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Construction of a β-glucosidase expression system using the multistress-tolerant yeast Issatchenkia orientalis

  • Applied Genetics and Molecular Biotechnology
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Abstract

We demonstrate the value of the thermotolerant yeast Issatchenkia orientalis as a candidate microorganism for bioethanol production from lignocellulosic biomass with the goal of consolidated bioprocessing. The I. orientalis MF-121 strain is acid tolerant, ethanol tolerant, and thermotolerant, and is thus a multistress-tolerant yeast. To express heterologous proteins in I. orientalis, we constructed a transformation system for the MF-121 strain and then isolated the promoters of TDH1 and PGK1, two genes that were found to be strongly expressed during ethanol fermentation. As a result, expression of β-glucosidase from Aspergillus aculeatus could be achieved with I. orientalis, demonstrating successful heterologous gene expression in I. orientalis for the first time. The transformant could convert cellobiose to ethanol under acidic conditions and at high temperature. Simultaneous saccharification and fermentation (SSF) was performed with the transformant, which produced 29 g l−1 of ethanol in 72 h at 40°C even without addition of β-glucosidase when SSF was carried out in medium containing 100 g l−1 of microcrystalline cellulose and a commercial cellulase preparation. These results suggest that using a genetically engineered thermotolerant yeast such as I. orientalis in SSF could lead to cost reduction because less saccharification enzymes are required.

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References

  • Ballesteros I, Ballesteros M, Cabañas A, Carrasco J, Martín C, Negro MJ, Saez F, Saez R (1991) Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol. Appl Biochem Biotechnol 28–29:307–315

    Article  Google Scholar 

  • Ballesteros I, Oliva JM, Ballesteros M, Carrasco J (1993) Optimization of the simultaneous saccharification and fermentation process using thermotolerant yeasts. Appl Biochem Biotechnol 39–40:201–211

    Article  Google Scholar 

  • Banat IM, Nigam P, Marchant R (1992) Isolation of thermotolerant, fermentative yeasts growing at 52°C and producing ethanol at 45°C and 50°C. World J Microbiol Biotechnol 8:259–283

    Article  CAS  Google Scholar 

  • Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346

    Article  CAS  Google Scholar 

  • Bollók M, Réczey K, Zacchi G (2000) Simultaneous saccharification and fermentation of steam-pretreated spruce to ethanol. Appl Biochem Biotechnol 84–86:69–80

    Article  Google Scholar 

  • Chen X, Fang H, Rao Z, Shen W, Zhuge B, Wang Z, Zhuge J (2008) An efficient genetic transformation method for glycerol producer Candida glycerinogenes. Microbiol Res 163:531–537

    Article  CAS  Google Scholar 

  • Daniel HM, Vrancken G, Takrama JF, Camu N, De Vos P, De Vuyst L (2009) Yeast diversity of Ghanaian cocoa bean heap fermentations. FEMS Yeast Res 9:774–783

    Article  CAS  Google Scholar 

  • De Backer MD, Maes D, Vandoninck S, Logghe M, Contreras R, Luyten WH (1999) Transformation of Candida albicans by electroporation. Yeast 15:1609–1618

    Article  Google Scholar 

  • Dmytruk OV, Dmytruk KV, Abbas CA, Voronovsky AY, Sibirny AA (2008) Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha. Microb Cell Fact 7:21

    Article  Google Scholar 

  • Faber KN, Haima P, Harder W, Veenhuis M, AB G (1994) Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr Genet 25:305–310

    Article  CAS  Google Scholar 

  • Fujita Y, Takahashi S, Ueda M, Tanaka A, Okada H, Morikawa Y, Kawaguchi T, Arai M, Fukuda H, Kondo A (2002) Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes. Appl Environ Microbiol 68:5136–5141

    Article  CAS  Google Scholar 

  • Fujita Y, Ito J, Ueda M, Fukuda H, Kondo A (2004) Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Appl Environ Microbiol 70:1207–1212

    Article  CAS  Google Scholar 

  • Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O’Shea EK, Weissman JS (2003) Global analysis of protein expression in yeast. Nature 425:737–741

    Article  CAS  Google Scholar 

  • Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34

    Article  CAS  Google Scholar 

  • Haas LO, Cregg JM, Gleeson MA (1990) Development of an integrative DNA transformation system for the yeast Candida tropicalis. J Bacteriol 172:4571–4577

    CAS  Google Scholar 

  • Hisamatsu M, Furubayashi T, Karita S, Mishima T, Isono N (2006) Isolation and identification of a novel yeast fermenting ethanol under acidic conditions. J Appl Glycosci 53:111–113

    CAS  Google Scholar 

  • Hisatomi T, Kuroyanagi S, Tsuboi M (1998) Identification of the orotidine-5′-phosphate decarboxylase gene and development of a transformation system in the yeast Saccharomyces exiguus Yp74L-3. Biosci Biotechnol Biochem 62:2280–2282

    Article  CAS  Google Scholar 

  • Hong J, Wang Y, Kumagai H, Tamaki H (2007) Construction of thermotolerant yeast expressing thermostable cellulase genes. J Biotechnol 130:114–123

    Article  CAS  Google Scholar 

  • Katemai W, Maneerat S, Kawai F, Kanzaki H, Nitoda T, H-Kittikun A (2008) Purification and characterization of a biosurfactant produced by Issatchenkia orientalis SR4. J Gen Appl Microbiol 54:79–82

    Article  CAS  Google Scholar 

  • Kawaguchi T, Enoki T, Tsurumaki S, Sumitani J, Ueda M, Ooi T, Arai M (1996) Cloning and sequencing of the cDNA encoding β-glucosidase 1 from Aspergillus aculeatus. Gene 173:287–288

    Article  CAS  Google Scholar 

  • Koh JH, Suh HJ (2009) Biological activities of thermo-tolerant microbes from fermented rice bran as an alternative microbial feed additive. Appl Biochem Biotechnol 157:420–430

    Article  CAS  Google Scholar 

  • Li Y, Shen W, Wang Z, Liu JQ, Rao Z, Tang X, Fang H, Zhuge J (2005) Isolation and sequence analysis of the gene URA3 encoding the orotidine-5′-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer. Yeast 22:423–430

    Article  CAS  Google Scholar 

  • Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577

    Article  CAS  Google Scholar 

  • Mandels M, Andreotti R, Roche C (1976) Measurement of saccharifying cellulase. Biotechnol Bioeng Symp 6:21–33

    CAS  Google Scholar 

  • Nonklang S, Abdel-Banat BM, Cha-aim K, Moonjai N, Hoshida H, Limtong S, Yamada M, Akada R (2008) High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast Kluyveromyces marxianus DMKU3-1042. Appl Environ Microbiol 74:7514–7521

    Article  CAS  Google Scholar 

  • Nonklang S, Ano A, Abdel-Banat BM, Saito Y, Hoshida H, Akada R (2009) Construction of flocculent Kluyveromyces marxianus strains suitable for high-temperature ethanol fermentation. Biosci Biotechnol Biochem 73:1090–1095

    Article  CAS  Google Scholar 

  • Prillinger H, Molnár O, Eliskases-Lechner F, Lopandic K (1999) Phenotypic and genotypic identification of yeasts from cheese. Antonie Van Leeuwenhoek 75:267–283

    Article  CAS  Google Scholar 

  • Rajoka MI, Shaukat F, Ghauri MT, Shahid R (2003) Kinetics of β-glucosidase production by Saccharomyces cerevisiae recombinants harboring heterologous bgl genes. Biotechnol Lett 25:945–948

    Article  CAS  Google Scholar 

  • Rajoka MI, Ferhan M, Khalid AM (2005) Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor-controlled bioreactor. Lett Appl Microbiol 40:316–321

    Article  CAS  Google Scholar 

  • Sakai Y, Kazarimoto T, Tani Y (1991) Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5′-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation. J Bacteriol 173:7458–7463

    CAS  Google Scholar 

  • Sánchez M, Iglesias FJ, Santamaría C, Domínguez A (1993) Transformation of Kluyveromyces lactis by electroporation. Appl Environ Microbiol 59:2087–2092

    Google Scholar 

  • Schwarz WH, Gräbnitz F, Staudenbauer WL (1986) Properties of a Clostridium thermocellum endoglucanase produced in Escherichia coli. Appl Environ Microbiol 51:1293–1299

    CAS  Google Scholar 

  • Seo SH, Rhee CH, Park HD (2007) Degradation of malic acid by Issatchenkia orientalis KMBL 5774, an acidophilic yeast strain isolated from Korean grape wine pomace. J Microbiol 45:521–527

    CAS  Google Scholar 

  • Shaw AJ, Podkaminer KK, Desai SG, Bardsley JS, Rogers SR, Thorne PG, Hogsett DA, Lynd LR (2008) Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc Natl Acad Sci U S A 105:13769–13774

    Article  CAS  Google Scholar 

  • Shimoda C, Itadani A, Sugino A, Furusawa M (2006) Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae. Genes Genet Syst 81:391–397

    Article  CAS  Google Scholar 

  • Sridhar M, Sree NK, Rao LV (2002) Effect of UV radiation on thermotolerance, ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains. Bioresour Technol 83:199–202

    Article  CAS  Google Scholar 

  • Suga M, Hatakeyama T (2001) High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation. Yeast 18:1015–1021

    Article  CAS  Google Scholar 

  • Suryawati L, Wilkins MR, Bellmer DD, Huhnke RL, Maness NO, Banat IM (2008) Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4. Biotechnol Bioeng 101:894–902

    Article  CAS  Google Scholar 

  • Tailliez P, Girard H, Millet J, Beguin P (1989) Enhanced cellulose fermentation by an asporogenous and ethanol-tolerant mutant of Clostridium thermocellum. Appl Environ Microbiol 55:207–211

    CAS  Google Scholar 

  • Thalagala TATP, Kodama S, Mishima T, Isono N, Furujyo A, Kawasaki Y, Hisamatsu M (2009) Study on ethanol fermentation using D-glucose rich fractions obtained from lignocelluloses by a two-step extraction with sulfuric acid and Issatchenkia orientalis MF 121. J Appl Glycosci 56:7–11

    CAS  Google Scholar 

  • Tokuhiro K, Ishida N, Kondo A, Takahashi H (2008) Lactic fermentation of cellobiose by a yeast strain displaying beta-glucosidase on the cell surface. Appl Microbiol Biotechnol 79:481–488

    Article  CAS  Google Scholar 

  • Tomás-Pejó E, García-Aparicio M, Negro MJ, Oliva JM, Ballesteros M (2009) Effect of different cellulase dosages on cell viability and ethanol production by Kluyveromyces marxianus in SSF processes. Bioresour Technol 100:890–895

    Article  Google Scholar 

  • Van Bogaert IN, De Maeseneire SL, De Schamphelaire W, Develter D, Soetaert W, Vandamme EJ (2007) Cloning, characterization and functionality of the orotidine-5'-phosphate decarboxylase gene (URA3) of the glycolipid-producing yeast Candida bombicola. Yeast 24:201–208

    Article  Google Scholar 

  • van Zyl WH, Lynd LR, den Haan R, McBride JE (2007) Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae. Adv Biochem Engin/Biotechnol 108:205–235

    Article  Google Scholar 

  • Wang Z, Zhuge J, Fang H (1999) A new osmotolerant and glycerol-highly-producing species—Candida glycerolgenesis Zhuge sp. nov. Wei Sheng Wu Xue Bao 39:68–74

    CAS  Google Scholar 

  • Wang X, Li G, Deng Y, Yu X, Chen F (2005) A site-directed integration system for the nonuniversal CUG(Ser) codon usage species Pichia farinosa by electroporation. Arch Microbiol 184:419–424

    Article  Google Scholar 

  • Watanabe I, Nakamura T, Shima J (2009) Characterization of a spontaneous flocculation mutant derived from Candida glabrata: a useful strain for bioethanol production. J Biosci Bioeng 107:379–382

    Article  CAS  Google Scholar 

  • Yamagata K, Kato J, Shimamoto A, Goto M, Furuichi Y, Ikeda H (1998) Bloom's and Werner's syndrome genes suppress hyperrecombination in yeast sgs1 mutant: implication for genomic instability in human diseases. Proc Natl Acad Sci U S A 95:8733–8738

    Article  CAS  Google Scholar 

  • Yang VW, Marks JA, Davis BP, Jeffries TW (1994) High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2. Appl Environ Microbiol 60:4245–4254

    CAS  Google Scholar 

  • Zhou J, Dong Z, Liu L, Du G, Chen J (2009) A reusable method for construction of non-marker large fragment deletion yeast auxotroph strains: a practice in Torulopsis glabrata. J Microbiol Methods 76:70–74

    Article  CAS  Google Scholar 

  • Zhuge J, Fang HY, Wang ZX, Chen DZ, Jin HR, Gu HL (2001) Glycerol production by a novel osmotolerant yeast Candida glycerinogenes. Appl Microbiol Biotechnol 55:686–692

    Article  CAS  Google Scholar 

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Acknowledgments

The pYO323 plasmid vector (NBRP ID number BYP563) of YEp type harboring the HIS3 marker was kindly donated by the National Bioresource Project (Yeast) of Japan (http://yeast.lab.nig.ac.jp/nig/index_en.html). We also thank Risa Nagura, Satoshi Katahira, and Takashi Matsuyama for technical assistance.

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Authors and Affiliations

  1. Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, 2-16-1, Tokiwadai, Ube, 755-8611, Japan

    Takao Kitagawa

  2. Biotechnology Laboratory, Toyota Central R&D Labs, Inc., 41-1 Yokomichi, Nagakute, Aichi, 480-1192, Japan

    Kenro Tokuhiro, Katsuhiro Kohda & Haruo Takahashi

  3. Environmental and Applied Biotechnology Laboratory, Toyota Central R&D Labs, Inc., 41-1 Yokomichi, Nagakute, Aichi, 480-1192, Japan

    Hidehiko Sugiyama & Takao Imaeda

  4. Laboratory of Food Science and Biotechnology, Department of Sustainable Resource Sciences, Graduate School of Bioresources, Mie University, 1577 Kurima, Tsu, Mie, 514-8507, Japan

    Naoto Isono & Makoto Hisamatsu

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  1. Takao Kitagawa
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  2. Kenro Tokuhiro
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  3. Hidehiko Sugiyama
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  4. Katsuhiro Kohda
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Correspondence to Takao Kitagawa.

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Takao Kitagawa and Kenro Tokuhiro contributed equally to this work.

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Kitagawa, T., Tokuhiro, K., Sugiyama, H. et al. Construction of a β-glucosidase expression system using the multistress-tolerant yeast Issatchenkia orientalis . Appl Microbiol Biotechnol 87, 1841–1853 (2010). https://doi.org/10.1007/s00253-010-2629-9

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