Advertisement

Archives of Microbiology

, Volume 196, Issue 1, pp 25–33 | Cite as

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

  • David J. Mendoza-Aguayo
  • Héctor M. Poggi-Varaldo
  • Jaime García-Mena
  • Ana C. Ramos-Valdivia
  • Luis M. Salgado
  • Mayra de la Torre-Martínez
  • Teresa Ponce-NoyolaEmail author
Original Paper

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.

Keywords

Cellulomonas flavigena β-Glucosidase Inhibition resistant Saccharomyces cerevisiae 

Notes

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.

References

  1. 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 54:266–270PubMedCrossRefGoogle Scholar
  2. Bayer EA, Raphael LR, Himmel ME (2007) The potential of cellulases and cellulosomes for cellulosic waste management. Curr Opin Biotech 18:237–245PubMedCrossRefGoogle Scholar
  3. 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 160:2036–2044PubMedCrossRefGoogle Scholar
  4. Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties and applications. Crit Rev Biotechnol 22:375–407PubMedCrossRefGoogle Scholar
  5. Chirico WI, Brown RD Jr (1985) Separation of [1-3H] cellooligosaccharides by thin-layer chromatography assay for cellulolytic enzymes. Anal Biochem 150:264–272PubMedCrossRefGoogle Scholar
  6. 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 25:23–30CrossRefGoogle Scholar
  7. 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 42:587–594PubMedCrossRefGoogle Scholar
  8. 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 40:1291–1299CrossRefGoogle Scholar
  9. Den Haan R, Rose SH, Lynd LR, van Zyl WH (2007b) Hydrolysis and fermentation of amorphous cellulose by recombinant S. cerevisiae. Metab Eng 9:87–94CrossRefGoogle Scholar
  10. 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 68:5136–5141PubMedCentralPubMedCrossRefGoogle Scholar
  11. Gietz D, Woods RA (2006) Methods in molecular biology. In: Xiao Wei (ed) Yeast protocols, 2nd edn. Human Press Inc, Totowa, New Jersey, pp 107–120Google Scholar
  12. 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 5:677–683PubMedCrossRefGoogle Scholar
  13. 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 4:1–16CrossRefGoogle Scholar
  14. Henrrissat B, Claeyssens M, Tomme P, Lemesle L, Mornon JP (1989) Cellulase families revealed by hydrophobic cluster-analysis. Gene 81:83–95CrossRefGoogle Scholar
  15. Hong J, Tamaki H, Kumagai H (2007) Cloning and functional expression of thermostable β-glucosidase gene from Thermoascus aurantiacus. Appl Microbiol Biotechnol 73:1331–1339PubMedCrossRefGoogle Scholar
  16. 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 342:1613–1623PubMedCrossRefGoogle Scholar
  17. 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 44:16529–16539PubMedCrossRefGoogle Scholar
  18. Lynd LR, Weimer PJ, van Zyl WH, Protorius IS (2002) Microbial cellulase utilization: fundamentals and biotechnology. Microbiol Mol Biol 66:506–577CrossRefGoogle Scholar
  19. Njokweni AP, Rose SH, van Zyl WH (2012) Fungal β-glucosidase expression in Saccharomyces cerevisiae. J Ind Microbiol Biotechnol 39:1445–1452PubMedCrossRefGoogle Scholar
  20. 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 24:1919–1925CrossRefGoogle Scholar
  21. Penttilä ME, Andre L, Lehtovaara P, Bailey M, Teeri TT, Knowles JK (1988) Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. Gene 63:103–112PubMedCrossRefGoogle Scholar
  22. 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 42:709–712CrossRefGoogle Scholar
  23. 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 78:285–291PubMedCrossRefGoogle Scholar
  24. 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 397:724–739PubMedCrossRefGoogle Scholar
  25. Sambrook JE, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New YorkGoogle Scholar
  26. 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 46:354–360PubMedCrossRefGoogle Scholar
  27. Schuster BG, Chinn MS (2012) Consolidated bioprocessing of lignocellulose feedstocks for ethanol fuel production. Bioenerg Res 6:416–435CrossRefGoogle Scholar
  28. 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 99:5099–5103PubMedCrossRefGoogle Scholar
  29. Skory CD, Freer SN, Bothast RJ (1996) Expression and secretion of the Candida wickerhamii extracellular β-glucosidase gene, bglB, in Saccharomyces cerevisiae. Curr Genet 30:417–422PubMedCrossRefGoogle Scholar
  30. Spiridonov NA, Wilson DB (2001) Cloning and biochemical characterization of BGLC, a β-glucosidase from the cellulolytic actinomycete Thermobifida fusca. Curr Microbiol 42:295–301PubMedGoogle Scholar
  31. Van Zyl WH, Lynd LR, Den Haan R, Mc Bride JE (2007) Consolidate bioprocessing for bioethanol production using Saccharomyces cerevisiae. Adv Biochem Engin Biotechnol 108:205–235Google Scholar
  32. Wildt S, Gerngross TU (2005) The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3:119–127PubMedCrossRefGoogle Scholar
  33. Yamada R, Tanaka T, Ogino C (2010) Gene copy number and polyploid on products formation in yeast. Appl Microbiol Biotechnol 88:849–857PubMedCrossRefGoogle Scholar
  34. 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 4:1–8CrossRefGoogle Scholar
  35. 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 46:431–437PubMedCrossRefGoogle Scholar
  36. 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 431:39–49PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • David J. Mendoza-Aguayo
    • 1
  • Héctor M. Poggi-Varaldo
    • 1
  • Jaime García-Mena
    • 2
  • Ana C. Ramos-Valdivia
    • 1
  • Luis M. Salgado
    • 3
  • Mayra de la Torre-Martínez
    • 4
  • Teresa Ponce-Noyola
    • 1
    Email author
  1. 1.Departamento de Biotecnología y BioingenieríaCentro de Investigación y de Estudios Avanzados del Instituto Politécnico NacionalZacatencoMéxico
  2. 2.Departamento de Genética y Biología MolecularCentro de Investigación y de Estudios Avanzados del Instituto Politécnico NacionalZacatencoMéxico
  3. 3.CICATA, Unidad QuerétaroInstituto Politécnico NacionalQuerétaroMéxico
  4. 4.Departamento de Análisis de los AlimentosCentro de Investigación en Alimentación y DesarrolloHermosilloMéxico

Personalised recommendations