of Biorechnology, 21 (1991) 283-288 0 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1656/91/$03.50
Genetic transformation of Aureobasidium pullulans D. Cullen ‘, V. Yang ‘, T. Jeffries I, J. Bolduc * and J.H. Andrews ’ ’ Institute for Microbial and Biochemical Technology, Forest Products Laboratory, Forest Seruice, U.S. Department of Agriculture, Madison, Wisconsin and 2 Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin, U.S.A.
(Received 13 June 1991; revision accepted 3 July 1991)
Summary Aureobasidium pullulans strain Y 117 was transformed to hygromycin resistance using plasmid pDH33, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the Aspergillus niger glucoamylase gene (gh4). Southern hybridizations of transformants revealed multiple, integrated copies of the vector. The glaA promoter was not induced by starch in A. pullulans as it is in A. niger; however, the transcriptional start points were the same in both species. Aureobasidium pullulans;
Introduction Aureobasidium pulluluns (de Bary) Arnaud, commonly known as the “black yeast” (Cooke, 19591, is a widespread, polymorphic fungus. It produces complex polysaccharides including pullulan (an a-glucan), which has many uses in medicine and industry (Leduy et al., 1988). A. pullulans does not possess cellulase activity, but it is frequently associated with the decomposition of plant products and with paint deterioration (Horvath et al., 1976). It has been isolated from kraft mill waste treatment ponds and has been shown to use various lignin-related aromatic acids as sole carbon sources. The organism will also cleave and metabolize certain phenolic dimers, making it of interest for studies of fungal aromatic metabolism to: D. Cullen, USDA Madison, WI 537052398, U.S.A.
FS, Forest Products Laboratory,
(Bourbonnais and Paice, 1987). A. pullulans secretes various enzymes including high levels of xylanases (Leathers, 1986, 1988, 19891, making it attractive as a potential system for the expression and secretion of heterologous proteins. A. pullulans is also of interest as a leaf inhabitant with biocontrol potential (Andrews et al., 1983). The ability to introduce selective markers into A. pullulans would facilitate tracking populations of genetically marked strains introduced into nature. In short, A. pullulans is of interest to a large and diverse audience of scientists and the availability of a transformation system would greatly facilitate a variety of studies. Transformation systems based upon resistance to the aminoglycoside antibiotic hygromycin B (Hyg) have been developed for several fungi including Saccharomyces cereuisiae (Gritz and Davies, 19831, Cephalosporium acremonium (Queener et al., 19851, Aspergillus nidulans (Cullen et al., 19871, Fusarium oxysporum (Kistler and Benny, 1988) and Ustilago maydis (Wang et al., 1988). Recently, Il. maydk has been transformed with vector pDH33, which contains the bacterial gene encoding hygromycin phosphotransferase (hph) fused to the promoter of the A. niger glucoamylase gene (glaA; Smith et al., 1990). In A. niger, glaA expression is induced several hundred-fold when cultures are grown on starch as opposed to xylose (Fowler et al., 1990). Regulated expression of glaA is also observed in A. nidulans (Cullen et al., 1987; Gwynne et al., 1987). We report here the successful transformation of A. pullulans with pDH33.
Protoplasts of A. pullulans strain Y117 (Andrews et al., 1983) were prepared and transformed as described (Smith et al., 1990; Wang et al., 1988) except that selection was on Holliday’s complete medium (Holliday, 1974) supplemented with 50-62.5 pg hygromycin per ml. Protoplasts (1.0 x 10’ per ml> were stored at -90” C in buffer II (25 mM CaCl,/25 mM Tris * HCI, pH 7.5/l M sorbitol) amended with 13% (w/v) polyethylene glycol (MW 33501, 5% dimethyl sulfoxide, and 1% P-mercaptoethanol (J. Wang, 1989, PhD thesis, Dept. Plant Pathology, University of Wisconsin, Madison). Selection plates were incubated at 28 OC for approx. 5-7 d. For Southern hybridizations, transformants were grown in 100 ml Holliday’s medium supplemented with 50 pg ml-’ hygromycin and starch, harvested by centrifugation, frozen in liquid N,, lyophilized, and the DNA extracted by the ‘gentle extraction method’ (Specht et al., 1982). Transformant DNA (approx. 5 pg) was digested with EcoRI, BglII, or EcoRV, size fractionated by electrophoresis on 0.6% agarose (SeaKern GTG, FMC, Rockland, ME), and blotted onto Nytran (Schleicher and Schuell, Keene, NH). A 1.5 kb EcoRI/XbaI fragment of pDH33 (Smith et al., 19901, which contains most of the hph coding region and the A. nidulans trpC terminator, was used as the probe. Following nick-translation of this fragment, 5 X 10’ cpm ml-’ hybridization buffer were used under moderate stringencies (42 o C; 40% formamide, 0.25 M Naf, 7% SDS).
Results and Discussion
After incubation for 7 d, putative transformants (approx. 1 per pg plasmid DNA) were transferred to Holliday’s complete medium amended with 150 pg ml-’ hygromycin. No differences in drug sensitivity were observed when minimal medium contained 0.5% xylose vs 0.5% starch as the sole carbon source. This observation suggested that the glaA promoter was not regulated as it is in Aspergillcrs. In contrast to previous studies (Smith et al., 1990; Wang et al., 19881, transformation frequency was not improved by linearizing pDH33 by digestion with XbaI, a unique restriction site located at the 3’ end of the trpC terminator. Southern analysis was performed on three transformants. The pattern and intensity of bands (Fig. 1) showed that all three transformants involved integration of the vector and that transformant number 2 contained multiple copies, probably A
Fig. 1. Southern hybridization analysis of Y117 transformants. Following washing at 42 o C with 0.25 M Na’, 2% SDS, 1 mM EDTA the blot was exposed to Kodak Ortho-G film for 3 d. Lane A contains pDH33 digested with EcoRI. Lanes B, C, D contain DNA from transformants 1, 2, 3, respectively, digested with EcoRI. Lanes E, F, G contain transformants 1, 2, 3 digested with &/II. Lanes H, I, J contain transformants 1, 2, 3 digested with EcoRV. Lanes K, L, M contain undigested DNA from transformants 1, 2, 3. Lane N contains Y117 DNA digested with EcoRI. Molecular size markers (Kbl are shown on left margin.
wt -RNA: Starch:
25 50 25 50 ++++
HyR 25 50 --
A B C D E F G H Fig. 2. Sl nuclease protection analysis of hph transcripts. Total RNA was extracted from transformant No. 2 grown on minimal medium containing 0.5% xylose or 0.5% maltose plus 0.5% starch as described (Timberlake and Barnard, 1981). Sl protection experiments were carried out essentially as described by Favaloro et al. (1980). An EcoRI/ClaI fragment of pDH33 spanning the transcriptional initiation points of &4 was radiolabelled at the C/a1 site and 4.5X 10’ cpm hybridized to 25 or 50 I.rg total RNA. Following hybridization overnight at 50 ’ C, 300 units of Sl nuclease (Amersham Inc, Arlington Heights, IL) were added to each sample and incubated 30 min at 37 o C. Samples were then extracted with phenol-chloroform and the nucleic acids ethanol precipitated. The pellets were washed with 70% ethanol, dried, and redissolved in 10 pl gel loading buffer (90% formamide, 10 mM EDTA, 10 mM NaOH, 0.2% bromophenol blue, 0.2% xylene cyanol). After heating to 80 o C for 5 min, samples were loaded onto a 40 cm denaturing gel (5% polyacrylamide/8 M urea), and subjected to electrophoresis at 25 W. The gel was exposed to Kodak XAR 5 film for 3 d at room temperature. Lanes A and B, RNA from wild-type Y117 grown on starch plus maltose; lanes C and D, RNA from transformant No. 2 grown on starch plus maltose; lanes E and F, RNA from transformant No. 2 grown on xylose; lane G, S. cereuiFiae-tRNA; lane H, molecular size marker [32P]-labeled pBR322 digested with Hinfl.
as tandem duplications. The latter point was supported by the presence of single, intense bands of large molecular weight for transformant No. 2 when digested with BglII and EcoRV, enzymes sites which are not present in pDH33. If transformant No. 2 had contained multiple insertions scattered throughout the genome, several low molecular weight bands would have been expected in lanes F and I. Transformants No. 1 and 3 contain fewer copies, perhaps one each, and integration has occurred at different genomic locations. Consistent with integration, no low molecular weight bands were observed in lanes with undigested DNA (K-M). No signal was observed for wild-type Y117 (lane N). The low transformation frequencies and the ineffectiveness of carbon source to alter the level of hygromycin resistance, suggested that the glaA promoter may not be functional in A. pullulans. To assess glaA function in A. pullulans, Sl nuclease protection analysis of glaA-hph transcripts was performed. Multiple transcriptional initiation points were found to exist approx. 80 bp upstream of the translational start codon (Fig. 2). This is similar to glaA transcriptional starts for native expression in A. niger (Boel et al., 19781, and for the closely related species A. awamori (Nunberg et al., 1984). Transcript levels of hph were independent of carbon source (Fig. 2; lanes D vs F). The pDH33 construction contains approx. 2.0 kb of the glad promoter, well beyond the minimal upstream distances required for regulated expression in Aspergillus (Fowler et al., 1990). Apparently, A. pullulans is unable to recognize the upstream activating sequences of the gld promoter.
Conclusions We have demonstrated genetic transformation of A. pullulans with a dominant selectable marker, hph. Constitutive expression of hph was obtained under the control of the promoter of A. niger glaA.
Acknowledgements We thank Phil Kersten and Sarah Covert for manuscript review and thoughtful comments. This work was supported in part by DOE grant DE-FG02-87ER13712 to D. Cullen and T.K. Kirk, and by USDA Hatch Grant 142216 and USDA CRGP 89-37151-4637 to J.H. Andrews.
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