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Biotechnol Lett (2007) 29:12031208

DOI 10.1007/s10529-007-9371-0

ORIGINAL RESEARCH PAPER

Efficient one-step starch utilization by industrial strains


of Saccharomyces cerevisiae expressing the glucoamylase
and a-amylase genes from Debaryomyces occidentalis
Dong-Myeong Ghang Li Yu Mi-Hyeon Lim
Hyun-Mi Ko Suhn-Young Im
Hwanghee Blaise Lee Suk Bai

Received: 8 February 2007 / Accepted: 8 March 2007 / Published online: 16 May 2007
 Springer Science+Business Media B.V. 2007

Abstract Amylolytic industrial polyploid strains of Introduction


Saccharomyces cerevisiae (ATCC 4126, ATCC 9763
and ATCC 24858) expressing a glucoamylase gene Saccharomyces cerevisiae lacks the amylolytic
(GAM1) or an a-amylase gene (AMY) from Debary- activity necessary for direct utilization of starch, a
omyces occidentalis were developed. The glucoam- widespread and relatively cheap carbon source.
ylase activity of S. cerevisiae ATCC 9763 expressing Manipulation of S. cerevisiae to produce glucoam-
the GAM1 gene was 3.7-times higher than that of ylase and a-amylase would contribute to the direct
D. occidentalis. On the other hand, a-amylase activity conversion of starch to ethanol and single cell
in the corresponding strain expressing the D. occi- proteins (Marin et al. 2001). Glucoamylase (EC
dentalis AMY gene increased 10-times relative to 3.2.1.3, 1,4-a-D-glucan glucohydrolase) is an exo-
D. occidentalis. These two recombinant yeast strains type enzyme that degrades starch and releases
expressing the GAM1 gene and AMY gene, respec- glucose, while a-amylase (EC 3.2.1.1, 1,4-a-D-glu-
tively were cultured simultaneously to produce both can glucanohydrolase) is an endo-type enzyme that
glucoamylase and a-amylase for efficient one-step cleaves a-1,4-glucosidic linkages of starch and
utilization of starch. Growth, substrate utilization and liberates maltose and oligosaccharides (Dohmen
enzyme activity of these strains are described. et al. 1990; Eksteen et al. 2003). Of more than
150 starch-assimilating yeast species, Debaryomyces
Keywords Debaryomyces occidentalis AMY  occidentalis (formerly Schwanniomyces occidentalis)
GAM1  Industrial polyploid strains of degrades starch efficiently as a result of the
Saccharomyces cerevisiae combined action of a-amylase and glucoamylase.
D. occidentalis expresses significant debranching
activity as a part of the glucoamylase, indicating
that it possesses the ability to hydrolyze both a-1,4
and a-1,6-glucosidic linkages, which is essential for
complete hydrolysis of starch (Dohmen et al. 1990).
Industrial strains of S. cerevisiae, which would
D.-M. Ghang  L. Yu  M.-H. Lim  H.-M. Ko  express D. occidentalis glucoamylase gene (GAM1),
S.-Y. Im  H. B. Lee  S. Bai (&) could be very valuable in the industrial fermenta-
Department of Biological Sciences, College of Natural
tions to use starch because of their high fermenta-
Sciences, Chonnam National University,
Gwangju 500-757, Korea tion rates and ethanol tolerance (Janse and Pretorius
e-mail: sukbai@chonnam.ac.kr 1995).

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1204 Biotechnol Lett (2007) 29:12031208

Transformation of industrial S. cerevisiae cells can et al. (1992). Isolation of total genomic DNA from
be achieved by integrating foreign genes into their yeast was performed according to the procedure
chromosomes by homologous recombination. The described by Zhu et al. (1993).
reiterated DNA sequences such as d-sequences of the
Ty retrotransposon and ribosomal DNA are used as
target sites for recombination which results in a Media and culture conditions
greater number of integrated genes, and therefore
higher expression level and mitotic stability (Cho Escherichia coli transformants were grown in Luria-
et al. 1999; Kang et al. 2003). Using this approach, Bertani media supplemented with 50 mg ampicil-
we recently demonstrated the expression of the lin ml1. Yeast cells were cultured in YPD medium
D. occidentalis a-amylase gene (AMY) in S. cerevisiae [1% (w/v) yeast extract, 1% (w/v) Bacto-peptone and
ATCC 4126, shown by the observation of integrants 2% (w/v) glucose]. Yeast transformants were grown
secreting 6 times the a-amylase as that of D. occi- on YPD plates containing aureobasidin A (1 mg ml1,
dentalis (Kang et al. 2003). In this paper, we TaKaRa, Japan) and then transferred onto YPDS3
constructed three industrial strains of S. cerevisiae plates [YPD containing 3% (w/v) soluble starch] and
that express high glucoamylase activity or a-amylase incubated for 4 days at 308C, followed by incubation
activity by d-integration of the D. occidentalis GAM1 at 48C for 2 days. Amylolytic clones were detected by
gene or AMY gene. The growth, substrate utilization observation of halos around the colonies. Buffered
and enzyme activity of these new strains are YPS medium (BYPS) containing 2% (w/v) soluble
analyzed. The efficacy of starch hydrolysis by the starch and 0.1 M sodium phosphate buffer (pH 6.0)
combination of an industrial strain of S. cerevisiae was used to assay amylase activities secreted by yeast
expressing the GAM1 gene and the same strain transformants. Starch hydrolysis (as indicated by the
expressing high a-amylase activity was compared to presence of residual starch) was assayed using a
that of D. occidentalis. starch-iodine reaction to measure the loss of iodine
[0.4% (w/v) iodine and 2% (w/v) KI] staining
capacity (Kim et al. 1988). Mitotic stability of the
Materials and methods GAM1 gene was determined using the method
described by Nieto et al. (1999).
Strains and plasmids

Escherichia coli JM83 was used for transformation Southern hybridization and enzyme assays
and plasmid constructions. A laboratory haploid
strain of S. cerevisiae ATCC 44771 (SHY3) and Nonradioactive colorometric hybridization and
industrial polyploid strains of S. cerevisiae ATCC detection was performed using the DIG DNA
4126, ATCC 9763 and ATCC 24858 (Ness et al. labeling and detection kit (Roche) according to the
1993) were used as hosts for the yeast transformation method described in the DIG Application manual.
experiment. D. occidentalis CBS 2863 (ATCC The GAM1 probe was a 0.8 kb BamHISacI
26077) was the source of the GAM1 gene. Plasmid D. occidentalis DNA fragment from pSGA that
pSBG (Kim et al. 2000) was used to clone the GAM1 included part of GAM1 gene. Glucoamylase activity
gene, and YIpdAURSAd (Kang et al. 2003) served as was quantified at pH 5.5 and 408C using the PGO/
the backbone of the d-integrative system. ODAD assay (Sigma). One unit of glucoamylase
activity was defined as the amount of enzyme that
DNA manipulation and yeast transformation liberated 1 mmol of glucose ml1 (Steyn and
Pretorius 1991). a-Amylase activity was determined
All procedures for the DNA manipulations and the as described by Park et al. (1999) at pH 5.5 and
transformation of E. coli were performed using the 408C. Assays were repeated three times and the
methods described by Sambrook and Russell (2001). means recorded. The reaction products were ana-
Integrative transformation of yeast was carried out lyzed by TLC using silica gel plates (Merck) as
using the lithium acetate method described by Gietz described by Lee et al. (2000).

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Biotechnol Lett (2007) 29:12031208 1205

Results and discussion (a)


Xho I Apa I Xho I Xho I Xho I

Construction of d-integrative systems AUR1-C ADC1p AMY CYC1T Ampr Ori

YIp AURSA (5.4 kb) (2.8 kb)


for expression of GAM1 gene
Xho I Apa I Xho I Xho I Xho I

PCR amplification of the D. occidentalis GAM1 gene AUR1-C ADC1p GAM1 CYC1T Ampr Ori

was carried out using the oligonucleotides 50 - YIp AURSG (6.8 kb) (2.8 kb)

GGAGGATCCACCATGATTTTTCTGAAGCT- (b) 1 2 3 4

G A T - 3 0 a n d 5 0- G G A G G A T C C C G G G A T G T
TAAAATTACCAAGT-30 . These primers were de-
signed using the published nucleotide sequences of
the D. occidentalis ATCC 26076 GAM1 gene (Doh-
men et al. 1990) and included BamHI (GGATCC)
sites which were introduced to facilitate the cloning
of the GAM1 gene. PCR resulted in 2.9 kb amplified
DNA fragments of the whole open reading frame
Fig. 1 Integration of the D. occidentalis GAM1 gene into
from genomic DNA of D. occidentalis CBS 2863, industrial S. cerevisiae genome. (a) Plasmid maps of linearized
which underwent digestion with BamHI and was then YIpdAURSAd and YIpdAURSGd showing relative size,
inserted into the same site of pSBG with deleted b- restriction sites and location of insert DNA. YIpdAURSAd
amylase gene, generating pDOG. S. cerevisiae ATCC and YIpdAURSGd were linearized by digesting with XhoI. (b)
Southern blot hybridization using 0.8 kb BamHISacI DNA
44771 transformed with pDOG were then screened fragment containing part of GAM1 gene as a probe. S. cerevi-
for amylolytic activity on YPDS3 medium. To obtain siae ATCC 9763 (lane 2) and ATCC 9763/YIpdAURSGd
industrial polyploid strains of S. cerevisiae with (lanes 3 and 4) genomic DNAs were digested with the
stable glucoamylase activity, a yeast integrating indicated enzymes. Numbers on the left indicate the size of
standard DNAs (in kb) and those on the right the size of
plasmid containing a dominant selection marker hybridizing DNAs (in kb)
exclusively derived from S. cerevisiae is required
(Hashida-Okado et al. 1996; Xie and Jimenez 1996).
A 2.9 kb DNA fragment containing the GAM1 gene number of integrations compared with the longer
from pDOG was inserted into the SmaI site down- construct (9.6 kb) by digesting AUR1-C gene with
stream of the ADC1 promoter (ADC1p) in YIpdAU- ApaI (Lee and Da Silva 1997; Kang et al. 2003). As
RSAd lacking the AMY gene, thereby generating expected, no signal was obtained in the wild-type
YIpdAURSGd (Fig. 1a). The double d system S. cerevisiae ATCC 9763 genomic DNA.
(YIpdAURSGd) could be linearized by digesting d To examine the mitotic stability, ATCC 9763/
sequences with XhoI and the unnecessary bacterial YIpdAURSGd was cultivated in nonselective YPD
plasmid DNA sequences containing the ampicillin media for 50 generations. On the YPS plate, 100% of
resistance gene and replication origin of pBR322 the integrant colonies still exhibited halos. This
excised before transformation (Fig. 1a). These result- suggests that the d-integrated GAM1 gene was stably
ing fragments (6.8 kb) containing the GAM1 gene maintained in the chromosomes of the integrants (Lee
flanked by d sequences were gel purified to transform and Da Silva 1997; Cho et al. 1999). Such stable
industrial polyploid strains of S. cerevisiae (Wang integrations of DNA containing GAM1 genes exclu-
et al. 1996; Lee and Da Silva 1997). sively derived from yeast facilitate the commercial
Southern blot analysis of the transformants exhib- application of recombinant DNA technology in the
iting glucoamylase activity was performed to confirm food and beverage industries (Nieto et al. 1999).
the GAM1 gene integration into the yeast chromo-
somes (Fig. 1b). The 9.6 kb lighter band and 6.8 kb Expression and secretion of glucoamylase
darker band visualized in the ApaI digestive DNA and a-amylase in industrial strains of S. cerevisiae
and XhoI digestive DNA from ATCC 9763/YIpdAU-
RSGd, respectively. The smaller construct (6.8 kb) Industrial polyploid strains of S. cerevisiae ATCC
lacking bacterial sequences resulted in a greater 4126, ATCC 9763 and ATCC 24858 were

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1206 Biotechnol Lett (2007) 29:12031208

transformed to GAM+ AUR+ with YIpdAURSGd, and a-amylase activity of ATCC 9763/YIpdAURSAd
generating ATCC 4126/YIpdAURSGd, ATCC 9763/ were simultaneously obtained by the mixed-culture of
YIpdAURSGd and ATCC 24858/YIpdAURSGd, ATCC 9763/YIpdAURSGd and ATCC 9763/
respectively. All transformants could utilize starch YIpdAURSAd. However, S. cerevisiae ATCC 9763
in the medium whereas the parental wild-type strains transformed with d-integrative system containing
could not. The glucoamylase activities were exam- both D. occidentalis AMY and GAM1 genes (9.3 kb)
ined in culture supernatants from transformants exhibited lower amylolytic activity and mitotic
grown in the BYPS media. The clones showing the stability, possibly because of low copy integrations
highest activity among the transformants were by long integrating constructs (data not shown).
selected. As shown in Table 1, ATCC 9763/YIpdAU- The activities of glucoamylase and a-amylase
RSGd exhibited the highest glucoamylase activity from ATCC 9763/YIpdAURSGd and ATCC 9763/
followed by ATCC 4126/YIpdAURSGd and ATCC YIpdAURSAd were assayed using various carbohy-
24858/YIpdAURSGd, suggesting the expression le- drate substrates (Table 2). Using each of the different
vel of GAM1 gene was affected by host strains. The starches as substrates resulted in similar amounts of
glucoamylase activity produced by ATCC 9763/ a-amylase from ATCC 9763/YIpdAURSAd, however
YIpdAURSGd was 3.7-times higher than that by the glucoamylase activities of ATCC 9763/YIpdAU-
D. occidentalis. Moreover, S. cerevisiae ATCC 9763 RSGd toward all the raw starches were low, similar to
transformed with YIpdAURSAd containing D. occi- what has been observed in Aspergillus glucoamylase
dentalis AMY gene produced approximately 10-times (Yamasaki et al. 1977). However, in this study,
the a-amylase as D. occidentalis. Aspergillus glucoamylase did not hydrolyze isomal-
Kang et al. (2003) have reported that the a- tose at all, whereas we observed glucoamylase
amylase activity of S. cerevisiae ATCC 4126 trans- activity of ATCC 9763/YIpdAURSGd when pullulan
formed with YIpdAURSAd (ATCC 4126/YIpdAU- hydrolysis was measured as well as when isomaltose
R S A d ) w a s 6- t i m e s h i g h e r t h a n t h a t o f hydrolysis was measured, indicating that this strain
D. occidentalis. The multicopy integration of GAM1 possesses debranching activity which is essential for
gene or AMY gene may be correlated with the high- complete hydrolysis of starch (Ma et al. 2000). As
level expression of the corresponding genes (Lee and shown in Fig. 2, the co-expression of GAM1 and AMY
Da Silva 1997; Cho et al. 1999). The glucoamylase genes in the mixed-culture of ATCC 9763/YIpdAU-
and a-amylase activities that were similar to the RSGd and ATCC 9763/YIpdAURSAd resulted in
glucoamylase activity of ATCC 9763/YIpdAURSGd 100% utilization of starch within 24 h. The industrial

Table 1 Glucoamylase and a-amylase activities in cell-free culture supernatants of S. cerevisiae transformants
Yeast strains Glucoamylase activitya (U ml1) a-Amylase activity (U ml1)

ATCC 4126/YIpdAURSGd 0.38b NDc


ATCC 9763/YIpdAURSGd 0.92 ND
ATCC 24858/YIpdAURSGd 0.21 ND
ATCC 4126/YIpdAURSAd ND 4.80
ATCC 9763/YIpdAURSAd ND 7.96
ATCC 24858/YIpdAURSAd ND 2.07
d
ATCC 9763/YIpd AURSGd + ATCC 9763/YIpdAURSAd 0.89 8.03
D. occidentalis CBS 2863 0.25 0.78
a
Yeast cells were grown in the BYPS media containing 0.1 M sodium phosphate buffer (pH 6.0) at 308C for 4 days
b
Values were means of results from triplicate experiments and were expressed in amylolytic activities present in the culture
supernatants
c
Not detected
d
ATCC 9763/YIpd AURSGd and ATCC 9763/YIpdAURSAd were mixed-cultured by a ratio of 1:1 (w/w) in the BYPS medium

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Biotechnol Lett (2007) 29:12031208 1207

Table 2 Hydrolysis of several carbohydrates by the action of


glucoamylase from ATCC 9763/YIpdAURSGd and a-amylase
from ATCC 9763/YIpdAURSAd
Substrates Glucoamylase activity a-Amylase activity
(U ml1) (U ml1)

Maltose 0.29 NDa


Isomaltose 0.1 ND
Matotriose 0.69 ND
Pullulan 0.32 ND
Soluble starch 0.92 7.96
Corn starch 0.12 8.02
Potato starch 0.14 8.07
Rice starch 0.13 8.12 Fig. 3 Thin-layer chromatography of the enzymatic products
from soluble starch by glucoamylase and a-amylase from
Wheat starch 0.1 7.85
S. cerevisiae transformants. The enzymatic products were
a
Not detected analyzed at different reaction times. Lane S, standards (G1:
glucose, G2: maltose, G3: maltotriose); 1, glucoamylase from
ATCC 9763/YIpdAURSGd (30 min); 2, a-amylase from
ATCC 9763/YIpdAURSAd (30 min); 38, glucoamylase and
a-amylase from ATCC 9763/YIpdAURSGd + ATCC 9763/
YIpdAURSAd (3, 30 min; 4, 60 min; 5, 120 min; 6, 180 min; 7,
240 min; 8, 300 min). Time (min) in parenthesis indicates the
enzymatic reaction time

a-amylase found in culture broths of ATCC 9763/


YIpdAURSGd and ATCC 9763/YIpdAURSAd was
glucose, indicating that glucoamylase and a-amylase
acted synergistically. The cooperative interaction of
glucoamylase and a-amylase resulted from the in-
Fig. 2 Growth curves, time course of starch hydrolysis and creased maltose and maltotriose produced from starch
extracellular glucoamylase activities and a-amylase activities by a-amylase, which in turn could serve as substrates
produced by the mixed-culture of ATCC 9763/YIpdAURSGd for glucoamylase, thereby increasing the free glucose
and ATCC 9763/YIpdAURSAd in the BYPS media. At
different days, growth was measured by the optical density at
liberation (Kim et al. 1988; Steyn and Pretorius 1991).
600 nm, and amylolytic activities and hydrolyzed starch were As shown in Table 2, since the high a-amylase
measured in the culture supernatants. The remaining starch activities against various raw starches were very
results were presented as percentages taking as 100% the starch similar to those against soluble starch, oligomeric
in the uninoculated medium. Each point represents the means
of three independent measurements with a standard deviation
products produced from raw starches by a-amylase
of 5%. D, O.D. 600 nm growth; j, U ml1 glucoamylase action were served as good substrates for glucoamy-
activities; h, U ml1 a-amylase activities; , starch % residual lase, reflecting the fact that the efficient one-step
starch utilization of raw starches can be achieved by
glucoamylase and a-amylase of ATCC 9763/YIpdAU-
S. cerevisiae strain reported by Marin et al. (2001) RSGd and ATCC 9763/YIpdAURSAd (Janse and
that produces D. occidentalis a-amylase hydrolyzes Pretorius 1995; Ma et al. 2000; Eksteen et al. 2003).
95% of starch within 35 h of growth. Janse and
Pretorius (1995) have reported that D. occidentalis Acknowledgement This study was financially supported by
Chonnam National University in the Program, 2005.
can complete starch hydrolysis after about 34 h.
According to the analysis of the enzymatic reaction
product by TLC (Fig. 3), glucoamylase degraded
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