Protoplast Generation from the Ascofuranone-Producing Fungus Acremonium Sclerotigenum

Protoplast Generation from the Ascofuranone-Producing Fungus Acremonium sclerotigenum

Motomichi Matsuzaki1,2*, Ryoko Tatsumi1† and Kiyoshi Kita1,2

1 Department of Biomedical Chemistry, Graduate School of Medicine, the University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan 2 School of Tropical Medicine and Global Health, Nagasaki University, 1–12–4 Sakamoto, Nagasaki 852–8523, Japan

Received February 13, 2017; accepted May 25, 2017

Summary Acremonium sclerotigenum (Bionectriaceae, Hypocreales, Sordariomycetes, Ascomycota) is a soil fungus found worldwide as an air and food contaminant. This fungus produces the antibiotic ascofuranone, a promising drug candidate against African trypanosomiasis; however, the details of ascofuranone biosynthesis have not yet been elucidated. Genetic manipulation techniques would greatly facilitate identification of the genes from ascofuranone biosynthetic pathway, but these techniques have not yet been established for this organism. Protoplast generation is a required step of the polyethylene glycol-mediated transformation of fungi. In this study, we aimed to establish a protocol for the isolation of protoplasts from A. sclerotigenum strain F-1392. The cell wall was digested with a commercial enzyme, Yatalase, to promote protoplast release. Spherical cells that were sensitive to osmotic shock were recovered only when the mycelia were pretreated with dithiothreitol. As a result, 2.0×107 protoplasts were obtained from a 40-mL liquid culture of A. sclerotigenum; the regeneration efficiency was estimated as 17%. This study comprises the first step toward development of genetic manipulation techniques in A. sclerotigenum, allowing future genetic dissection of this important microorganism.

Key words Ascofuranone, Dithiothreitol, Filamentous fungus, Protoplast, Yatalase.

Acremonium sclerotigenum Moreau & R. Moreau ex biosynthesis have not been established. We are currently Valenta (Bionectriaceae, Hypocreales, Sordariomycetes, using a comparative transcriptomic approach to identify Ascomycota) was originally isolated from sand dunes the genes that drive ascofuranone biosynthesis in strain in France and has since been found worldwide as a soil F-1392; however, the paucity of genetic manipulation fungus and a contaminant of air and food (Iwatsu et al. techniques prevents us from demonstrating the involve- 1987). The species has been reported as an endophyte ment of candidate genes in this biosynthetic pathway. of the holm oak and grapevine (Collado et al. 1999, Lo Polyethylene glycol-mediated transformation of pro- Piccolo et al. 2015, Summerbell and Scott 2015), and as toplasts is a simple and efficient method that is widely a pathogen of the apple fruit (Li et al. 2014). It was also used for genetic manipulation of filamentous fungi. suggested to be an opportunistic human pathogen (Sum- Protoplasts, cells devoid of the cell wall, can be prepared merbell and Scott 2015). Recently, we have reported that from fungal mycelia and spores by enzymatic digestion a fungal strain F-1392 that produces the antibiotic asco- of cell wall material (Davis 1985). However, the method furanone belongs to this species (Hijikawa et al. 2017). requires optimization depending on the fungal species Ascofuranone is a meroterpenoid with a strong in- or strain, to maximize protoplast yield and capacity to hibitory activity against cyanide-insensitive alternative regenerate into mycelia. Before the strain F-1392 was re- oxidases (Minagawa et al. 1997, Shiba et al. 2013, Wil- identified as A. sclerotigenum, we have unsuccessfully liams et al. 2010) and with demonstrated therapeutic attempted to generate its protoplasts. The generation of efficacy in rodent infection models of African try- protoplasts from the related species, Acremonium chry- panosomiasis (Yabu et al. 2003) and cryptosporidiosis sogenum (Hamlyn et al. 1981) and Acremonium diospyri (Suzuki et al. 2004). This promising drug candidate is (Deed et al. 1990), has been reported, with the yield supposed to be synthesized by prenylation of orsellinic greatly improved by a pretreatment with dithiothreitol acid and terminal cyclization via epoxidation (Hosono (DTT). In this study, we aimed to isolate the protoplasts et al. 2009, Kawaguchi et al. 2013), but the details of its from strain F-1392 using a protocol incorporating the DTT pretreatment.

* Corresponding author, e-mail: [email protected] Materials and methods † Present address: RIKEN Center for Sustainable Resource Science, 1–7–22 Suehiro, Tsurumi, Yokohama 230–0045, Japan DOI: 10.1508/cytologia.82.317 A. sclerotigenum strain F-1392 (previously referred 318 M. Matsuzaki et al. Cytologia 82(3)

Fig. 1. Protoplast generation from Acremonium sclerotigenum strain F-1392. Protoplasts were generated from mycelia with (A–F) or without (G–I) DTT pretreatment. A, mycelia after Yatalase treatment, with degenerated morphology, and spherical cells (arrowheads). B and C, cells recovered in TPC buffer stained with SYBR Green I (C). D and E, cells recovered in TPC buffer stained with Calcofluor White (E). The spherical cells (arrowheads) were negative, whereas the mycelial fragment was positive. F, cells recovered in ultrapure water. G, mycelia after Yatalase treatment. H and I, cells recovered in TPC buffer stained with Calcofluor White (I). Bright-field images (all panels except for C, E, and I) were visualized using differential interference contrasting. Bar: 20 µm. to as Ascochyta viciae) was kindly provided by aRi- gation at 600g for 5 min. Protoplasts were resuspended gen Pharmaceuticals, Inc. (Tokyo, Japan). Protoplast either in TPC buffer or in ultrapure water, and counted preparation was done based on a method used for A. using a Neubauer chamber. Three independent prepara- chrysogenum (Rodríguez-Sáiz et al. 2005), with minor tions were performed. modifications. The fungal strain was cultured in 5 mL Nuclear staining was performed by incubating the of Sabouraud liquid medium for seven days at 25°C, and cells in TPC buffer containing 1 : 1000 dilution of SYBR then inoculated into 100 mL of the same medium in a Green I Nucleic Acid Gel Stain (Thermo Fisher Scien- 300-mL conical flask. After 24-h incubation at 25°C in tific) for 5 min. Cell wall material was stained by mixing a reciprocal shaker at 200 rpm, the mycelia were har- the cell suspension with a tenth volume of Calcofluor vested by filtration through a 20-µm Nylon mesh, and White Stain (Sigma-Aldrich). The mycelium regenera- washed with TPC buffer (50 mM potassium phosphate tion was performed as follows: protoplast suspension 3 -1 [pH 7.0], 0.8 M NaCl, and 20 mM MgSO4). DTT pre- was first diluted to 10 cells mL in TPC buffer or sterile treatment was performed by gentle agitation for 1 h at water; it was then mixed with an equal volume of a top 25°C in TPC buffer supplemented with 10 mM DTT. agar medium (Bacto Tryptone [BD Biosciences] 10 g, After washing with TPC buffer, the mycelia were resus- glucose 150 g, and Bacto Agar [BD Biosciences] 10 g pended in the same buffer supplemented with 5 mg mL-1 per liter) pre-warmed to 50°C, and spread on a solid re- Yatalase (TaKaRa Bio Inc., Kusatsu, Japan), which was generation medium (Bacto Tryptone 10 g, glucose 150 g, filtered through a 0.45-µm filter before use. After gentle and Bacto Agar 20 g per liter), followed by incubation at agitation for 1 h at 25°C, the mycelium suspension was 25°C for three days. filtered through a 20-µm Nylon mesh, and the generated protoplasts were recovered from the filtrate by centrifu- 2017 Formation of Acremonium sclerotigenum Protoplasts 319

Table 1. Counts of cells recovered from 40 mL of fungal cultures. efficiency was around 17%. Counting medium With DTT pretreatment Control In conclusion, the DTT pretreatment enabled us to reproducibly prepare 2.0×107 protoplasts from a 40-mL 6 6 TPC buffer 20.3±7.4×10 7.6±0.4×10 culture. This amount is sufficient for a single transfor- Ultrapure water 0.1±0.1×106 5.3±1.5×106 mation reaction since 1–5×107 protoplasts are typically Contaminating mycelia were excluded from counting. The num- used per A. chrysogenum transformation (Rodríguez- bers are given as the mean±standard error (n=3). Sáiz et al. 2005), although there is scope for further optimization of culture and reaction conditions, in- cluding the choice of enzyme and osmoticum (Davis Results and discussion 1985). Polyethylene glycol-mediated transformation of protoplasts prepared by this method will promote the Protoplasts were prepared from A. sclerotigenum my- elucidation of the pathway and the underlying genes of celia using Yatalase, an enzyme mixture for the diges- ascofuranone biosynthesis in A. sclerotigenum. We also tion of fungal cell walls, with or without DTT pretreat- anticipate that this method will facilitate improving the ment. DTT pretreatment itself did not affect mycelium production yield of ascofuranone and related compounds morphology, but the mycelia were partly degenerated through genetic engineering. and spherical cells appeared after the Yatalase treatment (Fig. 1A). The majority of cells recovered from DTT- Acknowledgements treated mycelia was spherical and nucleated (Fig. 1B, C). The spherical cells lacked cell wall material, as indicated This work was financially supported by JSPS KA- by the absence of Calcofluor White staining, while the KENHI Grant Number 26253025 (to K. K.). contaminating mycelial fragments were stained with Calcofluor White (Fig. 1D, E). The cells were sensitive References to osmotic shock, and only cell debris was observed when the cells were diluted in ultrapure water (Fig. Collado, J., Platas, G., González, I. and Peláez, F. 1999. Geographical 1F). These observations indicate that the spherical cells and seasonal influences on the distribution of fungal endophytes in Quercus ilex. New Phytol. 144: 525–532. recovered from DTT-treated mycelia were protoplasts. Davis, B. 1985. Factors influencing protoplast isolation. In: Peberdy, In contrast, control mycelia without DTT pretreatment J. F. and Ferenczy, L. (eds.). Fungal Protoplasts: Applications appeared to be intact even after the Yatalase treatment in Biochemistry and Genetics. Marcel Dekker, New York. pp. (Fig. 1G), and the recovered cells were fusiform and Cal- 45–72. cofluor White-positive (Fig. 1H, I), suggesting that these Deed, A., Seviour, R. J. and Swift, I. E. 1990. Production of carot- enoids by protoplasts of the fungus Acremonium diospyri. En- were conidia. zyme Microb. Technol. 12: 940–944. The counts of cells recovered in isotonic or hypotonic Hamlyn, P. F., Bradshaw, R. E., Mellon, F. M., Santiago, C. M., Wil- solutions from 40-mL fungal cultures are shown in Table son, J. M. and Peberdy, J. F. 1981. Efficient protoplast isolation 1. More cells were recovered in TPC buffer from DTT- from fungi using commercial enzymes. Enzyme Microb. Tech- treated mycelia than from control mycelia. The latter nol. 3: 321–325. were relatively resistant to osmotic shock, and were Hijikawa, Y., Matsuzaki, M., Suzuki, S., Inaoka, D. K., Tatsumi, R., Kido, Y. and Kita, K. 2017. Re-identification of the asco- probably conidia (see above). 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