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Abstract Background Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains. Results The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy. Conclusion The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.

Abstract Background Fusarium Head Blight (FHB) is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusarium graminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists of identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration. Results Here, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities of Fg native promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations, cassette truncations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1. Conclusion The TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, protein secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work together with PCR amplification of the insert could be an alternative to Southern blotting, the gold standard technique used to identify ectopic integrations, cassette truncations and tandem insertions in fungal transformation.

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24–36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum. [ABSTRACT FROM AUTHOR]

There is an urgent need for new chemical structures to develop drugs as a result of the increasing number of viruses and pathogenic bacteria that have the ability to resist established treatments. Phytochemical screening on Pulicaria species have yielded a lot of compounds specially terpenoids, importantly though, some compounds have been found to have a potent bioactivity. Pulicaria undulata plants considered as a productive source for the effective biologically secondary metabolites. Herein, 2α-Hydroxyalantolactone (1), a major secondary metabolite, was enzymatically transformed by Rhizopus oryzae KX685359 fungal isolate. The fungal transformation affords a new transformed metabolite2-(alantolactonyloxy) propanoic acid (2) and know alantolactone-2α-acetate (3). Antimicrobial assessment of the substrate and transformed compounds disclosed the moderate antimicrobial activity of compound (3) against Gram-negative E. coli, Gram-positive L. cereus and the unicellular yeast C. albicans. [ABSTRACT FROM AUTHOR]

Fruit rot caused by Colletotrichum magnum is a crucial watermelon disease threatening the production and quality. To understand the pathogenic mechanism of C. magnum, we optimized the Agrobacterium tumefaciens-mediated transformation system (ATMT) for genetic transformation of C. magnum. The transformation efficiency of ATMT was an average of around 245 transformants per 100 million conidia. Southern blot analysis indicated that approximately 75% of the mutants contained a single copy of T-DNA. Pathogenicity test revealed that three mutants completely lost pathogenicity. The T-DNA integration sites (TISs) of three mutants were Identified. In mutant Cm699, the TISs were found in the intron region of the gene, which encoded a protein containing AP-2 complex subunit s, and simultaneous gene deletions were observed. Two deleted genes encoded the transcription initiation protein SPT3 and a hypothetical protein, respectively. In mutant Cm854, the TISs were found in the 5'-flanking regions of a gene that was similar to the MYO5 encoding Myosin I of Pyricularia oryzae (78%). In mutant Cm1078, the T-DNA was integrated into the exon regions of two adjacent genes. One was 5'-3' exoribonuclease 1 encoding gene while the other encoded a WD-repeat protein retinoblastoma binding protein 4, the homolog of the MSl1 of Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

The use of microorganisms in industry has enabled the (over)production of various compounds (e.g., primary and secondary metabolites, proteins and enzymes) that are relevant for the production of antibiotics, food, beverages, cosmetics, chemicals and biofuels, among others. Industrial strains are commonly obtained by conventional (non-GMO) strain improvement strategies and random screening and selection. However, recombinant DNA technology has made it possible to improve microbial strains by adding, deleting or modifying specific genes. Techniques such as genetic engineering and genome editing are contributing to the development of industrial production strains. Nevertheless, there is still significant room for further strain improvement. In this review, we will focus on classical and recent methods, tools and technologies used for the development of fungal production strains with the potential to be applied at an industrial scale. Additionally, the use of functional genomics, transcriptomics, proteomics and metabolomics together with the implementation of genetic manipulation techniques and expression tools will be discussed. [ABSTRACT FROM AUTHOR]

To produce an active metabolite of Chlordiazepoxide by fungal biotransformation in an easy and economic way and also to develop microbial models for drug metabolism studies. Chlordiazepoxide is metabolized in the liver by CYP3A4 and forms major active metabolite N-desmethyl chlordiazepoxide. The focus of the study was to explore the ability of six distinct fungi to biotransform the drug Chlordiazepoxide to its metabolites. Induction, Inhibition and kinetic studies were also conducted to find out the type and capability of enzyme involved in fungal biotransformation. The screening studies were performed and fermentation protocol was designed with two controls (culture control and drug control) and one sample. Extract metabolite samples were reconstituted and analysed using HPLC. Induction, Inhibition studies were conducted similarly by maintaining its respective controls using CYP3A4 inducer (Carbamazepine) and inhibitor (Fluoxetine), further kinetic studies were performed to find Km and Vmax of fungal biotransformation of Chlordiazepoxide. Among six organisms Aspergillus ochreus has shown an extra peak at 6.9 min. in HPLC when compared with its controls indicated the formation of metabolite. The metabolite thus formed was identified, isolated and structure was confirmed by mass spectrometry and NMR spectroscopy as Nor-chlordiazepoxide. During inhibition and induction studies, it was found that quantity of the metabolite was increased with inducer and decreased with inhibitor. The Km and Vmax of fungal metabolism of Chlordiazepoxide was 1.928 µg/ml and 0.1802 µg/ml/hr respectively. Aspergillus ochreus has the ability to biotransform the Chlordiazepoxide to its active metabolite by CYP3A4 like enzyme and it followed Michaelis-Menten kinetics.

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24–36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes’ integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum.

To produce an active metabolite of Chlordiazepoxide by fungal biotransformation in an easy and economic way and also to develop microbial models for drug metabolism studies. Chlordiazepoxide metabolized in the liver by CYP3A4 and forms major active metabolite N-desmethyl chlordiazepoxide. the focus of the study was to explore the ability of six distinct fungi to biotransform the drug Chlordiazepoxide to its metabolites. induction, inhibition and kinetic studies were also conducted to find out the type and capability of enzyme involved in fungal biotransformation. the screening studies were performed and fermentation protocol was designed with two controls (culture control and drug control) and one sample. extract metabolite samples were reconstituted and analysed using HPlC. induction, inhibition studies were conducted similarly by maintaining its respective controls using CYP3A4 inducer (Carbamazepine) and inhibitor (Fluoxetine), further kinetic studies were performed to find Km and Vmax of fungal biotransformation of Chlordiazepoxide. Among six organisms Aspergillus ochreus has shown an extra peak at 6.9 min. in HPLC when compared with its controls indicated the formation of metabolite. the metabolite thus formed was identified, isolated and structure was confirmed by mass spectrometry and NMR spectroscopy as Nor-chlordiazepoxide. During inhibition and induction studies, it was found that quantity of the metabolite was increased with inducer and decreased with inhibitor. the Km and Vmax of fungal metabolism of Chlordiazepoxide was 1.928 µg/ml and 0.1802 µg/ml/hr respectively. Aspergillus ochreus has the ability to biotransform the Chlordiazepoxide to its active metabolite by CYP3A4 like enzyme and it followed Michaelis-Menten kinetics. [ABSTRACT FROM AUTHOR]

The search for anticancer drugs is of continuous interest. Arecoline is an alkaloid with anticancer activity. Herein, the metabolism of arecoline through fungal transformation was investigated for the discovery of potential anticancer drugs with higher activity and selectivity. Compounds 1–5 were isolated, and their structures were fully elucidated using various spectroscopic analyses, including 1D and 2D NMR, ESIMS, and HRESIMS. This is the first report for the isolation of compounds 1 and 2. An MTT assay was performed to determine the cytotoxic activity of arecoline and its metabolites in vitro using non-small-cell lung cancer A549 and leukemia K562 cell lines compared to staurosporine and doxorubicin as positive controls. For the non-small-cell lung A549 cell line, arecoline hydrobromide, staurosporine, and doxorubicin resulted in IC50 values of 11.73 ± 0.71 µM, 10.47 ± 0.64 µM, and 5.05 ± 0.13 µM, respectively, while compounds 1, 3, and 5 exhibited IC50 values of 3.08 ± 0.19 µM, 7.33 ± 0.45 µM, and 3.29 ± 0.20 µM, respectively. For the leukemia K562 cell line, the IC50 values of arecoline hydrobromide, staurosporine, and doxorubicin were 15.3 ± 1.08 µM, 5.07 ± 0.36 µM, and 6.94 ± 0.21 µM, respectively, while the IC50 values of compounds 1, 3 and 5 were 1.56 ± 0.11 µM, 3.33 ± 0.24 µM, and 2.15 ± 0.15 µM, respectively. The selectivity index value of these compounds was higher than 3. These results indicated that compounds 1, 3, and 5 are very strong cytotoxic agents with higher activity than the positive controls and good selectivity toward the tested cancer cell lines. Cell cycle arrest was then studied by flow cytometry to investigate the apoptotic mechanism. Docking simulation revealed that most compounds possessed good binding poses and favorable protein-ligand interactions with muscarinic acetylcholine receptor M3-mAChR protein. In silico study of pharmacokinetics using SwissADME predicted compounds 1–5 to be drug-like with a high probability of good oral bioavailability. [ABSTRACT FROM AUTHOR]

The use of microorganisms in industry has enabled the (over)production of various compounds (e.g., primary and secondary metabolites, proteins and enzymes) that are relevant for the production of antibiotics, food, beverages, cosmetics, chemicals and biofuels, among others. Industrial strains are commonly obtained by conventional (non-GMO) strain improvement strategies and random screening and selection. However, recombinant DNA technology has made it possible to improve microbial strains by adding, deleting or modifying specific genes. Techniques such as genetic engineering and genome editing are contributing to the development of industrial production strains. Nevertheless, there is still significant room for further strain improvement. In this review, we will focus on classical and recent methods, tools and technologies used for the development of fungal production strains with the potential to be applied at an industrial scale. Additionally, the use of functional genomics, transcriptomics, proteomics and metabolomics together with the implementation of genetic manipulation techniques and expression tools will be discussed.

The microbial biotransformation is a robust procedure in developing steroids and fungi are practical tools in this process; therefore, the fungal modification of testosterone by Penicillium pinophilum was investigated. The three prominent metabolites, including 14α-hydroxyandrost-4-en-3,17-dione (II), 14α-hydroxytestosterone (III), and 11α-hydroxytestosterone (IV), were isolated and characterized by chromatographic and spectroscopic methods. The time course profile showed that the content of the metabolites II and III began to decrease after 96 and 24 h, respectively. In comparison, the content of the metabolite IV remained stable after 24 h. In silico studies showed that the probability of binding to the androgen receptor remains high for all three metabolites. However, the probability of binding to the estrogen receptors α and β increased for metabolite IV but decreased for metabolite III. Penicillium pinophilum as a potentially viable biocatalyst could hydroxylate C-11α and C-14α positions and oxidize the C-17β hydroxyl group to 17-ketone in testosterone molecule. [ABSTRACT FROM AUTHOR]

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of >90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens. [ABSTRACT FROM AUTHOR]

[Display omitted] • A method for the genetic transformation of Stachybotrys chartarum by protoplast-mediated and Agrobacterium-mediated transformation was established. • Developed method was successfully applied for the ectopic insertion of constructs, resulting in recombinant strains with GFP-labelling and overexpression of targeted genes. A full and partial gene deletion of a 6 kb polyketide synthase gene by homologous recombination was achieved. The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys , however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum , genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum , making this fungus accessible for genetic engineering. [ABSTRACT FROM AUTHOR]

Fungal transformation of a norethisterone (17a-ethynylestra-4-en-17ß-ol-3-one) (1) by using Macrophomina phaseolina and Paecilomyces variotii was studied. A new metabolite, 17a-hydroxymethyl-androst-4-en-11ß-ol-3-one-17ß-acetate (2) with novel changes and a known metabolite, 17a-ethynylestradiol (3) were obtained from 1 by using M. phaseolina and P. variotii, respectively. Based on various spectroscopic techniques, the structures of both metabolites were characterized. The antimicrobial activities of 1-3 were also evaluated. Compound 1 was found to be moderately active against Salmonella paratyphi while 1-3 were almost inactive against other microorganisms. [ABSTRACT FROM AUTHOR]

Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans. Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the DnkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants. [ABSTRACT FROM AUTHOR]

The search for anticancer drugs is of continuous interest. Arecoline is an alkaloid with anticancer activity. Herein, the metabolism of arecoline through fungal transformation was investigated for the discovery of potential anticancer drugs with higher activity and selectivity. Compounds 1–5 were isolated, and their structures were fully elucidated using various spectroscopic analyses, including 1D and 2D NMR, ESIMS, and HRESIMS. This is the first report for the isolation of compounds 1 and 2. An MTT assay was performed to determine the cytotoxic activity of arecoline and its metabolites in vitro using non-small-cell lung cancer A549 and leukemia K562 cell lines compared to staurosporine and doxorubicin as positive controls. For the non-small-cell lung A549 cell line, arecoline hydrobromide, staurosporine, and doxorubicin resulted in IC50 values of 11.73 ± 0.71 µM, 10.47 ± 0.64 µM, and 5.05 ± 0.13 µM, respectively, while compounds 1, 3, and 5 exhibited IC50 values of 3.08 ± 0.19 µM, 7.33 ± 0.45 µM, and 3.29 ± 0.20 µM, respectively. For the leukemia K562 cell line, the IC50 values of arecoline hydrobromide, staurosporine, and doxorubicin were 15.3 ± 1.08 µM, 5.07 ± 0.36 µM, and 6.94 ± 0.21 µM, respectively, while the IC50 values of compounds 1, 3 and 5 were 1.56 ± 0.11 µM, 3.33 ± 0.24 µM, and 2.15 ± 0.15 µM, respectively. The selectivity index value of these compounds was higher than 3. These results indicated that compounds 1, 3, and 5 are very strong cytotoxic agents with higher activity than the positive controls and good selectivity toward the tested cancer cell lines. Cell cycle arrest was then studied by flow cytometry to investigate the apoptotic mechanism. Docking simulation revealed that most compounds possessed good binding poses and favorable protein-ligand interactions with muscarinic acetylcholine receptor M3-mAChR protein. In silico study of pharmacokinetics using SwissADME predicted compounds 1–5 to be drug-like with a high probability of good oral bioavailability.

Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of >90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens.

• Diterpenes are a source of potential leading compounds to treat schistosomiasis. • Derivatives were obtained by fungal transformation with Aspergillus and Cunninghamella. • Three precursors and ten diterpene derivatives were evaluated against Schistosoma mansoni. • 7α-acetoxy- ent -kaur-16-en-19-oic acid (KA5) and ent -hardwickiic acid (HA) were the most active ones. • KA5 and HA are potential candidates for schistosomicidal drug discovery. Schistosomiasis continues to be a huge challenge for researchers, pharmaceutical companies, and governments in developing countries. Diterpene compounds are good source for the development of novel potential leading compounds to treat schistosomiasis. We are reporting herein the schistosomicidal activity of ent -kaurenoic acid, ent -copalic acid, ent -hardwickiic acid, isolated from oleoresins of Copaifera spp, and of their derivatives obtained by fungal transformation with strains of Aspergillus fumigatus , A. terreus , A. phoenicis and A. ochraceus , and of Cunninghamella echinulata e C. elegans. The in vitro antiparasitical assays were performed using adult worm pairs of Schistosoma mansoni for the evaluation of the worm pairing, egg production, and eggs development. Ten kaurane, labdane and clerodane-type diterpenes were obtained by fungal transformation and 7α-acetoxy- ent -kaur-16-en-19-oic acid and ent -hardwickiic acid were the most active ones by causing mortality of 100 % of the parasites within 24 h (concentrations of 100.0 and 200.0 μM) and displaying respective IC 50 values for 24, 48 and 72 h of 56.7, 37.6 and 29.2 μM and 29.6, 30.8 and 25.7 μM. Additionally, 7α-acetoxy- ent -kaur-16-en-19-oic acid and ent -hardwickiic acid highly reduced the number of laid eggs at 6.25 and 12.5 μM, respectively. These diterpenes should be further investigated as potential candidates for antiparasitic drug discovery. [ABSTRACT FROM AUTHOR]

Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans. Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the ΔnkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants.

• Fungal transformation of kaempulchraol E (1) was performed by Rhizopus oryzae KX685359. • Two new diterpenoids 2 and 3 along with roscorane B (4) were produced. • Compound 2 was confirmed via X-ray diffraction analysis. • Compound 4 showed potent toxicity against HSC-2 cell lines. Fungal transformation of the diterpenoid kaempulchraol E (1) by Rhizopus oryzae KX685359 afforded two trihydroxlated isopimaradienes: 2α,6β,14β-trihydroxy-isopimara-8(9),15-diene (2) and 2α,6β,14α-trihydroxy-isopimara-8(9),15-diene (3) in addition to the known metabolite roscorane B (4). Chemical structures were established by spectroscopic techniques including: HRMS, FT-IR and 1D- & 2D-NMR. The structure of 2 was confirmed by single-crystal, X-ray diffraction analysis. Bio-transformed metabolites as well as the substrate originally isolated from Kaempheria galanga were tested against two cancer cell lines: HSC-2 and B16-BL6. Compounds 4 showed greater toxicity than the original plant derived diterpene (IC 50 54.08 ± 0.05 versus 96.05 ± 0.03 μM, respectively) when assayed against the HSC-2 cell line. This study confirms the role of fungal transformations in developing novel natural products with efficacious biological activities. [ABSTRACT FROM AUTHOR]

Due to the rapidly increasing number of pathogenic bacteria, viruses, and tumor cells that possess resistance toward established therapies, lead structures for the development of new drugs are in high demand. The plant material of Tanacetum sinaicum is considered a prolific source of the potent biologically active sesquiterpene Tanacetolide A (1). In our research, we focused on the structural transformation of the substrate Tanacetolide A (compound 1) by suspended culture of the novel locally isolated terrestrial fungus Rhizopus oryzae KX685359. The fungal transformation resulted in the formation of 1β,3α‐dihydroxy‐6α‐hydroperoxy‐eudsm‐4(15)‐ene 12‐oic acid (2) with higher antiproliferative activity against colon cancer cell line (Caco‐2) than that of the parent compound (1). [ABSTRACT FROM AUTHOR]

Nigrospora is a diverse genus of fungi colonizing plants through endophytic, pathogenic, or saprobic interactions. Endophytic isolates can improve growth and development of host plants, as well as their resistance to microbial pathogens, but exactly how they do so remains poorly understood. Developing a reliable transformation method is crucial to investigate these mechanisms, in particular to identify pivotal genes for specific functions that correlate with specific traits. In this study, we identified eight isolates of Nigrospora sp. internally colonizing the leaves of switchgrass plants cultivated in North Carolina. Using an Agrobacterium tumefaciens -mediated transformation approach with control and GFP-expressing vectors, we report the first successful transformation of two Nigrospora isolates. Finally, we demonstrate that wild-type and transgenic isolates both negatively impact the growth of two plant pathogens in co-culture conditions, Bipolaris maydis and Parastagonospora nodorum , responsible for the Southern Leaf Blight and Septoria Nodorum Blotch diseases, respectively. The GFP-transformed strains developed here can therefore serve as accurate reporters of spatial interactions in future studies of Nigrospora and pathogens in the plant. Finally, the transformation method we describe lays the foundation for further genetic research on the Nigrospora genus to expand our mechanistic understanding of plant-endophyte interactions. • Identification of eight new Nigrospora sp. isolates from switchgrass leaves. • First Agrobacterium tumefaciens -mediated transformation of two Nigrospora isolates. • These two isolates inhibit the growth of the plant pathogens B. maydis and P. nodorum. • The genetic transformation does not affect the antagonistic ability of Nigrospora. [ABSTRACT FROM AUTHOR]

Microbial transformation of thymoquinone (5-isopropyl-2-methyl-cyclohexa-2,5-diene-1,4-dione) ( 1 ) by suspended cell-cultures of the plant pathogenic fungus Aspergillus niger resulted in the production of three metabolites. These metabolites were identified as 5-isopropyl-2-methyloxepin-1-one ( 2 ), 3-hydroxy-5-isopropyl-2-methylcyclohexa-2,5-diene-1,4-dione ( 3 ), and 5-isopropyl-2-methylbenzene-1,4-diol ( 4 ) by different spectroscopic methods. Metabolite 2 was found to be a new compound. Compound 4 showed a potent antioxidant activity. [ABSTRACT FROM AUTHOR]

Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization.

Fruit rot caused by Colletotrichum magnum is a crucial watermelon disease threatening the production and quality. To understand the pathogenic mechanism of C. magnum , we optimized the Agrobacterium tumefaciens -mediated transformation system (ATMT) for genetic transformation of C. magnum . The transformation efficiency of ATMT was an average of around 245 transformants per 100 million conidia. Southern blot analysis indicated that approximately 75% of the mutants contained a single copy of T-DNA. Pathogenicity test revealed that three mutants completely lost pathogenicity. The T-DNA integration sites (TISs) of three mutants were Identified. In mutant Cm699, the TISs were found in the intron region of the gene, which encoded a protein containing AP-2 complex subunit σ, and simultaneous gene deletions were observed. Two deleted genes encoded the transcription initiation protein SPT3 and a hypothetical protein, respectively. In mutant Cm854, the TISs were found in the 5'-flanking regions of a gene that was similar to the MYO5 encoding Myosin I of Pyricularia oryzae (78%). In mutant Cm1078, the T-DNA was integrated into the exon regions of two adjacent genes. One was 5'-3' exoribonuclease 1 encoding gene while the other encoded a WD-repeat protein retinoblastoma binding protein 4, the homolog of the MSl1 of Saccharomyces cerevisiae ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Guo, Wu, Peng, Kang, Liu, Luo and Gu.)

Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization. [ABSTRACT FROM AUTHOR]

The dimorphic fungal pathogen, Zymoseptoria tritici undergoes discrete developmental changes to complete its life cycle on wheat. Molecular mechanisms underlying morphogenesis during infection process of Z. tritici are poorly understood. In this study, we have investigated the role of ZtVf1 gene encoding a transcription factor belonging to C 2 -H 2 subfamily. In planta assays revealed that ZtVf1 is required for virulence. Reduced necrotic lesions and low pycnidia density within the lesions resulted in significantly reduced virulence of ZtVf1 mutants. Cytological analysis showed that the impaired virulence of ZtVf1 mutants attributed to reduced penetration and colonization along with hampered pycnidia differentiation. In vitro phenotyping showed that ZtVf1 deletion affects hyphal branching and biomass production suggesting that the reduced tissue colonization by the ZtVf1 mutant might be due to lower hyphal branching and less fungal biomass production. In addition, the majority of infected substomatal cavities by the ZtVf1 mutant filled with compacted mycelia mat that did not differentiate to mature pycnidia indicating that the impaired melanization negatively affected pycnidia formation and maturation. The ZtVf1 might target multiple genes belonging to different cellular processes whose identification is of eminent interest to increase our understanding of this pathosystem. Overall, the data provided in this study indicates that attenuated pathogenicity of ZtVf1 mutant is due to involvement of this gene in the regulation of both early and late stages of infection. [ABSTRACT FROM AUTHOR]

While select eudesmane sesquiterpenes exhibit anti-neoplastic activity, tumor-inhibition for costic-acids has not been established. Here biological activity of 3-oxo-γ-costic acid ( 1 ), previously isolated from Chiliadenus montanus , as well as new sesquiterpenes ( 2 – 5 ) and the known derivative, 3-oxoeudesma-1,4,11(13)-trien-7-1061αH-l2-oic acid ( 6 ), all produced from 1 by the fungus Athelia rolfsii , are reported. Structures were elucidated using MS and NMR spectroscopy with activity-screening utilizing human colon- and lung-tumor lines, Caco-2 and A549 respectively. Compound 1 exhibited anti-proliferative activity against Caco-2 (IC 50 39 µM) and 2 was active against A549 (IC 50 74 µM) suggesting therapeutic potential for the original substrate and a bio-transformed product. [ABSTRACT FROM AUTHOR]

In this study we describe the heterologous expression of the recently identified cyanobacterial pathway for long chain alkane biosynthesis, involving the reduction of fatty acyl-ACP to fatty aldehyde and the subsequent conversion of this into alkanes, in the filamentous fungus Aspergillus carbonarius ITEM 5010. Genes originating from Synechococcus elongatus strain PCC7942, encoding acyl-ACP/CoA reductase and aldehyde deformylating oxygenase enzymes, were successfully expressed in A. carbonarius, which lead to the production of pentadecane and heptadecane, alkanes that have not been previously produced by this fungus. Titers of 0.2, 0.5 and 2.7 mg/l pentadecane and 0.8, 1.6 and 10.2 mg/l heptadecane were achieved using glucose, Yeast malt and oatmeal media, respectively. Besides producing alkanes, we found elevated levels of internal free fatty acids and triglycerides in the alkane producing transformant. These findings can indicate that a yet unidentified, native fatty aldehyde dehydrogenase channels back the fatty aldehydes into the fatty acid metabolism, thus competing for substrate with the heterologously expressed fatty aldehyde deformylating oxygenase. These findings will potentially facilitate the future application of robust, fungal cell factories for the production of advanced biofuels from various substrates. [ABSTRACT FROM AUTHOR]

Fungal hyphae—and in some cases, spores—are multi-nucleate. During genetic transformation of these spores or mycelia, only one nucleus generally receives the transferred T-DNA generating heterokaryotic colonies. Characterization of genetic changes, such as the effects of gene disruption in the transformants, requires purified homokaryotic lines. Hyphal tip transfer has conventionally been used to isolate homokaryons. We developed an alternative method for purification of fungal homokaryons from transformed heterokaryotic lines of Sclerotinia sclerotiorum. Ultrasound pulses were used to generate bi-septate, unicellular hyphal fragments that regenerate under selection to produce homokaryotic lines that can be easily identified using colony PCR. This technique facilitates the purification of transformed lines, which allows for routine genome manipulation, and should be adaptable for other filamentous fungi.

The dominant mechanism to repair double-stranded DNA breaks in filamentous fungi is the non-homologous end joining (NHEJ) pathway, and not the homologous recombination (HR) pathway that operates in the mutation of genes by replacement of target DNA for selection cassettes. The key to improve HR frequency is the inactivation of the NHEJ pathway by eliminating components of its Ku70/80 heterodimeric complex. We have obtained ku70 mutants of Penicillium digitatum , the main citrus postharvest pathogen. The increased efficiency of HR in Δ ku70 strains was demonstrated by the generation of mutants in two different chitin synthase genes ( PdchsII and PdchsV ). P. digitatum Δ ku70 strains showed no differences from the parental strain in vegetative growth, asexual development or virulence to citrus fruit, when experiments were conducted at the optimal temperature of 24 °C. However, growth of Δ ku70 strains at temperatures higher than 24 °C demonstrated a detrimental effect in axenic growth and conidia production. These observations are in agreement with previous studies describing differences between ku70 mutants and their parental strains in some fungal species, and must be taken into account for future applications of the Δ ku approach to increase HR efficiency in fungi. [ABSTRACT FROM AUTHOR]

Armillaria mellea is a serious pathogen of horticultural and agricultural systems in Europe and North America. The lack of a reliable in vitro fruiting system for heterothallic A. mellea has hindered research and required dependence on intermittently available wild-collected basidiospores of endemic genotypes, necessitating the use of variable genetic material in transformation studies. Here we describe a reliable, reproducible in vitro fruiting method for heterothallic A. mellea from the western US. Isolates and growth conditions were evaluated to determine effective fruiting conditions. Following medium colonisation for 4 weeks, cultures were incubated under warm/bright conditions for 4–6 weeks before incubation in dim/cool conditions. Primordia emerged within 3–4 weeks following a temperature decrease and this was most efficient when coupled with a light reduction. Basidiocarps matured within 3–4 weeks and produced viable basidiospores. Agrobacterium tumefaciens and vectors were evaluated by transformation of in vitro -produced basidiospores and a versatile transformation vector was constructed to simplify promoter and marker gene exchange using homologous recombination in yeast. Fruiting bodies and viable basidiospores of A. mellea have been reliably produced in vitro which, coupled with the enhanced knowledge of suitable A. tumefaciens strains and vectors for transformation, will assist future genetic research into this important pathogen. [ABSTRACT FROM AUTHOR]

17α-Ethinylestradiol (EE2) is a commonly used synthetic estrogen, which is resistant to degradation and accumulates in all parts of the environment. In this work the capability of thirty eight fungal strains to remove this xenobiotic from culture medium was checked. Nine out of the tested strains were able to completely remove EE2 (at initial concentration 10 mg l −1 ) from the culture medium during 3 d of incubation. The most efficient were two Aspergillus strains ( Aspergillus fumigatus IM 6510 and Aspergillus versicolor IM 2161) which also possess the ability to eliminate the xenoestrogen in the presence of NaCl. A. versicolor seems to be less vulnerable to salinity and the transformation process was completed after 3 d of incubation in all tested NaCl concentrations. This strain transformed EE2 and formed its metabolites detected during liquid chromatography–(electrospray) triple quadrupole–mass spectrometry (LC–(ESI)MS–MS) analysis. The quantitative analyses revealed that the EE2 subsequent decrease in samples with or without salinity was accompanied by an increase in the concentrations of estrone (E1) and estradiol (E2). The presence of NaCl slightly inhibited E2 to E1 transformation during the first 24 h of incubation but at the end of the experiment the amount of nontoxic E1 prevailed in all tested samples. The results of this study suggest that Aspergillus sp. possesses an ability to utilize EE2 in the presence of NaCl. [ABSTRACT FROM AUTHOR]

Hypocrea jecorina , the sexual teleomorph of Trichoderma reesei , has long been favored as an industrial cellulase producer, first utilizing its native cellulase system and later augmented by the introduction of heterologous enzymatic activities or improved variants of native enzymes. Expression of heterologous proteins in H. jecorina was once considered difficult when the target was an improved variant of a native cellulase. Developments over the past nearly 30 years have produced strains, vectors, and selection mechanisms that have continued to simplify and streamline heterologous protein expression in this fungus. More recent developments in fungal molecular biology have pointed the way toward a fundamental transformation in the ease and efficiency of heterologous protein expression in this important industrial host. Here, 1) we provide a historical perspective on advances in H. jecorina molecular biology, 2) outline host strain engineering, transformation, selection, and expression strategies, 3) detail potential pitfalls when working with this organism, and 4) provide consolidated examples of successful cellulase expression outcomes from our laboratory. [ABSTRACT FROM AUTHOR]

Biologically active piperitone-derived racemic iodo-, bromo- and chlorolactones ( 1 – 3 ) were transformed with the use of microbial enzymatic systems. Four strains of filamentous fungi Absidia glauca AM254, Absidia cylindrospora AM336, Mortierella vinaceae AM149 and Nigrospora oryzae AM8 transformed halolactones ( 1 – 3 ) to four new halohydroxylactones ( 4 – 7 ). In all biotransformations the hydroxy group was incorporated in inactivated methine carbon atom at isopropyl substituent. In N. oryzae AM8 culture the bromolactone with additional hydroxy group in α-position, relative to C O bond in γ-lactone ring, was also formed as a product. The structures of new compounds were established on the basis of spectral data. [ABSTRACT FROM AUTHOR]

The white-rot fungus Phanerochaete chrysosporium secretes extracellular oxidative enzymes during secondary metabolism, but lacks versatile peroxidase, an enzyme important in ligninolysis and diverse biotechnology processes. In this study, we report the genetic modification of a P. chrysosporium strain capable of co-expressing two endogenous genes constitutively, manganese peroxidase ( mnp1) and lignin peroxidase ( lipH8), and the codon-optimized vpl2 gene from Pleurotus eryngii. For this purpose, we employed a highly efficient transformation method based on the use of shock waves developed by our group. The expression of recombinant genes was verified by PCR, Southern blot, quantitative real-time PCR (qRT-PCR), and assays of enzymatic activity. The production yield of ligninolytic enzymes was up to four times higher in comparison to previously published reports. These results may represent significant progress toward the stable production of ligninolytic enzymes and the development of an effective fungal strain with promising biotechnological applications. [ABSTRACT FROM AUTHOR]

Highlights: [•] Delivery of Cre recombinase by anastomosis excised fungal loxP-flanked marker genes. [•] The method is time and cost saving. [•] The method allows unlimited recycling of fungal marker genes. [•] The method results in marker gene-free excision strains. [•] The method was applied to Cryphonectria parasitica and Metarhizium robertsii. [Copyright &y& Elsevier]

Microbial transformation of mestanolone ( 1) using the plant pathogenic fungus, Rhizopus stolonifer, resulted in the production of two known metabolites, identified as 11α-hydroxymestanolone ( 3) and 6α-hydroxymestanolone ( 4). Transformation of 17-methyl-1-testosterone ( 2) by R. stolonifer yielded two known metabolites, methandrostenolone ( 5) and 11α,17β- dihydroxy-androsta-1,4-diene-3-one ( 6). These transformations included α-hydroxylations at C-11 and C-6, dehydrogenation at C-4, androsta and a demethylation at C-17 positions. Structures of transformed products were determined using spectroscopic techniques. [ABSTRACT FROM AUTHOR]