Vol. 170, No. 12JOURNAL OF BACTERIOLOGY, Dec. 1988, p. 5572-55780021-9193/88/125572-07$02.00/0Copyright © 1988, American Society for Microbiology

Transformation of Schwanniomyces occidentalis with an ADE2 geneCloned from S. occidentalisRONALD D. KLEIN* AND M. ANNE FAVREAU

Molecular Biology Research, The Upjohn Company, Kalamazoo, Michigan 49007

Received 2 May 1988/Accepted 1 September 1988

We have developed an efficient transformation system for the industrial yeast Schwanniomyces occidentalis(formerly Schwanniomyces castelliO. The transformation system is based on ade2 mutants of S. occidentalisdeficient for phosphoribosylaminoimidazole carboxylase that were generated by mutagenesis. As a selectablemarker, we isolated and characterized the S. occidentalis ADE2 gene by complementation in an ade2 strain ofSaccharomyces cerevisiae. S. occidentalis was transformed with the recombinant plasmid pADE, consisting ofa 4.5-kilobase-pair (kbp) DNA fragment from S. occidentalis containing the ADE2 gene inserted into the S.cerevisiae expression vector pYcDE8 by a modification of the spheroplasting procedure of Beggs (J. D. Beggs,Nature [London] 275:1044108, 1978). Intact plasmids were recovered in Escherichia coli from whole-cell lysatesofADE+ transformants, indicating that plasmids were replic;iting autonomously. High-molecular-mass speciesof pADE2 were found by Southern hybridization analysis of intact genomic DNA preparations. The shift tohigher molecular mass of these plasmids during electrophoresis in the presence of ethidium bromide afterexposure to shortwave UV suggests that they exist in a supercoiled form in the transformed host. Subclones ofthe 4.5-kbp insert indicated that ADE2-complementing activity and sequences conferring autonomousreplication in S. occidentalis were located within a 2.7-kbp EcoRI-SphI fragment. Plasmids containing thisregion cloned into the bacterial vector pUC19 complemented ade2 mutants of S. occidentalis with efficienciesidentical to those of the original plasmid pADE.

Advances in molecular biology have permitted the analy-sis of many non-Saccharomyces yeasts which possess qual-ities of both academic and industrial interest (16, 23). Theseinclude the ability to utilize a broad spectrum of carbonsources, the presence of complex regulatory mechanisms,and in many cases an ability to produce molecules ofcommercial interest (1). One of these yeasts, Schwan-niomyces occidentalis, efficiently converts inexpensive car-bon compounds, such as starch and inulin, to fuel-gradeethanol by means of a number of secreted amylolytic anddegradative enzymes (3, 6, 15, 22, 25). In fact, this facileconversion of biomass to ethanol as well as other attractivephysiological properties has prompted Ingledew (6) to referto the genus Schwanniomyces as "super yeast."Work in our laboratory has focused on S. occidentalis as

an efficient expression system for large-scale commercialproduction of heterologous gene products and as a model forprotein secretion and export through the cell wall. We havechosen this organism because it can secrete proteins greaterthan 140,000 daltons (Da) efficiently into the culture medium(3, 15, 22, 25), does not hyperglycosylate secreted proteins(3), and does not secrete measurable quantities of proteases(3). In addition, the genes for the secreted proteins aresubstrate induced and catabolite repressed (14, 25), and theorganism is capable of growth on inexpensive substrates (6).An earlier report from this laboratory described the isolationof two of the four major proteins present in culture super-natants when S. occidentalis is grown on starch, namelyglucoamylase (3) and a-amylase (3). We have also describedthe cloning and characterization of the S. occidentalis geneencoding orotidine 5'-phosphate decarboxylase (ODC) (9).Further development of Schwanniomyces spp. for commer-cial purposes has been hampered by the lack of an efficienttransformation and cloning system that will permit the

* Corresponding author.

maintenance and expression of both homologous and heter-ologous DNAs. Ideally such a system should permit themaintenance of these DNAs without selective pressure, animportant consideration for industrial scale-up procedures.In the present communication, we describe an efficienttransformation system for S. occidentalis based on ade2-deficient hosts that uses a modification of the spheroplast-mediated transformation procedure of Beggs (2).

MATERIALS AND METHODS

Materials. Materials, including restriction enzymes andDNA-modifying enzymes, have been described previously(9).

Strains, plasmids, and media. The wild-type Schwan-niomyces occidentalis strain used was ATCC 26076; it hasbeen described previously (9). S. occidentalis strains RKA-7(UC7647 [ade2]) and LRA-26 (UC7646 [ade2]) were gener-ated by UV and ethyl methanesulfonate (EMS) mutagenesis,respectively, as described below. Saccharomyces cerevisiaeUC 7586 (strain C2: a ade2 leu2-3 leu2-112 trpl sir3-8pep4:: URA3) was acquired from Zymogenetics Inc. (Seattle,Wash.). Escherichia coli DH5-a (Bethesda Research Labo-ratories Inc., Gaithersburg, Md.) was used in all experimentswhich required a bacterial host and was grown as describedbefore (9).The yeast expression plasmid pYcDE8 was acquired from

Zymogenetics Inc. with permission of the University ofWashington Research Foundation (Seattle, Wash.) and wasdescribed previously (9, 13). The E. coli plasmid pUC19 hasbeen described previously (24). The complete medium(YEPD) and minimal medium (YMM) for growing both S.cerevisiae and S. occidentalis were as described by Shermanet al. (21). When appropriate, YMM was supplemented withspecific amino acids at concentrations recommended bySherman et al. (21).

5572

on January 27, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

TRANSFORMATION OF S. OCCIDENTALIS 5573

TABLE 1. Transformation of ade2 strains with plasmidscarrying S. occidentalis ADE2

ADE Transformantsa

Plasmid Vector Instb S corevlslee a occidentalls

R R C SpADE pYcDE8 I I I 1 500 4000

S C R KpADE-6C pYcDE8 l l l l 15p 5000

R C SpADE-1 pYcDE8 l 150 5000

R R CpADE-7 pYcDE8 I 0 50

K R C SpADE-2 pUC19 l l l.J 15 400

pYcDE8 0 0

a Selection for adenine prototrophy was done on YMM supplemented withtryptophan and Casamino Acids as described before (9). Plates were kept at30°C for 4 days before counting was done. Strains used were S. cerevisiaeUC7586 and S. occidentalis RKA-7.

b The orientation of the insert for pADE is shown in Fig. 1. The letter codefor restriction enzymes is as described in the legend to Fig. 3, with Kindicating the KpnI site in the vector.

c Reversed orientation of pADE and deleted for ADHI promoter.

Plasmid pADE-1 was constructed from pADE by deletionof the 1.35-kilobase-pair (kbp) EcoRI fragment. PlasmidpADE-6 was constructed as follows. pADE was cleavedwith SphI and KpnI, and the 4.05-kbp DNA fragmentextending from the KpnI site contiguous with the ADHIpromoter to the SphI site was isolated. The vector pYcDE8was cleaved with SphI and KpnI, and a 6.3-kbp fragment,missing the ADH1 promoter, was isolated. The 4.05-kbpfragment containing ADE2 was then ligated into the 6.3-kbppYcDE8 vector. The resulting plasmid, pADE-6, is similarto pADE except that the ADHI promoter is missing and theinsert is in the opposite orientation. Plasmid pADE-7 wasconstructed by cloning the 3.05-kbp SphI-ClaI fragmentfrom pADE into the SphI and ClaI cloning sites of the vectorp2-lAA2200-2470 (9). The resulting plasmid is similar topADE but is missing the region from the ClaI site to the endof the insert (approximately 1.45 kbp). Plasmid pADE-2 wasconstructed by cloning the ADE2 fragment from the KpnIsite at + 1 bp to the SphI site at +4050 bp into the KpnI andSphI sites of pUC19. This plasmid contains no S. cerevisiaeDNA. The basic structure of these plasmids is shown inTable 1.DNA isolation and hybridization. Bacterial plasmid DNA

isolations were carried out by the alkaline lysis method (12).Yeast genomic DNA was isolated as described before (9).Small-scale plasmid DNA preparations from transformedyeast cells were made by the rapid boiling method asdescribed before (5). Isolation of DNAs from agarose gelswas by electroelution as described before (20). Approxi-mately 10 ,ug of each DNA sample was fractionated on 0.7%agarose gels and transferred to nitrocellulose (Schleicher &Schuell Inc., Keene, N.H.). The transfer, prehybridization,and wash conditions were as described before (12). PlasmidDNA or isolated restriction fragments were labeled with[a-32P]dATP (Amersham Corp.; 3,000 Ci/mmol) by randompriming with a Random Primed DNA Labeling Kit per the

manufacturer's instructions (Boehringer-Mannheim GmbH,Federal Republic of Germany).EMS mutagenesis. EMS (Sigma Chemical Company, St.

Louis, Mo.) mutagenesis was performed according to Fink(4), and 10 nonreverting, pigmented adenine auxotrophswere produced by this method.UV mutagenesis. YEPD (50 ml) was inoculated with 0.1 ml

of a saturated overnight culture of S. occidentalis. Cellswere harvested at an A550 of 1.3, washed twice with steriledistilled water, and suspended in sterile water to a concen-tration of 4 x 107 cells per ml. Samples (0.1 ml) of the finalsuspension were spread onto YEPD plates (25 by 100 mm)and allowed to dry. The plates were irradiated for 65 s, 43 cmfrom a shortwave UV lamp (GTE; 8 W). The dose at thesurface of the plates was 250 p.W/cm2 as measured with aUV intensity meter (Blak Ray ultraviolet meter J225; Ultra-violet Products, Inc., San Gabriel, Calif.). The plates werekept at 30°C for 72 h before being replicated to selectivemedium. Exposure time and distance were experimentallyadjusted to kill approximately 98% of the plated cells.Fifteen nonreverting, pigmented, adenine-requiring mutantswere produced by this procedure from 2.4 x 108 treatedcells.

Library construction. Genomic DNA from S. occidentaliswas partially digested with Sau3A. The DNA was fraction-ated in a 5 to 1.5 M NaCl linear gradient at 39,000 rpm in anSW41 rotor (Beckman, Fullerton, Calif.). Fractions contain-ing fragments from 6 to 10 kb long were pooled, dialyzedagainst 10 mM Tris hydrochloride-0.1 mM EDTA, pH 7.0,and concentrated by ethanol precipitation as described be-fore (12). The pooled fragments were ligated into the BamHIsite of the yeast expression vector pYcDE8. This DNA wasused to transform E. coli DH5-a, resulting in a total of S x104 transformants. Plasmid DNAs were isolated from 24 ofthese transformants as described before (9) and analyzed byrestriction enzyme digestion. The insertion frequency wasfound to be >95%; inserts ranged in size from 2.7 to 7.3 kbp,with an average size of 4.6 kbp. The remaining colonies werescraped from the plates and suspended in 10 ml of LBmedium supplemented with ampicillin to a final concentra-tion of 100 ,ug/ml, and the plasmid DNA was isolated asdescribed before (12).

Yeast transformations. S. cerevisiae spheroplasts and E.coli were transformed as described before (9).Transformation of S. occidentalis was done as described

by Beggs (2) with the following modifications. The concen-tration of the isoosmotic stabilizer sorbitol in all solutionswas 1.5 M. Once 95% of the cells were converted tospheroplasts, they were pelleted at 8,000 rpm for 8 s in aSavant microcentrifuge (Savant Instruments, Farmingdale,N.J.) and washed as described before (2). The incubationtime following the addition of spheroplast recovery (SOS)medium (2) was 45 min at 30°C. The recovered spheroplastswere suspended in top agar and incubated at 30°C for 48 h.Successful transformation of Schwanniomyces mutantscould be detected 20 h after plating. The number of sphero-plasts used in these studies was determined by direct countwith a hemacytometer (Reichert Scientific Instruments, Buf-falo, N.Y.).

RESULTS

Cloning of the ADE2 gene from S. occidentalis. An ade2-1trpl mutant of S. cerevisiae (UC7586) was transformed withthe Schwanniomyces library DNA, and TRP+ transformantswere selected. Approximately 103 tryptophan prototrophs

VOL. 170, 1988

on January 27, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

5574 KLEIN AND FAVREAU

HindillHindlill

FIG. 1. Restriction map of plasmid pADE. The thin line repre-sents pYcDE8 sequences, open boxes are important regions formaintenance in E. coli, stippled boxes denote important regionsfrom S. cerevisiae, and the heavy line indicates cloned S. occiden-talis genomic DNA. The arrow indicates the direction of transcrip-tion from the ADHI promoter. The polylinker containing multiplecloning sites is indicated near the ADHI promoter in pYcDE8. Theyeast origin of replication is from the 2,um plasmid, and the TRPIgene (containing ARSI) permits selection in S. cerevisiae. Thisplasmid was isolated by complementation of an S. cerevisiae ade2mutant. The DNA insert from S. occidentalis is -4.5 kbp.

were obtained per ,ug of DNA. The transformation mixturewas directly plated on YMM supplemented with tryptophanbut without adenine and resulted in selection of one colonyable to grow in the absence of adenine. DNA was preparedfrom this transformant and used to transform E. coli, select-ing for Ampr. An 11-kbp plasmid, pADE (Fig. 1), containinga 4.5-kbp insert was recovered from the bacterial transfor-mants. In order to confirm that the ade2-complementingactivity was plasmid mediated, the purified plasmid was usedto transform S. cerevisiae UC7586, selecting for TRP+transformants. All of the resulting tryptophan prototrophswere also able to grow without adenine, confirming that theade+ phenotype was cotransforming with the TRP markerand was plasmid mediated.

In order to demonstrate that the cloned fragment wasderived from S. occidentalis, we probed gel-fractionatedrestriction enzyme digests of genomic S. occidentalis DNAwith 32P-labeled pADE and subfragments of the 4.5-kbpinsert. We have previously demonstrated that the vectorpYcDE8 does not hybridize to S. occidentalis genomic DNAunder the conditions used in this study (9). Figure 2 showsthe results of Southern hybridization analysis of genomicDNA digested with a segment of the insert extending fromthe KpnI to the SphI site at approximately 4 kbp.The hybridization pattern was as predicted by the restric-

tion map of the insert (Fig. 1). For example, internal frag-ments of 1.5, 1.35, and 1.05 kbp from digests with tworestriction enzymes, EcoRI and ClaI (lane D), XbaI and ClaI(lane E), and EcoRI and HindIII (lane F), respectively, wereas expected from the mapping data given in Fig. 1. Inaddition, we were able to derive a restriction map of theADE2 locus and flanking regions (Fig. 3). Additional South-ern hybridization analyses with pADE and subfragments ofthe insert indicated that the region contiguous with the CYCIterminator was not contiguous with the ADE2 locus, aspresented in Fig. 1, and was probably obtained as a cloningartifact during ligation of the partially Sau3A-digested frag-ments into the vector. This region consisted of less than 200bp.Complementation of ade mutants. A total of 25 pigmented,

A B C D E F G

6.6-

2.3-2.0-

U-

FIG. 2. Southern hybridization analysis of total DNA from S.occidentalis with a 32P-labeled KpnI-SphI 4.05-kbp subfragment ofthe pADE insert DNA. DNA was digested with XbaI (lane A),HindIlI (lane B), ClaI (lane C), EcoRI and ClaI (lane D), XbaI andClaI (lane E), HindlIl and EcoRI (lane F), or HindlIl and ClaI (laneG). Molecular size markers (in kbp) were derived from a Hindllldigest of X DNA.

adenine-requiring mutants was generated by UV and EMSmutagenesis. Pigmentation indicated a lesion in the geneencoding either phosphoribosylcarboxyaminoimidazole syn-thase (ADEJ) or phosphoribosylaminoimidazole carboxylase(ADE2) (7). Sixteen mutants from each of the mutagenesistreatments were selected for further study; all were shown tohave a low spontaneous reversion frequency (10-' after 7days at 30°C). Eight independent mutants were randomlychosen to test for transformation and to determine whetherthey were adel or ade2. Since there may be a difference intransformation efficiency due to the nature of the DNA used,transformations were attempted with both linear (treatedwith KpnI) and supercoiled pADE DNA. Two of the mu-tants, RKA-7 and LRA-26, yielded ADE+ transformants at afrequency of >1,000 transformants per ,ug of DNA for bothlinear and supercoiled forms. None of the remaining sixstrains yielded ADE+ transformants. The mutant strainswere isolated independently; RKA-7 was a darker red thanLRA-26 and was generated by UV mutagenesis, whereasLRA-26 was obtained by treatment with EMS.

ADE-2

R SCI II

X RX H H C S CII I II I I

H R XI I

1 kbp

FIG. 3. Restriction map of the ADE2 locus of S. occidentalis.The limits of the cloned 4.25-kbp fragment containing ADE2-complementing activity are indicated by the thick line. Flankingregions are shown by the thin line. The locations of specificrestriction enzyme cleavage sites are indicated. These data werederived from Southern hybridization analysis of total S. occidentalisgenomic DNA. Restriction sites: HindII (H), ClaI (C), XbaI (X),EcoRI (R), and SphI (S). The location of the ADE2-complementingactivity as determined by deletion analysis is shown at the top, andthe scale (in kbp) is at the bottom. Not shown are the KpnI andBamHI sites that are located outside this region.

J. BACTERIOL.

on January 27, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

TRANSFORMATION OF S. OCCIDENTALIS 5575

A B

23 kbp -%. 40

_o

BA B C D E F G H I J

23-

9.4-

6.6-

-23

*.~~ ~ ~ ~ ~ ~ ~ ~ ~ -.

f.

9.4 kbp-

FIG. 4. (A) Southern hybridization analysis of DNA isolated from RKA-7-Scl and probed with 32P-labeled pYcDE8 (lane A) or pADE(lane B). Samples were electrophoresed at 30 V for 94 h. The location of genomic DNA as visualized by ethidium bromide staining is indicatedby the bracket. Electrophoresis was done without ethidium bromide in either the gel or running buffer, and the genomic DNA band was diffuseunder these conditions. (B) Autoradiogram of Southern hybridization analysis of S. occidentalis DNA isolated from strains RKA-7 andLRA-26 transformed with linear or supercoiled pADE and probed with 32P-labeled pYcDE8. Lanes B and C are RKA-7 transformed withlinear pADE (strains designated RKA-7-L1 and -L2, respectively); lanes D and E are RKA-7 transformed with supercoiled pADE (strainsdesignated RKA-7-Scl and -Sc2, respectively); lane A is untransformed RKA-7. Lanes G and H are LRA-26 transformed with linear pADE(strains designated LRA-26-L1 and -L2, respectively); lanes I and J are LRA-26 transformed with supercoiled pADE (strains designatedLRA-26-Scl and -Sc2, respectively). Lane F is untransformed LRA-26. The DNA samples were electrophoresed in the presence of ethidiumbromide (0.5 jig/ml) at 30 V for 36 h. The genomic DNA as identified by ethidium bromide staining comigrated with the 23-kbp marker in thissystem. The arrow indicates the origin.

Recovery of plasmids fromADE+ transformants. DNA wasprepared from two ADE+ transformants each of RKA-7 andLRA-26 transformed with linear (L) and supercoiled (Sc)pADE (Fig. 4). The DNA was used to transform E. coli,selecting for ampicillin resistance. Ampicillin-resistant E.coli cells were recovered from all transformants with theseextracts, and plasmid DNA was prepared from two repre-sentative clones of each bacterial transformation. The re-striction maps of the recovered plasmids were found to beidentical to that of the original plasmid pADE for all of therestriction enzymes tested (ClaI, XbaI, and EcoRI [data notshown]).Although it was apparent from these results that plasmids

were capable of autonomous replication in the transformedyeasts, we wished to know whether any of the transformantscontained DNA integrated into the host genome. To testwhether pADE existed as an extrachromosomal element,one transformant, RKA-7-Scl, was fractionated in a 0.7%agarose gel in the absence of ethidium bromide at 30 V for 94h. Southern hybridization analysis was performed on dupli-cate blots of this sample, probing with pADE and pYcDE8(Fig. 4A). The genomic DNA band, identified by ethidiumbromide staining and indicated by the brackets, was diffuseunder these conditions. Lane B shows hybridization of thepADE probe in the region of the ethidium bromide-stainedmaterial, i.e., genomic DNA. Lane A shows no hybridiza-tion in this region with pYcDE8 as a probe, suggesting that

pADE is not integrated in this transformant. Southern hy-bridization analysis was also performed on total DNA iso-lated from two strains of each set of transformants. Figure4B shows the results of probing uncut total DNA with thevector pYcDE8. Samples were fractionated on a 0.7% aga-rose gel in the presence of ethidium bromide at 30 V for 48 h.The genomic DNA band was not as diffuse under theseconditions. In all of the transformants several bands were

seen, one of which migrated very close to the genomic DNAnear the 23-kbp marker. The upper band of the doubletmigrating near the 23-kbp marker in lanes E and J and thelower band in lane H comigrated with genomic DNA, as

identified by ethidium bromide staining and probing withpADE (data not shown). The remaining bands migrated atmolecular masses greater than those of the intact genomicDNA and the 23-kbp marker. These higher-molecular-massspecies were at least four times larger than the plasmidsrecovered from E. coli. Further exposure of the autoradio-gram showed hybridizing material below the genomic band.Although these bands were very faint, they corresponded tothe size expected for supercoiled monomeric pADE (datanot shown). Lane D shows sample RKA-7-Scl. The differ-ence in the pattern of bands shown in panel A (lane A) andpanel B (lane D) is due to the presence or absence ofethidium bromide in the electrophoresis buffer.The DNAs from the ADE+ transformants were cleaved

with restriction enzymes that cut outside of the ADE2 gene

A

VOL. 170, 1988

..

*a .g.

on January 27, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

5576 KLEIN AND FAVREAU

A B CD E F G HI J A B C D E F G H I J

23-

23-

9.4-

6.6-

AFIG. 5. Autoradiogram of Southern hybridization of DNA from S. occidentalis ADE2+ transformants cleaved with KpnI and probed with

32P-labeled pYcDE8 or pADE. The lanes are as in Fig. 4. These samples were electrophoresed at 30 V for 12 (A) or 18 (B) h, transferred tonitrocellulose, and probed with pYcDE8 (A) or pADE (B). Molecular size markers (in kbp) are shown.

in the vector pYcDE8. The samples were fractionated inagarose gels, blotted, and probed with labeled pYcDE8 andpADE. The results of the Southern hybridization analysis forKpnI-digested DNAs are shown in Fig. 5. Samples probedwith pYcDE8 (Fig. 5A) showed a single band at approxi-mately 11 kbp (lanes B to E and G to J). This band was notpresent in the untransformed parent (lanes A and F). Probingwith pADE (Fig. 5B) revealed a single band in the parentalcontrols (lanes A and F). This band, migrating at 10 kbp,contained the ADE2 locus. As can be seen in Fig. 5B, in alllanes containing transformant DNA (lanes B to E and G toJ), the lower band of the doublet corresponded to the bandidentified in the parental controls as containing ade2; theupper band corresponded to the band identified by probingwith pYcDE8. In all cases, the plasmid band identified byprobing with pYcDE8 was more intensely labeled than thehost band containing ade2. This is consistent with thehypothesis that the plasmid consists of repeated units ofpADE in the ADE+ transformants.The ADE+ transformants were further characterized by

Southern hybridization analysis with additional restrictionenzymes. DNAs from the LRA-26 ADE+ transformantswere digested with ClaI and BamHI and analyzed as de-scribed above. Figure 6 shows the results of probing theseDNAs with 32P-labeled pYcDE8 or pADE. Only a singleband was seen at approximately 11 kbp for both enzymeswhen probed with pYcDE8. Probing the Clal-digested trans-formant DNAs with pADE revealed the same two bands at-5 and 1 kbp as seen in the untransformed LRA-26 control(Fig. 6B, lane A). The same bands are seen in three of thefour transformants tested (lanes C, D, and E) in addition tothe 11-kbp band seen when the same samples were probedwith pYcDE8. One sample, LRA-26-Sc1 (Fig. 6B, lane B),showed an alteration in the ClaI banding pattern with a lossof the 1-kbp band and the presence of additional bands at 1.8and >14 kbp. A similar result was seen with LRA-26-Sc2(Fig. 6B, lane C) but without the loss of the 1-kbp band. The

BamHI-treated DNAs showed a similar result. A single bandat approximately 11 kbp was seen for all of the ADE+transformants when probed with pYcDE8 (Fig. 6A, lanes Gto J). When probed with pADE, a single band at approxi-mately 14 kbp, containing the ADE2 locus, was seen in theuntransformed LRA-26 control (Fig. 6B, lane F). In all fourtransformants a doublet was seen (Fig. 6B, lanes G to J). Thelower band corresponded to that identified when thesesamples were probed with pYcDE8; the upper band comi-grated with the 14-kbp band seen in the untransformedcontrol. These results suggest that no significant modifica-tion of the ADE2 locus has taken place due to pADEintegration for the majority of transformants tested. (A shiftto a higher molecular mass or the presence of two new bandsdue to the introduction of new internal restriction sites dueto integration at the ADE2 locus would be expected for thesamples digested with BamHI or KpnI.) Modifications in thehybridization patterns in the case of LRA-26-Scl and LRA-26-Sc2 cleaved with ClaI suggest that integration of at leasta segment of pADE occurred. The generation of an 11-kbpband with restriction enzymes that cleave pADE onceshowed that only linearized pADE was being generated fromthe higher-molecular-mass material seen in Fig. 4.From these data, we conclude that the ADE+ phenotype

of the transformed mutants is plasmid mediated and thatpADE is present in the host as an extrachromosomal ele-ment. Since the hybridization results show that the extra-chromosomal DNA, which is of a high molecular mass, isreduced to a single species that is identical to linear pADE,it is likely that the material is a polymerized form of pADE.Extrachromosomal elements require autonomous replica-

tion sequences (ARSs), which could be located within thevector or the insert. In order to localize both ARS andADE-complementing activity, a number of derivatives ofpADE were constructed. Table 1 summarizes the structuresof these plasmids and the results of transformation experi-ments with the S. occidentalis mutant RKA-7 and S. cere-

9.4-6.6 -

J. BACTERIOL.

on January 27, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

TRANSFORMATION OF S. OCCIDENTALIS

A B C E F G H J

23-

9.4-

A B C 0 E F G H

6.6-

4.4-

2.3-

2.0-

A

_

;;4it-^EL ;G-_:...

*gF'.. .. .. ..

:.: :::......: Z

B :k

FIG. 6. Autoradiogram of Southern hybridization analysis of LRA-26 ade+ transformants treated with ClaI (lanes A-E) or BamHI (lanesF-J) and probed with 32P-labeled pYcDE8 (A) or pADE (B). The samples were as follows: untransformed LRA-26 (lanes A and F),LRA-26-Scl (lanes B and G), LRA-26-Sc2 (lanes C and H), LRA-26-L1 (lanes D and I), and LRA-26-L2 (lanes E and J). Conditions forelectrophoresis and sample designations are as described in the legend to Fig. 4; approximately 15 p.g of DNA was applied to lane F.

visiae UC7586. Transformants were selected on mediumlacking only adenine. All but plasmid pADE-7 comple-mented the ade2 mutation of RKA-7. The transformationefficiencies of RKA-7 with pADE and pADE-2 were identi-cal. Several ADE+ transformants from the pADE-2 transfor-mation were selected for further analysis. Plasmids were

recovered from E. coli transformed with total DNA extractsfrom these yeasts. From these data, we conclude thatsequences confirming ARS activity reside within the ADE2insert and not in the vector pYcDE8.

Since pADE-1 complemented RKA-7 with the same effi-ciency as pADE, the 1.35-kpb EcoRI-EcoRI region is notnecessary for complementation. The inability of pADE-7,which contains a deletion extending from the ClaI site to theSphI site (approximately 1 kbp), to efficiently complementthe ade2 mutation is consistent with our hypothesis thatDNA essential for complementation is contained within the2.75-kbp EcoRI-SphI fragment. The small number of trans-formants seen with pADE-7 may be due to integration if theinsert DNA only covers the ade lesion in RKA-7. If this istrue, then pADE-7 may only be missing the ARS. The lowertransformation efficiencies in UC7586 of plasmids containingthe 2p.m plasmid origin of replication or ARS1 (in TRPI)suggest that the ADE2 gene of S. occidentalis does notefficiently complement the ade2 mutation in S. cerevisiae.

DISCUSSION

We have developed an efficient transformation and cloningsystem for the yeast S. occidentalis based on ade2 mutantsand the ADE2 gene from S. occidentalis as a selectablemarker. This system uses a modification of the spheroplast-

ing procedure described by Beggs (2). All of the ADE+transformants analyzed by Southern hybridization showedthe presence of extrachromosomal DNA at a higher molec-ular mass than that of genomic DNA. Our data suggest thatthis material is a polymerized form of the vector pADE. Asimilar phenomenon has been reported for Schizosaccharo-myces pombe transformed with a (JRAI-based plasmid andCandida albicans transformed with an ADE2-based plasmid(10, 17, 18). Despite this fact, intact plasmids identical topADE were recovered in E. coli by standard transformationprocedures. Whether these plasmids were from the same

cells as those possessing the high-molecular-mass plasmidsor a subpopulation of transformants cannot be determined.Southern hybridization analysis of DNA isolated from thetransformants suggests that the pADE plasmid is (in mostcases) not integrated into the host genome.We have shown that plasmid pADE transforms ade2 S.

occidentalis with equal efficiency whether in linear or super-coiled form. This suggests that there is little discrimination inDNA uptake between the two forms. The analysis of thestructure of DNA isolated from the ADE+ transformantsshowed little difference, i.e., a preponderance of extrachro-mosomal elements with very little integration. These resultshave been reproduced and suggest that the linear moleculesare efficiently converted to a circular form by the host cell.The fact that there is little integrative transformation we findunusual for an ARS-based vector system, suggesting that theADE-replicating sequence is quite stable in S. occidentalis.The plasmid pADE-1 is missing the 1.35-kbp EcoRI frag-

ment, and pADE-6 has a deletion extending from the SphIsite to the end of the insert; both confer an ADE+ pheno-

I i

-23

-9.4- 6.6

-4.4

- 2.3

-2.0

VOL. 170, 1988 5577

on January 27, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

5578 KLEIN AND FAVREAU

type. Plasmid pADE-2 consists of pUC19 plus the ADE2insert from the KpnI site (contiguous to the ADH1 promoter)to the SphI site (approximately 4.05 kbp) and transformsade2 S. occidentalis to ADE2+ as efficiently as pADE,pADE-1, and pADE-6. These data allow us to locate thecomplementing activity to a 2.7-kbp EcoRI-SphI fragment.The transformation efficiencies of these plasmids were iden-tical, suggesting that the ARS is located in this region. If,however, the ARS was located within regions of the insertflanking this fragment, then the S. cerevisiae ARS1 (in TRPI)or the 2,um plasmid origin of replication would be as active inpADE-1 and pADE-6 as the S. occidentalis ARS in pADE-2.We consider this possibility unlikely. Alternatively, E. colisequences common to pYcDE8 and pUC19 could be respon-sible for ARS activity. We are currently investigating thesepossibilities.The ADE2 gene from S. cerevisiae has been cloned and

used in the successful transformation of a number of ade2mutants of other yeasts (14, 19). The published restrictionmap of ADE2 from S. cerevisiae differs frorp that of the S.occidentalis ADE2 gene. In addition, ade2 S. cerevisiaemutants transformed with the S. cerevisiae ADE2 gene arenot pigmented, whereas our S. occidentalis transformantswere pigmented, ranging in color from pink to dark red.An ADE2-based transformation system has also been

developed by Kirsch and co-workers for C. albicans withboth plasmids (10) and integration vectors (11). These re-searchers used the transformation system to overcome sev-eral obstacles in developing the genetics of C. albicans,namely its lack of a sexual cycle and the fact that theorganism is diploid. Defined mutations in the URA3 genehave been introduced by gene disruption techniques (8). Theexploitation of Schwanniomyces spp. is also hampered bythe absence of well-defined mutations and information aboutploidy, genetics, and sexual cycle, which limits the applica-tion of conventional genetic manipulations. The availabilityof the transformation system described in this report, clon-ing vectors such as pADE, pADE-1, and pADE-2, clonedgenes such as ADE2 and ODC (9), and the availability ofvectors (such as pYcDE8) for cloning genes by complemen-tation in S. cerevisiae open new possibilities for the explo-ration and exploitation of this organism. To the best of ourknowledge, this is the first report of an efficient transforma-tion and cloning system in the genus Schwanniomyces.

ACKNOWLEDGMENTS

We thank Kathy Hiestand for the preparation of this manuscript,L. Roof for preparing strain LRA-26, and L. Post, T. G. Geary, A.McNab, W. M. Ingledew, E. Olson, and P. Robbins for theircomments and suggestions.

LITERATURE CITED1. Abott, B. J., and W. E. Gledhill. 1971. The extracellular

accumulation of metabolic products by hydrocarbon-degradingmicroorganisms, p. 249-389. In D. Perlman (ed.), Advances inapplied microbiology. Academic Press, New York.

2. Beggs, J. D. 1978. Transformation of yeast by a replicatinghybrid plasmid. Nature (London) 275:104-108.

3. Deibel, M. R., R. R. Hiebsch, and R. D. Klein. 1988. Secretedamylolytic enzymes from Schwanniomyces occidentalis: purifi-cation by fast protein liquid chromatography (FPLC) and pre-liminary characterization. Prep. Biochem. 18:77-122.

4. Fink, G. R. 1970. The biochemical genetics of yeast. MethodsEnzymol. 17:59-78.

5. Hartig, A., J. Holly, G. Saari, and V. MacKay. 1986. Multiple

regulation of STE2 by a mating-type-specific gene of Saccha-romyces cerevisiae. Mol. Cell. Biol. 6:2106-2114.

6. Ingledew, W. M. 1987. Schwanniomyces: a potential superyeast? Crit. Rev. Biotechnol. 5:159-176.

7. Jones, E. W., and G. R. Fink. 1982. Regulation of amino acidand nucleotide biosynthesis in yeast, p. 181-300. In J. N.Strathern, E. W. Jones, and J. R. Broach (ed.), The molecularbiology of the yeast Saccharomyces: metabolism and geneexpression. Cold Spring Harbor Laboratory, Cold Spring Har-bor, N.Y.

8. Kely, R., S. M. Miller, M. B. Kurtz, and D. R. Kirsch. 1987.Directed mutagenesis in Candida albicans: one-step gene dis-ruption to isolate ura3 mutants. Mol. Cell. Biol. 7:199-207.

9. Klein, R. D., and L. L. Roof. 1988. Cloning of the oritidine5'-phosphate decarboxylase (ODC) gene of Schwanniomycesoccidentalis by complementation of the ura3 mutation in S.cerevisiae. Curr. Genet. 13:29-35.

10. Kurtz, M. B., M. W. Cortelyou, S. M. Miller, M. Lai, and D. R.Kirsch. 1987. Development of autonomously replicating plas-mids for Candida albicans. Mol. Cell. Biol. 7:209-217.

11. Kurtz, M. B., M. W. Cortelyou, and D. F. R. Kirsch. 1986.Integrative transformation of Candida albicans, using a clonedCandida ADE2 gene. Mol. Cell. Biol. 6:142-149.

12. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

13. McKnight, G. L., H. Kato, A. Upshall, M. D. Parker, G. Saari,and P. S. O'Hara. 1985. Identification and molecular analysis ofa third Aspergillus nidulans alcohol dehydrogenase gene.EMBO J. 4:2093-2099.

14. Neistat, M. A., V. V. Alenin, and I. I. Tolstorukov. 1986.Transformation of Hansenula polymorpha, Pichia guilliermon-dii and Williopsis saturnus by a plasmid containing ADE2 genefrom Saccharomyces cerevisiae. Mol. Gen. Mikrobiol. Virusol.12:19-23.

15. Oteng-Gyang, K., G. Moulin, and P. Galzy. 1981. A study of theamylolytic system of Schwanniomyces castellii. Z. Allg. Mikro-biol. 21:537-544.

16. Phaff, H. 1985. Biology of yeasts other than Saccharomyces, p.537-562. In A. L. Demain and N. A. Solomon (ed.), Biology ofindustrial microorganisms. Benjamin/Cummings PublishingCo., Menlo Park, Calif.

17. Sakaguchi, J., and M. Yamamoto. 1982. Cloned ural locus ofSchizosaccharomyces pombe propagates autonomously in thisyeast, assuming a polymeric form. Proc. Natl. Acad. Sci. USA79:7819-7823.

18. Sakai, K., J. Sakaguchi, and M. Yamamoto. 1984. High-fre-quency cotransformation by copolymerization of plasmids inthe fission yeast Schizosaccharomyces pombe. Mol. Cell. Biol.4:651-656.

19. Sasnauskas, K. V., G. K. Gedminene, V. I. Naktinis, D. B.Chitavichyus, and A. A. Yanulaitis. 1982. Cloning of the ade2gene of the yeast Saccharomyces cerevisiae. Dokl. Acad. NaukSSSR Biol. Sci. Sect. (Engl. Transi.) 263:224-227.

20. Schleif, R. F., and P. C. Wensink. 1981. Practical methods inmolecular biology, p. 124-125. Springer-Verlag, New York.

21. Sherman, F., G. R. Fink, and J. B. Hicks. 1981. Methods inyeast genetics. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

22. Sills, A. M., M. E. Sauder, and G. G. Stewart. 1984. Isolationand characterization of the amylolytic system of Schwan-niomyces castellii. J. Inst. Brew. 90:311-314.

23. Stewart, G. G., I. Russell, R. D. Klein, and R. R. Hiebsch (ed.).1987. Biological research on industrial yeasts. CRC Press, BocaRaton, Fla.

24. Vieira, J., and J. Messing. 1982. The pUC plasmids, anM13mp7-derived system for insertion mutagenesis and sequenc-ing with synthetic universal primers. Gene 19:258-268.

25. Wilson, J. J., and W. M. Ingledew. 1982. Isolation and charac-terization of the amylolytic enzymes of Schwanniomyces allu-vius. Appl. Environ. Microbiol. 44:301-307.

J. BACTERIOL.

on January 27, 2021 by guesthttp://jb.asm

.org/D

ownloaded from