Biotechnol Lett DOI 10.1007/s10529-014-1530-5
ORIGINAL RESEARCH PAPER
Construction of dextrin and isomaltose-assimilating brewer’s yeasts for production of low-carbohydrate beer Jin-Yeong Park • Ja-Yeon Lee • Seung-Hyun Choi • Hyun-Mi Ko • Il-Chul Kim Hwanghee Blaise Lee • Suk Bai
•
Received: 25 February 2014 / Accepted: 1 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer’s bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer’s yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both a-1,4- and a-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.
J.-Y. Park J.-Y. Lee S.-H. Choi I.-C. Kim H. B. Lee S. Bai (&) Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju 500-757, South Korea e-mail: [email protected] H.-M. Ko Department of Microbiology, College of Medicine, Seonam University, Namwon, Chunpook, South Korea
Keywords Brewer’s yeasts Dextrin and isomaltose assimilation Glucoamylase Low-carbohydrate beer Saccharomyces cerevisiae
Introduction Dextrin, which is the main non-fermentable sugar during beer fermentation, comprises 20–25 % of total wort carbohydrates. A reduction in wort dextrin content is a prerequisite to producing low-carbohydrate beers. Large amounts of exogenous enzymes that hydrolyze dextrin are added prior to fermentation to liberate fermentable sugars from wort dextrin during conventional beer fermentation (Hansen et al. 1990; Wang et al. 2010a, b). Alternative techniques to produce beer with decreased dextrin could be established by providing breweries with genetically-modified yeast strains that express amylolytic enzymes. The a-amylase gene (ALP1) from Saccharomycopsis fibuligera, the dextranase gene (LSD1) from Lipomyces starkeyi and the glucoamylase genes (STA, SGA1, and GLA) from Saccharomyces diastaticus, S. cerevisiae, and S. fibuligera have been used to develop brewer’s yeasts to reduce the caloric content of beer. However, these recombinant yeasts cannot completely degrade wort dextrins to fermentable sugars (Hansen et al. 1990; Liu et al. 2004, 2008; Wang et al. 2010a, b).
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The glucoamylases from Debaryomyces occidentalis (formerly Schwanniomyces occidentalis) and Aspergillus awamori possess activities of both a-1,4and a-1,6-debranching hydrolases. In particular, D. occidentalis is the only ascosporogenous yeast that exhibits significant debranching activity (Dohmen et al. 1990; Naim et al. 1991; Lin et al. 1998). In this study, we constructed two linearized double 18S rDNA-integrative vectors harboring both the CUP1 gene conferring resistance to copper and the D. occidentalis glucoamylase gene (GAM1) or A. awamori glucoamylase gene (GA1) to develop industrial brewer’s yeast strains that can completely degrade dextrin to fermentable sugars for brewing low-carbohydrate beers. We compared different brewer’s bottom and top yeast strains expressing GAM1 or GA1 using integrative vectors in this study in terms of enzyme activities and assimilation of dextrin and isomaltose.
YPI media [YP medium containing 2 % (w/v) dextrin or 2 % (w/v) isomaltose] at 24 °C for 5 days to assay the glucoamylase activities present in the culture supernatants. Residual dextrin and isomaltose were assayed via the phenol/sulfuric acid and dinitrosalicylic acid methods, respectively (Kim et al. 2011). All experiments were conducted three to five times independently. The results are shown as the mean ± SD of the mean of three different experiments. Reproducible results were obtained, and representative data are shown in the figures. DNA manipulation and yeast transformation All DNA manipulations and the E. coli transformation were conducted by standard methods. Integrative transformation of yeast was conducted via the lithium acetate method as described by Gietz et al. (1992). Construction of 2rDNA integrative systems
Materials and methods Strains and plasmids Escherichia coli DH5a was used for plasmid construction and transformation. The yeast strains used as hosts for the yeast transformation experiment were WLP 810, a bottom-fermenting brewer’s yeast (San Francisco lager yeast, White Labs) and S. cerevisiae ATCC 18824, a top-fermenting brewer’s yeast. S. cerevisiae ATCC 26108 was the source of the CUP1 gene. The YIpdAURSAdd plasmid (Kang et al. 2003) was used to clone the CUP1 gene. YIpSG2rD and YIpAG2rD (Kim et al. 2010) served as the backbones of the 2rDNA-integrative system. Media and culture conditions YPD medium [1 % (w/v) yeast extract, 2 % (w/v) Bacto-peptone and 2 % (w/v) glucose] was employed to propagate S. cerevisiae. The yeast transformants were grown on YNBD plates [0.67 % yeast nitrogen base, 2 % (w/v) glucose and 2 % (w/v) Bacto-agar] containing 0.35 mM CuSO4 (Domingues et al. 2000) and then transferred onto YPDS3 plates [YPD containing 2 % (w/v) soluble starch] and incubated for 4 days at 24 °C, after which they were incubated for 2 days at 4 °C. Yeast cells were grown in YPDex or
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The CUP1 gene was amplified by PCR using the oligonucleotides 50 -ACTGTCGACGGATCCCATT ACCGACATTTG-30 and 50 -TGAGTCGACGGTACC ATGAATAGCTTTTTT-30 . These primers were designed using the published nucleotide sequences of the S. cerevisiae ATCC 26108 CUP1 gene (GenBank accession number E00298). The PCR resulted in 0.9 kb amplified DNA fragments containing the entire open reading frame from genomic DNA of S. cerevisiae ATCC 26108, which underwent digestion with SalI, and was inserted into the same site of YIpdAURSAd with the deleted AMY gene to generate YIpdAURCUd. To construct the double 18S rDNA system containing the CUP1 gene, a 0.9 kb CUP1 gene excised from YIpdAURCUd was inserted into the SalI site in YIpSG2rD and YIpAG2rD lacking the G418 resistance gene, thereby generating YIpSGCU2rD and YIpAGCU2rD, respectively (Fig. 1). Enzyme assays Glucoamylase activity was quantified at pH 5.5 and 40 °C using the PGO/ODAD assay (Sigma). One unit of glucoamylase activity was defined as the amount of enzyme that liberated 1 lmol glucose ml-1 min-1 using 0.5 % (w/v) soluble starch, dextrin, or isomaltose as the enzyme substrate. All assays were repeated three times, and the means were calculated.
Biotechnol Lett
Fig. 1 Plasmid maps of YIpSGCU2rD and YIpAGCU2rD showing relative size, restriction sites, and location of the inserted DNA. Each rDNA-integration vector was linearized by digestion with SacII. The bacterial vector DNA sequences harboring the ampicillin resistance marker gene, the ColE1 (pUC) origin, and the URA3 gene (5.1 kb) were excised
Analysis of enzymatic reaction products by HPLC Culture supernatants (50 ll) were incubated with 0.5 % (w/v) isomaltose in 0.1 M sodium phosphate buffer (pH 5.5; 950 ll). After the start of the incubation, 100 ll was withdrawn at the indicated times and the products were analyzed by HPLC using a Rezex RSO-oligosaccharide column (200 9 10 mm, Phenomenex, Torrance, CA, USA). The operating conditions were 75 °C with water as mobile phase at 0.3 ml min-1. Sugars were monitored using a RI detector.
CU2rD were linearized by digesting the 18S rDNA sequences with SacII (Fig. 1), after which the fragments (7.4 or 6.6 kb) harboring the GAM1 and CUP1 gene cassettes or the GA1 and CUP1 gene cassettes flanked by the 18S rDNA sequences were integrated into the 18S rDNA sequences dispersed throughout the S. cerevisiae genome (Lin et al. 1998; Nieto et al. 1999). The integrative cassette (YIpSGCU2rD)-harboring yeasts were named ATCC 18824/YIpSGCU2rD and WLP 810/YIpSGCU2rD. The double 18S rDNA system generated a smaller unneeded fragment (5.1 kb) containing prokaryotic sequences and an ampicillin resistance marker, which was removed prior to transformation. The integrative cassette (YIpAGCU2rD)-harboring yeasts were designated ATCC 18824/YIpAGCU2rD and WLP 810/YIpAGCU2rD. According to Wang et al. (2010a, b), the ILV2 gene that encodes the synthase of a-acetolactate, the precursor of diacetyl, has been used as a recombination site to introduce exogenous amylolytic enzyme genes into brewer’s yeasts, and to reduce the diacetyl content in beer. However, multi-copy integration methods such as rDNA-integration or d-integration are the most suitable methods for overexpressing foreign glucoamylase genes in brewer’s yeasts (Kim et al. 2010). Several investigators have employed dominant selection markers such as the G418 resistance gene to select high-copy-number integrants (Wang et al. 1996). However, antibiotic-resistance genes such as the G418 resistance gene are absent in the integrative cassettes of commercially useful yeast strains (Nieto et al. 1999). Yeast metallothionein is encoded by the CUP1 gene and plays a role protecting yeast cells against oxidants, which correlates with beer flavor stability (Jamieson 1998; Wang et al. 2010a, b). Because the yeast-derived CUP1 gene as the dominant selection marker was introduced into industrial brewer’s yeasts, its use in commercial beverage production should be safe and therefore suitable for industrial applications (Wang et al. 2010a, b).
Results and discussion Introduction of GAM1 or GA1 genes into the brewer’s yeasts To achieve stable multiple-copy integration of the GAM1 or GA1 genes into the chromosomes of industrial brewer’s yeasts, YIpSGCU2rD and YIpAG
Assimilation of dextrin and isomaltose by brewer’s yeasts expressing the GAM1 or GA1 genes Brewer’s yeast transformants expressing the GAM1 or GA1 genes were grown in medium containing dextrin as the carbon source to study their capacities to hydrolyze and assimilate dextrin. The parental wild-
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type strains (S. cerevisiae ATCC 18824 and WLP 810) displayed a low level of growth with no dextrin hydrolysis. The brewer’s yeast transformants expressing GAM1 or GA1 grew substantially in medium containing dextrin. Moreover, growth of the yeast transformants expressing GAM1 occurred at relatively higher levels compared with that of the transformants expressing the GA1 gene (data not shown). This result suggests that the improved growth rates in the former yeast transformants may be correlated with their high glucoamylase activities at a low cultivation temperature suitable for brewer’s yeasts. The GAM1-encoded glucoamylase retained up to 75 % of its maximal activity at 20 and 25 °C, and more than 50 % at 15 °C (Sills et al. 1984). Glucoamylase activities were examined in the culture supernatants from transformants grown in YPDex media. The clones with the highest levels of activity were selected from the transformants and used for subsequent analyses. The results (Table 1) show that the bottom yeast transformant WLP 810/YIpSGCU2rD (secreting the GAM1-encoded glucoamylase) had the highest activity of glucoamylase at 0.5 U ml-1. Glucoamylase activity exhibited by WLP 810/YIpSGCU2rD was 2.5-times higher than that of WLP 810/YIpAGCU2rD secreting the GA1-encoded glucoamylase (0.2 U ml-1; Table 1 Glucoamylase activities in cell-free culture supernatants of brewer’s yeasts and their transformants Yeast strains
Glucoamylase activity (U ml-1)a Substrate Soluble starch
Dextrin
S. cerevisiae ATCC 18824
ND
ND
San Francisco lager yeast WLP 810
ND
ND
ATCC 18824/YIpSGCU2rD (GAM1)
0.24 ± 0.02
0.22 ± 0.04
ATCC 18824/YIpAGCU2rD (GA1)
0.13 ± 0.03
0.12 ± 0.01
WLP 810/YIpSGCU2rD (GAM1)
0.52 ± 0.03
0.48 ± 0.02
WLP 810/YIpAGCU2rD (GA1)
0.21 ± 0.02
0.19 ± 0.05
Data (mean ± SD) were from three independent experiments ND not detected a
Yeast cells were grown in medium containing dextrin (YPDex) at 24 °C for 4 days
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Kim et al. 2010). In contrast, the bottom yeast transformants showed glucoamylase activities about twice that of the top yeast transformants. Time course analyses of the glucoamylase activities, cell growth, and dextrin hydrolysis for the bottom yeast transformants (WLP 810/YIpSGCU2rD and WLP 810/YIpAGCU2rD) over 5 days are shown in Fig. 2. Cell growth was positively affected by enzyme activities, reaching almost maximal values after 3 days of cultivation. WLP 810/YIpSGCU2rD utilized 96 % of the dextrin in culture medium containing 2 % (w/v) dextrin during 5 days of growth, whereas WLP 810/YIpAGCU2rD utilized 75 % of the dextrin in the corresponding culture medium. These findings indicate that the rate-limiting step in growth from dextrin is the efficient degradation of dextrin to glucose by glucoamylase (Hansen et al. 1990). To elucidate the ability of the WLP 810/YIpSGCU2rD and WLP 810/YIpAGCU2rD glucoamylases to digest a-1,6-glycosidic and a-1,4 bonds in dextrin, these bottom yeast transformants were grown in medium containing isomaltose (disaccharide consisting of glucose units joined by a-1,6-glycosidic linkage) as the sole carbon source. The growth rate, glucoamylase activity and isomaltose hydrolysis for WLP 810/YIpSGCU2rD over 5 days were compared with those of WLP 810/YIpAGCU2rD. As shown in Fig. 3, WLP 810/YIpSGCU2rD hydrolyzed 98 % of the isomaltose in a culture medium containing 2 % (w/ v) isomaltose during 5 days of growth, whereas WLP 810/YIpAGCU2rD displayed a relatively low growth rate with no distinct decrease in isomaltose. Thus, the GAM1-encoded glucoamylase of WLP 810/YIpSGCU2rD has significant a-1,6-debranching activity and hydrolyzed isomaltose to glucose, whereas the GA1-encoded glucoamylase of WLP 810/YIpAGCU2rD showed a very low activity towards the a-1,6-linkage, which could lead to incomplete hydrolysis of isomaltose in culture (Lin et al. 1998). According to the enzymatic reaction product analyses by HPLC (Fig. 4), isomaltose served as a substrate for the GAM1-encoded glucoamylase, thereby increasing free glucose liberation with the decrease of isomaltose as reaction time passed, whereas the GA1-encoded glucoamylase gave a low level of glucose with no significant hydrolysis of isomaltose. Up to now, several attempts have been made to express a-amylase and glucoamylase genes in
Biotechnol Lett
Fig. 2 Time courses of growth, dextrin degradation, and extracellular glucoamylase activity produced by WLP 810/YIpSGCU2rD (a) and WLP 810/YIpAGCU2rD (b) at 24 °C in YPDex medium. Growth was measured on different days based on cell dry weight, and glucoamylase activities were measured in culture supernatants. The remaining dextrin values are
presented as percentages considering the dextrin in the uninoculated medium as 100 %; mg ml-1 cell mass (circles); U ml-1 glucoamylase activities (squares); % residual dextrin (triangles). Data (mean ± SD) were from three independent experiments performed in triplicate
Fig. 3 Growth curves, time courses of isomaltose hydrolysis and extracellular glucoamylase activity produced by WLP 810/YIpSGCU2rD (a) and WLP 810/YIpAGCU2rD (b) at 24 °C in medium containing isomaltose (YPI). Growth was measured on different days based on cell dry weight, and glucoamylase activities were measured in culture supernatants.
The remaining isomaltose values are presented as percentages considering the isomaltose in the uninoculated medium as 100 %; mg ml-1 cell mass (circles); U ml-1 glucoamylase activities (squares); % residual isomaltose (triangles). Data (mean ± SD) were from three independent experiments performed in triplicate
brewer’s yeasts to degrade dextrins for low-carbohydrate beers. However, due to their low ability or inability to hydrolyze a-1,6-bonds, the dextrins were not completely degraded to fermentable sugars (Liu et al. 2004, 2008; Wang et al. 2010a, b). Hansen et al.
(1990) reported that brewer’s yeasts can completely degrade wort dextrins and attain an 100 % apparent degree of attenuation only when an enzyme such as GAM1-encoded glucoamylase from D. occidentalis, that hydrolyzes a-1,6 and a-1,4-bonds, was added.
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Biotechnol Lett Fig. 4 HPLC analyses of the enzymatic products from isomaltose by glucoamylases from WLP 810/YIpSGCU2rD (a) and WLP 810/YIpAGCU2rD (b). Time (h) in parentheses indicates the enzymatic reaction times. I isomaltose, G glucose
The new bottom yeast producing GAM1-encoded glucoamylase (WLP 810/YIpSGCU2rD) can be used for prolonged bottom beer fermentation because the glucoamylase retains up to 30 % of its maximal activity at much lower temperature less than 10 °C. Moreover, this enzyme retains up to 90 % of its maximal activity at pH 4.0, which is the average pH during beer fermentation (Sills et al. 1984). Thus the brewer’s yeast strains secreting D. occidentalis glucoamylase may be useful for reducing wort dextrins at low pHs and temperatures during the brewing process and are suitable for producing low-carbohydrate beers. Further studies are currently underway to introduce the D. occidentalis a-amylase gene (Kim et al. 2011) into brewer’s yeast strains to improve the rate and efficiency of wort dextrin hydrolysis, which would be suitable for producing diabetic beers. Acknowledgments Jin-Yeong Park was supported by the second stage of the Brain Korea 21 Project.
References Dohmen RJ, Strasser AWM, Dahlems UM, Hollenberg CP (1990) Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in Saccharomyces cerevisiae. Gene 95:111–121
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Domingues L, Onnela ML, Teixeira JA, Lima N, Penttila M (2000) Construction of a flocculent brewer’s yeast strain secreting Aspergillus niger b-galactosidase. Appl Microbiol Biotechnol 54:97–103 Gietz D, St. Jean A, Woods R, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425 Hansen M, Rocken W, Emeis CC (1990) Construction of yeast strains for the production of low-carbohydrate beer. J Inst Brew 96:125–129 Jamieson D (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 14:1511–1527 Kang NY, Park JN, Chin JE, Lee HB, Im SY, Bai S (2003) Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis a-amylase gene. Biotechnol Lett 25:1847–1851 Kim JH, Kim HR, Lim MH, Ko HM, Chin JE, Lee HB, Kim IC, Bai S (2010) Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, a-amylase and debranching enzyme. Biotechnol Lett 32:713–719 Kim HR, Im YK, Ko HM, Chin JE, Kim IC, Lee HB, Bai S (2011) Raw starch fermentation to ethanol by an industrial distiller’s strain of Saccharomyces cerevisiae expressing glucoamylase and a-amylase genes. Biotechnol Lett 33:1643–1648 Lin LL, Ma YJ, Chien HR, Hsu WH (1998) Construction of an amylolytic yeast by multiple integration of the Aspergillus awamori glucoamylase gene into a Saccharomyces cerevisiae chromosome. Enzym Microb Technol 23:360–365 Liu Z, Zhang G, Liu S (2004) Integrative expression of glucoamylase gene in a brewer’s yeast Saccharomyces pastorianus strain. Food Technol Biotechnol 46:32–37
Biotechnol Lett Liu Z, Zhang G, Li J, Chen G (2008) Construction of an amylolytic brewing yeast Saccharomyces pastorianus suitable for accelerated brewing. J Biosci Bioeng 98:414–419 Naim HY, Niermann T, Kleinhans U, Hollenberg CP, Strasser AWM (1991) Striking structural and functional similarities suggest that intestinal sucrase-isomaltase, human lysosomal a-glucosidase and Schwanniomyces occidentalis glucoamylase are derived from a common ancestral gene. FEBS Lett 294:109–112 Nieto A, Prieto JA, Sanz P (1999) Stable high-copy number integration of Aspergillus oryzae a-amylase cDNA in an industrial Baker’s yeast strain. Biotechnol Prog 15:459– 466 Sills AM, Sauder ME, Stewart GG (1984) Isolation and characterization of the amylotic system of Schwanniomyces castellii. J Inst Brew 90:311–314
Wang X, Wang Z, Da Silva NA (1996) G418 selection and stability of cloned genes integrated at chromosomal d sequences of Saccharomyces cerevisiae. Biotechnol Bioeng 49:45–51 Wang JJ, Wang ZY, He XP, Zhang BR (2010a) Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor. J Microbiol Biotechnol 20:1539–1545 Wang JJ, Wang ZY, Liu XF, Guo XN, He XP, Wensel PC, Zhang BR (2010b) Construction of an industrial brewing yeast strain to manufacture beer with low caloric content and improved flavor. J Microbiol Biotechnol 20:767–774
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ORIGINAL RESEARCH PAPER
Construction of dextrin and isomaltose-assimilating brewer’s yeasts for production of low-carbohydrate beer Jin-Yeong Park • Ja-Yeon Lee • Seung-Hyun Choi • Hyun-Mi Ko • Il-Chul Kim Hwanghee Blaise Lee • Suk Bai
•
Received: 25 February 2014 / Accepted: 1 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer’s bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer’s yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both a-1,4- and a-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.
J.-Y. Park J.-Y. Lee S.-H. Choi I.-C. Kim H. B. Lee S. Bai (&) Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju 500-757, South Korea e-mail: [email protected] H.-M. Ko Department of Microbiology, College of Medicine, Seonam University, Namwon, Chunpook, South Korea
Keywords Brewer’s yeasts Dextrin and isomaltose assimilation Glucoamylase Low-carbohydrate beer Saccharomyces cerevisiae
Introduction Dextrin, which is the main non-fermentable sugar during beer fermentation, comprises 20–25 % of total wort carbohydrates. A reduction in wort dextrin content is a prerequisite to producing low-carbohydrate beers. Large amounts of exogenous enzymes that hydrolyze dextrin are added prior to fermentation to liberate fermentable sugars from wort dextrin during conventional beer fermentation (Hansen et al. 1990; Wang et al. 2010a, b). Alternative techniques to produce beer with decreased dextrin could be established by providing breweries with genetically-modified yeast strains that express amylolytic enzymes. The a-amylase gene (ALP1) from Saccharomycopsis fibuligera, the dextranase gene (LSD1) from Lipomyces starkeyi and the glucoamylase genes (STA, SGA1, and GLA) from Saccharomyces diastaticus, S. cerevisiae, and S. fibuligera have been used to develop brewer’s yeasts to reduce the caloric content of beer. However, these recombinant yeasts cannot completely degrade wort dextrins to fermentable sugars (Hansen et al. 1990; Liu et al. 2004, 2008; Wang et al. 2010a, b).
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The glucoamylases from Debaryomyces occidentalis (formerly Schwanniomyces occidentalis) and Aspergillus awamori possess activities of both a-1,4and a-1,6-debranching hydrolases. In particular, D. occidentalis is the only ascosporogenous yeast that exhibits significant debranching activity (Dohmen et al. 1990; Naim et al. 1991; Lin et al. 1998). In this study, we constructed two linearized double 18S rDNA-integrative vectors harboring both the CUP1 gene conferring resistance to copper and the D. occidentalis glucoamylase gene (GAM1) or A. awamori glucoamylase gene (GA1) to develop industrial brewer’s yeast strains that can completely degrade dextrin to fermentable sugars for brewing low-carbohydrate beers. We compared different brewer’s bottom and top yeast strains expressing GAM1 or GA1 using integrative vectors in this study in terms of enzyme activities and assimilation of dextrin and isomaltose.
YPI media [YP medium containing 2 % (w/v) dextrin or 2 % (w/v) isomaltose] at 24 °C for 5 days to assay the glucoamylase activities present in the culture supernatants. Residual dextrin and isomaltose were assayed via the phenol/sulfuric acid and dinitrosalicylic acid methods, respectively (Kim et al. 2011). All experiments were conducted three to five times independently. The results are shown as the mean ± SD of the mean of three different experiments. Reproducible results were obtained, and representative data are shown in the figures. DNA manipulation and yeast transformation All DNA manipulations and the E. coli transformation were conducted by standard methods. Integrative transformation of yeast was conducted via the lithium acetate method as described by Gietz et al. (1992). Construction of 2rDNA integrative systems
Materials and methods Strains and plasmids Escherichia coli DH5a was used for plasmid construction and transformation. The yeast strains used as hosts for the yeast transformation experiment were WLP 810, a bottom-fermenting brewer’s yeast (San Francisco lager yeast, White Labs) and S. cerevisiae ATCC 18824, a top-fermenting brewer’s yeast. S. cerevisiae ATCC 26108 was the source of the CUP1 gene. The YIpdAURSAdd plasmid (Kang et al. 2003) was used to clone the CUP1 gene. YIpSG2rD and YIpAG2rD (Kim et al. 2010) served as the backbones of the 2rDNA-integrative system. Media and culture conditions YPD medium [1 % (w/v) yeast extract, 2 % (w/v) Bacto-peptone and 2 % (w/v) glucose] was employed to propagate S. cerevisiae. The yeast transformants were grown on YNBD plates [0.67 % yeast nitrogen base, 2 % (w/v) glucose and 2 % (w/v) Bacto-agar] containing 0.35 mM CuSO4 (Domingues et al. 2000) and then transferred onto YPDS3 plates [YPD containing 2 % (w/v) soluble starch] and incubated for 4 days at 24 °C, after which they were incubated for 2 days at 4 °C. Yeast cells were grown in YPDex or
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The CUP1 gene was amplified by PCR using the oligonucleotides 50 -ACTGTCGACGGATCCCATT ACCGACATTTG-30 and 50 -TGAGTCGACGGTACC ATGAATAGCTTTTTT-30 . These primers were designed using the published nucleotide sequences of the S. cerevisiae ATCC 26108 CUP1 gene (GenBank accession number E00298). The PCR resulted in 0.9 kb amplified DNA fragments containing the entire open reading frame from genomic DNA of S. cerevisiae ATCC 26108, which underwent digestion with SalI, and was inserted into the same site of YIpdAURSAd with the deleted AMY gene to generate YIpdAURCUd. To construct the double 18S rDNA system containing the CUP1 gene, a 0.9 kb CUP1 gene excised from YIpdAURCUd was inserted into the SalI site in YIpSG2rD and YIpAG2rD lacking the G418 resistance gene, thereby generating YIpSGCU2rD and YIpAGCU2rD, respectively (Fig. 1). Enzyme assays Glucoamylase activity was quantified at pH 5.5 and 40 °C using the PGO/ODAD assay (Sigma). One unit of glucoamylase activity was defined as the amount of enzyme that liberated 1 lmol glucose ml-1 min-1 using 0.5 % (w/v) soluble starch, dextrin, or isomaltose as the enzyme substrate. All assays were repeated three times, and the means were calculated.
Biotechnol Lett
Fig. 1 Plasmid maps of YIpSGCU2rD and YIpAGCU2rD showing relative size, restriction sites, and location of the inserted DNA. Each rDNA-integration vector was linearized by digestion with SacII. The bacterial vector DNA sequences harboring the ampicillin resistance marker gene, the ColE1 (pUC) origin, and the URA3 gene (5.1 kb) were excised
Analysis of enzymatic reaction products by HPLC Culture supernatants (50 ll) were incubated with 0.5 % (w/v) isomaltose in 0.1 M sodium phosphate buffer (pH 5.5; 950 ll). After the start of the incubation, 100 ll was withdrawn at the indicated times and the products were analyzed by HPLC using a Rezex RSO-oligosaccharide column (200 9 10 mm, Phenomenex, Torrance, CA, USA). The operating conditions were 75 °C with water as mobile phase at 0.3 ml min-1. Sugars were monitored using a RI detector.
CU2rD were linearized by digesting the 18S rDNA sequences with SacII (Fig. 1), after which the fragments (7.4 or 6.6 kb) harboring the GAM1 and CUP1 gene cassettes or the GA1 and CUP1 gene cassettes flanked by the 18S rDNA sequences were integrated into the 18S rDNA sequences dispersed throughout the S. cerevisiae genome (Lin et al. 1998; Nieto et al. 1999). The integrative cassette (YIpSGCU2rD)-harboring yeasts were named ATCC 18824/YIpSGCU2rD and WLP 810/YIpSGCU2rD. The double 18S rDNA system generated a smaller unneeded fragment (5.1 kb) containing prokaryotic sequences and an ampicillin resistance marker, which was removed prior to transformation. The integrative cassette (YIpAGCU2rD)-harboring yeasts were designated ATCC 18824/YIpAGCU2rD and WLP 810/YIpAGCU2rD. According to Wang et al. (2010a, b), the ILV2 gene that encodes the synthase of a-acetolactate, the precursor of diacetyl, has been used as a recombination site to introduce exogenous amylolytic enzyme genes into brewer’s yeasts, and to reduce the diacetyl content in beer. However, multi-copy integration methods such as rDNA-integration or d-integration are the most suitable methods for overexpressing foreign glucoamylase genes in brewer’s yeasts (Kim et al. 2010). Several investigators have employed dominant selection markers such as the G418 resistance gene to select high-copy-number integrants (Wang et al. 1996). However, antibiotic-resistance genes such as the G418 resistance gene are absent in the integrative cassettes of commercially useful yeast strains (Nieto et al. 1999). Yeast metallothionein is encoded by the CUP1 gene and plays a role protecting yeast cells against oxidants, which correlates with beer flavor stability (Jamieson 1998; Wang et al. 2010a, b). Because the yeast-derived CUP1 gene as the dominant selection marker was introduced into industrial brewer’s yeasts, its use in commercial beverage production should be safe and therefore suitable for industrial applications (Wang et al. 2010a, b).
Results and discussion Introduction of GAM1 or GA1 genes into the brewer’s yeasts To achieve stable multiple-copy integration of the GAM1 or GA1 genes into the chromosomes of industrial brewer’s yeasts, YIpSGCU2rD and YIpAG
Assimilation of dextrin and isomaltose by brewer’s yeasts expressing the GAM1 or GA1 genes Brewer’s yeast transformants expressing the GAM1 or GA1 genes were grown in medium containing dextrin as the carbon source to study their capacities to hydrolyze and assimilate dextrin. The parental wild-
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type strains (S. cerevisiae ATCC 18824 and WLP 810) displayed a low level of growth with no dextrin hydrolysis. The brewer’s yeast transformants expressing GAM1 or GA1 grew substantially in medium containing dextrin. Moreover, growth of the yeast transformants expressing GAM1 occurred at relatively higher levels compared with that of the transformants expressing the GA1 gene (data not shown). This result suggests that the improved growth rates in the former yeast transformants may be correlated with their high glucoamylase activities at a low cultivation temperature suitable for brewer’s yeasts. The GAM1-encoded glucoamylase retained up to 75 % of its maximal activity at 20 and 25 °C, and more than 50 % at 15 °C (Sills et al. 1984). Glucoamylase activities were examined in the culture supernatants from transformants grown in YPDex media. The clones with the highest levels of activity were selected from the transformants and used for subsequent analyses. The results (Table 1) show that the bottom yeast transformant WLP 810/YIpSGCU2rD (secreting the GAM1-encoded glucoamylase) had the highest activity of glucoamylase at 0.5 U ml-1. Glucoamylase activity exhibited by WLP 810/YIpSGCU2rD was 2.5-times higher than that of WLP 810/YIpAGCU2rD secreting the GA1-encoded glucoamylase (0.2 U ml-1; Table 1 Glucoamylase activities in cell-free culture supernatants of brewer’s yeasts and their transformants Yeast strains
Glucoamylase activity (U ml-1)a Substrate Soluble starch
Dextrin
S. cerevisiae ATCC 18824
ND
ND
San Francisco lager yeast WLP 810
ND
ND
ATCC 18824/YIpSGCU2rD (GAM1)
0.24 ± 0.02
0.22 ± 0.04
ATCC 18824/YIpAGCU2rD (GA1)
0.13 ± 0.03
0.12 ± 0.01
WLP 810/YIpSGCU2rD (GAM1)
0.52 ± 0.03
0.48 ± 0.02
WLP 810/YIpAGCU2rD (GA1)
0.21 ± 0.02
0.19 ± 0.05
Data (mean ± SD) were from three independent experiments ND not detected a
Yeast cells were grown in medium containing dextrin (YPDex) at 24 °C for 4 days
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Kim et al. 2010). In contrast, the bottom yeast transformants showed glucoamylase activities about twice that of the top yeast transformants. Time course analyses of the glucoamylase activities, cell growth, and dextrin hydrolysis for the bottom yeast transformants (WLP 810/YIpSGCU2rD and WLP 810/YIpAGCU2rD) over 5 days are shown in Fig. 2. Cell growth was positively affected by enzyme activities, reaching almost maximal values after 3 days of cultivation. WLP 810/YIpSGCU2rD utilized 96 % of the dextrin in culture medium containing 2 % (w/v) dextrin during 5 days of growth, whereas WLP 810/YIpAGCU2rD utilized 75 % of the dextrin in the corresponding culture medium. These findings indicate that the rate-limiting step in growth from dextrin is the efficient degradation of dextrin to glucose by glucoamylase (Hansen et al. 1990). To elucidate the ability of the WLP 810/YIpSGCU2rD and WLP 810/YIpAGCU2rD glucoamylases to digest a-1,6-glycosidic and a-1,4 bonds in dextrin, these bottom yeast transformants were grown in medium containing isomaltose (disaccharide consisting of glucose units joined by a-1,6-glycosidic linkage) as the sole carbon source. The growth rate, glucoamylase activity and isomaltose hydrolysis for WLP 810/YIpSGCU2rD over 5 days were compared with those of WLP 810/YIpAGCU2rD. As shown in Fig. 3, WLP 810/YIpSGCU2rD hydrolyzed 98 % of the isomaltose in a culture medium containing 2 % (w/ v) isomaltose during 5 days of growth, whereas WLP 810/YIpAGCU2rD displayed a relatively low growth rate with no distinct decrease in isomaltose. Thus, the GAM1-encoded glucoamylase of WLP 810/YIpSGCU2rD has significant a-1,6-debranching activity and hydrolyzed isomaltose to glucose, whereas the GA1-encoded glucoamylase of WLP 810/YIpAGCU2rD showed a very low activity towards the a-1,6-linkage, which could lead to incomplete hydrolysis of isomaltose in culture (Lin et al. 1998). According to the enzymatic reaction product analyses by HPLC (Fig. 4), isomaltose served as a substrate for the GAM1-encoded glucoamylase, thereby increasing free glucose liberation with the decrease of isomaltose as reaction time passed, whereas the GA1-encoded glucoamylase gave a low level of glucose with no significant hydrolysis of isomaltose. Up to now, several attempts have been made to express a-amylase and glucoamylase genes in
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Fig. 2 Time courses of growth, dextrin degradation, and extracellular glucoamylase activity produced by WLP 810/YIpSGCU2rD (a) and WLP 810/YIpAGCU2rD (b) at 24 °C in YPDex medium. Growth was measured on different days based on cell dry weight, and glucoamylase activities were measured in culture supernatants. The remaining dextrin values are
presented as percentages considering the dextrin in the uninoculated medium as 100 %; mg ml-1 cell mass (circles); U ml-1 glucoamylase activities (squares); % residual dextrin (triangles). Data (mean ± SD) were from three independent experiments performed in triplicate
Fig. 3 Growth curves, time courses of isomaltose hydrolysis and extracellular glucoamylase activity produced by WLP 810/YIpSGCU2rD (a) and WLP 810/YIpAGCU2rD (b) at 24 °C in medium containing isomaltose (YPI). Growth was measured on different days based on cell dry weight, and glucoamylase activities were measured in culture supernatants.
The remaining isomaltose values are presented as percentages considering the isomaltose in the uninoculated medium as 100 %; mg ml-1 cell mass (circles); U ml-1 glucoamylase activities (squares); % residual isomaltose (triangles). Data (mean ± SD) were from three independent experiments performed in triplicate
brewer’s yeasts to degrade dextrins for low-carbohydrate beers. However, due to their low ability or inability to hydrolyze a-1,6-bonds, the dextrins were not completely degraded to fermentable sugars (Liu et al. 2004, 2008; Wang et al. 2010a, b). Hansen et al.
(1990) reported that brewer’s yeasts can completely degrade wort dextrins and attain an 100 % apparent degree of attenuation only when an enzyme such as GAM1-encoded glucoamylase from D. occidentalis, that hydrolyzes a-1,6 and a-1,4-bonds, was added.
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Biotechnol Lett Fig. 4 HPLC analyses of the enzymatic products from isomaltose by glucoamylases from WLP 810/YIpSGCU2rD (a) and WLP 810/YIpAGCU2rD (b). Time (h) in parentheses indicates the enzymatic reaction times. I isomaltose, G glucose
The new bottom yeast producing GAM1-encoded glucoamylase (WLP 810/YIpSGCU2rD) can be used for prolonged bottom beer fermentation because the glucoamylase retains up to 30 % of its maximal activity at much lower temperature less than 10 °C. Moreover, this enzyme retains up to 90 % of its maximal activity at pH 4.0, which is the average pH during beer fermentation (Sills et al. 1984). Thus the brewer’s yeast strains secreting D. occidentalis glucoamylase may be useful for reducing wort dextrins at low pHs and temperatures during the brewing process and are suitable for producing low-carbohydrate beers. Further studies are currently underway to introduce the D. occidentalis a-amylase gene (Kim et al. 2011) into brewer’s yeast strains to improve the rate and efficiency of wort dextrin hydrolysis, which would be suitable for producing diabetic beers. Acknowledgments Jin-Yeong Park was supported by the second stage of the Brain Korea 21 Project.
References Dohmen RJ, Strasser AWM, Dahlems UM, Hollenberg CP (1990) Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in Saccharomyces cerevisiae. Gene 95:111–121
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Domingues L, Onnela ML, Teixeira JA, Lima N, Penttila M (2000) Construction of a flocculent brewer’s yeast strain secreting Aspergillus niger b-galactosidase. Appl Microbiol Biotechnol 54:97–103 Gietz D, St. Jean A, Woods R, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425 Hansen M, Rocken W, Emeis CC (1990) Construction of yeast strains for the production of low-carbohydrate beer. J Inst Brew 96:125–129 Jamieson D (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 14:1511–1527 Kang NY, Park JN, Chin JE, Lee HB, Im SY, Bai S (2003) Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis a-amylase gene. Biotechnol Lett 25:1847–1851 Kim JH, Kim HR, Lim MH, Ko HM, Chin JE, Lee HB, Kim IC, Bai S (2010) Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, a-amylase and debranching enzyme. Biotechnol Lett 32:713–719 Kim HR, Im YK, Ko HM, Chin JE, Kim IC, Lee HB, Bai S (2011) Raw starch fermentation to ethanol by an industrial distiller’s strain of Saccharomyces cerevisiae expressing glucoamylase and a-amylase genes. Biotechnol Lett 33:1643–1648 Lin LL, Ma YJ, Chien HR, Hsu WH (1998) Construction of an amylolytic yeast by multiple integration of the Aspergillus awamori glucoamylase gene into a Saccharomyces cerevisiae chromosome. Enzym Microb Technol 23:360–365 Liu Z, Zhang G, Liu S (2004) Integrative expression of glucoamylase gene in a brewer’s yeast Saccharomyces pastorianus strain. Food Technol Biotechnol 46:32–37
Biotechnol Lett Liu Z, Zhang G, Li J, Chen G (2008) Construction of an amylolytic brewing yeast Saccharomyces pastorianus suitable for accelerated brewing. J Biosci Bioeng 98:414–419 Naim HY, Niermann T, Kleinhans U, Hollenberg CP, Strasser AWM (1991) Striking structural and functional similarities suggest that intestinal sucrase-isomaltase, human lysosomal a-glucosidase and Schwanniomyces occidentalis glucoamylase are derived from a common ancestral gene. FEBS Lett 294:109–112 Nieto A, Prieto JA, Sanz P (1999) Stable high-copy number integration of Aspergillus oryzae a-amylase cDNA in an industrial Baker’s yeast strain. Biotechnol Prog 15:459– 466 Sills AM, Sauder ME, Stewart GG (1984) Isolation and characterization of the amylotic system of Schwanniomyces castellii. J Inst Brew 90:311–314
Wang X, Wang Z, Da Silva NA (1996) G418 selection and stability of cloned genes integrated at chromosomal d sequences of Saccharomyces cerevisiae. Biotechnol Bioeng 49:45–51 Wang JJ, Wang ZY, He XP, Zhang BR (2010a) Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor. J Microbiol Biotechnol 20:1539–1545 Wang JJ, Wang ZY, Liu XF, Guo XN, He XP, Wensel PC, Zhang BR (2010b) Construction of an industrial brewing yeast strain to manufacture beer with low caloric content and improved flavor. J Microbiol Biotechnol 20:767–774
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