6ene. 95 (1990) 111-121 Elsevier

11 !

GENE 03720

Cloning of the Schwanniomyces occidentalis glucoamylase gene ( G A M I ) and its expression in S a c c h a m myces cerevisiae

(Recombinant DNA; gene disruption; glucose repression; promoter fusions; dextrin; pullulan; secretion; genomic library)

R. Jtirgen Dohmen'*, Alexander W.M. Strasser b, Ulrike M. Dahlems b and ComeHs P. Hollenberg" ° Institutfiir Mikrobiologie, Heinrich-Heine-Universit~t Diisseldo~ Dtisseldo~(F.R.G.), and b Rhein Biotech GmbH.. 4000 D~sseldorf I (F,R, G.) Tel. (211)736436 Received by J.K.C. Knowles: 19 September 1989 Revised: 21 February 1990 Accepted: ! March 1990

SUMMARY

The Schwanniomyces occidentalis glucoamylase (GAM)-encoding gene (GAMI) was isolated from a ACharon4A genomic library using synthetic oligodeoxynucleotides as probes. GAMI contains an ORF of 2874 nucleotides (nt) coding for 958 amino acids. SI mapping revealed that the transcript has only a very short 5'-untranslated leader of 8-12 nt. Disruption and displacement of the GAMI gene in Sc. occidentalis resulted in loss of the ability to grow on starch efficiently. The gaml strains still exhibit low GAM activity suggesting that at least a second weakly expressed GAM-encoding gene (GAM2) is present in Sc. occidentalis. Expression of the Sc. occidentalis OAMI gene in Saccharomyces cerevisiae was achieved after promoter exchange. $. cerevisiae cells transformed with centromere plasmids carrying the GAMI gene fused to promoters of different S. cerevisiae genes, namely GALl, PDCI and ADHI, efficiently secrete GAM and are able to grow with soluble starch as a sole carbon source. The essential enzymatic properties of the GAMs secreted from S. cerevisiae and Sc. occidentalis are identical, although the modifications of the proteins are different.

INTRODUCTION

Starch-containing raw materials are widely available and are frequently used as feedstock for industrial production of ethanol, potable spirits or beer (Tubb, 1986). The most widely used organism for commercial alcoholic fermentation is Saccharomyces cerevisiae, which, however, lacks amylolytic activity necessary for direct starch utilization. Correspondence to: Dr. C. Hollenberg, Institut fiir Mikrobiologie, Heinrich-Heine-Universitftt D0sseldorf, Universitiitsstrasse I, Gebttude 26.12, 4000 D0sseldorf I (F.R.G.) Tel. (211) 311.4720; Fax (211 ) 342229. * Present address: Massachusetts Institute of Technology, Department of Biology, 77 Massachusetts Avenue, Cambridge, MA 02139 (U.S.A.) Tel. (617) 253-6788. Abbreviations: A ~ , absorbance at 600 nm; aa, amino acid(s); AMYI, ~.amylase-encoding gene; Ap R, ampicillin resistance gene; bp, base pair(s); BSA, bovine serum albumin; DEAE, diethylaminoethyl; EndoH, 0378-1119/90/$03.50 © 1990Elsevier Science Publishers BN. (BiomedicalDivision)

S. cerevisiae strains, which would secrete u-amylase and GAM with debranching activity would contribute to the improvement of a variety of biotechnological processes since they combine high fermentation rates and ethanol tolerance with the ability ~o use starch completely (Sills and Stewart, 1982). Mainly because oftheir close relationship to S. cerevisiae other yeast species are favorable candidates as donors for endoglycosidase H; EtdBr, ethidium bromide; GAM, glueoamylase; GAM, gene(s) encodin~ GAM; HPLC, high performance liquid chromatography; kb, kilobas¢.'~s) or 1000bp; HAD, nicotinamideadenine dinucleotide; nt, nucleotide(s); oligo, oligodeoxynucleotide; ORF, open reading frame; PA, polyacrylamide; PAGE, PA-gel electrophoresis; $., gaccharomyces; $c. (Sw), Schwanniomyces; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/0.15 M Nas" citrate pH 7.0; tsp, transcription start point(s); u, unit(s); UV, ultraviolet; YEP, yeast extract (I %)/peptone (2%); YNB, yeast nitrogen base (0.67%).

112 genes encoding amylolytj'c enzymes. There are about 100 yeast species known, which are able to grow on starch (Lodder and Krieger-van Rij, 1970). However, only very few degrade starch with high efficiency as a result of the combined action of n,-amylase (E.C,3.2.11), GAM (EC3,2,13) and debranching activity (Sills and Stewart, 1982). Such strains are: Lipomyces kononenkoae and L. starkeryi (Spencer-Martins and van Uden, 1979), Endomycopsis flbuligera and E, capsularis, Pichia burtonii and Schwanniomyces occidentalb (Sills and Stewart, 1982), comprising the former Schwanniomyces species castellii, alluvius, persoonii and occidentalis (Price et al., 1978). Of these strains, Sc, occidentalis expresses the most significant debranching activity (Sills and Stewart, 1982), as a part of the GAM enzyme (Sills et al., 1984a; Wilson and lngledew, 1982). The 0t-amylaseand the GAM secreted by this species moreover have the advantage to be inactivated during pasteurization conditions usually employed in brewing (Sills et al., 1967). In this article we report the cloning and sequencing of the Sc. occidentalis GAMI gene and find that it bears no •homology to all other GAMs which are sequenced thus f~r and have highly conserved regions in common (Itoh et al., 198"/). We also describe for the first time the analysis ofgene function in $c. occidemalis by creating null alleles via homologous recombination. The possibility of deleting genes and introducing altered versions of them either on autonomously replicating plasmids (Dohmen et al., 1989) or via homologous recombination make $c. occidentalis, a yeast for which classical genetic methods are not available up until now, an organism also of interest for more general studies, The expression of the 6AMI gene in S, cerevislae resulted in strains that secreted GAM and thus were able to grow with soluble starch as a sole carbon source. The construction and analysis of 8. cerevisiae strains expressing both the AMYI gene of $c. occidentalis (Strasser et al., 1989) and the 6AMI gene will be presented elsewhere. The most remarkable property of these strains, compared to strains secreting either enzyme, is the improved utilization of crude starch preparations.

RESULTS AND DISCUSSION

(a) Cloning and mapping of the glueoamylase-encoding gene GAMI A ~.Charon4A clone bank of $c, occidentalis ATCC26076 was screened by plaque hybridization using a mixture of 256 different labeled oligos (oligo 1 in Fig. 1), all coding for the same 8 aa sequence found in a 19 aa tryptic pepfide of the glucoamylase protein (Fig. 2, nt 2356-2412). Using the synthetic oligo mixture, two different strongly hybridizing clones were isolated out of 104 clones. The

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Fig. 1. Restriction maps of the So. occidentalis DNA inserts of the ~.Charon4A clones GAM183 and GAM 187 and of the plasmid subclooc pBRGAMI3. ~.GAM183 and ;tGAMIg? were isolated from a ZCharon4A (Blattner et al., 19"/7) library of Sc. occidentalis DNA. The library (10 s clones) was constructed by ligation of partial EcoRI fragments with the two ~.Charon4A arms produced by EcoRl digestion, in vitro packaging (Hohn, 1980) and transfection into E. coil K802 (Wood, 1986). The library was screened by plaque hybridization with a mixture of 5'-labeled oligos (oligo 1) coding for the same 8 an ofa tryptic peptide ofpurified GAM (Fig. 2). The hybridization solution was 6 × SSC/0.25% nonfat dry milk (Johnson et al., 1984). Stringent washing was performed according to Wood et al. (1985). The fragments hybridizing with oligo ! and another oligo mixture (oligo 2) deduced from another tryptic peptide of GAM (Fig. 2) are indicated by bars. The location of an element (SwAR52) that confers autonomous replication to plasmids is indicated by boxing. The overlapping areas of the phage inserts are indicated by dashed lines. The map of the 5.8-kb BamHl-£coRI fragment, which was analyzed in more detail a~er subcloning into plasmid pBR322, is enlarged relative to the maps of the phage inserts. An arrow indicates the transcribed region as it was deduced from S I mapping experiments (data not shown).

inserts are 16 kb in the case of phage ),GAMIG3 and 17.3 kb in the case of phage ~,GAM187. The two clones were mapped with BamHl, EcoRI and Sail and were found to have a 12,3 kb overlap (Fig. 1), Both clones also hybridized to a second oligo mixture deduced from another tryptic peptide of the GAM protein (oligo 2 in Fig. 1; nt 970-989 in Fig. 2). The $.8-kb BamHIEcoRl fragment present in both clones was subcloned into pBR322 and analyzed in more detail (plasmid pBRGAMI3, Fig. 1), Sl nuelease mapping (data not shown) revealed that a region of 3 kb within the 3.7-kb Bglll fragment ofplasmid pBRGAM 13 (Fig. l) is protected by RNA isolated from So. ocddentaib ATCC26076 grown under inducing conditions (2% maltose), This was in agreement with results from Northern blot experiments where a 3-kb RNA hybridized to GAMI probes (data not shown), No protection was observed when the cells were grown in medium with 2~o glucose indicating that transcription of GAMI is subject to strong catabolite repression (see Fig. 3), Further subcloning revealed that the l.l-kb BfllI-EcoRl fragment downstream from the GAM] gene harbours a sequence (SwARS2) conferring autonomous replication to plasmids in Sc. occidentalb and S. cerevbiae. The transformation frequencies, plasmid stabilities and copy numbers of plasmids containing this sequence (data not shown) were

!!3 "~L~O "300 "280 "~O "240 "220 AGATCTACATTTTAAACCCCAGTCTACTCCAGATATTGGAGTAT/LACCCCATTCTTACCGTTATATCCATGACC'~ICATCGNtATT TTCAAAGGATTTCGAGMMTTCTTT~T~ -200 -1W -160 -140 -120 -100 ATN.~U~GTGTTATTGGTGATT~/tTTACTAO~dUU~r/~TCATATGGTAGTAC~,0TT~Tf~MGTA~GrJUtTTGTA/tTTTG~AGT TAT~MT~T~~T -00 -60 -40 -20 1 29 TTTTCATTATTGGGAAAATAT/~TN~AGGCAA6TATCCATTG/tAAT TTTANUUGAACTCATGACTGTATTATAACAAGr.AA~T~TTTTTC~~MAT~TMTT ~" T l 'It T MetI lePheLeuLysL~! tekysSerl teVat I t e 40 60 80 100 120 140 GGTTTSGSATTAGTTAGTGCTATCCAAQI~t~CI~TGCCTCTTCGAT T6GATCTAGTI~TTCAI~ATCTAGT TCAAGTGAGAG~TA~TT~T~TGT~TTA~T 6tyLeuStyLeuVMSerA|MteGLnAtaAtIProAtaSerSerI t eStySerSerAlmSerAtaSerSerSerSerGtuSerSerGtnMaThr I |eProAsrv~spVatThrLeuSty 160

180

200

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240

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GTTAAACNtMTCCfAATATCTTTAATGACT~T~CT~TC~T6CTAAT~CAGCTGCTAAA6~GTATGA~TTGGTNU~T~TT~T~TA~T~T~AT~T9~TTA VatLysGtnl |eProAsnl t ePheAsnAsDSerA|aVa[AsnAtaAsNt|eAt aAtaLvsGtyTyrAspi.euVMAsnVMThrA~mThrProArgGlyLeuThrG|yl teLeuLysLe~a 280 3OO 32O 34O 36O 38O NtAGAAGcTA~cAATATTTAT~TTATGATTTTGATTATTTAAACTTAA~T~TT~AATAC~GCT~ATA~AGATTAAAc~TTCATATT~AA~cT~TTTATC~T~TATTTGTT LysSt~AIeThrAsnl teTyrG|yTyrAspPheAspTyrLeuAsnLeuThrVmtGtuTyrGtnAtMspThrArgLeuAsr#a|llislteGtuProThrAspl.euSerAspVa|PheVM ~00 420 440 ~0 480 500 TTA~CAGA~CATTTA~TTGTTNtACCACT~GT~AAGGTGAT~CACAATCTTATAACTTC~ACAATTCCGATTT~GTTTTC~NkTA~TCT~TA~TT~TTT~GTTATT~ LeUProGtuHisLeuV~a|Lys~r~L~V~i~UG~yAspA~nSerTyrAsnPheAspAr.nSerAs~LeuVa~pheG~uTyr~erAshThrAs~d~he~erpheG~UVa~ ! tear9 520 ~0 560 580 600 620 TCAT(:TACTAAASAAGTTTTATTTTCTACTAAAGGTAATCCATTGGTTT TTTCAAATC~TTCATTCAATTC~TTCGTCM TGCCA~GAACCATGTTATTACT~TCTTGGT~TCT SerSerThrLysS|UVstLeuPheSerThrLysO|yAsr~roLeuVetPheSerAmS|nPhelteStnPheA~nSerSerLeuProLysAsnllisVa|I |eThrGtyt.euSty~tuSer 64O 66O 68O ~ 72O 1'40 ATTCA~GTTTAGTTAACGAA~CA~GTAG~GTT/L~ACATTATTTGCTAATGMGTTG6TGATCCAATC6M6GTAMATTTATGGTGTCCMCCAGTTTATCTTGAT~TM~C ! t eH|sStyLeuVmlAsnStUProStySerValLysThrLeuPheAtaAs,AsnV~t GtvAIProl leAsnGtvAsnlt eTvr6| Ws I H~spraY,,t Tyrl~euAsPG|nArqTyrAsp 760 78O BOO 820 84O 860 ACTGAAACTACCCATGCT•TTTATT6GA6AACTTCT•CTATTCAA6AAGTATTAMCG6T6AGGAATCTATTACTTGGA6AGCTCTTTCAGGTGTTATTGATTTATACTT•TTTAGT66T ThrStuThrThrHisAlaVatTyrTrpAroThrSerA|alteStnGLuVelLeulleGLyStuSLuSerlLeTKrTrpArgAlaLeuSer6LyVe| I teAspl.euTyrPhePheSerGt y 88O 9OO 920 9~0 96O 980 CCTAr,ACC/UtAASATG(:CATTCAACAGTATOTCAAAGAGATTGGTT TACCASCTTTCCAACCATA~TGGTCOTTASGTTACCAT~TGTA~T~TTA~TACTMC~TTA ProThrProLyeAsnAtalleSlnOlnTvrVaLLvsG|ulteGt yLeuPr~AlePheSLnProTyrTrpSerLee6lyTyrHisStnCysAraTr~tvTvrAsoThr! ~eStuLvsLeu

t000 1020 10&O 1060 1080 1100 TCTGAAGTTGTTSAA~CTTCAASAAATTTAATATTCCAT TASNtACTATCTOSTCASACATTSATTACATGGACTCTTATAAAGATTTCACTTATSMC~TTCC~CTA~T SerG|uVllt Vat 6tt~enPheLylLysPheAsnlteProLeuGiuThrl leTrnSerAenlt eAsDTvrNetAsnSerTvrLvsAspPheThrTyr&spProHi sArgPheProLe~sp 1120 1140 1160 1160 1;:00 1220 GAATATCGTkAATTCCTTSATSASTTSCAC,S_a_a_a_ATAAT CAACACTATSTTCCTATTTTGGATGCTGCTATT TACGTTC C A A A C ~ T G C T A C ~ T ~ C ~ T A C ~ T TTC GluTyrAroLysPheLet~Sl:6LuLe~dlI eLysA|n/~sfl6| nil ! 8TyrVMProl teLeuAq)MeAtel teTyrVaLProAsr~roAsnAsnALaThrAspAsnGLuTyrGtnProPhe 1240 1260 1280 1300 1320 1~0 CACTATGGTAATON~ACCSATOTCT TCTTAMSAATCCASATSGTTCATTATATMTOGTGCTGTTTGGCA66T TACACTGTTTTCCASATTTCTTAGCA~U~AACMT~TAT~T HI sTyrStyAsnStuThrAepVa|PheLeuLysAsnProAspStyhrLeuTyrI test yAtever TrpStnVe|ThrLeuPheSerAroPheLeuSerArgLysHI sSerAsplletAep 1360 1380 1&00 1420 14~0 1440 AAA0TCATTAAA~ATT~TAT6AATTAA~TC~TTTT6AT~TATTTGGn~T6ATAT6N~T6AA~TCTCATCATT~T~T~TT6~TTCTT~T6GTACT66T/L~TACT TCGAAAACCCAGCA LyeV~|I ~eLwAenTrMvrOt ul.euThrPr~heAm,V:-tvlteTrnAt eAqnMetAen6|LNa|SerSerPheCvsVatOtvhrCvsn|vThrG|vLveTvrPheGt~sr~ProAte

1480 1500 1~20 1540 1560 1580 I'ATCC1'CCATTi'ACTOT'rOSAASI'AAAQCTACCI'CTTAi'CCAOTl"0G' TTCSATOTTTCI'AACQ~TCT~T~TCTATTC~GCT r C~TTTCTGCTACT~T~CTTCTT~ TvrPr~#r~#tteThrVstOtv~=erLvaAtaThrhrTvrPrsVatOlvPhoAenVaihrAsrd~tderatuTroLveSerl teStnSerSerl t eSerAtaThrAteLysThrSerSer 1600 1(~,0 1640 ltMO 1580 1700 ACTTCTTC(:QTMCGTCOTCTTCATCCACAATCSATTATMOJ~ACACTT TAQCTCCA66TAAAnnT~TMT~TTMC~C~TMQCTM~TA~TG~ST~CTCC~TCT I ThrSerSerVa|SerhehrSerSerThr I teAepTyrMetAinThrLe~tePro6iyl.ysStyAenl t eAenTyrProProTyrAre! t eTyrAtfll4et6tr~ WAel~erAsl0LS~ 1720 1740 11'60 1780 1800 1820 QCTACTCATQCAGTATCTCCAAATGCTACACATGCTGAT66TACA~TT6AATAT6ATATTCACAATCTTTATQGTTACT TQCAA~AATGCTACTTATCATGCATTATTG~TTTT T AtaThrlfl eAtsVe|hrProA~nAt eThrlll~At ~ep6t yThrVatGtuTyrAeplt eHIsAenLe~TyrOtyTyrLeuG|nGtt~AsrAtaThrTyrHI~A|aLe~.e~AtuVetPhe 1840 1860 1880 1990 1920 1%0 ~CTAAC~6A6A~CATTCAT6ATTTCCA6ATCAA~CTTT~CAc6C~CT~GTAMT~6ACC6~CCMT~TG6TGAcAA~GCT~T TGGGCTTMGCTTACIT~TCTATCCCT~ ProAenLysAroProPhstletI teSerArgSerThrPheProArgALa~-t~ysTrpThrG|ytIlsTrpSty~|yAspAsnThrAteAspTrlMtaTyrAteTYrpheserl teProStn

1960 1980 2000 2020 2040 2060 ~CATTCTCAATGGGTATT6CT6~CCTTCCATTCTTT6GT6~C6AT~TTTGTG6TTTCAAT6GTAATTCT6ATTCT6AATTAT~TTCAA6ATG6AT~CAMTAG~TT~TTT~T T~TTC AlePheSerMetSlylteMeGtyl.euProPh~heStyAteAepVetCysGtyPheAenGWAenSerAspSerGtuL~ysSerArOTrl~etotN-eu6 tyse~pheph~r~he

(l~g. 2 congnued on page il4)

comparable to those obtained for SwARS1 plasmids (Dohmen etal., 1989). (b) Sequence analysis The 3.7-kb Bglll fragment containing the 3-kb region protected by $c. occidentalis mRNA was sequenced entirely. The nt sequence (Fig. 2) revealed an open reading frame of 2874 nt coding for a 958 aa protein. The gene contains

a perfect TATA-box (TATAAA) a t r position -66 and a termination signal (TAA...T-rich...TAG...TATGT...ATrich...TTT) as postulated by Zaret and Sherman (1982) 3' to the coding region. There is a short ORF of 12 nt from positions -23 to -11 with respect to the A of the initiation codon of the GAMI ORF. This suggests that transcription starts 3' to position -23. The N-terminal aa sequence e11coded by GAMI is

114 2060 :MOO 2120 2140 2t~) 2180 T~cAcAA~T&T~TA6~T6CTMT~Mr`AG6AA~ATMGT~TGr~AAT~A~TT6cT6~A~CTAcTA~AAcTTCTAT66C~ATTAGATAcTTATTATTAc~TATTA~TA~T TyrArsAslVlisAsnTyrLeu6tyA t a! LeAspGtnGtuProTyrVetTrl~tuSerValAtaGluAtaThrArsThrSerMetAtelteArgTyrLeul.eui.euProTyrTyrTyrThr 2200 2220 2240 2260 2280 2300 TTATTACAT~VtATCTCATACTACT~GTTTA~CAATCTTAAGAGCTTTCT~TGGCAATTCCCTKACaATC~TT~TTAAGT~TGT~TM~TTTTTTGT~T~T~G~T~AGT~ LeuLe~t~~atuSerHisThrThrGtyt.~Prol t eLeuArgAtaPheSerTrp6 t nPheProAsnAsl~r9SerLeuSer6tyVat aspAsnGtnPhePheVat Gt yAsp6t yL~Va t 2320 2340 ~ 2380 2400 2420 ~TTA~CCT~TCTTAGAA~T~GTGTT~ATAA~GTTK~AGGT~TTTTCCCAGGAGCTGGTAAAGAGGAAG~TTACTA~GACTGGT~CA~C~GTT~TTT~C~TMG VatThrProvaLLeuGtUPro6tyVaLaspLysVat Ly:SGIWIstPheProGtyAtaGtvLysGtug t uVat TyrTyrAsoT~T~ThrG t ~rgG t ~ a t Hi sPheL~As~ t yLys 2&40 ~ 2460 2500 2520 2540 AM~AAN~TTTA~ATGCACCATTA6GTCATATT~CATTACACATTA~AGGTGGTAACGTCTTGccAA~TCAAGAGC~TAT~c~TT~T~~T~TTT~TTTA asnStuThrLeuAsl~tId)roLeu6tyllis I teProLeuilisl t eArgGtvGtvAShVatLeUP.roThrGtnSlUProGtvTvrThrVatAt aGtuSer~rgGtNLsrOroPheGtyL~ 2560 2580 2600 2620 2640 2660 ATTGTCGCTTTAGATAA~GAT6GCAAAGCTrJ~A~GTAGcTTATA~CTTGAT6AT~GTG/~ATCATT~TAGT~GA~T~TTCATT~TTGGTTAGTTT~T~TGTTT~TGATAACA~TTA~ I tevetataLeuAspAshAspGtyLysAtaGtratySerLeuTyrLeuAslMspG tyG|uSerLeuVatVatA~erSerL~atSerPheSerVatSerAs~nThrL~er 2680 2700 2720 =7'40 2760 2780 GcAT~TCCAT~T~TGACTATAAAGCTGAT~AAC~TTTAGCTAAT~TTA~CAT~TTAGG~GTTGGCCMAAA~CA/~AATCA~1T~TTT~TMT~T T~TTT~CCTA~G AtaSerProSerGtyasDTyrLysAtaAsp(;tnProLeuAteAsnVetThr: t eLeuatyVatatytl| sLysProLysSerV|t LvsPheGtuAsnAtaAsnVat asuPheThrTvrLvs 28OO 2820 284O 285O 288O 29OO AAMCAACCGTTTTCaTTACTGGCTTAGMAAATACACCAAGGATGGT GCATTTTCT/~AGGATTTCACCATTACT TGGTAATTTTAACATCCACTTAGTTCAATTCCAT TCTTTTCTTTT LvsSerThrVatPheVatThratyLeuAspLysTyrThrLysAupOtyAtaPheSerLysAspPheThrlteThrTrp 2920 2%0 2960 2980 3000 3020 T~C~TGAMTTCTGAATTTCJU~ATTATTT6MTGATATCATTTTA~TTTTCTTCA~cTTAT~TATGTTTATTTcGATTTTA~ATGTTAAAAGTTTTTTATGTTTAT~TTGTTTTATTG ttttQ

~kt/ktt

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3040 3060 3080 3100 3120 3140 ATGTAGTTGATAAAATATAGCAAATACATCGAAk~TTTGCGATaAAATTTTGCAGCTCATTAGAAATGTAGTCMTCAT TAGTCA~TTG~C~CTATATMC~C~C~CTATTC 3160 3180 3200 3220 3240 3200 CAA6AAAAATATATGTAAGGATACTAG~TCATAAMTCT TATTGACTTTfiTTTTTTTTMCAATAGTTACAT~GC,AATATTCGTTTACTACAAAACCATTGGTCTTGTAAAGAAGCAGA 3280 3300 3320 3340 3360 3380 C~AGGCGTATGT TTGTGGTTGCGGCCGCAMACTAGTTTACkqAGCTCACCT TATCATTGTGCTAGACT TTCTTTTCAACGAAAGG/U~AGCAAAAGTAGkTGACGTGCTCTACTTTAATT 3400 3~20 CAATTAAGATAAAT TTGGTATTTTGCTAGATCT Fig. 2. Nucleotide sequence of the

GAMI

gene, Shown is the complete sequence of the 3,7-kb

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of the GAMI gene, This sequence data will appear in the EMBL, OenBank and DDBJ nt sequence databases under the accession number M34666. The analysis was performed according to Sanger et al. (1977) using the MI3 system (Vieira and Messing, 1982) and with the method described by Maxam and Gilbert (1980) to obtain overlapping sequences from both strands. The potential TATA box and the potential termination signal . . . T A G . . . T A T G T . . ~ . . . are indicated by asterisks. The tgp obtained in SI.protection experiments (Fig. 3) are indicated by upward arrows (double.stem arrow for main tgp). The sequences of tryptic peptides and the hybridization sites of the synthetic oligo mixtures are underlined. Last digits of numbers are aligned with corresponding nt.

enriched for hydrophobic aa and exhibits the properties of a signal sequence (yon Heijne, 1986) with the highest probability for signal peptidase cleavase between Aia== and Pro =~. The calculated molecular weight of the unmodified precursor is 106,5 kDa resulting in a 104 kDa protein if the first 22 aa are cleaved. The protein has ten potential N-glycosylation sites (Asn-Xaa-Thr/Ser).

(c) Comparison of the GAMI sequence with the amino acid sequences of GAMs from other organisms The aa sequence of the Sc, occidentalis GAM was compared with the aa sequences of the GAMs of Aspergillus awamori (Nunberg et al., 1984), Rhizopus oryzae (Ashikari et al., 1986), E.flbuligera (Itch et al., 1987) and Saccharomyces diastaticus (Yamashita et al., 1985), Surprisingly no significant homology between the Sc. occidentalix GAM and the other GAMs could be found although significant similarities exist amongst the others (Itch et al., 1987). This suggests that the Sc. occidemalis GAMI encodes a novel type of GAM, which may have evolved in parallel or convergently with the class which all other described GAMs belong to, In contrast, the ~-amylase of $c, occidentalis

shows extensive homology to several ~-amylases of other organisms (Strasser et al., 1989).

(d) Sl mapping of the tsp for the GAM! gene In order to map the tsp of the GAMI gene an S 1 nuclease protection experiment was performed (Fig. 3). The strongest signals obtained, map the tsp around nt -8 and -11 with respect to the A of the translation initiation codon (Fig. 3, lane 3). Weaker signals were obtained for nt -19 and -24. This is consistent with the sequence data which suggested that the tsp is between the ATGs at nt -23 and +1, (e) Differences at the GAMI.loci of various $ckwanniomyces oecidentalis strains The GAMI loci of the Sc. occidentalis strains ATCC26076 (formerly Sc. castellii) and RJDll (formerly Sc. alluvius) were analyzed by Southern blotting and hybridization with GAMi-specifc probes (Fig, 4A and B). On the basis of the bands obtained, restriction maps of the GAMI loci of both strains were constructed (Fig. 4C). The maps ofthe GAMI loci ofthese strains are almost identical.

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Fig, 3. SI-mapping of the tsp ofOAMl. RNA was isolated from Sc. occldentallsATCC26076grown toA6oonm= ! in YEP with 2~ glucoseor 2% maltose. Cells were broken with glass beads in 100 mM "Iris. HCi pH ?.$/20 mM NaCI/10mM MgCI2/0.4% SDS, containing 100/~g cycloheximide and 200 #g heparin, both per ml. Nucleicacids were purifiedby phenol/chloroform extractions and ethanol precipitation. DNA was removed after selective precipitation of RNA with LiCI (Wallace, 1987). Plasmid pBRGAM13 (Fig. I) was digestedwith Pvull and labeled at the 5' ends with [?J'P]ATP. Al~er digestion with Bg/ll the 529-bp BgliIPmll fragment was isolated from an agarose gel and hybridized with 60 #g of total RNA from Sc. occidentalis26076 grown under inducing (lane 3)or repressingconditions (lane 1) at 42°C as described by Maniatis et ai. (1982). After treatment with S I nuclease (Maniatis et ai., 1982)the fragments were separated in an 8~ polyacrylamide/7M urea gel together with the untreated fragment (lane 2) and the G.reaction of Maxam and Gilbert sequencing of the same fragment (lane 4). The positions of the corresponding C's in the sequence ofthe codingstrand (Fig. 4) are given with respect to the A oftbe translation start codon ( - , denotes numbers upstream and +, downstream). Arrows of d.;fferentsize (corresponding to those in Fig. 2) indicate the positions ofbands of different intensityin lane 3, which are the result of the S1 protection assay.

The main difference between them could be explained by a deletion in strain PODI 1 or altematively by an insertion in strain ATCC26076 of about 0.3 kb containing a BamHl site. The position of this difference is about 1.5 kb 5' to the GAM! coding region. The cloning of a GAM-encoding gene from Sc. occidentalis ATCC26077 having a similar restriction map was reported recently (Lancashire et al., 1989). However, this gene differs from the GAMI genes of both Sc. occidentalis 26076 and R J D I I in that it has a Sail site within the coding region. In the case of strain ATCC26076, and with consideration of the deletion also in the case of strain PODII, only the bands expected from the maps of the two overlapping clones (Fig. 1) were obtained by the Southern analysis. This indicated that there is only a single copy ofthe gene present in the genome of both strains. (f) Disruption and displacement of the GAM! gene of Schwanniomyces occidentalis In order to confh'm the identity of GAMI, its function in $c. occidentalis strain PODII was eliminated using the cloned sequences by two different approaches. First, the gene was disrupted according to the method of Shortle et al. (1982) by integration of a plasmid containing the S. cerevisiae TRP5 gene as a selectable marker and the internal 2. l-kb Dral fragment from GAMI (Fig. 5A). The plasmid was linearized within the DraI fragment with Sstl, and transformed into Sc. occidentalis POD11. Some 50-100 Trp ÷ colonies were obtained per pg of plasmid, about 70% of which grew only very slowly on amylolytic substrates such as maltose, soluble starch, dextrin and pullulan (Table I). Using a second approach, the Bglli fragment carrying the entire structural gene and 321 bp of the 5'.nontranslated region was displaced by the S. cerevi. siae TRP3 gene according to the method of Rothstein (1983). $c. occidentalis R J D I l transformed with about i #g of a BamHI-Pstl fragment containing the TRP~ gene in place of the OAMI gene (Fig. $A, lower part) gave rise to several hundred colonies, most of which had a fissured edge. Under nonselective conditions, the Trp ÷ phenotype of these transformants was highly unstable. Thirty colonies had a slightly larger diameter, were more fiat and had a smooth colony form. The Trp ÷ phenotype of these transformants was 100% stable after about 15 generatinns in nonselective culture. Four of tbe stable transformants showed the same growth features on plates containing amylolytic substrates as the disruption strain (Table I). One of these gaml ° strains (PODI l-gdpl) was in addition grown in YEP with 2% soluble starch and was found to grow more poorly than the original strain PODI 1 (Table i). The G A M activity in the medium of the gaml ° strain was in the range of the detection limit and accounts for less than 1% of the activity found in the medium of the original strain, in con-

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Fig. 4. Southern analysis of the GAMI loci of the $¢. occldentalls strains ATCC26076 and RJDI I. About 30/~g of yeast DNA prepared as described by Sherman et al. (1983)were digested with different restriction enzymes, separated in 0.8 % qarose gels and blotted to nRroceHuloseaccording to Maniatis et al. (1982~ (Panel A) Southern blot hybridized with nick-translated 5.8-kbBamHl.£coRI probe FrompBRGAMI3 (Fig. 1) and J. DNA. ~lHindlll Oane !), 26076 DNA (lanes 2-5) and PJDI 1 DNA (lanes 6-9) digested with BamHI 0anes 2 and 6), B&III (lanes 3 and 7), EcoRl (lanes 4 and 8) and Sail (lanes S and 9). (Panel B) Southern blot hybridized with nick-translated 8.3-kb EcoRl Fragmentfrom ~.GAM183(Fig. 1). ~lEcoRI + HindllI (lane 1, the positions of the bands indicated to the leR were deduced from the EtdBr stained gel), 26076 DNA (lanes 2-7) and RJD! ! DNA 0anes 8-13) digested with $stl (lanes 2 and 8), $sd + BamHI (lanes 3 and 9), $stI + EcoRl (lanes 4 and 10), $sd + Sail (lanes S and 11), £coRl + Bgl[l (lanes 6 and 12), )

117

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(compare Fig. 5A and B) indicating that integration and displacement had taken place via homologous recombination. The fact that both gaml strains show residual ability to grow on different amylolytic substrates including substrates with a high content of ~,-1,6 glycosidic bonds, such as dextrin or pullulan, suggests the presence of a second GAM enz3~ne, which has been described previously (OtengGyang et el., 1981; Moranelli et el., 1987; Lancashire et el., ! 089). This enzyme accounts for less than 1~ ofthe GAM activity of Sc. occidentalb (Table I). Sc. occidentalis gaml cells, being dependent on the activity of this enzyme grow only poorly on amylolytic substrates. Interestingly, the secretion of u-amylase is increased in this strain. This phe-

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trast, the ,,-amylase activity in the medium of the gaml ° strain was increased twofold over that of the original strain, although the optical density of the culture was fourfold lower (Table I). The integration (RJDll-grdl) and displacement (RID 1 l-gdp 1) events were confn'med by Southern analysis

-0,94 -0.83 -0.56

Fig. 5. Disruption and displacement ofthe GAMI gene ofSc. occidentalis RIDll. (Panel A)Strategy of gene disruption (upper part) and gene displacement (lower part). Plasmid pJDgd3 was constructed in two steps.

First, the 3.2-kb BamH! TRP$ fragment from plasmid pYe(trp$)l-$3 (Walz et el., 1978) was inserted into the BamHl site of pATI$3 (Twig8 and Sherrat, 1980) resulting in pAT153-TRP$. The 2.I-kb internal Drnl frngment ofthe GAMi structural gem (nt 318-2424, Fig. 27was inserted into the Rrul site ofpATI53-TRP$ resulting in plDgd3. The BamHl.Pstl fragment used in the gene displacement experiment was isolated from plasmid pJDGkS, which was constructed in two steps. First of all, an EcoRI-H/ndlll fragment including a portion of the M I3mp8 polylinker (Vieira and Messing, 1982) was ligated to the large EcoRI.H/ndlll fragment of pBRGAMI3 (Fig. I). As a result of this, a Psti site from the M 13rap8 polylinker was linked to the H/ndlll site 3' to the GAMI coding region, in a second step, the 3.7-kb Bg/I! fragment containing the GAMI gene was displaced by the 3.2-kb BamHI TRPJ fragment (see above). Restriction sites as in Fig. 6; in addition: P, Pstl. (Panel B) Southern blot analysis ofPJDI I (lanes 2, $, 8, !1); IUDI I-gdrl (lanes 3, 6, 9,12) and RJDI l-gdpl (lanes 4, 7,10,13). The blot was probed with nick.translated plasmid pJDI3, which was constructed by inserting the 3,2-kb BamH! fragment containing the 8. cerev~ae TRP$ gene.into the BamHI site of plasmid pBRGAMI3. X/EcoRl + Hindlll (lane I), yeast DNA digested with BamHI (lanes 2, 3, 4), with Bglll (lanes $, 6, "/), with EcoRl (lanes 8, 9,10) or with SstI (lanes II, 12, 13). The origin ofthe gel is indicated by a large arrow. Bands indicated by asterisks are due to cross hybridization with the 8. cerevblae TRP5 gene. Bands explained by the presence of small amounts of an autonomous circular form of the transforming fragment (lanes "/and 10) are marked by arrowheads. Weak bands from small fragments in lane 9 are indicated by small arrows.

EcoRi + Clal (lanes'/and 13). The origins ofthe gels are indicated by arrows. (Panel C) Restriction maps of the 6AM! loci of$c. occidentalb ATCC26076 (formerly $c. castellii)and of$c. occldentalis IUDI I (formerly $c. alluvius). The continuous lines represent areas where the map for the given restriction enzymes is complete, whereas the dashed lines represent areas where only the restriction sites next to the regions hybridizing with the used probes are shown. The solid bar indicates the BamHI.EcoR! fragment used as probe in panel A. The open bar indicates the EcoR! fragment used as probe in panel B. The triangle indicates the position where a deletion in strain RID I i or an insertion in strain 26076 may explain the differences in the restriction maps. The boxes between the restriction maps represent the fragments found in Southern analysis shown in panel B. The numbers to the left correspond to the respective lanes. The boxes contain the fragment sizes in kb. Restriction endonucleases are: B, BamHl; Bg, Bg/ll; C, Clal; E, EcoRl; S, Sail; S,, Sstl.

118 TABLE i Comparison of Sc. occidentalisOAMI and gain! strains Strain a

RID1 ! RJDll.gdrl RJDII-gdpl

Growth on YNB solid media b

Growth in YEP 2% starch liquid culture d

Glucose

Ae~¢~,. in stationary phase

GAM

~,-amylase

27 n.d. 6

399 n.d.