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Agric. Biol Chem,, 50 (4), 957~964, 1986 957 Rhizopus Raw-Starch-Degrading Glucoamylase: Its Cloning and Expression in Yeast Toshihiko Ashikari, Norihisa Nakamura, Yoshikazu Tanaka, Naoko Kiuchi, Yuji Shibano, Takaharu Tanaka, Teruo Amachi and Hajime Yoshizumi Laboratory of Applied Microbiology, Research Center, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan Received October 1, 1985 A glucoamylase gene has been cloned from a Rhizopus genomic DNAlibrary using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a CDNAlibrary prepared from Rhizopus mRNA.Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the CDNAgene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grewat almost the normalrate on a mediumcontaining starch as the sole carbon Glucoamylases a(l,4)- (EC 3.2.1.3) and a(l,6)-glycosidic hydrolyze linkages from the hydrolyzing activity toward raw starch com- pared to other glucoamylases. A strain of yeast These which could produce glucoamylase would be very useful in industrial alcohol produc- enzymeshave been isolated from the culture fluid of a variety of fungi and yeasts, and their tion, because it could directly convert starch nonreducing ends of starch and related maltooligosaccharides to release properties and biological been studied.1} Generally, glucose. significance have fungal glucoamy- into ethanol. In this report we describe the cloning and the expression of the Rhizopus glucoamylase gene in yeast. Several features lases are knownto exist in multiple forms of the glucoamylase gene are also described. varying in size. A few distinct forms of As- pergillus glucoamylase are produced by differential RNA splicing events.2'3* On the other hand the multiple forms of Rhizopus glucoamylase are thought to arise from limited proteolysis.4'5) Recently, the genes coding for the glucoamylases of Asp. awamori2) and Asp. niger3) were cloned and it was discovered that their structures were the same. Glucoamylases are especially useful in saccharifying starch in commercial alcohol production. It is unfortunate for this commer- cial use that Rhizopus produces large amounts of glucoamylase in solid culture but not in submerged culture despite extremely strong MATERIALS AND METHODS (a) Microorganisms. Rhizopus oryzae SAM0034, a strain producing high-level glucoamylase, was selected from our culture collection. E. coli WA802(metb-1 lacY galK-2 galT-22 X~ supE44 hsd-3) was used as a bacterial host. Saccharomyces cerevisiae XS-30-2B (MATaIeu2 his3 ura3 trpl), consturcted in our laboratory, was used as a yeast host and yeast cells were transformed by the method of Ito etal.6) (b) Amino acid analysis ofglucoamylase. Glucoamylase was purified from Rhizopus oryzae by the method of Tsujisaka et al.1] Aminoacid composition and N-terminal sequence of the purified glucoamylase were determined as described previously.8) Glucoamylase was cleaved by 958 T. Ashkari et al. BrCN and succinylated, followed by digestion with trypsin, and the peptides were purified from the digest and sequenced as described.8) The C-terminal residue of glucoamylase was identified by carboxypeptidase A digestion. specific mutagenesis as described by Morinaga et al (c) Construction of the Rhizopus genomic DNAlibrary. Chromosomal DNAwas isolated from fully sporulated (a)Some Chemicaltryptophan synthesis ofcontaining DNAprobes peptides Rhizopus oryzae. The spores were disrupted by a Dinomill derived from Rhizopus glucoamylase were sequenced. One of these sequences, Thr-TrpSer-His-Ala-Ser-Leu-Ile-Thr-Ala-Ser, was used for designing the synthetic oligonucleotide probes. The oligonucleotides, 5 -ACNTGGTCNCAQGC-3', were synthe- apparatus (Willy A. Bachofen, in Basel, Switzerland) with glass beads (0.25~0.5mm) an 0.15m NaCl-0.05M EDTAbuffer at 4°C for 15 seconds and DNAwas isolated by the method of Cryer et al.9) The molecular weights of the isolated DNAswere estimated by agarose gel electrophoresis to be 1 x 106 to 107 daltons. The DNAswere digested with HindlW and fractionated by sucrose density gradient centrifugation. A sample of each fraction was analyzed by both agarose gel electro- phoresis and dot-hybridization to 14-mer synthetic probes. The strongest hybridizing fraction, containing approxi- mately 4kb DNAfragments, was used to construct the genomic gene library in E. coli using pBR322as a vector. (d) Construction of the Rhizopus CDNAlibrary. The total cellular RNAwas isolated and purified from R. oryzae mycelia by the method of Chirgurin et al.10) with slight modifications. Thirty grams (wet weight) of mycelia grown on YpSs agar (0.4% yeast extract, 0.1% KH2PO4, 0.05% MgSO4-7H2O, 1.5% soluble starch, and 2% agar) was suspended in 50 ml guanidium thiocyanate stock solution. After addition of deionized water to a total volume of 100ml with stirrig at room temperature, 50ml of phenol-chloroform-isoamylalcohol (20 : 19 : 1) was added to the suspension with vigorous stirring with a Bio-mixer (Nihonseiki, Tokyo, Japan). The homogenate was centrifuged for 30min at 1500xg and the aqueous phase RESULTS sized by the modified triester method (Q; T or C, N; any base).17) One of these sequences was specifically hybridized to the glucoamy- lase gene in the region coding for Thr-Trp-SerHis-Ala. (b)TheCloning of the genomic glucoamylase gene Rhizopus genomic gene library was screened by colony hybridization using the 32P-labeled 14-mer mixed probes. Among approximately 1000 transformants, clones 39, 83, and 93, which contained an identical 4.3 kb Hindlll fragment, were selected and clone 39 was used for further analysis. Figure 1 shows the restriction map of the 4.3 kb Hindlll insert of pRGA39isolated from clone 39. To elucidate the region hybridizing the 14-mer probes, containing the total RNAwas pooled. The RNAwas pRGA39was digested with various restriction recovered from the guanidium thiocyanate homogenate by ultracentrifugation through a dense cushion of cesium chloride and mRNAwas purified by oligo-(dT)-cellulose column chromatography as described by Maniatis et al.n) The CDNA library was constructed by the method of Okayama and Berg12) in E. coli WA802 using 15^g of poly(A) RNAand 4.2 jig of vector-primer. 100,000 ampicillin-resistant Approximately transformants were obtained. (e) Other materials and methods. Preparation of E. coli plasmids, transformation, analysis by restriction enzymes, nick translation of probes, and Southern hybridization were done as previously described.ll) The synthetic 14-mer probes were labeled by the methods of Donis-Keller et al.13) Colony hybridization was carried out by the method of Grunstein et al.1A) The hybridization conditions were 18hr at 40°C for the 14-mer mixed probes and 65°C for the probe derived from the genomic gene. The nucleotide sequence was found by the dideoxy method using M13mpl0 and M13mpll.15) In vitro mutagenesis was done by the method for oligonucleotide-directed site- enzymes and Southern hybridization experiments were performed. The result indicates that the region hybridized to probes was located on a 0.3kb fragment between Kpnl and Dral sites (Fig. 1). Single stranded M13 DNAswere prepared from M13mpl0and M13mpll RF DNAsinserted by an 0.8kb BgHl-Dral fragment at the Smal-BamHl sites. Only the MBmpll single-stranded DNA hybridized to the 14-mer mixed probes, suggesting that the transcription of the gluco- amylase gene occurs from the BgUlsite to the Dral site (Fig. 1). (c)TheCloning of CDNAglucoamylase gene CDNA library constructed from Rhizopus mRNAwas screened by the hybridization with a 2.0kb Dral fragment of the 959 Cloning and Expression of Rhizopus Glucoamylase M. H M I-I , , I-I I I W I '-i »-t T3 i_( I-i I II-I >I -IPBR322 transcription F0 1 -I 1.0Kb 1 2.0Kb 1 3.0Kb 4.0Kb 1 Fig. 1. The Physical Map of the Genomic Glucoamylase Gene. The restriction map of the 4.3 kb Hindlll insert ofpRGA39is presented. The closed box indicates the region hybridized to glucoamylase-specific synthetic 14-mer probes. The arrow shows the direction of transcription found by the method described in the text. Plasmid a -h| |] s ± ££ 0 | £ ^ l|||T'^å «**»à" 51 i i i in m\\ \ B -Y//k/////)F\WL ATG ni H I I 500 1000 . 1 I å 1500 , ^i^^ 1 1 I1 ]- pRGA39 TAA 2000 bp , Fig. 2. The Restriction Maps of Glucoamylase Genes. The restriction sites are shown for the CDNA(A) in pCGA239, and the genomic gene (B) in pRGA39. The shaded boxes represent the region lacking in the CDNAregion and closed box represents the intervening sequences. ATG and TAA indicate respectively. Rhizopus genomic glucoamylase the initiation gene. Several clones containing glucoamylase CDNAwere obtained. One of these clones contained pCGA239, which had the largest CDNAinsert and termination codons of the glucoamylase gene, sequences reveals that the genomic gene contains 4 intervening sequences and the CDNA gene lacks 145 nucleotides coresponding to the N-terminal aminoacid sequence. A large open (2.0kb), covering almost the entire coding reading frame which started from ATGat sequence of the glucoamylase. The restriction position 115, continuing to TAAat position map of the glucoamylase CDNAin pCGA239 2160 and interrupting by four intervening seis shown in Fig. 2 with that of the genomic glucoamylase gene in pRGA39. Comparison of these two maps revealed that both genes had almost the same restriction gene sizes were different. sites although the These results sug- gested that the glucoamylase gene had at least one intervening sequence. (d) The nucleotide sequences of glucoamylase genes The nucleotide sequences of the glucoamy- quences was the best candidate for the gluco- amylase. The deduced amino acid sequence corresponds completely to the known amino acid sequence of the peptides from glucoamylase (Fig. 3). For instance, the N-terminal amino acid sequence of the mature glucoamylase, Ala-Ser-Ile-Pro-, was found at residues 26 ~29, suggesting that the glucoamylase contains a signal peptide. The putative signal peptide contains 16 hydrophobic amino acids includ- lase genes derived from genomic DNAand ing N-terminus methionine. The basic amino CDNAare shown in Fig. 3. Comparison of the acid lysine is located at the 9th position from 960 T. Ashikari 10 GATCTCAATT 120 CAA GLN GTC Val 20 TGTGTTGTGA CTG TTC LEU PHE AAT ASN CAG CTT Gin Leu gctgtag GTC Val TCT TAC SerTyr TCT Ser GAT Asp 30 TATATTCAGA TTG LEU CCA TTG PRO LEU AAA LYS 341 AAG AAC ATT GCT Lys Asn lieAla 431 GCT CCT Ala Pro ATT lie GTT TCA VAL SER 150 TTC PHE TTT PHE TAC Tyr TCC AAG AAG GTT SerLys Lys Val TCT GGA TCA Ser Gly Ser AAT TAC Asn Tyr GTC VAL CTC LEU attaactaac 636 740 ACC gcctacaact 461 ACA TTC Thr Phe ccttttttct ctatag attcacattt ttatagTA GGT Gly ATC He 830 AGC CGC TTT GCT ATG SerArg PheAla Met 920 TAC Tyr TAT Tyr GCT TGG ACT Ala TrpThr CGT GAT GCT Arg AspAla CTC Leu AAG Lys GAC TAT GTT Asp Tyr Val ACA TTC TCA Thr Phe Ser TAT Tyr ACT Thr GGT GCT TGG Gly Ala Trp 1130 GGA AGA CCT CAA AAT GAT GGA CCT GCT Gly Arg ProGin Asn AspGly ProAla GAA GAA 1310 GTC 1220 TAT GTC Tyr Val CCC CCC TCA GTT Val ACT Thr GGT Gly GGT Gly 1520 GTC AGT AAA AAG GGT Val Ser Lys Lys Gly TAT Tyr CCT Pro ,»..,;": 1710 ATC AAC AAA AAC CTT He Asn Lys Asn Leu GGA AAC GlyAsn 1800 TCT TGG SerTrpPhe TTC ATA AGT He Ser TAA CCC Leu Pro TTC Phe ATT He GCA Ala CTC GCT GCT LeuAlaAlaAspArg TCC Ser ACC Thr GGT GCT Gly Ala 2190 AAAGCATTAC TTG GCT LeuAla GAT GTC Asp Val GAC CGT CCA Pro CTT Leu TCT TAC Ser Tyr ACC Thr CTTTTTCAAA 2010 TTG TCC ACT Leu SerThrVal TGG TCT Trp Ser CAC His TAAAAACATA 2230 TTGATATGTT AAT TAC AsnTyr TCA TGG ATT AAG AAG CAA GAA GGT TTC Phe ATT lie 890 GCT GCC Ala Ala TCA CTC TCT ACC GCT Ser LeuSerThrAla GGT CCC Gly Pro GAT TAC AspTyr 980 AAC ACT Asn Thr ACT TTG Thr Leu TCC GGT Ser Gly TAC GAA TAC Tyr Glu Tyr 1070 AAC TGC CTT Asn Cys Leu GGT GAG CCT Gly Glu Pro AAT Asn AAG ACT Lys Thr ATC CTC He Leu 1010 AAC GTC Asn Val AAG TTC Lys Phe AAT CCT Asn Pro 1100 GAT GCT AspGly TCT GGC Ser Gly GAC AGT Asp Ser 1190 TAT CTT Tyr Leu ACT CAA Thr Gin 1250 AAG GAC TTG Lys Asp Leu GAC TAT Asp Tyr GTC Val GTC AAT GTC TGG Val Asn Val Trp 1280 TCT Ser CTT CTT 1370 GGT GCA GAT TTC GCT AAG GGT TTG TTG Leu CAT GCT His Ala AAT GGC TGT TTC AsnGlyCys Phe AAA CGT 1460 GTT Val TCT TCT Ser Ser AAT AAC TGG Asn Asn Trp 1550 AAC CTT Asn Leu GGT Gly AGT GTT Ser Val GAT GAT GGA TTC AspAspGly Phe GCT GCT Ala Ala , ATC CTT CCC ACT CCT CTT He Leu Ala Thr Ala Val ATT GGT AGA TAT He Gly Arg Tyr CGT GCC CAC His TTT GCT Phe Ala AGC TTC TGG Ser Phe Trp ATC He ACA TCT GGA AAG AAG TAC Thr Ser Gly Lys Lys Tyr TTA Leu 611 TCT GCC Ser Ala 800 AAC TCT ACA ATC TCC 1830 GCT GAG CTC TAT TAC Glu LeuTyrTyrArgAla GCT TCT Ala Ser tt ACT GGT tttct.=«=. GTC CAG CTC Gin Leu GCT Ala TTC CCA 1740 GGT AAC TCT Gly Asn Ser TCT GCT Ser Ala ATT AAG gtaacttatt lie Lys ac ACT ATT TTG He Leu ACA TTG Thr Leu t..tt=l^ GTA ACT GGT TAC Val ThrGlyTyrAla TTC Phe TCC Ser TAC Tyr GCT Ala TCT Ser ACC GCT Thr Ala GCA AAC AAG ATC TCT Ala Asn Lys He Ser ataacattac 1920 TTC AAG AAG TTT GAT TCA Phe Lys Lys Phe"Asp Ser AGA GAC TTG Arg Asp Leu AGCTTATTTT TTG Leu ttcatgctgt317 aatattaatg 581 AGT GGA AAA ACA TAC TAT GAT AAC AAC AAT SerGly Lys ThrTyrTyrAspAsnAsnAsn TTC Phe 1340 ACT TTA ATG GTT ATG CGT 1430 ACT ATT Thr He 491 ATC AAG GAG TTC lie Lys Glu Phe ACT ACC ThrThr CAC TTC TAT AGT Ser 401 AAT AAT GGA AAC ACC ATT Asn Thr lie 1160 GCT Ala AAC GGT GTT AGT Ser ACT ACT Thr Thr GAA CGT Glu Arg AAG CCT GCT Lys Pro Ala CCT Pro ATG MET ACT Thr ATT GTT He Val GGT ACA CTC Gly Thr Leu ATT lie 2^97 acattaaa'aa tataattcaa AAT GGT Asn Gly GCA AGC Ala Ser 210 GCT Ala 710 ACT GCT Thr Ala AAG ACC CAA TCA ACT TCT ACC GTC TGT Lys Thr Gin Ser Thr Ser Thr Val Cys ACC TAT AGC AGC ACT GCA TCC Thr Tyr Ser Ser Thr Ala Ser GCT ALA TCTA ACT ACT ACT GCT Thr Thr.Thr Ala GTA Val TCT Ser TCT SER 110 TTGATCTTTC ACT Thr TCC AAT Ser Asn ATC TTC He Phe GTT VAL TCT GAC AAC TGG AAT Ser AspAsnTrpAsnAsnAsnGly ATT lie 100 TGTCTTCAAA AAG CCT Lys Pro 950 ACC Thr 1040 GTC Val 180 CTC LEU 90 AATCATCACT 680 TCT ACA TCC Ser Thr Ser CCT GGA AGC GCT ACC ProGly SerAlaThrGly GCA TTA Ala Leu TTG LEU 277 gtaggttgca GCC TCC Ala Ser 860 AAT CCT Asn Pro GCA Ala TTG Leu TCT Ser CTT CGA AAC ATC LeuArg Asn He CGT Arg TCC Ser AGC TCT ACT Thr CAA AAC ,f..t^.t,=.tM.. GluLys TCT SER TAT GAG GTC TyrGluVal 770 GAG CCA GCT ACT TCA 80 GTTTTTCAAA 371 GCC GAT GGC AlaAspGly ACT GTA ATT TAC Thr Val lieTyr TCC TCC Ser ACG ACT TAC TTT TYR PHE TCA GGA AAA ATT TAT Ser Gly Lys lie Tyr CCT GAT GCT Asp Ala TCT SER 656 atatacaaat 70 TTATTTCCTC 563 cttatactta aaagataata CTC LEU 60 TAAGACGCGT GAA TAC TGG Glu Tyr Trp 543 gatatttgcc CAA Ggt Gin Val 50 CAAACATATA 240 TAC AAT TAC GAT GGC TCT ACT TTT Tyr Asn Tyr Asp Gly Ser Thr Phe TCA Ser 523 tactttat 40 TTTAAAATTT et al. 1770 CCT GAA GAC ACT TAC ProGlu AspThr Tyr ACA AAG Thr Lys GAT Asp TTA Leu AAC GGT GAC TGG Trp TCT ACT ATT CAA GTC He Gin Val AGT Ser 1490 CAA AGC Gin Ser 1580 TTC ACT Phe Thr CCT Pro GGC TCT Gly Ser CCT^T CAA CAC TCC Ala Val Glu AspSer TTC CCT Phe Ala AAT GGT Asn Gly AAC GGA AAC Asn Gly Asn TCT CAA Ser Gin 1860 AAG GAA TGG ATC GGC AAC GGT Lys GluTrp HeGlyAsnGlyGly GGT GTC Val ACT GTC ThrVal AGC AGC SerSer 1550 ACT GTT Thr Val AAC AAC CTT GCT Asn Asn Leu Ala 1980 CAA AAT Gin Asn 2040 AAC AAT GGA TCT Asn AsnGly Ser GGT Gly ACC TCC Thr Ser GAC TTT Asp Phe CTT GCT GAA GAG TTT LeuAla GluGlu Phe 2070 GAC CGC ACC AspArgThrThr ACT GGT TTA Gly Leu ATC He CATAACATTT TCTTGTTTGT TGTACTGTAA 2270 TATGGGTAAC CACATAAGCA TAAACAGCAA Fig. 3. The Nucleotide Sequence for the Rhizopus Glucoamylase Gene. The intervening sequences are shownin small letters. The predicted amino acid sequence is shownunder the nucleotide sequence. (The putative signal peptide is shown by capital letters.) The amino acid sequences found for peptides derived glucoamylase-specific from the glucoamylase are underlined. synthetic 14-mer probes are overlined. the N-terminus of the signal peptide and the signal peptide is cleaved between Ala-Ala at the 25 ~26th position. These features are consistent with those of other signal peptides.18) The nucleotide sequences hybridized with The C-terminal sequence, Ala-Ala, is located just before the stop codon TAA.The 3'noncoding region is A-T rich and contains the nucleotide sequence AATAAA that has been J 961 Cloning and Expression of Rhizopus Glucoamylase CM IV H J cU I AAATTTATCTACGTTjnC InSA|---TTTAAATACATCCAACG---I pRGA39 mutagenesis I AAATTTATGTCAAGAAC ^1---TTTAAATACAGTTGTTC---P\ \ ^_Ap_ ) \ rautagenesis IYVD" I H"T _Ag^pCGA239 -i_x 1LDvitro C\ PiYVKNPV I 1p J .ji_nvitro y P!YVDNPV 1--^Hindlll ^ 1 \^V S P Jn I-aaatttatg|tcgacaac-I à"**"~"]rGA| TTTAAATAGAGGTJGTTG f~^X_ ( ?al1 1 ^ I_&+ V_) y p PCGA439 J .In vitro j mutagenesis H å¼ t i K aaatttatgtcaagaac Py i ^HPca|-tttaaatIcagttcttg( _Ap^ PCGA449 ) K P Fig. 4. The Construction of the Glucoamylase Gene to be Expressed in Yeast. Boxes represented the fragments containing the glucoamylase genes. I, Y, K, N, and D are one letter descriptions of amino acids. Sail sites were created in pRGA39and pCGA239by in vitro mutagenesis (see Materials and Methods) resulting in pRGA39sand pCGA239s, respectively. pCGA439was constructed by recombination ofpRGA39s and pCGA239sat Hindlll and sail sites. The altered amino acid (D) was restored to the original amino acid (K) by in vitro mutagenesis. h f I h PJDB219 |r Ih / / ^ \h I PBR322 I j V PYGA201? / JHindlll \ IP / \ H ^ ^ PYE2O7 ^R /HindlllX 1 ^-W p^7 ^N. /^ P -^-^T ^ ^X I"2»\^{/ Fig. 5. The Construction of a Shuttle Vector Containing the Glucoamylase Gene. Therestriction sites used are shown.The thin line represents the pBR322moiety and the thick line represents the yeast DNAmoiety. IR indicates the inverted repeat sequence of 2 /im DNA.The restriction sites P, H, B, and Pv indicates Pstl, Hindlll, BamHl and Pvull, respectively. 962 T. Ashikari proposed to be necessary for polyadenylation.19) The glucoamylase gene encodes 604 amino acids and the mature glucoamylase consists of 579 amino acids. The molecular weight of the mature glucoamylase is calculated to be 62,197 daltons which correlates well with the molecular size reported by Takahashi et alA) The four intervening sequences identified within the Rhizopus genomic glucoamylase gene are short (from 48 to 66bp) and A-T rich (71 to 77%). These features are similar to those of Aspergillus2^ and Trichoderma20) secreted enzymes. (e) Expression of the glucoamylase gene in yeast The genomic glucoamylase gene was not expressed in yeast, probably due to the exis- et al. consensus sequences, TACTAAC23} and PuCTPuAC2) in the intervening sequences of yeast and fungi, are not found in the Rhizopus glucoamylase gene. There were no more than 6 nucleotides in commonbetween four intervening sequences of the Rhizopus glucoamy- lase gene. The mRNAtranscribed from the genomic glucoamylase gene in yeast was not accurately spliced due to the difference of splicing systems between Saccharomyces and Rhizopus, resulting in a failure of the gene to be expressed in Saccharomyces cells. The bias of codon selection for the Rhizopus glucoamylase gene was compared with those of filamentous ascomycetes and Saccharomyces cerevisiae (Table I). Preference in codon usage for Rhizopus glucoamylase gene is not consistent with those of Aspergillus awamori 2) tence of the intervening sequences. The CDNA Trichoderma reesei20) or Neurospora crassa 2^ glucoamylase gene also is not expressed due to These filamentous ascomycetes preferred G and C at the third position of codons. The the lack of the N-terminal coding sequence. Therefore weconstructed a glucoamylase gene pattern of codon selection of Rhizopus glucowhich could be expressed in yeast cells (Fig. 4). amylase is rather similar to that of SacchaThe resulting recombinant plasmid pCGA449 romyces cerevisiae codon selection.25) In parcontains both the 5'- and the S'-noncoding ticular, acid phosphatase,26) one of the secre- sequences and the entire glucoamylase coding sequence without the intervening sequences. To examine the expression of the constructed glucoamylase gene, the shuttle vector pYGA201 was constructed (Fig. 5). pYGA201 consists of the glucoamylase gene from pCGA449, a replication origin derived from tion enzymes of yeast, is in good agreement yeast 2 fim DNA, and selectable markers (ApR for E. coli and LEU2for yeasts). This plasmid was introduced into Saccharomyces cerevisiae XS-30-2B (MATa trp\ leul his3 ura3). Transformants grew at nearly the normal rate in a mediumcontaining starch as the sole carbon source and secreted the glucoamylase into the culture fluid. These results have already been reported in another paper.21) peptide fragments. These peptide fragments were isolated as fragment H and L.4) These results suggest that there is a single gene DISCUSSION The Rhizopus genomic glucoamylase contains four intervening sequences. gene The with Rhizopus glucoamylase. Rhizopus glucoamylase exists in multiple forms, Glucl, Gluc2, and Gluc3, differing in molecular size.3) It is thought that Gluc2 and Gluc3 are produced by limited proteolysis of Glucl accompanied by the loss of N-terminal coding for Rhizopus glucoamylase, and this is confirmed by the hybridization experiment. Only one band was detected in the HindlW fragments of Rhizopus DNAwhen the DNA fragment derived from the genomic gluco- amylase gene was used as a probe (data not shown). The N-termini of Glucl, Gluc2, and Gluc3 were alanine, glutamic acid, and lysine, respectively. contents Gluc2 Judging from both carbohydrate and amino acid compositions and Gluc3,4'5) the N-termini of can be consensus splice junction sequence, GT-AG,22) assigned to glutamic acid at the 134th residue is found in these intervening sequences/Other for Gluc2 and lysine at the 85th or the 91st 963 Cloning and Expression of Rhizopus Glucoamylase Table I. The Codon Usage for Various Glucoamylase Genes and Yeast PHO5Gene The table lists Aspergillus oryzae the codon usages in following secretion enzymes: rh, Rhizopus glucoamylase gene; as glucoamylase gene (2); sa, Saccharomyces diastaticus glucoamylase gene (27) and Saccharomyces cerevisiae PHO5gene (16). G l u c o a m y la s e P H O 5 rh as sa 4 17 8 u u c U I JA U U G 21 6 1 18 0 6 14 6 19 19 9 20 C C C C U C A G 12 10 0 0 3 17 2 20 5 6 6 14 A U U A U C A U A 16 12 1 12 ll 1 G G G G 13 18 4 0 6 15 2 19 U U U U U U U U U C A G G lu c o a m y la s e P H O 5 sa G lu c o a m y la s e P H O 5 rh as sa sa U C U 34 16 29 9 U A U U C C U C A U C G 15 ll 0 19 4 14 16 19 9 6 U A C U A A U A G 0 0 4 2 C C C C C C C C U C A G 15 4 4 0 4 10 0 8 15 5 21 3 5 1 7 0 C C C C A A A A 22 13 8 13 A A A A C C C C U C A G 36 15 9 1 20 39 5 10 48 27 32 19 16 20 2 0 A A A A 19 19 ll 7 9 16 2 1 G G G G C C C C U C A G 42 7 8 0 25 19 10 ll 16 14 7 7 13 14 2 0 G G G G 1 rh G lu c o a m y la s e P H O 5 as sa sa rh as sa sa 13 6 12 7 22 .- 21 - 14 - 25 - U G U 2 3 4 10 U G C U G A U G G 1 19 ll U C A G 1 4 9 2 0 4 4 13 6 7 13 9 5 3 12 2 C C C C G G G G U C A G 7 2 1 0 A A A A U C A G 20 25 6 25 6 19 0 13 14 27 ll ll 9 22 8 16 A A A A G G G G U C A G 10 3 0 A A A A U C A G 15 12 ll 6 21 23 9 17 16 19 20 ll 20 21 23 2 G G G G G G G G U C A G 28 6 12 0 12 4. 7 4 - 4 1 2 0 0 23 1 1 10 2 14 22 7 4 13 19 ll 22 1 residue for Gluc3. All three forms of glucoamylase hydrolyze gelatinized starch at similar amylase in yeast and efficient ethanol production from starch materials are nowin progress and will be reported elsewhere. substrate. It seems likely that the N-terminal Acknowledgments. Wethank Dr. Hiroshi Matsubara rates, but only the largest one (Glucl) is able to adsorb to raw starch and degrade this region of the Rhizopus glucoamylase must be involved in raw starch adsorption and degradation. The constructed Rhizopus glucoamylase gene containing the 5'-flanking sequence and and Dr. Sadao Wakabayashi, sequencing of the glucoamylase. Osaka University, We also thank for Dr. Takehiro Oshima for helpful discussions. REFERENCES 1) S. Ueda, Trends Biochem. Sci., 6, 89 (1981). the entire coding sequence without the intervening sequences was expressed in the yeast 2) J. H. Nunberg, J. H. Meade, G. Cole, F. C. Lawyer, P. MacCabe, V. Schwerckart, R. Tal, V. P. Wittman, cells and glucoamylase was secreted into the J. E. 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