Hygromycin b

DFT1 and DFT2 cell line C5065 and JV, respectively, were transfected with plasmid pCO2 to generate stable cell lines that overexpress CIITA. DNA transfections were performed using polyethylenimine (PEI) (1 mg ml⁻¹, linear, 25 kDa; Polysciences, Warrington, FL, USA) at a 3 : 1 ratio of PEI to DNA (w/w) as previously described [8 (link)]. Briefly, DFT cells were co-transfected with pCO2 and SB transposase vector pCMV(CAT)T7-SB100 [33 (link)] (a gift from Zsuzsanna Izsvak; Addgene plasmid no. 34879) at a ratio of 3 : 1 in µg, respectively. One microgram of total plasmid DNA was used per millilitre of culture volume. The cells were incubated with the transfection solution overnight at 35°C with 5% CO₂. The media was removed and replaced with fresh complete RPMI medium. Forty-eight hours of post-transfection, the cells were observed for expression of reporter gene mTagBFP. Positively transfected cells were selected with 1 mg ml⁻¹ hygromycin B (Sigma-Aldrich) for 7 days before being maintained in 200 µg ml⁻¹ hygromycin B in complete RPMI medium. The two tumour cell lines were also transfected with empty vector pSBbi-BH as controls.

NIH3T3 cells were seeded 24 h prior to transfection, and 4.5 μg of the respective plasmid DNA was transfected using TurboFect transfection reagent (Catalog no. R0534, Thermo Fisher Scientific). Cells were cultured for 24 h in DMEM with 10% FCS and 100 IU, ml penicillin/100 μg/ml streptomycin (Catalog no. 15140122, Thermo Fisher Scientific). Subsequently, the culture medium was replaced, fresh medium containing 500 μg/ml hygromycin B (Catalog no. H3274, Sigma-Aldrich) was added to the cells, and this step was repeated after 24 h. Before analysis, all transfected cells were cultured for a minimum of 4 weeks in DMEM containing 500 μg/ml hygromycin B.

P. hygromyciniae sp. nov. SDM007^T was isolated from a sealed bottle of lyophilized hygromycin B purchased from Sigma-Aldrich Co. (product number H3274-1G, lot number SLBZ3956, St. Louis, Missouri, USA) in November 2019. A second bottle of hygromycin B of the same product and lot number was purchased Sigma-Aldrich Co. in February 2020. Other hygromycin B vials were purchased from Dot Scientific, Inc. (product number DSH75020-1, lot number 94491-105646) and Gold Biotechnology (product number H-270-1). Ampicillin was purchased from Acros Organics. E. coli, P. aeruginosa, P. fluorescens, and SDM007^T were grown aerobically (250 RPM) in LB broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) or on LB agar plates (LB broth supplemented with 16 g/L agar). M9 medium was made as follows: Na₂HP0₄ 6.9 g/L, KH₂PO₄ 3 g/L, NaCl 0.5 g/L, NH₄Cl 1 g/L, CaCl₂ 0.1 mM, MgSO₄ 2 mM, and glucose 0.5%. A549 (male, human lung epithelial-like) cells were obtained from ATCC and grown at 37°C in DMEM (Gibco) supplemented with 10% FBS (GE Healthcare Life Science) under 5% CO₂. All strains used in this study are listed in Table S4.

The ldhA (lactate dehydrogenase A gene) sequence of R. oryzae (Genbank accession number AF226154) was codon-optimized for expression in Aspergillus and synthesized by Life Technologies. The construct contained a NcoI and HindIII at the 5′ and 3′ ends, respectively, for cloning into pAN52.3 (kindly supplied by Pr. P. J. Punt, TNO, The Netherlands). The recombinant gene inserted into the expression vector was sequence-checked before transformation. Fungal co-transformation of A. brasiliensis BRFM103 was carried out as previously described [44 (link)] using pAN52.3 ldhA vector and pAN7.1 co-vector (kindly supplied by Pr. P. J. Punt, TNO, The Netherlands). The pAN7.1 co-vector contained the hph selection marker conferring Hygromycin B resistance. Hygromycin B inhibition tests were carried out with the wild-type A. brasiliensis BRFM103 strain grown on MM solid medium supplemented with 10, 30, 50, 75, 100, 150, 200, 300, and 400 μg/mL Hygromycin B (Sigma Aldrich) inoculated with spore suspensions. Growth inhibition tests showed that low Hygromycin B concentration (10 μg/mL) slowed fungal growth and higher (above 10 μg/mL) Hygromycin B concentrations prevented growth. Transformant screening was therefore done on MM solid medium supplemented with 20 μg/mL of Hygromycin B.

To generate the ump1 mutant with the split-marker approach^{63 (link)}, the flanking sequences of UMP1 were amplified and linked to the hph cassette amplified from pCX63^{64 (link)} by overlapping PCR with the primers listed in Supplementary Table 2. The resulting gene replacement fragments were transformed into strain JL3 by PEG-mediated protoplast transformation. Transformants resistant to 30 µg/ml hygromycin B (Sigma-Aldrich) were isolated and screened by PCR with primer pairs 5F/6R, H850/H852, 7F/HY-R, and YG-F/8R (Fig. S6b) to identify ump1 deletion mutants.
For complementation assays, a 2574-bp fragment containing the entire UMP1 coding region (except the TAG stop codon) and its promoter was cloned between the KpnI and HindIII sites on pKNTG that carries the geneticin resistance marker and GFP^{65 (link)} to generate the in-frame UMP1-GFP fusion construct pUmp1GFP. A 382-bp fragment containing the 3’-UTR and terminator sequences UMP1 was then cloned into the BamHI site on pUmp1GFP. The resulting UMP1-GFP construct was transformed into the ump1 mutant as described^{9 (link)}. Transformants resistant to both 30 µg/ml hygromycin B and 30 µg/ml geneticin (Sigma-Aldrich) were isolated and confirmed by PCR analysis.

The T. reesei strains QM6a Δtmus53 (Steiger et al. [2011 (link)]) and Rut-C30 (ATCC 56765), which was derived from the wild-type strain QM6a by one UV-light and two N-methyl-N’-nitro-N-nitrosoguanidine mutation steps (Montenecourt and Eveleigh [1979 (link)]), were maintained on 3% malt extract (MEX) agar. The recombinant T. reesei strains QPEC1, QBEC2, RPEC1, and RBEC2 generated during this study, were maintained on MEX agar containing 250 μl/l hygromycin B (Merck, Darmstadt, Germany).
Purification of transformed strains by streak out of spores was done on MEX agar containing 250 μl/l hygromycin B and 500 μl/l IGEPAL®; CA-630 (Sigma-Aldrich, St. Louis, MO, USA).
Cultivation in shake flasks was performed in 250-ml-Erlenmeyer flasks containing 50 ml Mandels-Andreotti (MA) medium (Mandels [1985 (link)]) supplemented with 1% (w/v) D-xylose or 1% (w/v) birch-wood xylan. For inoculation 10⁹ conidia per liter were used. Growth conditions were pH 5, 30℃, and 160 rpm shaking rate. Mycelia and supernatant were seperated by filtration. For short-term storage, harvested mycelia were shock-frozen and kept in liquid nitrogen, supernatants were kept at -20℃.

P. hygromyciniae sp. nov. SDM007^T was isolated from a sealed bottle of lyophilized hygromycin B purchased from Sigma-Aldrich Co. (product number H3274-1G, lot number SLBZ3956, St. Louis, Missouri, USA) in November 2019. A second bottle of hygromycin B of the same product and lot number was purchased Sigma-Aldrich Co. in February 2020. Other hygromycin B vials were purchased from Dot Scientific, Inc. (product number DSH75020-1, lot number 94491-105646) and Gold Biotechnology (product number H-270-1). Ampicillin was purchased from Acros Organics. E. coli, P. aeruginosa, P. fluorescens, and SDM007^T were grown aerobically (250 RPM) in LB broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) or on LB agar plates (LB broth supplemented with 16 g/L agar). M9 medium was made as follows: Na₂HP0₄ 6.9 g/L, KH₂PO₄ 3 g/L, NaCl 0.5 g/L, NH₄Cl 1 g/L, CaCl₂ 0.1 mM, MgSO₄ 2 mM, and glucose 0.5%. A549 (male, human lung epithelial-like) cells were obtained from ATCC and grown at 37°C in DMEM (Gibco) supplemented with 10% FBS (GE Healthcare Life Science) under 5% CO₂. All strains used in this study are listed in Table S4.