Fungal Biology and Biotechnology最新文献

{"title":"Functional production of human antibody by the filamentous fungus <i>Aspergillus oryzae</i>.","authors":"Hung Hiep Huynh, Naoki Morita, Toshihiro Sakamoto, Takuya Katayama, Takuya Miyakawa, Masaru Tanokura, Yasunori Chiba, Reiko Shinkura, Jun-Ichi Maruyama","doi":"10.1186/s40694-020-00098-w","DOIUrl":"10.1186/s40694-020-00098-w","url":null,"abstract":"<p><strong>Background: </strong>Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. <i>Aspergillus oryzae</i>, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNFα antibody adalimumab, one of the world's best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by <i>A. oryzae</i>. Generally, <i>N</i>-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to first delete the <i>Aooch1</i> gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa.</p><p><strong>Results: </strong>Adalimumab was expressed in <i>A. oryzae</i> by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by <i>A. oryzae</i> were comparable with those of a commercial product Humira^®. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the <i>N</i>-glycan structure using the <i>Aooch1</i> deletion strain, which suggests only a little additional activity of immune effector functions.</p><p><strong>Conclusion: </strong>These results demonstrated an alternative low-cost platform for human antibody production by using <i>A. oryzae</i>, possibly offering a reasonable expenditure for patient's welfare.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"7 ","pages":"7"},"PeriodicalIF":0.0,"publicationDate":"2020-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7257131/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38024038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transport systems, intracellular traffic of intermediates and secretion of β-lactam antibiotics in fungi.","authors":"Juan F Martín","doi":"10.1186/s40694-020-00096-y","DOIUrl":"https://doi.org/10.1186/s40694-020-00096-y","url":null,"abstract":"<p><p>Fungal secondary metabolites are synthesized by complex biosynthetic pathways catalized by enzymes located in different subcellular compartments, thus requiring traffic of precursors and intermediates between them. The β-lactam antibiotics penicillin and cephalosporin C serve as an excellent model to understand the molecular mechanisms that control the subcellular localization of secondary metabolites biosynthetic enzymes. Optimal functioning of the β-lactam biosynthetic enzymes relies on a sophisticated temporal and spatial organization of the enzymes, the intermediates and the final products. The first and second enzymes of the penicillin pathway, ACV synthetase and IPN synthase, in <i>Penicillium chrysogenum</i> and <i>Aspergillus nidulans</i> are cytosolic. In contrast, the last two enzymes of the penicillin pathway, phenylacetyl-CoA ligase and isopenicillin N acyltransferase, are located in peroxisomes working as a tandem at their optimal pH that coincides with the peroxisomes pH. Two MFS transporters, PenM and PaaT have been found to be involved in the import of the intermediates isopenicillin N and phenylacetic acid, respectively, into peroxisomes. Similar compartmentalization of intermediates occurs in <i>Acremonium chrysogenum;</i> two enzymes isopenicillin N-CoA ligase and isopenicillin N-CoA epimerase, that catalyse the conversion of isopenicillin N in penicillin N, are located in peroxisomes. Two genes encoding MFS transporters, <i>cefP</i> and <i>cefM,</i> are located in the early cephalosporin gene cluster. These transporters have been localized in peroxisomes by confocal fluorescence microscopy. A third gene of <i>A. chrysogenum</i>, <i>cefT</i>, encodes an MFS protein, located in the cell membrane involved in the secretion of cephalosporin C, although <i>cefT</i>-disrupted mutants are still able to export cephalosporin by redundant transporters. The secretion of penicillin from peroxisomes to the extracellular medium is still unclear. Attempts have been made to identify a gene encoding the penicillin secretion protein among the 48 ABC-transporters of <i>P. chrysogenum</i>. The highly efficient secretion system that exports penicillin against a concentration gradient may involve active penicillin extrusion systems mediated by vesicles that fuse to the cell membrane. However, there is no correlation of pexophagy with penicillin or cephalosporin formation since inactivation of pexophagy leads to increased penicillin or cephalosporin biosynthesis due to preservation of peroxisomes. The penicillin biosynthesis finding shows that in order to increase biosynthesis of novel secondary metabolites it is essential to adequately target enzymes to organelles.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"7 ","pages":"6"},"PeriodicalIF":0.0,"publicationDate":"2020-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-020-00096-y","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37887305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Growing a circular economy with fungal biotechnology: a white paper.","authors":"Vera Meyer,&nbsp;Evelina Y Basenko,&nbsp;J Philipp Benz,&nbsp;Gerhard H Braus,&nbsp;Mark X Caddick,&nbsp;Michael Csukai,&nbsp;Ronald P de Vries,&nbsp;Drew Endy,&nbsp;Jens C Frisvad,&nbsp;Nina Gunde-Cimerman,&nbsp;Thomas Haarmann,&nbsp;Yitzhak Hadar,&nbsp;Kim Hansen,&nbsp;Robert I Johnson,&nbsp;Nancy P Keller,&nbsp;Nada Kraševec,&nbsp;Uffe H Mortensen,&nbsp;Rolando Perez,&nbsp;Arthur F J Ram,&nbsp;Eric Record,&nbsp;Phil Ross,&nbsp;Volha Shapaval,&nbsp;Charlotte Steiniger,&nbsp;Hans van den Brink,&nbsp;Jolanda van Munster,&nbsp;Oded Yarden,&nbsp;Han A B Wösten","doi":"10.1186/s40694-020-00095-z","DOIUrl":"https://doi.org/10.1186/s40694-020-00095-z","url":null,"abstract":"<p><p>Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation's sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"7 ","pages":"5"},"PeriodicalIF":0.0,"publicationDate":"2020-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-020-00095-z","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37825343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen <i>Parastagonspora nodorum</i>.","authors":"Haseena Khan,&nbsp;Megan C McDonald,&nbsp;Simon J Williams,&nbsp;Peter S Solomon","doi":"10.1186/s40694-020-00094-0","DOIUrl":"https://doi.org/10.1186/s40694-020-00094-0","url":null,"abstract":"<p><strong>Background: </strong>The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (sgRNA) which interact to form a Cas9-sgRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus <i>Parastagonospora nodorum</i>.</p><p><strong>Results: </strong><i>Parastagonospora nodorum</i> protoplasts were transformed with the Cas9 protein and sgRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the <i>Tox3</i> effector gene. Subsequent screening of the <i>P. nodorum</i> transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a <i>Tox3</i>-Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a <i>Tox3</i>-Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved with 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations.</p><p><strong>Conclusion: </strong>These data highlight the significant potential that CRISPR/Cas9 has in expediting transgene-free gene knockouts in <i>Parastagonospora nodorum</i> and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"7 ","pages":"4"},"PeriodicalIF":0.0,"publicationDate":"2020-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-020-00094-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37810260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Promoter tools for further development of <i>Aspergillus oryzae</i> as a platform for fungal secondary metabolite production.","authors":"Maiko Umemura,&nbsp;Kaoru Kuriiwa,&nbsp;Linh Viet Dao,&nbsp;Tetsuya Okuda,&nbsp;Goro Terai","doi":"10.1186/s40694-020-00093-1","DOIUrl":"https://doi.org/10.1186/s40694-020-00093-1","url":null,"abstract":"<p><strong>Background: </strong>The filamentous fungus <i>Aspergillus oryzae</i> is widely used for secondary metabolite production by heterologous expression; thus, a wide variety of promoter tools is necessary to broaden the application of this species. Here we built a procedure to survey <i>A. flavus</i> genes constitutively highly expressed in 83 transcriptome datasets obtained under various conditions affecting secondary metabolite production, to find promoters useful for heterologous expression of genes in <i>A. oryzae</i>.</p><p><strong>Results: </strong>To test the ability of the promoters of the top 6 genes to induce production of a fungal secondary metabolite, ustiloxin B, we inserted the promoters before the start codon of <i>ustR</i>, which encodes the transcription factor of the gene cluster responsible for ustiloxin B biosynthesis, in <i>A. oryzae</i>. Four of the 6 promoters induced ustiloxin B production in all tested media (solid maize, liquid V8 and PDB media), and also <i>ustR</i> expression. Two of the 4 promoters were those of <i>tef1</i> and <i>gpdA</i>, which are well characterized in <i>A. oryzae</i> and <i>A. nidulans</i>, respectively, whereas the other two, those of AFLA_030930 and AFLA_113120, are newly reported here and show activities comparable to that of the <i>gpdA</i> promoter with respect to induction of gene expression and ustiloxin B production.</p><p><strong>Conclusion: </strong>We newly reported two sequences as promoter tools for secondary metabolite production in <i>A. oryzae</i>. Our results demonstrate that our simple strategy of surveying for constitutively highly expressed genes in large-scale transcriptome datasets is useful for finding promoter sequences that can be used as heterologous expression tools in <i>A. oryzae</i>.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"7 ","pages":"3"},"PeriodicalIF":0.0,"publicationDate":"2020-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-020-00093-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37770411","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enabling community-based metrology for wood-degrading fungi.","authors":"Rolando Perez, Marina Luccioni, Rohinton Kamakaka, Samuel Clamons, Nathaniel Gaut, Finn Stirling, Katarzyna P Adamala, Pamela A Silver, Drew Endy","doi":"10.1186/s40694-020-00092-2","DOIUrl":"10.1186/s40694-020-00092-2","url":null,"abstract":"<p><strong>Background: </strong>Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi could enable more direct means of manufacture. However, basic methods for cultivating wood-degrading fungi are often ad hoc and not readily reproducible. Here, we developed standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products in simple laboratory settings.</p><p><strong>Results: </strong>We show that a widely-available and globally-regularized consumer product (Pringles™) can support the growth of wood-degrading fungi, and that growth on Pringles™-broth can be correlated with growth on media made from a fully-traceable and compositionally characterized substrate (National Institute of Standards and Technology Reference Material 8492 Eastern Cottonwood Whole Biomass Feedstock). We also establish a Relative Extension Unit (REU) framework that is designed to reduce variation in quantification of radial growth measurements. So enabled, we demonstrate that five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay, and that our REU-based approach reduced variation in reported measurements by up to ~ 75%.</p><p><strong>Conclusions: </strong>Reliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial. Our community-based measurement methods incentivize practitioners to coordinate the reuse of standard materials, methods, strains, and to share information supporting work with wood-degrading fungi.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"7 ","pages":"2"},"PeriodicalIF":0.0,"publicationDate":"2020-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081594/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37765966","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Towards a better understanding of the role of nectar-inhabiting yeasts in plant-animal interactions.","authors":"Joon Klaps,&nbsp;Bart Lievens,&nbsp;Sergio Álvarez-Pérez","doi":"10.1186/s40694-019-0091-8","DOIUrl":"https://doi.org/10.1186/s40694-019-0091-8","url":null,"abstract":"<p><p>Flowers offer a wide variety of substrates suitable for fungal growth. However, the mycological study of flowers has only recently begun to be systematically addressed from an ecological point of view. Most research on the topic carried out during the last decade has focused on studying the prevalence and diversity of flower-inhabiting yeasts, describing new species retrieved from floral parts and animal pollinators, and the use of select nectar yeasts as model systems to test ecological hypotheses. In this primer article, we summarize the current state of the art in floral nectar mycology and provide an overview of some research areas that, in our view, still require further attention, such as the influence of fungal volatile organic compounds on the foraging behavior of pollinators and other floral visitors, the analysis of the direct and indirect effects of nectar-inhabiting fungi on the fitness of plants and animals, and the nature and consequences of fungal-bacterial interactions taking place within flowers.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"7 ","pages":"1"},"PeriodicalIF":0.0,"publicationDate":"2020-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-019-0091-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37528318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of synthetic formaldehyde and methanol assimilation pathways in <i>Yarrowia lipolytica</i>.","authors":"Eija Vartiainen,&nbsp;Peter Blomberg,&nbsp;Marja Ilmén,&nbsp;Martina Andberg,&nbsp;Mervi Toivari,&nbsp;Merja Penttilä","doi":"10.1186/s40694-019-0090-9","DOIUrl":"https://doi.org/10.1186/s40694-019-0090-9","url":null,"abstract":"<p><strong>Background: </strong>Crude glycerol coming from biodiesel production is an attractive carbon source for biological production of chemicals. The major impurity in preparations of crude glycerol is methanol, which is toxic for most microbes. Development of microbes, which would not only tolerate the methanol, but also use it as co-substrate, would increase the feasibility of bioprocesses using crude glycerol as substrate.</p><p><strong>Results: </strong>To prevent methanol conversion to CO₂ via formaldehyde and formate, the formaldehyde dehydrogenase (FLD) gene was identified in and deleted from <i>Yarrowia lipolytica.</i> The deletion strain was able to convert methanol to formaldehyde without expression of heterologous methanol dehydrogenases. Further, it was shown that expression of heterologous formaldehyde assimilating enzymes could complement the deletion of FLD. The expression of either 3-hexulose-6-phosphate synthase (HPS) enzyme of ribulose monosphosphate pathway or dihydroxyacetone synthase (DHAS) enzyme of xylulose monosphosphate pathway restored the formaldehyde tolerance of the formaldehyde sensitive Δ<i>fld1</i> strain.</p><p><strong>Conclusions: </strong>In silico, the expression of heterologous formaldehyde assimilation pathways enable <i>Y. lipolytica</i> to use methanol as substrate for growth and metabolite production. In vivo, methanol was shown to be converted to formaldehyde and the enzymes of formaldehyde assimilation were actively expressed in this yeast. However, further development is required to enable <i>Y. lipolytica</i> to efficiently use methanol as co-substrate with glycerol.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"6 ","pages":"27"},"PeriodicalIF":0.0,"publicationDate":"2019-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-019-0090-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37501808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction to: Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in <i>Aspergillus niger</i> submerged culture.","authors":"Timothy C Cairns,&nbsp;Claudia Feurstein,&nbsp;Xiaomei Zheng,&nbsp;Li Hui Zhang,&nbsp;Ping Zheng,&nbsp;Jibin Sun,&nbsp;Vera Meyer","doi":"10.1186/s40694-019-0087-4","DOIUrl":"https://doi.org/10.1186/s40694-019-0087-4","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1186/s40694-019-0081-x.].</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"6 ","pages":"26"},"PeriodicalIF":0.0,"publicationDate":"2019-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-019-0087-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37502273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A new vector system for targeted integration and overexpression of genes in the crop pathogen <i>Fusarium solani</i>.","authors":"Mikkel Rank Nielsen,&nbsp;Anna Karolina Rilana Holzwarth,&nbsp;Emmett Brew,&nbsp;Natalia Chrapkova,&nbsp;Samba Evelyne Kabemba Kaniki,&nbsp;Kenneth Kastaniegaard,&nbsp;Trine Sørensen,&nbsp;Klaus Ringsborg Westphal,&nbsp;Reinhard Wimmer,&nbsp;Teis Esben Sondergaard,&nbsp;Jens Laurids Sørensen","doi":"10.1186/s40694-019-0089-2","DOIUrl":"https://doi.org/10.1186/s40694-019-0089-2","url":null,"abstract":"<p><strong>Background: </strong>Besides their ability to produce several interesting bioactive secondary metabolites, members of the <i>Fusarium solani</i> species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation.</p><p><strong>Results: </strong>To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in <i>F. solani</i> through <i>Agrobacterium tumefaciens</i>-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of <i>F. solani</i> and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)₂Cys₆ transcriptional factor <i>fsr6</i> controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin.</p><p><strong>Conclusion: </strong>By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen <i>F. solani</i> in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (<i>PKS3</i>: <i>fsr</i>) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in <i>F. solani</i> via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the <i>Fusarium solani</i> species complex, and is obtainable from Addgene catalog #133094.</p><p><strong>Graphic abstract: </strong></p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"6 ","pages":"25"},"PeriodicalIF":0.0,"publicationDate":"2019-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-019-0089-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37501807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}