Biotechnol Lett (2007) 29:1203–1208

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 Æ Li Yu Æ Mi-Hyeon Lim Æ
Hyun-Mi Ko Æ Suhn-Young Im Æ
Hwanghee Blaise Lee Æ 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).

123

123 . cerevisiae ATCC detection was performed using the DIG DNA 4126. (1999) at pH 5. starch-iodine reaction to measure the loss of iodine [0. shown by the observation of integrants 2% (w/v) glucose]. followed by incubation activity by d-integration of the D. 2000) was used to clone the GAM1 included part of GAM1 gene. (2000). Buffered and enzyme activity of these new strains are YPS medium (BYPS) containing 2% (w/v) soluble analyzed. occidentalis. 1988). In this paper. occidentalis DNA fragment from pSGA that pSBG (Kim et al.1204 Biotechnol Lett (2007) 29:1203–1208 Transformation of industrial S. occidentalis CBS 2863 (ATCC The GAM1 probe was a 0. substrate utilization observation of halos around the colonies. The growth. 2003). 1999. occidentalis a-amylase gene (AMY) in S. cerevisiae [1% (w/v) yeast extract. Kang et al. 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. 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. coli were performed using the 408C.4% (w/v) iodine and 2% (w/v) KI] staining capacity (Kim et al. 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. occi. a-Amylase activity was determined All procedures for the DNA manipulations and the as described by Park et al. on YPD plates containing aureobasidin A (1 mg ml1. The described by Zhu et al. cerevisiae plates [YPD containing 3% (w/v) soluble starch] and that express high glucoamylase activity or a-amylase incubated for 4 days at 308C. cerevisiae ATCC 44771 (SHY3) and Nonradioactive colorometric hybridization and industrial polyploid strains of S. (1993). Yeast transformants were grown secreting 6 times the a-amylase as that of D.8 kb BamHI–SacI 26077) was the source of the GAM1 gene. Assays were repeated three times and the methods described by Sambrook and Russell (2001). A laboratory haploid strain of S. 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). Glucoamylase activity gene. cerevisiae cells can et al. we TaKaRa. ODAD assay (Sigma). and YIpdAURSAd (Kang et al. 1% (w/v) Bacto-peptone and ATCC 4126. 2003) served as was quantified at pH 5. experiment. 2003). means recorded. 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. The efficacy of starch hydrolysis by the starch and 0. dentalis (Kang et al. Japan) and then transferred onto YPDS3 constructed three industrial strains of S. labeling and detection kit (Roche) according to the 1993) were used as hosts for the yeast transformation method described in the DIG Application manual. (1992). and therefore higher expression level and mitotic stability (Cho Escherichia coli transformants were grown in Luria- et al. ATCC 9763 and ATCC 24858 (Ness et al.1 M sodium phosphate buffer (pH 6.0) combination of an industrial strain of S. Bertani media supplemented with 50 mg ampicil- we recently demonstrated the expression of the lin ml1.5 and transformation of E. occidentalis GAM1 at 48C for 2 days. cerevisiae was used to assay amylase activities secreted by yeast expressing the GAM1 gene and the same strain transformants. Yeast cells were cultured in YPD medium D. Using this approach. Strains and plasmids Escherichia coli JM83 was used for transformation Southern hybridization and enzyme assays and plasmid constructions. D. Amylolytic clones were detected by gene or AMY gene. Plasmid D.5 and 408C using the PGO/ the backbone of the d-integrative system. Mitotic stability of the Materials and methods GAM1 gene was determined using the method described by Nieto et al. (1999).

1a). cerevisiae with (lanes 3 and 4) genomic DNAs were digested with the stable glucoamylase activity. 1999). occidentalis GAM1 gene AUR1-C ADC1p GAM1 CYC1T Ampr Ori was carried out using the oligonucleotides 50 .8 kb) Industrial polyploid strains of S.9 kb amplified DNA fragments of the whole open reading frame Fig. PCR resulted in 2. cerevisiae (Wang integrations of DNA containing GAM1 genes exclu- et al. cerevisiae and XhoI digestive DNA from ATCC 9763/YIpdAU- RSGd. occidentalis GAM1 gene into from genomic DNA of D. 1996. The double d system S. S. 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 ATCC 9763 genomic DNA. YIpdAURSAd amylase gene. This excised before transformation (Fig. These result. Cho et al.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. ATCC 9763 and ATCC 24858 were 123 .9 kb DNA fragment containing the GAM1 gene number of integrations compared with the longer from pDOG was inserted into the SmaI site down. cerevisiae ATCC and YIpdAURSGd were linearized by digesting with XhoI. a yeast integrating indicated enzymes. thereby generating expected. 1990) and included BamHI (GGATCC) sites which were introduced to facilitate the cloning of the GAM1 gene.Biotechnol Lett (2007) 29:1203–1208 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. cerevisiae is required (Hashida-Okado et al. 2003). occidentalis CBS 2863. cerevisiae genome. cerevisiae ATCC lacking bacterial sequences resulted in a greater 4126. Xie and Jimenez 1996). 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.8 kb) for expression of GAM1 gene Xho I Apa I Xho I Xho I Xho I PCR amplification of the D. The smaller construct (6.G G A G G A T C C C G G G A T G T TAAAATTACCAAGT-30 . restriction sites and location of insert DNA. generating pDOG. Lee and Da Silva 1997).8 kb) (2. occidentalis ATCC 26076 GAM1 gene (Doh- men et al. industrial S. application of recombinant DNA technology in the iting glucoamylase activity was performed to confirm food and beverage industries (Nieto et al. The 9. 1 Integration of the D. cerevi- for amylolytic activity on YPDS3 medium. no signal was obtained in the wild-type YIpdAURSGd (Fig. 1996. 1b). suggests that the d-integrated GAM1 gene was stably ing fragments (6. construct (9. ApaI (Lee and Da Silva 1997. A 2. inserted into the same site of pSBG with deleted b.6 kb) by digesting AUR1-C gene with stream of the ADC1 promoter (ADC1p) in YIpdAU. Kang et al. These primers were de- signed using the published nucleotide sequences of the D.8 kb Expression and secretion of glucoamylase darker band visualized in the ApaI digestive DNA and a-amylase in industrial strains of S. Such stable industrial polyploid strains of S. the GAM1 gene integration into the yeast chromo- somes (Fig. sively derived from yeast facilitate the commercial Southern blot analysis of the transformants exhib. (b) Southern blot hybridization using 0.6 kb lighter band and 6.8 kb) GGAGGATCCACCATGATTTTTCTGAAGCT.4 kb) (2.3 0 a n d 5 0. As RSAd lacking the AMY gene. 1999). (b) 1 2 3 4 G A T . On the YPS plate. To obtain siae ATCC 9763 (lane 2) and ATCC 9763/YIpdAURSGd industrial polyploid strains of S. (YIpdAURSGd) could be linearized by digesting d To examine the mitotic stability. S.8 kb BamHI–SacI DNA 44771 transformed with pDOG were then screened fragment containing part of GAM1 gene as a probe. 1a). (a) Plasmid maps of linearized which underwent digestion with BamHI and was then YIpdAURSAd and YIpdAURSGd showing relative size. 100% of resistance gene and replication origin of pBR322 the integrant colonies still exhibited halos. YIp AURSG (6. respectively.

21 ND ATCC 4126/YIpdAURSAd ND 4. occidentalis CBS 2863 0. the a-amylase as D. the co-expression of GAM1 and AMY Da Silva 1997.03 D. selected. occidentalis AMY and GAM1 genes (9. 2. occidentalis. The glucoamylase activities were exam. hydrolysis was measured as well as when isomaltose R S A d ) w a s 6. However. cerevisiae ATCC 9763 in the medium whereas the parental wild-type strains transformed with d-integrative system containing could not. similar to transformed with YIpdAURSAd containing D. tose at all. As level expression of the corresponding genes (Lee and shown in Fig.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 123 .80 ATCC 9763/YIpdAURSAd ND 7. cerevisiae ATCC 9763 RSGd toward all the raw starches were low. suggesting the expression le.t i m e s h i g h e r t h a n t h a t o f hydrolysis was measured.96 ATCC 24858/YIpdAURSAd ND 2. ATCC 9763/ were simultaneously obtained by the mixed-culture of YIpdAURSGd and ATCC 24858/YIpdAURSGd.78 a Yeast cells were grown in the BYPS media containing 0. All transformants could utilize starch YIpdAURSAd. The starches as substrates resulted in similar amounts of glucoamylase activity produced by ATCC 9763/ a-amylase from ATCC 9763/YIpdAURSAd.89 8. Aspergillus glucoamylase did not hydrolyze isomal- Kang et al. what has been observed in Aspergillus glucoamylase dentalis AMY gene produced approximately 10-times (Yamasaki et al. activity of ATCC 9763/YIpdAURSGd when pullulan formed with YIpdAURSAd (ATCC 4126/YIpdAU.92 ND ATCC 24858/YIpdAURSGd 0. in this study. cerevisiae ATCC 4126 trans. The multicopy integration of GAM1 possesses debranching activity which is essential for gene or AMY gene may be correlated with the high.25 0. complete hydrolysis of starch (Ma et al. ATCC 9763/YIpdAURSGd and ATCC 9763/ respectively. However. 2000). (2003) have reported that the a. S.1206 Biotechnol Lett (2007) 29:1203–1208 transformed to GAM+ AUR+ with YIpdAURSGd. cerevisiae transformants Yeast strains Glucoamylase activitya (U ml1) a-Amylase activity (U ml1) ATCC 4126/YIpdAURSGd 0. Using each of the different vel of GAM1 gene was affected by host strains. occidentalis. occi. Cho et al. indicating that this strain D. whereas we observed glucoamylase amylase activity of S. occidentalis. As shown in Table 1. ATCC 9763/YIpdAU.1 M sodium phosphate buffer (pH 6. S. Moreover.07 d ATCC 9763/YIpd AURSGd + ATCC 9763/YIpdAURSAd 0. and a-amylase activity of ATCC 9763/YIpdAURSAd generating ATCC 4126/YIpdAURSGd. 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. 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. 1977). possibly because of low copy integrations highest activity among the transformants were by long integrating constructs (data not shown). however YIpdAURSGd was 3.7-times higher than that by the glucoamylase activities of ATCC 9763/YIpdAU- D. The industrial Table 1 Glucoamylase and a-amylase activities in cell-free culture supernatants of S. drate substrates (Table 2).38b NDc ATCC 9763/YIpdAURSGd 0.3 kb) ined in culture supernatants from transformants exhibited lower amylolytic activity and mitotic grown in the BYPS media. both D. The clones showing the stability.

time course of starch hydrolysis and creased maltose and maltotriose produced from starch extracellular glucoamylase activities and a-amylase activities by a-amylase. U ml1 glucoamylase action were served as good substrates for glucoamy- activities. 6.12 Fig.32 ND Soluble starch 0. indicating that glucoamylase and a-amylase acted synergistically. Ma et al.02 Potato starch 0. 2000. 7. The enzymatic products were a Not detected analyzed at different reaction times. 180 min. glucoamylase degraded References soluble starch to glucose. occidentalis Acknowledgement This study was financially supported by Chonnam National University in the Program. while a-amylase broke down starch into maltose and maltotriose.D. The major starch Cho KM.92 7. standards (G1: glucose. Each point represents the means of three independent measurements with a standard deviation products produced from raw starches by a-amylase of ±5%. occidentalis a-amylase hydrolyzes Pretorius 1995. 5. Yoo YJ. 600 nm growth. U ml1 a-amylase activities. O. Lane S. Eksteen et al. starch % residual lase. G3: maltotriose). 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.69 ND Pullulan 0. (2001) RSGd and ATCC 9763/YIpdAURSAd (Janse and that produces D. •. cerevisiae strain reported by Marin et al. 240 min. Steyn and Pretorius 1991).1 ND Matotriose 0. glucoamylase and a-amylase from ATCC 9763/YIpdAURSGd + ATCC 9763/ YIpdAURSAd (3. growth was measured by the optical density at liberation (Kim et al. a-amylase from ATCC 9763/YIpdAURSAd (30 min). According to the analysis of the enzymatic reaction product by TLC (Fig. which in turn could serve as substrates produced by the mixed-culture of ATCC 9763/YIpdAURSGd for glucoamylase. 600 nm. 95% of starch within 35 h of growth. 2. 3–8. and amylolytic activities and hydrolyzed starch were As shown in Table 2. can complete starch hydrolysis after about 34 h. j.96 Corn starch 0. Kang HS (1999) d-Integration of endo/ hydrolysis product produced by glucoamylase and exo-glucanase and b-glucosidase genes into the yeast 123 .85 S. h. 300 min). 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. since the high a-amylase measured in the culture supernatants.Biotechnol Lett (2007) 29:1203–1208 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. 2005.14 8. glucoamylase from ATCC 9763/YIpdAURSGd (30 min). 3).29 NDa Isomaltose 0. 8. 30 min. At different days. 1988. The cooperative interaction of glucoamylase and a-amylase resulted from the in- Fig. 4. D. 120 min. 2 Growth curves. 3 Thin-layer chromatography of the enzymatic products from soluble starch by glucoamylase and a-amylase from Wheat starch 0.1 7. thereby increasing the free glucose and ATCC 9763/YIpdAURSAd in the BYPS media.13 8. 1. G2: maltose. Janse and Pretorius (1995) have reported that D.07 Rice starch 0. 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. 60 min. 2003).12 8. oligomeric in the uninoculated medium. cerevisiae transformants.

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