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Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae

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Abstract

The catalytic fraction of the Cellulomonas flavigena PN-120 oligomeric β-glucosidase (BGLA) was expressed both intra- and extracellularly in a recombinant diploid of Saccharomyces cerevisiae, under limited nutrient conditions. The recombinant enzyme (BGLA15) expressed in the supernatant of a rich medium showed 582 IU/L and 99.4 IU/g dry cell, with p-nitrophenyl-β-d-glucopyranoside as substrate. BGLA15 displayed activity against cello-oligosaccharides with 2–5 glucose monomers, demonstrating that the protein is not specific for cellobiose and that the oligomeric structure is not essential for β-d-1,4-bond hydrolysis. Native β-glucosidase is inhibited almost completely at 160 mM glucose, thus limiting cellobiose hydrolysis. At 200 mM glucose concentration, BGLA15 retained more than 50 % of its maximal activity, and even at 500 mM glucose concentration, more than 30 % of its activity was preserved. Due to these characteristics of BGLA15 activity, recombinant S. cerevisiae is able to utilize cellulosic materials (cello-oligosaccharides) to produce bioethanol.

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References

  • Barrera-Islas GA, Ramos-Valdivia AC, Salgado LM, Ponce-Noyola T (2007) Characterization of a β-glucosidase produced by a high-specific growth-rate mutant of Cellulomonas flavigena. Curr Microbiol 54:266–270

    Article  CAS  PubMed  Google Scholar 

  • Bayer EA, Raphael LR, Himmel ME (2007) The potential of cellulases and cellulosomes for cellulosic waste management. Curr Opin Biotech 18:237–245

    Article  CAS  PubMed  Google Scholar 

  • Benoliel B, Poças-Fonseca MJ, Gonçalves Torres FA, Pepe de Moraes LM (2010) Expression of a glucose-tolerant β-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae. Appl Biochem Biotechnol 160:2036–2044

    Article  CAS  PubMed  Google Scholar 

  • Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties and applications. Crit Rev Biotechnol 22:375–407

    Article  CAS  PubMed  Google Scholar 

  • Chirico WI, Brown RD Jr (1985) Separation of [1-3H] cellooligosaccharides by thin-layer chromatography assay for cellulolytic enzymes. Anal Biochem 150:264–272

    Article  CAS  PubMed  Google Scholar 

  • Cho KM, Yoo YJ, Kanq HS (1999) δ-integration of endo/exo-glucanase and β-glucosidase genes into the yeast chromosomes for direct conversion of cellulose to ethanol. Enzyme Microb Technol 25:23–30

    Article  CAS  Google Scholar 

  • Choi ES, Sohn JH, Rhee SK (1994) Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 42:587–594

    Article  CAS  PubMed  Google Scholar 

  • Den Haan R, McBride JE, La Grange DC, Lynd LR, Van Zyl W (2007a) Functional expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. Enzyme Microb Technol 40:1291–1299

    Article  Google Scholar 

  • Den Haan R, Rose SH, Lynd LR, van Zyl WH (2007b) Hydrolysis and fermentation of amorphous cellulose by recombinant S. cerevisiae. Metab Eng 9:87–94

    Article  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  PubMed Central  PubMed  Google Scholar 

  • Gietz D, Woods RA (2006) Methods in molecular biology. In: Xiao Wei (ed) Yeast protocols, 2nd edn. Human Press Inc, Totowa, New Jersey, pp 107–120

    Google Scholar 

  • Görgens JF, Passoth V, van Zyl WH, Knoetze JH, Hahn-Hägendal B (2005) Amino acids supplementation controlled oxygen limitation and sequential double induction improves heterologous xylanase production by P. stipites. FEMS Yeast Res 5:677–683

    Article  PubMed  Google Scholar 

  • Hahn-Hägerdal B, Karhumaa K, Larsson CU, Gorwa-Grauslund M, Görgens J, van Zyl W (2005) Role of cultivation media in the development of yeast strain for large scale industrial use. Microb Cell Fact 4:1–16

    Article  Google Scholar 

  • Henrrissat B, Claeyssens M, Tomme P, Lemesle L, Mornon JP (1989) Cellulase families revealed by hydrophobic cluster-analysis. Gene 81:83–95

    Article  Google Scholar 

  • Hong J, Tamaki H, Kumagai H (2007) Cloning and functional expression of thermostable β-glucosidase gene from Thermoascus aurantiacus. Appl Microbiol Biotechnol 73:1331–1339

    Article  CAS  PubMed  Google Scholar 

  • Hrmova M, Fichner BG (2007) Dissecting the catalytic mechanism of a plant β-D-glucan glucohydrolase through structural biology using inhibitors and substrate analogues. Carbohydr Res 342:1613–1623

    Article  CAS  PubMed  Google Scholar 

  • Hrmova M, Streltson AV, Smith JB, Vasella A, Varghese NJ, Fincher BG (2005) Structural rationale for low-nanomolar binding of transition state mimics to a family GH3 β-D-glucan glucohydrolase from barley. Biochemistry 44:16529–16539

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Njokweni AP, Rose SH, van Zyl WH (2012) Fungal β-glucosidase expression in Saccharomyces cerevisiae. J Ind Microbiol Biotechnol 39:1445–1452

    Article  CAS  PubMed  Google Scholar 

  • Pack SP, Park K, Yoo YJ (2002) Enhancement of β-glucosidase stability and cellobiose-usage using surface engineered recombinant Saccharomyces cerevisiae in ethanol production. Biotechnol Lett 24:1919–1925

    Article  CAS  Google Scholar 

  • Penttilä ME, Andre L, Lehtovaara P, Bailey M, Teeri TT, Knowles JK (1988) Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. Gene 63:103–112

    Article  PubMed  Google Scholar 

  • Ponce-Noyola T, de la Torre M (1995) Isolation of a high-specific-growth-rate mutant of Cellulomonas flavigena on sugar cane bagasse. Appl Microbiol Biotechnol 42:709–712

    Article  CAS  Google Scholar 

  • Ponce-Noyola T, de la Torre M (2001) Regulation of cellulases and xylanases from a derepressed mutant of Cellulomonas flavigena growing on sugar-cane bagasse in continuous culture. Bioresour Technol 78:285–291

    Article  CAS  PubMed  Google Scholar 

  • Pozzo T, Pasten LJ, Karlsson NE, Logan DT (2010) Structural and functional analyses of β-glucosidase 3B Thermotoga neapolitana: a thermostable three-domain representative of glycoside hydrolase 3. J Mol Biol 397:724–739

    Article  CAS  PubMed  Google Scholar 

  • Sambrook JE, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York

    Google Scholar 

  • Sánchez-Torres P, González-Candelas L, Ramón D (1998) Heterologous expression of a Candida molischiana anthocyanin-β-glucosidase in a wine yeast strain. J Agric Food Chem 46:354–360

    Article  PubMed  Google Scholar 

  • Schuster BG, Chinn MS (2012) Consolidated bioprocessing of lignocellulose feedstocks for ethanol fuel production. Bioenerg Res 6:416–435

    Article  Google Scholar 

  • Shen Y, Zhang Y, Ma T, Bao X, Du F, Zhuang G, Qu Y (2008) Simultaneous saccharification and fermentation of acid-pretreated corncobs with a recombinant Saccharomyces cerevisiae expressing β-glucosidase. Bioresour Technol 99:5099–5103

    Article  CAS  PubMed  Google Scholar 

  • Skory CD, Freer SN, Bothast RJ (1996) Expression and secretion of the Candida wickerhamii extracellular β-glucosidase gene, bglB, in Saccharomyces cerevisiae. Curr Genet 30:417–422

    Article  CAS  PubMed  Google Scholar 

  • Spiridonov NA, Wilson DB (2001) Cloning and biochemical characterization of BGLC, a β-glucosidase from the cellulolytic actinomycete Thermobifida fusca. Curr Microbiol 42:295–301

    CAS  PubMed  Google Scholar 

  • Van Zyl WH, Lynd LR, Den Haan R, Mc Bride JE (2007) Consolidate bioprocessing for bioethanol production using Saccharomyces cerevisiae. Adv Biochem Engin Biotechnol 108:205–235

    Google Scholar 

  • Wildt S, Gerngross TU (2005) The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3:119–127

    Article  CAS  PubMed  Google Scholar 

  • Yamada R, Tanaka T, Ogino C (2010) Gene copy number and polyploid on products formation in yeast. Appl Microbiol Biotechnol 88:849–857

    Article  CAS  PubMed  Google Scholar 

  • Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A (2011) Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression. Biotechnol Biofuels 4:1–8

    Article  Google Scholar 

  • Yan T, Lin Y, Lin C (1998) Purification and characterization of an extracellular β-glucosidase II with high hydrolysis and transglucosylation activities from Asperillus niger. J Agric Food Chem 46:431–437

    Article  CAS  PubMed  Google Scholar 

  • Yoshida E, Hidaka M, Foshinobu S, Koyanagi T, Minami H, Tamakis H, Kitaoka M, Katayama T, Kumagi H (2010) Role of a PA14 domain in determining substrate specificity of a glucoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus. Biochem J 431:39–49

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Consejo Nacional de Ciencia y Tecnología México (CONACYT) (Grant 104333). D. J. Mendoza-Aguayo received a scholarship number 204305 from CONACYT México. María Isabel Pérez- Montfort corrected the English version of the manuscript.

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Authors and Affiliations

  1. Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, AV. IPN 2508, 07300, Zacatenco, DF, México

    David J. Mendoza-Aguayo, Héctor M. Poggi-Varaldo, Ana C. Ramos-Valdivia & Teresa Ponce-Noyola

  2. Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, AV. IPN 2508, 07300, Zacatenco, DF, México

    Jaime García-Mena

  3. CICATA, Unidad Querétaro, Instituto Politécnico Nacional, Cerro blanco 141, 76090, Querétaro, México

    Luis M. Salgado

  4. Departamento de Análisis de los Alimentos, Centro de Investigación en Alimentación y Desarrollo, Carretera a la Victoria Km 6, 83304, Hermosillo, Sonora, México

    Mayra de la Torre-Martínez

Authors
  1. David J. Mendoza-Aguayo
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  2. Héctor M. Poggi-Varaldo
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  3. Jaime García-Mena
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  4. Ana C. Ramos-Valdivia
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  5. Luis M. Salgado
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  6. Mayra de la Torre-Martínez
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  7. Teresa Ponce-Noyola
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Corresponding author

Correspondence to Teresa Ponce-Noyola.

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Communicated by Erko Stackebrandt.

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Mendoza-Aguayo, D.J., Poggi-Varaldo, H.M., García-Mena, J. et al. Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae . Arch Microbiol 196, 25–33 (2014). https://doi.org/10.1007/s00203-013-0935-1

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  • DOI: https://doi.org/10.1007/s00203-013-0935-1

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