Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 45
Filtrar
1.
Chem Sci ; 14(14): 3826-3833, 2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-37035691

RESUMO

Pleuromutilin is an antibiotic diterpenoid made by Clitopilus passeckerianus and related fungi, and it is the progenitor of a growing class of semi-synthetic antibiotics used in veterinary and human medicine. To harness the biotechnological potential of this natural product class, a full understanding of its biosynthetic pathway is essential. Previously, a linear pathway for pleuromutilin biosynthesis was established. Here we report two shunt pathways involving Pl-sdr and Pl-atf that were identified through the rational heterologous expression of combinations of pleuromutilin biosynthetic genes in Aspergillus oryzae. Three novel pleuromutilin congeners were isolated, and their antimicrobial activity was investigated, alongside that of an additional derivative produced through a semi-synthetic approach. It was observed that the absence of various functional groups - 3 ketone, 11 hydroxyl group or 21 ketone - from the pleuromutilin framework affected the antibacterial activity of pleuromutilin congeners. This study expands our knowledge on the biosynthesis of pleuromutilin and provides avenues for the development of novel pleuromutilin analogues by combining synthetic biology and synthetic chemistry.

2.
Nat Prod Rep ; 40(1): 128-157, 2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-36129067

RESUMO

Covering: up to early 2022Maleidrides are a family of polyketide-based dimeric natural products isolated from fungi. Many maleidrides possess significant bioactivities, making them attractive pharmaceutical or agrochemical lead compounds. Their unusual biosynthetic pathways have fascinated scientists for decades, with recent advances in our bioinformatic and enzymatic understanding providing further insights into their construction. However, many intriguing questions remain, including exactly how the enzymatic dimerisation, which creates the diverse core structure of the maleidrides, is controlled. This review will explore the literature from the initial isolation of maleidride compounds in the 1930s, through the first full structural elucidation in the 1960s, to the most recent in vivo, in vitro, and in silico analyses.


Assuntos
Produtos Biológicos , Policetídeos , Anidridos/metabolismo , Fungos/metabolismo , Dimerização , Vias Biossintéticas , Policetídeos/metabolismo , Produtos Biológicos/química
3.
PLoS One ; 17(8): e0271622, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35913938

RESUMO

Armillaria mellea is an important fungal pathogen worldwide, affecting a large number of hosts in the horticulture and forestry industries. Controlling A. mellea infection is expensive, labour intensive and time-consuming, so a new, environmentally friendly management solution is required. To this effect, endophytic Trichoderma species were studied as a potential protective agent for Armillaria root rot (ARR) in strawberry and privet plants. A collection of forty endophytic Trichoderma isolates were inoculated into strawberry (Fragaria × ananassa) plants and plant growth was monitored for two months, during which time Trichoderma treatment had no apparent effect. Trichoderma-colonised strawberry plants were then inoculated with A. mellea and after three months plants were assessed for A. mellea infection. There was considerable variation in ARR disease levels between plants inoculated with different Trichoderma spp. isolates, but seven isolates reduced ARR below the level of positive controls. These isolates were further tested for protective potential in Trichoderma-colonized privet (Ligustrum vulgare) plants where five Trichoderma spp. isolates, including two highly effective Trichoderma atrobrunneum isolates, were able to significantly reduce levels of disease. This study highlights the potential of plants pre-colonised with T. atrobrunneum for effective protection against A. mellea in two hosts from different plant families.


Assuntos
Armillaria , Fragaria , Ligustrum , Trichoderma , Fragaria/microbiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Plantas/microbiologia
4.
Microb Biotechnol ; 15(10): 2547-2561, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35829647

RESUMO

Burkholderia have potential as biocontrol agents because they encode diverse biosynthetic gene clusters (BGCs) for a range of antimicrobial metabolites. Given the opportunistic pathogenicity associated with Burkholderia species, heterologous BGC expression within non-pathogenic hosts is a strategy to construct safe biocontrol strains. We constructed a yeast-adapted Burkholderia-Escherichia shuttle vector (pMLBAD_yeast) with a yeast replication origin 2 µ and URA3 selection marker and optimised it for cloning BGCs using the in vivo recombination ability of Saccharomyces cerevisiae. Two Burkholderia polyyne BGCs, cepacin (13 kb) and caryoynencin (11 kb), were PCR-amplified as three overlapping fragments, cloned downstream of the pBAD arabinose promoter in pMLBAD_yeast and mobilised into Burkholderia and Paraburkholderia heterologous hosts. Paraburkholderia phytofirmans carrying the heterologous polyyne constructs displayed in vitro bioactivity against a variety of fungal and bacterial plant pathogens similar to the native polyyne producers. Thirteen Paraburkholderia strains with preferential growth at 30°C compared with 37°C were also identified, and four of these were amenable to genetic manipulation and heterologous expression of the caryoynencin construct. The cloning and successful heterologous expression of Burkholderia biosynthetic gene clusters within Paraburkholderia with restricted growth at 37°C opens avenues for engineering non-pathogenic biocontrol strains.


Assuntos
Burkholderia , Arabinose/metabolismo , Agentes de Controle Biológico/metabolismo , Burkholderia/genética , Clonagem Molecular , Família Multigênica , Poli-Inos/metabolismo , Saccharomyces cerevisiae/metabolismo
5.
Fungal Genet Biol ; 161: 103715, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35709910

RESUMO

The fungus Zymoseptoria tritici causes Septoria Tritici Blotch (STB), which is one of the most devastating diseases of wheat in Europe. There are currently no fully durable methods of control against Z. tritici, so novel strategies are urgently required. One of the ways in which fungi are able to respond to their surrounding environment is through the use of photoreceptor proteins which detect light signals. Although previous evidence suggests that Z. tritici can detect light, no photoreceptor genes have been characterised in this pathogen. This study characterises ZtWco-1, a predicted photoreceptor gene in Z. tritici. The ZtWco-1 gene is a putative homolog to the blue light photoreceptor from Neurospora crassa, wc-1. Z. tritici mutants with deletions in ZtWco-1 have defects in hyphal branching, melanisation and virulence on wheat. In addition, we identify the putative circadian clock gene ZtFrq in Z. tritici. This study provides evidence for the genetic regulation of light detection in Z. tritici and it open avenues for future research into whether this pathogen has a circadian clock.


Assuntos
Ascomicetos , Triticum , Ascomicetos/fisiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Triticum/microbiologia , Virulência/genética
6.
Front Bioeng Biotechnol ; 9: 567384, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34109161

RESUMO

Natural products with novel chemistry are urgently needed to battle the continued increase in microbial drug resistance. Mushroom-forming fungi are underutilized as a source of novel antibiotics in the literature due to their challenging culture preparation and genetic intractability. However, modern fungal molecular and synthetic biology tools have renewed interest in exploring mushroom fungi for novel therapeutic agents. The aims of this study were to investigate the secondary metabolites of nine basidiomycetes, screen their biological and chemical properties, and then investigate the genetic pathways associated with their production. Of the nine fungi selected, Hypholoma fasciculare was revealed to be a highly active antagonistic species, with antimicrobial activity against three different microorganisms: Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. Genomic comparisons and chromatographic studies were employed to characterize more than 15 biosynthetic gene clusters and resulted in the identification of 3,5-dichloromethoxy benzoic acid as a potential antibacterial compound. The biosynthetic gene cluster for this product is also predicted. This study reinforces the potential of mushroom-forming fungi as an underexplored reservoir of bioactive natural products. Access to genomic data, and chemical-based frameworks, will assist the development and application of novel molecules with applications in both the pharmaceutical and agrochemical industries.

7.
Mol Plant Pathol ; 22(3): 382-389, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33471956

RESUMO

Plant viruses typically have highly condensed genomes, yet the plant-pathogenic viruses Cassava brown streak virus, Ugandan cassava brown streak virus, and Euphorbia ringspot virus are unusual in encoding an enzyme not yet found in any other virus, the "house-cleaning" enzyme inosine triphosphatase. Inosine triphosphatases (ITPases) are highly conserved enzymes that occur in all kingdoms of life and perform a house-cleaning function by hydrolysing the noncanonical nucleotide inosine triphosphate to inosine monophosphate. The ITPases encoded by cassava brown streak virus and Ugandan cassava brown streak virus have been characterized biochemically and are shown to have typical ITPase activity. However, their biological role in virus infection has yet to be elucidated. Here we review what is known of viral-encoded ITPases and speculate on potential roles in infection with the aim of generating a greater understanding of cassava brown streak viruses, a group of the world's most devastating viruses.


Assuntos
Manihot/virologia , Doenças das Plantas/virologia , Potyviridae/enzimologia , Pirofosfatases/metabolismo , Potyviridae/genética , Pirofosfatases/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Inosina Trifosfatase
8.
Front Fungal Biol ; 2: 655323, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-37744150

RESUMO

Interrogation of an EST database for Clitopilus passeckerianus identified a putative homolog to the unusual stress response gene from yeast; ddr48, as being upregulated under pleuromutilin production conditions. Silencing of this gene, named cprp, produced a population of transformants which demonstrated significantly reduced pleuromutilin production. Attempts to complement a Saccharomyces cerevisiae ddr48 mutant strain (strain Y16748) with cprp were hampered by the lack of a clearly identifiable mutant phenotype, but interestingly, overexpression of either ddr48 or cprp in S. cerevisiae Y16748 led to a conspicuous and comparable reduction in growth rate. This observation, combined with the known role of DDR48 proteins from a range of fungal species in nutrient starvation and stress responses, raises the possibility that this family of proteins plays a role in triggering oligotrophic growth. Localization studies via the production of a Cprp:GFP fusion protein in C. passeckerianus showed clear localization adjacent to the hyphal septa and, to a lesser extent, cell walls, which is consistent with the identification of DDR48 as a cell wall-associated protein in various yeast species. To our knowledge this is the first study demonstrating that a DDR48-like protein plays a role in the regulation of a secondary metabolite, and represents the first DDR48-like protein from a basidiomycete. Potential homologs can be identified across much of the Dikarya, suggesting that this unusual protein may play a central role in regulating both primary and secondary metabolism in fungi.

9.
PLoS One ; 15(4): e0229925, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251434

RESUMO

Basidiomycete fungi are a rich source of natural products with a diverse array of potentially exploitable bioactivities. Two dimeric sesquiterpenes, bovistol B (1) and D (2), and one monomeric sesquiterpene, strossmayerin (7), were isolated from the culture filtrate of the basidiomycete fungus Coprinopsis strossmayeri. The structures were determined through a combination of MS and 1D/2D NMR spectroscopic techniques. Likely monomeric precursors, identified on the basis of HRMS analysis, allow a plausible biosynthetic pathway to be proposed for the biosynthesis of 1 and 2, involving the dimerisation of the monomer through a hetero-Diels-Alder mechanism. A gene cluster, including a putative sesquiterpene 1-11 cyclase, was identified through phylogenetic and RNA-seq analysis, and is proposed to be responsible for the biosynthesis of 1 and 2.


Assuntos
Agaricales/química , Basidiomycota/química , Redes e Vias Metabólicas , Sesquiterpenos/química , Agaricales/ultraestrutura , Basidiomycota/ultraestrutura , Produtos Biológicos/química , Filtração , Espectroscopia de Ressonância Magnética , Estrutura Molecular
10.
Front Microbiol ; 10: 2210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632366

RESUMO

The ascomycete fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB), a major disease of wheat across Europe. Current understanding of the genetic components and the environmental cues which influence development and pathogenicity of this fungus is limited. The velvet B gene, velB, has conserved roles in development, secondary metabolism, and pathogenicity across fungi. The function of this gene is best characterised in the model ascomycete fungus Aspergillus nidulans, where it is involved in co-ordinating the light response with downstream processes. There is limited knowledge of the role of light in Z. tritici, and of the molecular mechanisms underpinning the light response. We show that Z. tritici is able to detect light, and that the vegetative morphology of this fungus is influenced by light conditions. We also identify and characterise the Z. tritici velB gene, ZtvelB, by gene disruption. The ΔztvelB deletion mutants were fixed in a filamentous growth pattern and are unable to form yeast-like vegetative cells. Their morphology was similar under light and dark conditions, showing an impairment in light-responsive growth. In addition, the ΔztvelB mutants produced abnormal pycnidia that were impaired in macropycnidiospore production but could still produce viable infectious micropycnidiospores. Our results show that ZtvelB is required for yeast-like growth and asexual sporulation in Z. tritici, and we provide evidence for a role of ZtvelB in integrating light perception and developmental regulation in this important plant pathogenic fungus.

11.
Virus Genes ; 55(6): 825-833, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31388891

RESUMO

Cassava brown streak disease (CBSD) is a leading cause of cassava yield losses across eastern and central Africa and is having a severe impact on food security across the region. Despite its importance, relatively little is known about the mechanisms behind CBSD viral infections. We have recently reported the construction of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) infectious clones (IC), which can be used to gain insights into the functions of viral proteins and sequences associated with symptom development. In this study, we perform the first reporter gene tagging of a CBSV IC, with the insertion of green fluorescent protein (GFP) sequence at two different genome positions. Nicotiana benthamiana infections with the CBSV_GFP ICs revealed active CBSV replication in inoculated leaves at 2-5 days post inoculation (dpi) and systemic leaves at 10-14 dpi. We also constructed the chimera CBSV_UCP IC, consisting of the CBSV genome with a UCBSV coat protein (CP) sequence replacement. N. benthamiana infections with CBSV_UCP revealed that the CBSV CP may be associated with high levels of viral accumulation and necrosis development during early infection. These initial manipulations pave the way for U/CBSV ICs to be used to understand U/CBSV biology that will inform vital CBSD control strategies.


Assuntos
Manihot/genética , Doenças das Plantas/virologia , Potyviridae/genética , Replicação Viral/genética , Evolução Clonal/genética , Abastecimento de Alimentos , Genoma Viral/genética , Manihot/virologia , Filogenia , Doenças das Plantas/genética , Folhas de Planta/virologia , Potyviridae/patogenicidade , Uganda , Proteínas Virais/genética
12.
Mol Biotechnol ; 61(10): 754-762, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392585

RESUMO

Naematolin is a biologically active sesquiterpene produced by Hypholoma species. Low titres and complex structure constrain the exploitation of this secondary metabolite. Here, we de novo sequenced the H. fasciculare genome to identify a candidate biosynthetic gene cluster for production of naematolin. Using Aspergillus oryzae as a heterologous host for gene expression, the activity of several sesquiterpene synthases were investigated, highlighting one atypical sesquiterpene synthase apparently capable of catalysing the 1,11 and subsequent 2,10 ring closures, which primes the synthesis of the distinctive structure of caryophyllene derivatives. Co-expression of the cyclase with an FAD oxidase adjacent within the gene cluster generated four oxidised caryophyllene-based sesquiterpenes: 5ß,6α,8ß-trihydroxycariolan, 5ß,8ß-dihydroxycariolan along with two previously unknown caryophyllene derivatives 2 and 3. This represents the first steps towards heterologous production of such basidiomycete-derived caryophyllene-based sesquiterpenes, opening a venue for potential novel antimicrobials via combinatorial biosynthesis.


Assuntos
Agaricales/genética , Vias Biossintéticas , Sesquiterpenos Policíclicos/metabolismo , Sequenciamento Completo do Genoma/métodos , Agaricales/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/crescimento & desenvolvimento , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Família Multigênica
13.
Mol Plant Pathol ; 20(8): 1080-1092, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31154674

RESUMO

Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep-sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild-type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.


Assuntos
Mutagênicos/metabolismo , Nucleotídeos/metabolismo , Potyviridae/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Fluoruracila/farmacologia , Hidrólise , Taxa de Mutação , Necrose , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas , Saccharomyces cerevisiae/metabolismo , Nicotiana/genética , Nicotiana/virologia , Proteínas Virais/química
14.
Microbiol Resour Announc ; 8(19)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072880

RESUMO

The fungus Lecanicillium fungicola causes dry bubble disease in the white button mushroom Agaricus bisporus Control strategies are limited, as both the host and pathogen are fungi, and there is limited understanding of the interactions in this pathosystem. Here, we present the genome sequence of Lecanicillium fungicola strain 150-1.

15.
Front Microbiol ; 9: 1859, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30154771

RESUMO

Zymoseptoria tritici is the causal agent of septoria tritici blotch, a devastating fungal disease of wheat which can cause up to 40% yield loss. One of the ways in which Z. tritici spreads in the field is via rain splash-dispersed asexual pycnidiospores, however there is currently limited understanding of the genetic mechanisms governing the development of these propagules. In order to explore whether the existing models for conidiation in ascomycete fungi apply to Z. tritici, homologs to the well-characterized Aspergillus nidulans genes abacus (abaA), bristle (brlA), fluffy B (flbB), fluffy C (flbC), and stunted (stuA) were identified and knocked-out by Agrobacterium-mediated transformation. Although deletion of the ZtAbaA, ZtBrlA1, and ZtFlbB genes had no apparent effect on Z. tritici asexual sporulation or on pathogenicity, deletion of ZtFlbC or ZtBrlA2 resulted in mutants with reduced pycnidiospore production compared to the parental IPO323 strain. Deletion of ZtStuA gave non-pigmented mutants with altered vegetative growth and eliminated asexual sporulation and pathogenicity. These findings suggest that the well-established A. nidulans model of asexual sporulation is only partially applicable to Z. tritici, and that this pathogen likely uses additional, as yet uncharacterized genes to control asexual sporulation.

16.
Mol Plant Pathol ; 19(5): 1282-1294, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28887856

RESUMO

Cassava is the second most important staple food crop in terms of per capita calories consumed in Africa and holds potential for climate change adaptation. Unfortunately, productivity in East and Central Africa is severely constrained by two viral diseases: cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). CBSD was first reported in 1936 from northeast Tanzania. For approximately 70 years, CBSD was restricted to coastal East Africa and so had a relatively low impact on food security compared with CMD. However, at the turn of the 21st century, CBSD re-emerged further inland, in areas around Lake Victoria, and it has since spread through many East and Central African countries, causing high yield losses and jeopardizing the food security of subsistence farmers. This recent re-emergence has attracted intense scientific interest, with studies shedding light on CBSD viral epidemiology, sequence diversity, host interactions and potential sources of resistance within the cassava genome. This review reflects on 80 years of CBSD research history (1936-2016) with a timeline of key events. We provide insights into current CBSD knowledge, management efforts and future prospects for improved understanding needed to underpin effective control and mitigation of impacts on food security.


Assuntos
Manihot/virologia , Doenças das Plantas/virologia , Potyviridae/fisiologia , Evolução Biológica , Resistência à Doença , Geografia , Potyviridae/genética
17.
Plant Biotechnol J ; 16(4): 832-843, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29271098

RESUMO

Plant virus infectious clones are important tools with wide-ranging applications in different areas of biology and medicine. Their uses in plant pathology include the study of plant-virus interactions, and screening of germplasm as part of prebreeding programmes for virus resistance. They can also be modified to induce transient plant gene silencing (Virus Induced Gene Silencing - VIGS) and as expression vectors for plant or exogenous proteins, with applications in both plant pathology and more generally for the study of plant gene function. Plant viruses are also increasingly being investigated as expression vectors for in planta production of pharmaceutical products, known as molecular farming. However, plant virus infectious clones may pose a risk to the environment due to their ability to reconstitute fully functional, transmissible viruses. These risks arise from both their inherent pathogenicity and the effect of any introduced genetic modifications. Effective containment measures are therefore required. There has been no single comprehensive review of the biosafety considerations for the contained use of genetically modified plant viruses, despite their increasing importance across many biological fields. This review therefore explores the biosafety considerations for working with genetically modified plant viruses in contained environments, with focus on plant growth facilities. It includes regulatory frameworks, risk assessment, assignment of biosafety levels, facility features and working practices. The review is based on international guidance together with information provided by plant virus researchers.


Assuntos
Contenção de Riscos Biológicos/normas , Microrganismos Geneticamente Modificados , Vírus de Plantas/genética , Plasmídeos/genética , Equipamentos e Provisões , Vetores Genéticos , Laboratórios , Vírus de Plantas/patogenicidade , Medição de Risco/métodos , Virologia/legislação & jurisprudência
18.
Nat Commun ; 8(1): 1831, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29184068

RESUMO

The rise in antibiotic resistance is a major threat for human health. Basidiomycete fungi represent an untapped source of underexploited antimicrobials, with pleuromutilin-a diterpene produced by Clitopilus passeckerianus-being the only antibiotic from these fungi leading to commercial derivatives. Here we report genetic characterisation of the steps involved in pleuromutilin biosynthesis, through rational heterologous expression in Aspergillus oryzae coupled with isolation and detailed structural elucidation of the pathway intermediates by spectroscopic methods and comparison with synthetic standards. A. oryzae was further established as a platform for bio-conversion of chemically modified analogues of pleuromutilin intermediates, and was employed to generate a semi-synthetic pleuromutilin derivative with enhanced antibiotic activity. These studies pave the way for future characterisation of biosynthetic pathways of other basidiomycete natural products in ascomycete heterologous hosts, and open up new possibilities of further chemical modification for the growing class of potent pleuromutilin antibiotics.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/química , Basidiomycota/genética , Basidiomycota/metabolismo , Antibacterianos/farmacologia , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Vias Biossintéticas/genética , Clonagem Molecular , DNA Fúngico , Diterpenos/química , Diterpenos/metabolismo , Diterpenos/farmacologia , Resistência Microbiana a Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Inativação Gênica , Genes Fúngicos/genética , Engenharia Genética , Humanos , Engenharia Metabólica , Compostos Policíclicos , Pleuromutilinas
19.
Artigo em Inglês | MEDLINE | ID: mdl-28955474

RESUMO

The implementation of Agrobacterium tumefaciens as a transformation tool revolutionized approaches to discover and understand gene functions in a large number of fungal species. A. tumefaciens mediated transformation (AtMT) is one of the most transformative technologies for research on fungi developed in the last 20 years, a development arguably only surpassed by the impact of genomics. AtMT has been widely applied in forward genetics, whereby generation of strain libraries using random T-DNA insertional mutagenesis, combined with phenotypic screening, has enabled the genetic basis of many processes to be elucidated. Alternatively, AtMT has been fundamental for reverse genetics, where mutant isolates are generated with targeted gene deletions or disruptions, enabling gene functional roles to be determined. When combined with concomitant advances in genomics, both forward and reverse approaches using AtMT have enabled complex fungal phenotypes to be dissected at the molecular and genetic level. Additionally, in several cases AtMT has paved the way for the development of new species to act as models for specific areas of fungal biology, particularly in plant pathogenic ascomycetes and in a number of basidiomycete species. Despite its impact, the implementation of AtMT has been uneven in the fungi. This review provides insight into the dynamics of expansion of new research tools into a large research community and across multiple organisms. As such, AtMT in the fungi, beyond the demonstrated and continuing power for gene discovery and as a facile transformation tool, provides a model to understand how other technologies that are just being pioneered, e.g. CRISPR/Cas, may play roles in fungi and other eukaryotic species.

20.
J Microbiol Methods ; 142: 4-9, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28843436

RESUMO

The basidiomycete fungi Hypholoma fasciculare and H. sublateritium are both prolific producers of sesquiterpenes and triterpenes, some of which have relevant pharmaceutical properties. Although H. sublateritium has been transformed in the past, the low reported efficiencies highlighted the need for establishing an effective simple transformation system for these valuable species. We have optimized Agrobacterium tumefaciens-mediated transformation through testing various parameters in these two Hypholoma species, showing that a mixture of homogenized mycelia and Agrobacterium (strain LBA4404) co-cultivated for 84h at 25°C is optimal for efficient transformation in these basidiomycetes. This study also reveals the requirements for transgene expression, with the first report of GFP expression in these Hypholoma, the need for an intron for such transgene expression, and further demonstrates the functionality of the expression vector by its use in Clitopilus passeckerianus. This development of transformation system and expression constructs, can facilitate further genetic investigation such as gene functionality in these fungi.


Assuntos
Agaricales/genética , Agrobacterium tumefaciens/genética , Proteínas de Fluorescência Verde/genética , Transformação Genética/genética , Técnicas de Cocultura , Engenharia Genética , Íntrons/genética , Regiões Promotoras Genéticas/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA