The filamentous fungus T. reesei QM9414 (ATCC 26921) was used as the parental strain for construction of all the deletion strains in this study. Relative strains were grown on potato dextrose agar (PDA) plates (200 g/L potato, 20 g/L glucose, 20 g/L agar) at 30 °C for 5–7 days to harvest spores. Then, the spores were collected and then 108 of spores were pre-cultured in 150 mL of minimal medium or induction medium (CPM, Minimal uridine medium with 2% Lactose or Avicel) at 30 °C for 36 h and, subsequently, 1g of mycelia was transferred into 150 mL of CPM for cellulase production [39 (link)]. The plasmid T-hph was used as the template to amplify the hygromycin B-resistant gene hph. The pyrithiamine-resistant gene ptrA was amplified from the plasmid T-ptrA. Minimal medium supplemented with 0.3 μg/mL pyrithiamine (Sigma, St. Louis, Missouri, USA) or 300 μg/mL hygromycin B was applied as a selective medium for screening the fungal transformants. The skim-milk agar plates were used to investigate the capacity of T. reesei strains to secrete proteases. The composition of skim-milk medium was MM supplemented with 2% skim milk plus different carbon sources or different nitrogen sources. The CMC plates were used to screen the strains showing cellulase (EG) activity. The CMC medium composition was as follows (g/L): 10 CMC–Na, 1 yeast extract, 5 triton X-100, and 20 agar.
Pyrithiamine
Manufactured by Merck Group
Sourced in Germany, United States
Pyrithiamine is a laboratory product manufactured by Merck Group. It is a chemical compound used in scientific research and experimentation. The core function of Pyrithiamine is to serve as a research tool, but a detailed description of its intended use cannot be provided in an unbiased and factual manner without extrapolation.
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Lab products found in correlation
Hygromycin, by InvivoGen (2 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (2 mentions)
Glufosinate, by Bayer (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Thiamine, by Thermo Fisher Scientific (1 mentions)
Cyanocobalamin, by Merck Group (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Clariostar plate reader, by BMG Labtech (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Sangon (1 mentions)
Thiamine, by Merck Group (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Hygromycin b, by Merck Group (1 mentions)
Kanamycin sulfate, by AppliChem (1 mentions)
Oxythiamine, by Merck Group (1 mentions)
Itc200 calorimeter, by Malvern Panalytical (1 mentions)
Nano itc calorimeter, by TA Instruments (1 mentions)
Hygromycin b, by InvivoGen (1 mentions)
Hygromycin, by Merck Group (1 mentions)
15 protocols using pyrithiamine
1
Cellulase Production by T. reesei Mutants
2
Isothermal Titration Calorimetry of Thiamine Analogs
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For the experiments presented in Fig. 3 and Table 1 , ITC experiments were performed using Nano ITC calorimeter (TA Instruments) at the constant temperature of 25°C. ITC cells were loaded with 200 µl of 10–18 µM PnuT in buffer F (50 mM Tris, pH 8.0, 200 mM NaCl, 0.03% DDM). Thiamine (3–5 µM), TMP (thiamine monophosphate; 5 µM), TPP (thiamine pyrophosphate; 5 µM), pyrithiamine (1.5–3 µM), and oxythiamine (5 µM; Sigma-Aldrich) were dissolved in buffer F at indicated concentrations and were titrated into the cell with protein in 1-µl steps. Data were analyzed using software provided by TA instruments.
For the experiments presented inTable 2 , ITC measurements were conducted with an ITC200 calorimeter (MicroCal) at 25°C. Thiamine, TMP, TPP, pyrithiamine, and oxythiamine (Sigma-Aldrich) were dissolved in the buffer used for purification at a concentration of 1 mM to 5 mM. The ligand solutions were added in the indicated steps into the temperature-equilibrated ITC cells. The final protein concentration used for ITC measurements was 10–20 µM. Data were analyzed with the ORIGIN-based software (MicroCal).
For the experiments presented in
3
Cultivating Fungal Strains for Research
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A. fumigatus, A. niger, and S. cerevisiae strains that were used in this study are listed in Table S2 and S3. A. fumigatus strains were grown at 37°C on Aspergillus minimal media (AMM). A. fumigatus CEA17 ΔakuB strain (47 (link)) was grown on media supplemented with uracil and uridine with the final concentration of 20 µg/mL. When necessary, the media were supplemented with pyrithiamine (Sigma-Aldrich, Merck, Darmstadt, Germany) or hygromycin B (InvivoGen Europe, Toulouse, France) with the final concentration of 0.1 µg/mL and 200 µg/mL, respectively. S. cerevisiae strains were grown on YPD or selective SD at 30°C.
4
Fungal Strains and Culture Conditions
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The fungal species used in this work are T. reesei strain QM9414 and A. nidulans. The A. nidulans strains used in this study are listed in Table 1 . Strains were grown in complete (CMA) or minimal (MMA) media containing the appropriate supplements (Cove 1966 (link)). Experiments performed with thermosensitive strains were carried out at 30 or 42° as indicated. Glucose was used as sole carbon source, and ammonium tartrate was used as sole nitrogen source. Strains were obtained by following standard procedures (Pontecorvo et al. 1953 (link)). Pyrithiamine was obtained from Sigma and used at 0.1 µg/mL. Glufosinate was prepared from Basta (Bayer CropScience) and used as previously described (Nayak et al. 2006 (link)).
5
Inducible Transformation of A. fumigatus
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Transformation of the A. fumigatus wild-type strain (Af293) was performed as previously described (50 (link)). Antifungal-resistant transformants were selected using 0.5 μg/ml pyrithiamine (Sigma). Verification of the presence of the linear Tet-ON-brlA construct and the absence of a circular autonomously replicating Tet-ON-brlA plasmid within pyrithiamine-resistant transformants was accomplished by PCR analysis of genomic DNA.
6
Aspergillus fumigatus Culture and Manipulation
7
Cultivation of Microalgal Species
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Phaeodactylum tricornutum CCAP 1055/1 was grown in f/2 minus silica without vitamins at 18°C and 30 μmol m−2 s−1 under a 16 h : 8 h, day : night cycle. Thalassiosira pseudonana 1085/12 was grown in f/2 plus silica and 0.6 μM cyanocobalamin (Millipore‐Sigma) at the same temperature and light regime. Chlamydomonas reinhardtii UVM4 (Neupert et al., 2009 (link)) was grown in TAP without vitamins at 24°C and same light regime. Cultures were supplemented with thiamine (Acros Organics, Geel, Belgium), pyrithiamine (Sigma‐Aldrich), or 4‐amino‐5‐hydroxymethyl‐2‐methylpyrimidine (HMP; Sigma‐Aldrich), at the indicated concentrations for each of the experiments. Zeocin (InvivoGen, San Diego, CA, USA) at 75 mg l−1 was used to select transgenic P. tricornutum cells and at 10 mg l−1 to select for C. reinhardtii transformants. Cell growth was measured as optical density (OD)730 with a ClarioStar plate reader (BMG Labtech, Ortenberg, Germany).
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Phaeodactylum tricornutum CCAP 1055/1 was grown in f/2 minus silica without vitamins at 18°C and 30 μmol m−2 s−1 under a 16 h : 8 h, day : night cycle. Thalassiosira pseudonana 1085/12 was grown in f/2 plus silica and 0.6 μM cyanocobalamin (Millipore‐Sigma) at the same temperature and light regime. Chlamydomonas reinhardtii UVM4 (Neupert et al., 2009 (link)) was grown in TAP without vitamins at 24°C and same light regime. Cultures were supplemented with thiamine (Acros Organics, Geel, Belgium), pyrithiamine (Sigma‐Aldrich), or 4‐amino‐5‐hydroxymethyl‐2‐methylpyrimidine (HMP; Sigma‐Aldrich), at the indicated concentrations for each of the experiments. Zeocin (InvivoGen, San Diego, CA, USA) at 75 mg l−1 was used to select transgenic P. tricornutum cells and at 10 mg l−1 to select for C. reinhardtii transformants. Cell growth was measured as optical density (OD)730 with a ClarioStar plate reader (BMG Labtech, Ortenberg, Germany).
8
Overexpression of fgnA in A. fumigatus
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For overexpression of fgnA, the tetracycline-controlled transcriptional activation system (tetOn) was used (Helmschrott et al., 2013 (link)) and integrated in locus directly upstream of the fgnA gene. For this purpose, 2000-bp flanking regions were amplified from A. fumigatus ATCC 46645 genomic DNA with the primer pairs P7/P8 and P9/P10, while the tetOn-system was amplified from plasmid pSK562 (Sven Krappmann, personal communication) with primers P11/P12 and the ptrA cassette was amplified from plasmid pSK275 with the primer pair P5/P6. The above generated PCR fragments were assembled together with the HindIII-linearized pUC18 vector using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Frankfurt, Germany) according to the manufacturer’s instructions, yielding plasmid pUC18-tetOn-fgnA. The final transformation construct was generated by PCR amplification from plasmid pUC18-tetOn-fgnA using primers P7/P10. The obtained linear DNA fragment, comprising the 2 kb coding regions upstream and downstream of the fgnA (Afu1g1g01010) ATG start codon, flanking the tetOn promoter and the ptrA cassette, was used for transformation of A. fumigatus ATCC 46645. Transformants were selected on AMM agar plates supplemented with 0.1 µg/mL pyrithiamine (Sigma-Aldrich, Hamburg, Germany).
9
MMEJ-CRISPR Genome Editing in A. fumigatus
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For editing the A. fumigatus gene, the MMEJ-CRISPR system was used as described in our previous published papers [45 (link),49 (link)]. sgRNA targeted to the appropriate site of the target gene was synthesized in vitro using the MEGAscript T7 Kit (Life Technologies, AM1333). The corresponding repair template (DNA) with microhomology arms was amplified by PCR. Then, the repair template fragments and sgRNA were cotransformed into a Cas9-expressing A. fumigatus recipient strain. The primers and annotations for sgRNAs and repair templates are listed in Table S2. Transformation procedures were performed as previously described. Transformants were selected in medium lacking uridine or uracil or in the presence of 150 μg ml−1 hygromycin B (Sangon) or 0.1 μg ml−1 pyrithiamine (Sigma). For the recycling usage of the selectable marker pyr4, 1 mg ml−1 5-FOA was used for screening recipient strains. All primers used are listed in the supplementary data Table S2. All transformant isolates were verified by diagnostic PCR analysis using mycelia as the source of DNA. Primers were designed to probe upstream and downstream of the expected cleavage sites as labeled in Figure S1D.
10
Construction of A. fumigatus fgnA Deletion Strain
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The transformation cassettes for the A. fumigatus fgnA deletion strain were constructed essentially as previously described (Szewczyk et al., 2006 (link)). 2000 bp sequences homologous to the regions upstream and downstream of fgnA (Afu1g01010) were amplified using primer pairs P1/P2 and P3/P4. The pyrithiamine (ptrA) resistance cassette was amplified from plasmid pSK275 (Szewczyk and Krappmann, 2010 (link)) with the primer pair P5/P6. The above PCR-amplified DNA fragments were assembled together with the HindIII-linearized pUC18 vector using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Frankfurt, Germany) according to the manufacturer’s instructions, yielding plasmid pUC18-ptrA::fgnA. The final fgnA deletion construct was generated by PCR amplification of the 2 kb upstream and downstream coding regions of fgnA flanking the pyrithiamine resistance cassette from plasmid pUC18-ptrA::fgnA using primers P1/P4. A. fumigatus strain ATCC 46645 was transformed with the resulting linear DNA product as previously described (Unkles et al., 2014 (link)). Colonies of transformant strains were selected on AMM agar plates supplemented with 0.1 µg/mL pyrithiamine (Sigma-Aldrich, Hamburg, Germany).
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