Download Rhizopus Raw-Starch-Degrading Glucoamylase: Its

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.
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M
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I-I
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-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
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1000
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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
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K
aaatttatgtcaagaac
Py
i
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_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
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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. Flatgaard and M. A. Innis, Molec. Cell. BioL, 4,
2306 (1984).
culture mediumas shown previously. These
results indicate that the 5'-flanking region of 3) E. Boel, I. Hjort, B. Svensson, F. Norris, K. E.
Rhizopus glucoamylase gene promoted gene Norris and N. P. Fiil, EMBO /., 3, 1097 (1984).
4) T. Takahashi, Y. Tsuchida and M. Irie, /. Biochem.,
expression and the signal peptide functioned
84, 1183
(1978).
properly in yeast cells. Howeverthe expression
level of glucoamylase in these yeast cells is not
sufficiently high enough to allow the practical
production
of glucoamylase and ethanol
from
raw starch. High level expression
of gluco-
5) T. Takahashi,
92,
1623
Y. Tsuchida and M. Irie,
(1982).
J. Biochem.,
6) H. Ito, Y. Fukuda, K. Murata and A. Kimura, J.
7)
Bacteriol.,
153,
Y. Tsujisaka,
163 (1983).
N. Hamada
and
S.
Takenishi,
T. Ashikari
964
8)
Proceedings
of the Symposium
on Amylase (in
Japanese),
10th meeting of amylase kenkyu kai,
Osaka, Japan, October, 1975, p. 61.
Y. Tanaka, Y. Fukumori and T. Yamanaka,
Biochim. Biophys. Ada, 707, 14
9) D. R. Cryer, R. Ecdeshall and
in Cell Biology," Vol. 12, ed.
Academic Press Inc., New York,
10)
J. M. Chirgwin,
A. E. Przybyla,
(1982).
J. Marmur, "Methods
by D. M. Prescott,
1975, p. 39.
R. J. MacDonald
Laboratory,
pp.
188-209.
Cloning,"
Cold
Spring
Harbor
Cold Spring Harbor, New York, 1982,
12) H. Okayama and P. Berg, Molec. Cell. BioL, 2, 161
(1982).
Y. Morinaga,
BioITechnology,
T. Franceshini,
S. Inouye
and
M.
2, 636
(1984).
K. Miyoshi, R. Arentzen, T. Huang and K. Itakura,
Nucl. Acids Res., 8, 5509 (1980).
M. E. E. Watson, Nucl. Acids. Res., 12, 5145 (1984).
M. Fitzgerald
and T. Shenk, Cell, 24, 251 (1981).
S. Shoemaker,
V. Schweickart,
M. Ladner,
D.
Gelfand,
S. Kwok, K. Myambo and M. Innis,
Bio/Technology,
1, 691 (1983).
Y.
N. Nakamura, Y. Tanaka, N. Kiuchi,
Shibano,
T.
Tanaka,
T. Amachi
Yoshizumi, Agric. Biol. Chem., 49, 2521
M. R. Lerner, J. A. Boyle, S. M. Mount,
and J. A. Stertz, Nature, 283, 220 (1980).
C. J. Langford and D. Gallwitz, Cell, 33,
M. G. Schechman and C. Yanofsky, J.
Genet.,
13) H. Donis-Keller,
A. M. Maxam and W. Gilbert,
Nucl. Acids. Res., 4, 2527 (1977).
14) M. Crunstein
and J. Walls,
"Methods
in
Enzymology," Vol. 68, Academic Press Inc., New
York, 1979, p. 379.
15) J. Messing and J. Vieira, Gene, 19, 269 (1982).
16)
Inouye,
T. Ashikari,
and W. J. Rutter, Biochmistry, 18, 5294 (1979).
ll) T. Maniatis, E. F. Fritsch and J. Sambrook,
"Molecular
et al.
1, 83
H.
519 (1983).
Mol. Appl.
(1983).
J. L. Bennetzen and B. D. Hall, J. Biol.
3036
and
(1985).
S. L. Wolin
Chem., 257,
(1982).
K. Arima, T. Oshima, I. Kubota> N. Nakamura, T.
Mizunaga and A. Toh-e, Nucl. Acids Res., ll, 1657
(1983).
I. Yamashita, K. Suzuki and S. Fukui, J. Bacteriol,
161,
567
(1985).