Transformation ofAspergillus awamori andA. niger by electroporation

EXPERIMENTAL

MYCOLOGY

13, 289-293

(1989)

BRIEF NOTE Transformation MICHAEL

WARD,

Genencor,

Inc.,

of Aspergillus awamori by Electroporation

and A. niger

KATHERINE H. KODAMA,AND 180 Kimball

Accepted

Way,

for publication

South

LORI J. WILSON

Sun Francisco,

November

CA 94080

16, 1988

WARD, M., KODAMA, K. H., AND WILSON, L. J. 1989. Transformation of Aspergilfus awamori and A. niger by electroporation. Experimental Mycology 13, 289-293. A method is described which allows transformation of Aspergillus awamori and A. niger mediated by electroporation. This procedure gave transformation frequencies similar to those obtained with polyethylene glycol. For A. niger no differences were observed between the two procedures with respect to the number of integrated plasmid copies or the frequency of homologous integration. Q 1989 Academic Press, Inc.

INDEX

DESCRIPTORS:

Aspergillus

awamori;

A. niger;

electroporation;

transformation.

Transformation of filamentous fungi, in- plasts or cells in a buffer containing cluding species of Aspergillus, Neurospora The mechanism of uptake is presu crassa, and several plant pathogens, generinvolve formation of transient pores in the plasma membrane allowing access t ally relies on the use of polyethylene glycol (PEG) treatment of protoplasts in the pres- cytoplasm for large molecules like and Vienken, 1982). The ence of Ca”’ and DNA (Case et al., 1979; (Zimmermann Ballance et al., 1983). Other methods of Bio-Rad Gene Pulser apparatus used in this transformation have been used with N. study discharges a pulse with ~xpQ~~ntia~ crassa, e.g., the use of liposomes (Radford decay from a capacitor. Important parameet al., 1981) or lithium acetate treatment of ters which can be varied experim intact mycelium (Dhawale et al., 1984). elude the field strength (V/cm), However, these techniques have not been the capacitor used, and the time constant of widely adopted for transformation of other the pulse (the time taken for the voltage filamentous fungi. Previous reports of drop to lie, or approximately 37%, of PEG-mediated transformation of Aspergilinitial voltage). The latter can be influenc Iws niger, using various selection systems, by the resistance of the buffer throu which the pulse is discharged as well as have indicated that approximately 4-20 transformants per microgram can be ob- initial voltage of the pulse and capacitor tained (Kelly and Hynes, 1985; Buxton et size. al., 1985; Punt et al., 1987). Initial experiments were conducte Electroporation has previously been A. awamori strain GC12 (pyrG5; a used in transformation of higher plant pro- rivative of UVK143f, a glut duction strain derived from toplasts (Fromm et al., 1985), mammalian Transformation and CQmplerne~t~ti~~ to cells (Potter et al., 1984), yeast protoplasts (Karube et al., 1985), and intact yeast cells give uridine prototrophy were (Hashimoto et al., 1985). Transformation plished with the plasmid pANGl~ T by this method involves application of an mid consists of pUClO0 (a electrical pulse to a suspension of protopUC18 (Yanisch-Perron et al., 4985) wit 289 0147-597.5189 Copyright All rights

$3.00

0 1989 by Academic Press. Inc. of reproduction in any %rm reserved.

290

WARD,

KODAMA,

an oligonucleotide inserted into the multiple cloning site adding recognition sites for BglII, Xhol, and CZaI) and a 5.5-kb BamHI fragment containing a pyrG gene obtained from A. niger (Wilson et al., 1988). Protoplast isolation from 16-h shake flask cultures using Novozyme 234 was performed, with slight modification, by the method of Cullen et al. (1987); 0.7 M KC1 replaced the 0.6 M KC1 used for A. nidulans. After being washed twice in 0.7 M KC1 and once in electroporation buffer (7 mM sodium phosphate buffer, pH 7.2, 1 mM MgS04, 1.2 M sorbitol) 2 x lo7 protoplasts were finally resuspended in 0.8 ml electroporation buffer. Electroporation was performed in Bio-Rad electroporation cuvettes having an interelectrode distance of 0.4 cm. A IO-min incubation on ice was allowed before and after delivery of the pulse, the DNA being added immediately prior to the pulse. Following transformation protoplasts were added to molten Aspergillus minimal medium (Rowlands and Turner, 1973) with 2% agar and 1.2 M sorbitol, and plated onto the same medium. Transformed colonies were observed following 2-5 days incubation at 37°C. Preliminary experiments demonstrated that highest transformation frequencies could be obtained using the buffer described above, the 25 PFD capacitor and field strengths between 1250 and 3750 V/ cm. A more detailed study was conducted to measure both protoplast viability and transformation frequency within this range of conditions. Ten micrograms of plasmid DNA was used for each transformation and time constants for the pulses were typically IO-13 ms. Figure 1 gives the results of three separate experiments and several points emerge from these data. (1) The field strengths giving the highest transformation frequency varied between experiments. In order to attain the maximum transformation frequency in any one experiment several different aliquots of the

AND

WILSON

-60

FIG. 1. Effects of pulse strength on transformation frequency and protoplast viability of A. awamori GC12. Viability after electroporation is expressed as a percentage of the viability of an untreated protoplast preparation. x , transformation frequency; 0, viability .

same batch of protoplasts would need to be given pulses of differing field strengths. (2) In each experiment the field strength which gave the maximum transformation frequency resulted in a loss of viability of approximately 95% compared to untreated protoplasts. Since untreated protoplasts generally had a viability of approximately 10% the final protoplast population after electroporation had a viability of approximately 0.5%. (3) Although frequencies varied between experiments up to 10 transformants per mi-

TRANSFORMATION

crogram DNA could be obtained using this method. In order to compare the results of this method of transformation with PEGmediated transformation on the same batch of protoplasts nine separate experiments were conducted (Table 1). In three >f these (experiments I-3) the electroporation protocol was identical to that described above, whereas in the other six (experiments 4-9) the sorbitol concentration in the electroporation buffer was increased to 1.4 M because some protoplast lysis had previously been observed. Pulses of three different field strengths were employed in some of these experiments and the time constants for the pulses fell within the range of 9.913.0 ms. For experiments 6-9 lowgelling-temperature agarose (SeaPlaque, FMC BioProducts) was used in the overlays in which the treated protoplasts were embedded. In these cases protoplasts could

TABLE 1 Comparison of Transformation Frequencies Obtained with PEG or Electroporation Transformation conditions Expt 1

EP” 21SSVicm PEG

Expt 2 Expt 3 Expt 4

Expt5

EP 2155Vkm PEG EP 2155V/cm PEG EP 2175Vkm EP 2325Vicm EP 2540Vicm PEG EP 2175Vkm EP 2325Vicm EP 24SOVicm PEG

Expt 6

Expt 7 Expt 8 Expt 9

EP 2175Vicm EP 2325Vlcm EP 24SOVicm PEG EP 2375Vkm PEG EP 2375Vlcm PEG EP 2375Vfcm PEG

* EP, electroporation.

A. awamori

strain GC12 GC12 GC12 GC12 GC12 GCi2 GC5

GC5 GCS GC5 GC5 GC5 GCS GCS

GC5 GC5 GCS GCS

GC12 GC12 GC12 GC12 GC12 GC12

Transformants per p,g DNA 4.9 0.3 2.8 3.8 2.0 5.4 2.2 3.2 3.0 0.6 0.5 1.1 0.7 8.5 0.4 0.6 0.9 12.3 1.3 2.9 12.1 0.7 9.8 2.8

BY ELECTRQPOlUTION

291

be added to molten medium at 37” posed to approximately 50°C use ular agar. The lower temperatur has been shown to give increased viability and transformation frequencies (u~p~blished observations). All experiments employed plasmid pBH2 (a 2.4-kb Hind111 fragment containing the pyrG gene in pUC18) with either strain CC12 or strain GC5, a pyrG.5 derivitive of UVK143f. Transformation m PEG was performed by the method lance and Turner (1985) except KC1 replaced 0.6 M KC1 in the tions and was also added to the PEG sdution. Additionally 10 pg/ml aurin tricarboxylic acid was included in the final prot~p~~st wash prior to PEG treatment. This ~~~~eas~ inhibitor has been shown to in formation frequencies by 2- to have little effect on protoplast this concentration (L. 9. Wi Ward, unpublished observations). It is interesting to note that in several instances the frequency obtain method of transformation was the frequency with the other low. The high frequency of cotransformation previously observed for filarn~~~ fungi (Wernars el al., 1987; Kelly a Hynes, 1985; Mattern et al., 1987) has suggested that a small subpopulation plasts is competent for DNA upt is the size of this subpopulaticm its transformation frequency. It a different subpopulation is competes electroporation compared t mediated transformation and that protoplast isolation conditions cou one transformation method as oppo the other. As other fungal species are some may be found to be more amen PEG-mediated transformation whereas others may give higher .transformati~~l frequencies by electroporatik A sig~i~~a~t difference between the two me transformation may be that,

292

WARD,

KODAMA,

electroporation should not induce protoplast fusion in addition to DNA uptake. Similar transformation frequencies have been obtained with Aspergillus niger strain GC44 @y&44 derivative of ATCC 10864). Transformants were obtained using either PEG or electroporation and plasmid pMW30 (containing the N. crassa pyr4 and A. niger oliC3 genes) (Ward et al., 1988). Total DNA was isolated from six transformants generated by either method and subjected to Southern blotting and hybridization analysis (results not shown). No obvious differences were observed with respect to copy number of integrated plasmids. In addition, integration of pMW30 either at the homologous oliC locus or elsewhere in the genome could be distinguished phenotypically (Ward et al., 1988). Homologous integration occurred in 3 of 18 (16.6%) transformants obtained by PEG-mediated transformation and in 13 of 104 (12.5%) transformants obtained by electroporation. Thus the proportions of homologous and nonhomologous integrations were similar regardless of the transformation protocol used. Similar investigations were not conducted with A. awamori because homologous integration of circular plasmid has not been observed in our hands. Various modifications to the electroporation protocol were tested but no increase in transformation frequency was observed. These preliminary tests included addition of 1.5 mg/ml proteinase K or 75 pg/ml endoglycosidase H to the novozyme solution for the final 30 minutes of protoplast isolation in an attempt to remove surface glycoproteins which might hinder DNA uptake. PEG 4000 (8 or 12%) was added to the electroporation buffer used for pulse delivery, as used to good effect by Shillito et al. (1985) with tobacco protoplasts, but this had no obvious effect on our system. Initial attempts to transform intact fragments of mycelium by electroporation also failed. Electroporation may not be an appropriate method of transformation for species such

AND

WILSON

as A. nidulans or Neurospora crassa for which highly efficient PEG-mediated transformation is possible. However, transformation systems are being developed for many filamentous fungal species, including plant pathogens and species of industrial importance, and evaluation of the electroporation method may prove useful in these cases. REFERENCES BALLANCE,

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TRANSFORMATION 1987. Transformation

BY ELECTROPORATIQN

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