fewer proposed BGC-MS/MS links to manually inspect. We anticipate that our addition to the NPLinker platform will aid integrative omics mining workflows in discovering novel NPs and understanding complex metabolic interactions in the microbiome. Video Abstract.


Assuntos
Vias Biossintéticas , Espectrometria de Massas em Tandem , Vias Biossintéticas/genética , Genômica , Metabolômica/métodos , Família Multigênica
18.
J Am Chem Soc ; 145(3): 1886-1896, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36634356

RESUMO

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or ß-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and ß-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a ß-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 µM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.


Assuntos
Streptomyces , Estrutura Molecular , Lactamas Macrocíclicas/farmacologia , Lactamas Macrocíclicas/química , Streptomyces/metabolismo , Genômica , Reação em Cadeia da Polimerase , Família Multigênica
19.
Nat Commun ; 14(1): 343, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36670101

RESUMO

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.


Assuntos
Diterpenos , Lamiaceae , Lamiaceae/genética , Lamiaceae/metabolismo , Diterpenos/metabolismo , Terpenos/metabolismo , Família Multigênica , Vias Biossintéticas/genética
20.
Int J Mol Sci ; 24(2)2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36674776

RESUMO

Growth-regulating factor (GRF) is a kind of transcription factor unique to plants, playing an important role in the flowering regulation, growth, and development of plants. Melastoma dodecandrum is an important member of Melastomataceae, with ornamental, medicinal, and edible benefits. The identification of the GRF gene family in M. dodecandrum can help to improve their character of flavor and continuous flowering. The members of the GRF gene family were identified from the M. dodecandrum genome, and their bioinformatics, selective pressure, and expression patterns were analyzed. The results showed that there were 20 GRF genes in M. dodecandrum. Phylogenetic analysis showed that the 71 GRF genes from M. dodecandrum, Arabidopsis thaliana, Camellia sinensis, and Oryza sativa can be divided into three clades and six subclades. The 20 GRF genes of M. dodecandrum were distributed in twelve chromosomes and one contig. Furthermore, the gene structure and motif analysis showed that the intron and motif within each clade were very similar, but there were great differences among different clades. The promoter contained cis-acting elements related to hormone induction, stress, and growth and development. Different transcriptomic expression of MdGRFs indicated that MdGRFs may be involved in regulating the growth and development of M. dodecandrum. The results laid a foundation for further study on the function and molecular mechanism of the M. dodecandrum GRF gene family.


Assuntos
Melastomataceae , Melastomataceae/química , Filogenia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Família Multigênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
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