Korean Journal of Microbiology (2016) Vol. 52, No. 2, pp. 220-225 pissn 0440-2413 DOI http://dx.doi.org/10.7845/kjm.2016.6021 eissn 2383-9902 Copyright c 2016, The Microbiological Society of Korea 단보 유전자형질전환을통한쌀전분분해효소재조합효모균주의개발과발효특성조사 이자연 1 진종언 2 배석 1 * 1 전남대학교자연과학대학생물학과, 2 동강대학교뷰티미용과 Construction of the recombinant yeast strain with transformation of rice starch-saccharification enzymes and its alcohol fermentation Ja-Yeon Lee 1, Jong-Eon Chin 2, and Suk Bai 1 * 1 Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju 61186, Republic of Korea 2 Department of Cosmetology, Dongkang College, Gwangju 61200, Republic of Korea (Received April 4, 2016; Revised May 9, 2016; Accepted May 10, 2016) ABSTRACT: To improve antioxidant glutathione (GSH) content and saccharification ability in sake yeasts of Saccharomyces cerevisiae, the γ-glutamylcysteine synthetase gene (GSH1 ) from S. cerevisiae, glucoamylase gene (GAM1 ) and α-amylase gene (AMY ) from Debaryomyces occidentalis were co-expressed in sake yeasts for manufacturing a refreshing alcoholic beverage abundant in GSH from rice starch. The extracellular GSH content of the recombinant sake yeasts increased 1.5-fold relative to the parental wide-type strain. The saccharification ability by glucoamylase of the new yeast strain expressing both GAM1 and AMY genes was 2-fold higher than that of the yeast strain expressing only GAM1 gene when grown in the culture medium containing 2% (w/v) rice starch. It generated 11% (v/v) ethanol from 20% (w/v) rice starch and consumed up to 90% of the starch content after 7 days of fermentation. Key words: Saccharomyces cerevisiae, fermentation, glutathione, rice starch, saccharification 곡물을이용하는발효주제조공정에널리사용되는공급원은쌀을비롯하여밀, 보리, 감자, 고구마등의많은전분작물들이포함된다. 이들중특히쌀은가장선호하는원료로서구성성분으로는대부분이전분으로되어있다 (Suresh et al., 1999; Fujieda et al., 2012; Wang et al., 2014). 맥주효모와와인효모, 그리고청주효모를포함하는 Saccharomyces cerevisiae 는전분을직접이용하는데필요한전분분해효소가결여되어있다. 그러므로누룩이나다량의 α-amylase와 glucoamylase 를첨가하는액화 (liquefaction) 와당화 (saccharification) 공정으로전분을포도당으로분해해야발효에이용할수있다 (Eksteen et al., 2003; Shigechi et al., 2004; Ghang et al., 2007). 이러한많은비용과복잡한과정을줄이기위해많은연구자들은전분분해효소능이우수한누룩곰팡이 Aspergillus awamori *For correspondence. E-mail: sukbai@chonnam.ac.kr; Tel.: +82-62-530-3412; Fax: +82-62-530-3409 를비롯한 Aspergillus 종들의 glucoamylases [α-(1,4), (1,6)-Dglucan glucohydrolase (EC 3.2.1.3)] 와 α-amylase [α-(1,4)-dglucan glucanohydrolase (EC 3.2.1.1)] 유전자와효모 Lipomyces, Saccharomycopsis 그리고 Debaryomyces (Schwanniomyces) 종들의 glucoamylases와 α-amylases 유전자를발현시키는재조합 S. cerevisiae를개발하였다 (Dohmen et al., 1990; Lin et al., 1998; Nieto et al., 1999; Eksteen et al., 2003). 이중 D. occidentalis는 α-amylase와함께 α-1,4 결합은물론 Aspergillus 종들의 glucoamylases를비롯한타 glucoamylases보다높은 α-1,6 결합의분해활성즉절지 (debranching) 활성을가진 glucoamylase (GAM) 를생산하는데 α-1,4 결합과함께 α-1,6 배당결합이풍부한전분당화에최적으로알려져있다 (Lin et al., 1998; Park et al., 2014). 또한, 맥주효모나와인효모와달리곡물발효에주로이용되는청주효모는전분액화의주산물인이당류맥아당자화능이매우낮아액화에필요한 α-amylase
쌀전분분해효모개발과발효 221 보다전분당화능이우수한 glucoamylase가전분발효에더필수적이다 (Baba et al., 1974; Saito et al., 1996; Asano et al., 2001). 한편, glutathione (GSH) 은발효과정중중요한방향화합물의안전성을증가시킴으로써알코올음료의풍미를유지, 향상시킬수있는주요산화방지제로작용한다 (Wei et al., 2003; Fan et al., 2004). GSH의합성에중요한효소중하나인 γ-glutamylcysteine synthetase의유전자 (GSH1) 를과발현시켜 GSH 함량을증진시킬수있다 (Wang et al., 2010a; Chen et al., 2013). 본연구에서는누룩이나전분분해효소없이쌀전분을원료로직접효모를이용하여 GSH가풍부한알코올음료의제조를위해, GSH 생산능이증진되고고효율전분당화능을가진청주효모를개발하는것을목표로하였다. 먼저, GSH 생산능을증진시키기위해 S. cerevisiae의 GSH1 유전자를 δ- integration system (Lee and Da Seliva, 1997; Kang et al., 2003) 을이용하여청주효모에서발현시켰다. 다음으로전분당화능과 GSH 생산능의증진을위해 D. occidentalis α-amylase 유전자 (AMY) 와 GSH1 유전자를 δ-integration system을이용하여 D. occidentalis glucoamylase 유전자 (GAM1) 를발현하는청주효모균주 (Lee et al., 2015) 에공동발현시켰다. 제조된여러청주효모균주들의전분분해능력과효소의활성, GSH 함량그리고알코올생산능을조사하였다. Escherichia coli DH5α는형질전환숙주와 plasmids 제조및증폭에사용하였고 DNA의조작과 E. coli 형질전환은표준프로토콜에따라실시하였다. 청주효모 (sake yeasts) 인 Saccharomyces cerevisiae NBRC 2346 (Kyokai No. 6) 와 GAM1 유전자와선별유전자로 S. cerevisiae의구리저항성유전자 (CUP1) 를포함하고있는 YIpSGCU2rD (Park et al., 2014) 로형질전환된재조합균주 NBRC 2346/YIpSGCU2rD (NBRC 2346SG) 를효모형질전환의숙주균주로이용하였으며, YIpδ AURSAδ, YIpδSGSAδ 그리고 YIpδAGSGδ (Kang et al., 2003; Kim et al., 2011) 는본연구에서제작된 2δ-integrative systems 의골격으로이용하였다. 효모형질전환은 Gietz 등 (1992) 의방법에따라실시하였다. GSH1 유전자를증폭하기위해 S. cerevisiae GSH1 유전자 (GenBank accession number: NM_ 001181534) 의염기서열을이용하여제작한 PCR primers 의정방향염기서열은 5'-TCAGGTACCATGGGACTCTTAGCTT TGGGC-3' 이고역방향서열은 5'-CGATCTAGATTAACAT TTGCTTTCTATTGAAGG-3' 이었다. S. cerevisiae의 genomic DNA로부터위 primers를이용하여 PCR로증폭된 2.0 kb GSH1 유전자의말단부분을 KpnI과 XbaI로절단하여 YIpδ AURSAδ에서 AMY 유전자가제거된부분인 ADC1p의뒤부위에삽입하여 YIpδAURGSHδ를제조하였고, 제조된벡터안 에 GSH1 유전자의삽입여부를 PCR로확인하였다. 다음으로 GSH1와 AMY 유전자혹은 GSH1와 GAM1 유전자의공동발현을목적으로두유전자가포함된 2δ-integration systems을구축하기위해, YIpδAGSGδ와 YIpδSGSAδ에서 ApaI과 SacI 으로절단하여각각 GA1과 GAM1 유전자가제거된부분에 YIpδAURGSHδ로부터동일한효소들로절단하여얻은 GSH1 유전자를도입하여 YIpδGSHSGδ와 YIpδGSHSAδ를제조하였다 (Fig. 1). YIpδGSHSAδ와 YIpδGSHSGδ를선형화하기위해 XhoI을처리하여불필요한 2.8 kb 크기의세균유전자와 ampicillin 저항성유전자부분은형질전환전에제거하고나머지 δ서열이양쪽말단에존재하는 ADC1p-GSH1과 ADC1p-GAM1 (9.4 kb) 혹은 ADC1p-GSH1과 ADC1p-AMY 유전자 cassettes (8.1 kb) 를 S. cerevisiae NBRC 2346 염색체내에반복서열로 100 copies 이상존재하는 δ서열 (Saito et al., 1996; Lee and Da Silva, 1997) 에상동접합으로다수도입시켜 multicopies로존재하는형질전환체를제조하였고, 이들을각각 NBRC 2346/ YIpδGSHSAδ (NBRC 2346GSHSA) 와 NBRC 2346/YIpδ GSHSGδ (NBRC 2346GSHSG) 라고명명하였다. 이러한 integration cassettes를이용하여효모염색체내에다중도입된후발현되는유전자는비선택배지에서장기간발효에도높은안정성을유지해야하고또한, 유전자도입시에원핵성세균유전자와항생제저항성유전자가제거되어야하는데본연구의청주효모를이용하여안전하고유용한재조합효모균주의제조는위의두가지조건에적합하였다 (Nieto et al., 1999). 한편, S. cerevisiae 균주들중청주효모는높은발효능과풍미를가지고있으므로곡물을이용한양조용효모로선호되고있으나 (Lee et al., 2015), 대부분의 S. cerevisiae 균주들과달리맥아당 (maltose) 자화능이매우낮다 (Baba et al., 1974; Kang et al., 2003). AMY 유전자발현결과로전분을대부분 maltose로분해시키는 α-amylase 활성만으로는청주효모들이전분을유일 Fig. 1. Schematic plasmid maps of YIpδGSHSGδ and YIpδGSHSAδ showing the relative size, restriction sites, and locations of the insert DNA. YIpδGSHSGδ and YIpδGSHSAδ were linearized by digestion with XhoI. The bacterial vector DNA sequences (2.8 kb) were excised before transformation. Korean Journal of Microbiology, Vol. 52, No. 2
222 Lee et al. 한기질로이용할경우생장및발효율이매우낮다 (Lee et al., 2015). 그러므로 α-amylase 활성에이어 maltose를포도당으로분해시키는 glucoamylase 활성이청주효모에서는필수적이다 (Saito et al., 1996). 그러므로 GAM1 그리고 AMY 유전자공동발현을위해 GAM1 유전자를함유한 rdna integration cassette인 YIpSGCU2rD로형질전환된균주인 NBRC 2346SG (Lee et al., 2015) 의염색체 δ서열부위에이차적으로 YIpδ GSHSAδ가다수도입된형질전환체를제조하여 GAM1, AMY 그리고 GSH1유전자가공동발현되는이균주를 NBRC 2346SG/ YIpδGSHSAδ (NBRC 2346GSHSGSA) 라고명명하였다. 효모배양에는 YPD 배지 [1% (w/v) yeast extract, 2% (w/v) Bacto-peptone 그리고 2% (w/v) glucose] 를사용하였다. 효모형질전환체는 1 μg/ml aureobasidin A (TaKaRa) 를첨가한 YPD 평판배지또는 0.4 mm CuSO 4 를첨가한 YNBD 평판배지 [0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose 그리고 2% (w/v) Bacto-agar] (Domingues et al., 2000) 에서배양하였다. 그다음 YPDS3 배지 [3% (w/v) soluble starch를함유한 YPD] 로옮겨 30 C에서 4일간배양한후 4 C에서 2일간유지시켜콜로니주위의투명환으로전분분해효소활성을확인하였다 (Lin et al., 1998). 형질전환체의유지보존은 1 μg/ml aureobasidin A과 5 mm CuSO 4 (Wang et al., 2010b) 를첨가한 YPDS3를사용하였다. 효소활성능을측정하기위해서 YPS 액체배지 [2% (w/v) soluble starch 혹은 2% (w/v) rice starch (Sigma) 를함유한 YP] 를이용하였다. Glucoamylase 와 α-amylase 활성측정은 ph 6.0, 40 C에서 30분간반응시켰고각각 PGO assay kit (Sigma) 와 dinitrosalicylic acid 방법을이용하여측정하였다 (Ghang et al., 2007). Glucoamylase와 α-amylase 효소활성 1 unit (U) 는각각 ml당 1 μmol 의포도당과환원당을유리시키는양으로정의하였다. 배지의잔존전분함량은전분- 요오드반응 (Kim et al., 1988) 에의해측정하였다. 환원형 GSH 의함량분석하기위해 Quantichrom glutathione assay kit (BioAssay Systems) 을사용하여 YPD 배지에서 3일간자란효모의배양액으로부터세포외 GSH 함량을분석하였다. 발효를통한에탄올생산은 Ma등 (2000) 의방법에따라 S. cerevisiae 형질전환체의콜로니를 5 ml YPS에접종하여 30 C, 200 rpm 에서 24 48시간호기적으로전배양하였다. 이배양액을 20% (w/v) 쌀전분이함유된 50 ml 발효액에첨가하여이산화탄소의배출구가있는 100 ml 플라스크에서 30 C, 100 rpm으로 10 일간유지하면서발효를수행하였다 (Shigechi et al., 2004). 에탄올함량은 Quantichrom ethanol assay kit (Bioassay Systems) 를사용하여측정하였다. YPD 배지에서배양 3일째에세포외로분비되는 GSH의 함량은원균주인청주효모 S. cerevisiae NBRC 2346의경우 23.5 mg/l인데반해 (Wei et al., 2003), 형질전환청주효모들 (NBRC 2346GSHSA, NBRC 2346 GSHSG 그리고 NBRC 2346GSHSGSA) 의경우 34.2 34.6 mg/l으로 1.5배증진되었다 (Table 1). Wang 등 (2010a) 은맥주제조시재조합맥주효모에서 GSH1 유전자의과발현으로 GSH 생산이증진되고이로인해풍미를저해하는화합물의양을감소시킴으로써안정성을증진시키는데기여한다는보고를한바있는데 (Wang et al., 2010b) GSH 양의증진이쌀전분을원료로제조한발효음료의신선도유지와풍미안정성에도기여할것으로기대된다. 2% (w/v) soluble starch 배지에서 3일동안자란형질전환효모균주들의배양상등액으로부터 glucoamylase 활성을조사한결과 (Table 1), glucoamylase와 α-amylase를동시에생산분비하는 NBRC 2346GSHSGSA에서 glucoamylase 활성이 glucoamylase 만을생산하는 NBRC 2346GSHSG, 혹은 NBRC 2346SG와비교하여 1.9배증진되었는데이는 glucoamylase 와전분액화에필요한 α-amylase의시너지효과로사료되었다 (Kim et al., 2010; Im et al., 2013). 2% (w/v) 쌀전분을탄소원으로형질전환청주효모들의생장과전분이용을비교조사하였다 (Fig. 2). Glucoamylase와 α-amylase 모두생산분비하는 NBRC 2346GSHSGSA는 glucoamylase만을생산하는 7904GSHSG과비교하였을때전분분해능이좋아더빠른생장을보였고배양 3일째에 100% 전분을가수분해하였다. α- Amylase만을생산분비하는 NBRC 2346GSHSA는전분당화능이없어낮은생장을보였다 (Im et al., 2013). NBRC 2346GSHSGSA, NBRC 2346GSHSG, 그리고 NBRC 2346GSHSA 를 2% (w/v) 쌀전분배지에서배양했을때시간별로효소활성의증가를비교하였다 (Fig. 3). 효소활성은생장에비례하여배양 3일째최대에이르렀는데 NBRC 2346GSHSGSA의 Table 1. Glutathione content and glucoamylase activities in the culture broth of S. cerevisiae transformants GSH content a Glucoamylase activity b Yeast strains (mg/l) (U/ml) S. cerevisiae NBRC 2346 23.5 ± 0.5 ND c NBRC 2346SG 23.3 ± 0.3 d 0.38 ± 0.04 NBRC 2346GSHSA 34.5 ± 0.3 ND NBRC 2346GSHSG 34.6 ± 0.4 0.37 ± 0.07 NBRC 2346GSHSGSA 34.2 ± 0.6 0.72 ± 0.05 a Yeast cells were grown in the YPD at 30 C for 3 days b Yeast cells were grown in the YP containing 2% (w/v) soluble starch at 30 C for 3 days c Not detected d Values are means of results from three independent experiments 미생물학회지제 52 권제 2 호
쌀전분분해효모개발과발효 223 glucoamylase 활성은 NBRC 2346GSHSG의 2배이었고, α- amylase 활성은 7904GSHSGSA에비해 24배증가하였다. 특히 α-amylase 활성이증가된이유로는이균주가 glucoamylase Fig. 2. Time courses of growth and rice starch degradation by NBRC 2346GSHSGSA (squares), NBRC 2346GSHSG or NBRC 2346SG (circles) and NBRC 2346GSHSA (triangles). Yeast cells were grown in the YP media containing 2% (w/v) rice starch at 30 C for 5 days. Growth was measured on different days based on the cell dry weight. The remaining starch values were presented as percentages considering the starch in the uninoculated medium as 100%. Filled and unfilled symbols represent cell concentration and relative residual starch content, respectively. Data (mean ± SD) are from three independent experiments performed in triplicate. 활성으로전분으로부터포도당을생성하여생장하면서 glucoamylase는물론 α-amylase 생산도증가되었고, 다시 α-amylase 분해산물인 maltose, isomaltose, maltotriose그리고 dextrin이 glucoamylase의기질로포도당으로분해되어이를탄소원으로청주효모의빠른생장에기여하였고그결과 α-amylase 효소생산도동시에크게증가되었다고사료되었다 (Eksteen et al., 2003; Shigechi et al., 2004; Kim et al., 2010). 20% (w/v) 쌀전분을함유한발효액에 NBRC 2346GSHSGSA 와 NBRC 2346GSHSG를접종하여에탄올생산과전분감소를조사하였다. Fig. 4에서보는바와같이, glucoamylase만생산하는 NBRC 2346GSHSG는전분당화능은있으나, 액화능이없어점도가낮은 2% (w/v) 쌀전분을기질로배양한경우 (Fig. 3) 와달리점도가매우높은 20% (w/v) 쌀전분을가수분해하지못해에탄올생산이원균주인 S. cerevisiae NBRC 2346같이거의없었다. 반면에 glucoamylase와 α-amylase 모두생산분비하는 NBRC 2346GSHSGSA에서는 glucoamylase 와 α-amylase에의해높은점도의쌀전분이액화되어점도가낮은액상발효액으로전환되고이어서포도당으로분해되어발효에이용됨으로써발효 2일째에 6% (v/v) 이상에탄올이생산되었고이후에도에탄올생산이계속증가하여 7일째에거의최대치에이르게되어에탄올함량이 11% (v/v) 로증가하였고 90% 이상의전분이분해되어에탄올로전환되었음을알수있었다. 이와같은결과로청주효모가 glucoamylase와 α -amylase활성을모두갖게되면누룩의첨가없이도직접쌀전 Fig. 3. Extracellular glucoamylase and α-amylase activities produced by NBRC 2346GSHSGSA, NBRC 2346GSHSG, and NBRC 2346GSHSA at 30 C in the YP medium containing 2% (w/v) rice starch. The graph marked with filled circles represents α-amylase activities of NBRC 2346GSHSGSA; filled squares, glucoamylase activities of NBRC 2346GSHSGSA; unfilled squares, glucoamylase activities of NBRC 2346GSHSG; unfilled circles, α-amylase activities of NBRC 2346GSHSA. Data (mean ± SD) are from three independent experiments performed in triplicate. Fig. 4. Time course of ethanol fermentation from 20% (w/v) rice starch by NBRC 2346GSHSGSA (squares), NBRC 2346GSHSG (triangles), and S. cerevisiae NBRC 2346 (circles). Filled and unfilled symbols represent ethanol concentration and relative residual starch content, respectively. Data are averages from three independent experiments. Korean Journal of Microbiology, Vol. 52, No. 2
224 Lee et al. 분을당화발효하여청주와같은알코올음료를제조할수있고신선도유지를위해 GSH가증가된발효음료생산이가능할것으로사료되었다 (Wang et al., 2010a). 현재다수의형질전환을통해 GSH 함량과 glucoamylase와 α-amylase 활성이보다증진된청주효모균주들을찾는연구가진행중이다. 적요 쌀전분을원료로효모를직접이용하여항산화제 glutathione (GSH) 이풍부한알코올음료를제조할목적으로 GSH 함량과 당화능을증진시키기위하여 Saccharomyces cerevisiae의 γ- glutamylcysteine synthetase 유전자 (GSH1), Debaryomyces occidentalis 의 glucoamylase 유전자 (GAM1), 그리고 α-amylase 유전자 (AMY) 를청주효모 S. cerevisiae 에서공동발현시켰다. 재조합청주효모의세포외 GSH 함량은원균주에비해 1.5 배 증가하였다. 2% (w/v) 쌀전분이함유된배지에서배양하였을 때 GAM1 과 AMY 유전자모두발현하는효모균주의 glucoamylase 에의한당화능은 GAM1 유전자만발현하는균주와 비교하여 2 배증가하였다. 이새로운균주는쌀전분이 20% (w/v) 함유된배지에서 7 일간발효를통해에탄올 11% (v/v) 를 생산하였고, 전분함유량의 90% 이상을소비하였다. References Asano, T., Kurose, N., and Tarumi, S. 2001. Isolation of high-malateproducing sake yeasts from low-maltose-assimilating mutants. J. Biosci. Bioeng. 92, 429 433. Baba, S., Oguri, I., Fukuzawa, M., Moriyama, K., Lida, T., Kobayashi, I., and Imai, K. 1974. Maltose assimilation by Saccharomyces sake. J. Brew. Soc. Jpn. 69, 453 455. Chen, J.L., Xie, L., Cai, J.J., Yang, C.S., and Duan, X.H. 2013. Enzymatic synthesis of glutathione using engineered Saccharomyces cerevisiae. Biotechnol. Lett. 35, 1259 1264. Dohmen, R.J., Strasser, A.W.M., Dahlems, U.M., and Hollenberg, C.P. 1990. Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in Saccharomyces cerevisiae. Gene 95, 111 121. Domingues, L., Onnela, M.L., Teixeira, J.A., Lima, N., and Penttila, M. 2000. Construction of a flocculent brewer s yeast strain secreting Aspergillus niger β-galactosidase. Appl. Microbiol. Biotechnol. 54, 97 103. Eksteen, J.M., van Renseburg, P., Cordero Otero, R.R., and Pretorius, I.S. 2003. Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the α-amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol. Bioeng. 84, 639 646. Fan, X., He, X., Guo, X., Qu, N., Wang, C., and Zhang, B. 2004. Increasing glutathione formation by functional expression of the γ-glutamylcysteine synthetase gene in Saccharomyces cerevisiae. Biotechnol. Lett. 26, 415 417. Fujieda, T., Kitamura, Y., Yamasaki, H., Furuishi, A., and Motobayashi, K. 2012. An experimental study on whole paddy saccharification and fermentation for rice ethanol production. Biomass Bioeng. 44, 135 141. Ghang, D.M., Yu, L., Lim, M.H., Ko, H.M., Im, S.Y., Lee, H.B., and Bai, S. 2007. Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis. Biotechnol. Lett. 29, 1203 1208. Gietz, D., St. Jean, A., Woods, R., and Schiestl, R.H. 1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425. Im, Y.K., Park, J.Y., Lee, J.Y., Choi, S.H., Chin, J.E., Ko, H.M., Kim, I.C., and Bai, S. 2013. Construction of amylolytic industrial strains of Saccharomyces cerevisiae for improved ethanol production from raw starch. Korean J. Microbiol. 40, 200 204. Kang, N.Y., Park, J.N., Chin, J.E., Lee, H.B., Im, S.Y., and Bai, S. 2003. Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis α-amylase gene. Biotechnol. Lett. 25, 1847 1851. Kim, H.R., Im, Y.K., Ko, H.M., Chin, J.E., Kim, I.C., Lee, H.B., and Bai, S. 2011. Raw starch fermentation to ethanol by an industrial distiller s strain of Saccharomyces cerevisiae expressing glucoamylase and α-amylase genes. Biotechnol. Lett. 33, 1643 1648. Kim, J.H., Kim, H.R., Lim, M.H., Ko, H.M., Chin, J.E., Lee, H.B., Kim, I.C., and Bai, S. 2010. Construction of a direct starchfermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, α-amylase and debranching enzyme. Biotechnol. Lett. 32, 713 719. Kim, K., Park, C.S., Mattoon, J.R. 1988. High-efficiency, one-step utilization by transformed Saccharomyces cerevisiae cells which secrete both yeast glucoamylase and mouse α-amylase. Appl. Environ. Microbiol. 54, 966 971. Lee, F.W. and Da Silva, N.A. 1997. Improved efficiency and stability of multiple cloned gene insertions at the δ sequences of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 48, 339 345. Lee, J.Y., Im, Y.K., Ko, H.M., Chin, J.E., Kim, I.C., Lee, H.B., and Bai, S. 2015. Direct utilization of purple sweet potato by sake yeasts to produce an anthocyanin-rich alcoholic beverage. Biotechnol. Lett. 37, 1439 1445. Lin, L.L., Ma, Y.J., Chien, H.R., and Hsu, W.H. 1998. Construction of an amylolytic yeast by multiple integration of the Aspergillus awamori glucoamylase gene into a Saccharomyces cerevisiae chromosome. Enzyme Microb. Technol. 23, 360 365. 미생물학회지제 52 권제 2 호
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