Fungal Biology and Biotechnology最新文献

{"title":"Genome sequencing and molecular networking analysis of the wild fungus Anthostomella pinea reveal its ability to produce a diverse range of secondary metabolites.","authors":"R Iacovelli, T He, J L Allen, T Hackl, K Haslinger","doi":"10.1186/s40694-023-00170-1","DOIUrl":"10.1186/s40694-023-00170-1","url":null,"abstract":"<p><strong>Background: </strong>Filamentous fungi are prolific producers of bioactive molecules and enzymes with important applications in industry. Yet, the vast majority of fungal species remain undiscovered or uncharacterized. Here we focus our attention to a wild fungal isolate that we identified as Anthostomella pinea. The fungus belongs to a complex polyphyletic genus in the family of Xylariaceae, which is known to comprise endophytic and pathogenic fungi that produce a plethora of interesting secondary metabolites. Despite that, Anthostomella is largely understudied and only two species have been fully sequenced and characterized at a genomic level.</p><p><strong>Results: </strong>In this work, we used long-read sequencing to obtain the complete 53.7 Mb genome sequence including the full mitochondrial DNA. We performed extensive structural and functional annotation of coding sequences, including genes encoding enzymes with potential applications in biotechnology. Among others, we found that the genome of A. pinea encodes 91 biosynthetic gene clusters, more than 600 CAZymes, and 164 P450s. Furthermore, untargeted metabolomics and molecular networking analysis of the cultivation extracts revealed a rich secondary metabolism, and in particular an abundance of sesquiterpenoids and sesquiterpene lactones. We also identified the polyketide antibiotic xanthoepocin, to which we attribute the anti-Gram-positive effect of the extracts that we observed in antibacterial plate assays.</p><p><strong>Conclusions: </strong>Taken together, our results provide a first glimpse into the potential of Anthstomella pinea to provide new bioactive molecules and biocatalysts and will facilitate future research into these valuable metabolites.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"11 1","pages":"1"},"PeriodicalIF":0.0,"publicationDate":"2024-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10763133/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139089286","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":"Mechanical, physical and thermal properties of composite materials produced with the basidiomycete Fomes fomentarius.","authors":"Bertram Schmidt, Carsten Freidank-Pohl, Justus Zillessen, Lisa Stelzer, Tamara Núñez Guitar, Carsten Lühr, Henri Müller, Fangxing Zhang, Jörg U Hammel, Heiko Briesen, Sascha Jung, Hans-Jörg Gusovius, Vera Meyer","doi":"10.1186/s40694-023-00169-8","DOIUrl":"10.1186/s40694-023-00169-8","url":null,"abstract":"<p><strong>Background: </strong>To achieve climate neutrality, fundamentally new concepts of circularity need to be implemented by the building sector as it contributes to 40% of anthropogenic CO₂ emission. Fungal biotechnology can make a significant contribution here and help eliminate fossil dependency for building material production. Recently, we have shown that the medicinal polypore Fomes fomentarius feeds well on renewable lignocellulosic biomass and produces composite materials that could potentially replace fossil fuel-based expanded polystyrene as insulation material.</p><p><strong>Results: </strong>In this study, we explored the mechanical, physical, and thermal properties of F. fomentarius-based composite materials in more detail and determined key performance parameters that are important to evaluate the usability of F. fomentarius-based composite materials in the construction sector. These parameters were determined according to European standards and included compressive strength, modulus of elasticity, thermal conductivity, water vapour permeability, and flammability of uncompressed composites as well as flexural strength, transverse tensile strength, and water absorption capacity of heat-pressed composites, among others. We could show that uncompressed composites obtained from F. fomentarius and hemp shives display a thermal conductivity of 0.044 W (m K)^-1 which is in the range of natural organic fibres. A water vapour permeability of 1.72 and classification into flammability class B1 clearly surpasses fossil-based insulation materials including expanded polystyrene and polyurethane. We could furthermore show that heat-pressing can be used to reliably generate stiff and firm particleboards that have the potential to replace current wood-based particleboards that contain synthetic additives. X-ray microcomputed tomography finally visualized for the first time the growth of hyphae of F. fomentarius on and into the hemp shive substrates and generated high-resolution images of the microstructure of F. fomentarius-based composites.</p><p><strong>Conclusion: </strong>This study demonstrates that fungal-based composites produced with F. fomentarius partially meet or even exceed key performance parameters of currently used fossil fuel-based insulation materials and can also be used to replace particleboards.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"22"},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10694974/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138483417","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":"Compatible solutes determine the heat resistance of conidia.","authors":"Sjoerd J Seekles, Tom van den Brule, Maarten Punt, Jan Dijksterhuis, Mark Arentshorst, Maryam Ijadpanahsaravi, Winfried Roseboom, Gwendolin Meuken, Véronique Ongenae, Jordy Zwerus, Robin A Ohm, Gertjan Kramer, Han A B Wösten, Johannes H de Winde, Arthur F J Ram","doi":"10.1186/s40694-023-00168-9","DOIUrl":"10.1186/s40694-023-00168-9","url":null,"abstract":"<p><strong>Background: </strong>Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle.</p><p><strong>Results: </strong>In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance.</p><p><strong>Conclusions: </strong>Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"21"},"PeriodicalIF":0.0,"publicationDate":"2023-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10644514/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92157284","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":"Identification and evaluation of suitable reference genes for RT-qPCR analyses in Trichoderma atroviride under varying light conditions.","authors":"Daniel Flatschacher, Alexander Eschlböck, Susanne Zeilinger","doi":"10.1186/s40694-023-00167-w","DOIUrl":"10.1186/s40694-023-00167-w","url":null,"abstract":"<p><strong>Background: </strong>Trichoderma atroviride is a competitive soil-borne mycoparasitic fungus with extensive applications as a biocontrol agent in plant protection. Despite its importance and application potential, reference genes for RT-qPCR analysis in T. atroviride have not been evaluated. Light exerts profound effects on physiology, such as growth, conidiation, secondary metabolism, and stress response in T. atroviride, as well as in other fungi. In this study, we aimed to address this gap by identifying stable reference genes for RT-qPCR experiments in T. atroviride under different light conditions, thereby enhancing accurate and reliable gene expression analysis in this model mycoparasite. We measured and compared candidate reference genes using commonly applied statistical algorithms.</p><p><strong>Results: </strong>Under cyclic light-dark cultivation conditions, tbp and rho were identified as the most stably expressed genes, while act1, fis1, btl, and sar1 were found to be the least stable. Similar stability rankings were obtained for cultures grown under complete darkness, with tef1 and vma1 emerging as the most stable genes and act1, rho, fis1, and btl as the least stable genes. Combining the data from both cultivation conditions, gapdh and vma1 were identified as the most stable reference genes, while sar1 and fis1 were the least stable. The selection of different reference genes had a significant impact on the calculation of relative gene expression, as demonstrated by the expression patterns of target genes pks4 and lox1.</p><p><strong>Conclusion: </strong>The data emphasize the importance of validating reference genes for different cultivation conditions in fungi to ensure accurate interpretation of gene expression data.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"20"},"PeriodicalIF":0.0,"publicationDate":"2023-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10546744/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41177463","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":"Non-canonical two-step biosynthesis of anti-oomycete indole alkaloids in Kickxellales.","authors":"Johannes Rassbach, Nathalie Hilsberg, Veit G Haensch, Sebastian Dörner, Julia Gressler, Robin Sonnabend, Caroline Semm, Kerstin Voigt, Christian Hertweck, Markus Gressler","doi":"10.1186/s40694-023-00166-x","DOIUrl":"10.1186/s40694-023-00166-x","url":null,"abstract":"<p><strong>Background: </strong>Fungi are prolific producers of bioactive small molecules of pharmaceutical or agricultural interest. The secondary metabolism of higher fungi (Dikarya) has been well-investigated which led to > 39,000 described compounds. However, natural product researchers scarcely drew attention to early-diverging fungi (Mucoro- and Zoopagomycota) as they are considered to rarely produce secondary metabolites. Indeed, only 15 compounds have as yet been isolated from the entire phylum of the Zoopagomycota.</p><p><strong>Results: </strong>Here, we showcase eight species of the order Kickxellales (phylum Zoopagomycota) as potent producers of the indole-3-acetic acid (IAA)-derived compounds lindolins A and B. The compounds are produced both under laboratory conditions and in the natural soil habitat suggesting a specialized ecological function. Indeed, lindolin A is a selective agent against plant-pathogenic oomycetes such as Phytophthora sp. Lindolin biosynthesis was reconstituted in vitro and relies on the activity of two enzymes of dissimilar evolutionary origin: Whilst the IAA-CoA ligase LinA has evolved from fungal 4-coumaryl-CoA synthetases, the subsequently acting IAA-CoA:anthranilate N-indole-3-acetyltransferase LinB is a unique enzyme across all kingdoms of life.</p><p><strong>Conclusions: </strong>This is the first report on bioactive secondary metabolites in the subphylum Kickxellomycotina and the first evidence for a non-clustered, two-step biosynthetic route of secondary metabolites in early-diverging fungi. Thus, the generally accepted \"gene cluster hypothesis\" for natural products needs to be reconsidered for early diverging fungi.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"19"},"PeriodicalIF":0.0,"publicationDate":"2023-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10478498/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10177785","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":"CRISPR/Cas9 mediated gene editing of transcription factor ACE1 for enhanced cellulase production in thermophilic fungus Rasamsonia emersonii.","authors":"Varinder Singh, Yashika Raheja, Neha Basotra, Gaurav Sharma, Adrian Tsang, Bhupinder Singh Chadha","doi":"10.1186/s40694-023-00165-y","DOIUrl":"10.1186/s40694-023-00165-y","url":null,"abstract":"<p><strong>Background: </strong>The filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed.</p><p><strong>Results: </strong>A gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), β-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, β-xylosidase, xylanase, β-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3.</p><p><strong>Conclusions: </strong>Current work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"18"},"PeriodicalIF":0.0,"publicationDate":"2023-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10472679/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10514631","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":"Basidiomycete non-reducing polyketide synthases function independently of SAT domains.","authors":"Nikolai A Löhr,&nbsp;Malik Rakhmanov,&nbsp;Jacob M Wurlitzer,&nbsp;Gerald Lackner,&nbsp;Markus Gressler,&nbsp;Dirk Hoffmeister","doi":"10.1186/s40694-023-00164-z","DOIUrl":"https://doi.org/10.1186/s40694-023-00164-z","url":null,"abstract":"<p><strong>Background: </strong>Non-reducing polyketide synthases (NR-PKSs) account for a major share of natural product diversity produced by both Asco- and Basidiomycota. The present evolutionary diversification into eleven clades further underscores the relevance of these multi-domain enzymes. Following current knowledge, NR-PKSs initiate polyketide assembly by an N-terminal starter unit:acyl transferase (SAT) domain that catalyzes the transfer of an acetyl starter from the acetyl-CoA thioester onto the acyl carrier protein (ACP).</p><p><strong>Results: </strong>A comprehensive phylogenetic analysis of NR-PKSs established a twelfth clade from which three representatives, enzymes CrPKS1-3 of the webcap mushroom Cortinarius rufoolivaceus, were biochemically characterized. These basidiomycete synthases lack a SAT domain yet are fully functional hepta- and octaketide synthases in vivo. Three members of the other clade of basidiomycete NR-PKSs (clade VIII) were produced as SAT-domainless versions and analyzed in vivo and in vitro. They retained full activity, thus corroborating the notion that the SAT domain is dispensable for many basidiomycete NR-PKSs. For comparison, the ascomycete octaketide synthase atrochrysone carboxylic acid synthase (ACAS) was produced as a SAT-domainless enzyme as well, but turned out completely inactive. However, a literature survey revealed that some NR-PKSs of ascomycetes carry mutations within the catalytic motif of the SAT domain. In these cases, the role of the domain and the origin of the formal acetate unit remains open.</p><p><strong>Conclusions: </strong>The role of SAT domains differs between asco- and basidiomycete NR-PKSs. For the latter, it is not part of the minimal set of NR-PKS domains and not required for function. This knowledge may help engineer compact NR-PKSs for more resource-efficient routes. From the genomic standpoint, seemingly incomplete or corrupted genes encoding SAT-domainless NR-PKSs should not automatically be dismissed as non-functional pseudogenes, but considered during genome analysis to decipher the potential arsenal of natural products of a given fungus.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"17"},"PeriodicalIF":0.0,"publicationDate":"2023-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10401856/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10000098","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":"Antimicrobial activities of metabolites isolated from endophytic Aspergillus flavus of Sarcophyton ehrenbergi supported by in-silico study and NMR spectroscopy.","authors":"Abdel Nasser B Singab,&nbsp;Yasmin A Elkhawas,&nbsp;Eman Al-Sayed,&nbsp;Ahmed M Elissawy,&nbsp;Iten M Fawzy,&nbsp;Nada M Mostafa","doi":"10.1186/s40694-023-00161-2","DOIUrl":"https://doi.org/10.1186/s40694-023-00161-2","url":null,"abstract":"<p><strong>Background: </strong>Endophytic Aspergillus species produce countless valuable bioactive secondary metabolites. In the current study, Aspergillus flavus an endophyte from the soft coral Sarcophyton ehrenbergi was chemically explored and the extracted phytoconstituents were subsequently evaluated for antimicrobial activity. This is accomplished by employing nuclear magnetic resonance (NMR) spectroscopy and computational techniques. Additionally, An in vitro anticancer analysis of A. flavus total extract against breast cancer cells (MCF-7) was investigated.</p><p><strong>Result: </strong>Six compounds were separated from the crude alcohol extract of the endophytic Aspergillus flavus out of which anhydro-mevalonolactone was reported for the first time. The anti-fungal and anti-Helicobacter pylori properties of two distinct compounds (Scopularides A and B) were assessed. Additionally, computational research was done to identify the binding mechanisms for all compounds. Both the compounds were found to be active against H. pylori with minimum inhibitory concentration (MIC) values ranging from 7.81 to 15.63 µg/ mL as compared with clarithromycin 1.95 µg/ mL. Scopularides A was potent against both Candida albicans and Aspergillus niger with MIC values ranging from 3.9 to 31.25 µg/ mL, while scopularides B only inhibits Candida albicans with MIC value of 15.63 µg/ mL and weak inhibitory activity against A. niger (MIC = 125 µg/ mL). Furthermore, cytotoxic activity showed a significant effect (IC₅₀: 30.46 mg/mL) against MCF-7 cells.</p><p><strong>Conclusion: </strong>Our findings report that cytotoxic activity and molecular docking support the antimicrobial activity of Aspergillus flavus, which could be a promising alternative source as a potential antimicrobial agent.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"16"},"PeriodicalIF":0.0,"publicationDate":"2023-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10394880/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9929822","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":"Rapid and robust squashed spore/colony PCR of industrially important fungi.","authors":"Guoliang Yuan,&nbsp;Jeffrey J Czajka,&nbsp;Ziyu Dai,&nbsp;Dehong Hu,&nbsp;Kyle R Pomraning,&nbsp;Beth A Hofstad,&nbsp;Joonhoon Kim,&nbsp;Ana L Robles,&nbsp;Shuang Deng,&nbsp;Jon K Magnuson","doi":"10.1186/s40694-023-00163-0","DOIUrl":"https://doi.org/10.1186/s40694-023-00163-0","url":null,"abstract":"<p><strong>Background: </strong>Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals.</p><p><strong>Results: </strong>In this study we developed a rapid and robust technique called \"Squash-PCR\" to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains.</p><p><strong>Conclusion: </strong>The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"15"},"PeriodicalIF":0.0,"publicationDate":"2023-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10329332/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10166712","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":"Bifurcate evolution of quinone synthetases in basidiomycetes.","authors":"Paula Sophie Seibold, Stefanie Lawrinowitz, Ihar Raztsou, Markus Gressler, Hans-Dieter Arndt, Pierre Stallforth, Dirk Hoffmeister","doi":"10.1186/s40694-023-00162-1","DOIUrl":"10.1186/s40694-023-00162-1","url":null,"abstract":"<p><strong>Background: </strong>The terphenylquinones represent an ecologically remarkable class of basidiomycete natural products as they serve as central precursors of pigments and compounds that impact on microbial consortia by modulating bacterial biofilms and motility. This study addressed the phylogenetic origin of the quinone synthetases that assemble the key terphenylquinones polyporic acid and atromentin.</p><p><strong>Results: </strong>The activity of the Hapalopilus rutilans synthetases HapA1, HapA2 and of Psilocybe cubensis PpaA1 were reconstituted in Aspergilli. Liquid chromatography and mass spectrometry of the culture extracts identified all three enzymes as polyporic acid synthetases. PpaA1 is unique in that it features a C-terminal, yet catalytically inactive dioxygenase domain. Combined with bioinformatics to reconstruct the phylogeny, our results demonstrate that basidiomycete polyporic acid and atromentin synthetases evolved independently, although they share an identical catalytic mechanism and release structurally very closely related products. A targeted amino acid replacement in the substrate binding pocket of the adenylation domains resulted in bifunctional synthetases producing both polyporic acid and atromentin.</p><p><strong>Conclusions: </strong>Our results imply that quinone synthetases evolved twice independently in basidiomycetes, depending on the aromatic α-keto acid substrate. Furthermore, key amino acid residues for substrate specificity were identified and changed which led to a relaxed substrate profile. Therefore, our work lays the foundation for future targeted enzyme engineering.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"14"},"PeriodicalIF":0.0,"publicationDate":"2023-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10316625/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10127967","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}