Archives of Microbiology

, Volume 199, Issue 4, pp 605–611 | Cite as

Expression of a codon-optimized β-glucosidase from Cellulomonas flavigena PR-22 in Saccharomyces cerevisiae for bioethanol production from cellobiose

  • Francisco Javier Ríos-Fránquez
  • Enrique González-Bautista
  • Teresa Ponce-Noyola
  • Ana Carmela Ramos-Valdivia
  • Héctor Mario Poggi-Varaldo
  • Jaime García-Mena
  • Alfredo Martinez
Original Paper


Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding β-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active β-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L−1 and 222 IU g−1, respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L−1) as the sole carbon source for 48 h. Ethanol production was 5 g L−1 after 96 h of culture, which represented a yield of 0.41 g g−1 of substrate consumed (12 g L−1), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the β-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.


Saccharomyces cerevisiae Cellulomonas flavigena β-Glucosidase expression Cellobiose Bioethanol 



This work was supported by the Mexican Council of Science and Technology (CONACyT), Grant CB-2014/236895, and the Bioenergy Thematic Network (Red Temática de Bioenergía), Grant 260457. The authors thank Odilia Pérez-Avalos and Gustavo Medina-Mendoza for technical assistance. FJ Ríos-Fránquez received a Ph. D scholarship from CONACyT-México. The authors have no conflict of interest to declare.

Supplementary material

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Supplementary material 1 (TIFF 349 KB)
203_2016_1333_MOESM2_ESM.docx (22 kb)
Supplementary material 2 (DOCX 22 KB)


  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 54:266–270CrossRefPubMedGoogle Scholar
  2. 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 yeast strain displaying cellulolytic enzymes. Appl Environ Microbiol 68:5136–5141CrossRefPubMedPubMedCentralGoogle Scholar
  3. Gietz RD, Woods RA (2006) Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol 313:107–120PubMedGoogle Scholar
  4. Gurgu L, Lafraya A, Polaina J, Marín-Navarro J (2011) Fermentation of cellobiose to ethanol by industrial Saccharomyces strains carrying the β-glucosidase gene (BGL1) from Saccharomycopsis fibuligera. Bioresour Technol 102:5229–5236CrossRefPubMedGoogle Scholar
  5. Liu GL, Fu GY, Chi Z, Chi ZM (2014) Enhanced expression of the codon-optimized exo-inulinase gene from the yeast Meyerozyma guilliermondii in Saccharomyces sp. W0 and bioethanol production from inulin. Appl Microbiol Biotechnol 98:9129–9138CrossRefPubMedGoogle Scholar
  6. Lynd LR, Weimer PJ, vanZyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577CrossRefPubMedPubMedCentralGoogle Scholar
  7. McBride JE, Zietsman JJ, van Zyl WH, Lynd LR (2005) Utilization of cellobiose by recombinant β-glucosidase-expressing strains of Saccharomyces cerevisiae: characterization and evaluation of the sufficiency of expression. Enzyme Microb Tech 37:93–101CrossRefGoogle Scholar
  8. Mendoza-Aguayo DJ, Poggi-Varaldo HM, García-Mena J, Ramos-Valdivia AC, Salgado LM, de la Torre-Martínez M, Ponce-Noyola T (2014) Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae. Arch Microbiol 196:25–33CrossRefPubMedGoogle Scholar
  9. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  10. Ponce-Noyola T, de la Torre M (1993) Interactions in a mixed culture composed of Cellulomonas flavigena and Xanthomonas sp. growing in continuous culture on sugar cane bagasse. Appl Microbiol Biotechnol 40:531–534CrossRefGoogle Scholar
  11. Raele R, Boaventura JMG, Fischmann AA, Sarturi G (2014) Scenarios for the second generation ethanol in Brazil. Technol Forecast Soc Change 87:205–223CrossRefGoogle Scholar
  12. Rojas-Rejón OA, Poggi-Varaldo HM, Ramos-Valdivia AC, Martinez-Jimenez A, Cristiani-Urbina E, de la Torre-Martinez M, Ponce-Noyola T (2011) Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena. J Ind Microbiol Biotechnol 38:257–264CrossRefPubMedGoogle Scholar
  13. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual (2nd edition). Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  14. 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 99:5099–5103CrossRefPubMedGoogle Scholar
  15. Shim JH, Withers SG (2013) Improvement of the expression level of β-glucosidase from Agrobacterium sp. in Escherichia coli by rare codon optimization. Food Sci Biotechnol 22:269–273CrossRefGoogle Scholar
  16. Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 83:1–11CrossRefPubMedGoogle Scholar
  17. Tye YY, Lee KT, Abdullah WNW, Leh CP (2016) The world availability of non-wood lignocellulosic biomass for the production of cellulosic ethanol and potential pretreatments for the enhancement of enzymatic saccharification. Renew Sustain Energy Rev 60:155–172CrossRefGoogle Scholar
  18. van Rooyen R, Hahn-Hagerdal B, La Grange DC, van Zyl WH (2005) Construction of cellobiose-growing and fermenting Saccharomyces cerevisiae strains. J Biotechnol 120:284–295CrossRefPubMedGoogle Scholar
  19. Wiedemann B, Boles E (2008) Codon-optimized bacterial genes improve L-arabinose fermentation in recombinant Saccharomyces cerevisiae. Appl Environ Microbiol 74:2043–2050CrossRefPubMedPubMedCentralGoogle Scholar
  20. Wilde C, Bawa N, Gold ND, Tambor H, Mougharbel L, Storms R, Martin VJJ (2012) Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain. Appl Microbiol Biotechnol 95:647–659CrossRefPubMedGoogle Scholar
  21. 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 46:431–437CrossRefPubMedGoogle Scholar
  22. Yanase S, Yamada R, Kaneko S, Noda H, Hasunuma T, Tanaka T (2010) Ethanol production from cellulosic materials using cellulase-expressing yeast. Biotechnol J 5:449–455CrossRefPubMedGoogle Scholar
  23. Yang F, Cao M, Jin Y, Yang X, Tian S (2013) Construction of a novel а-agglutinin expression system on the surface of wild-type Saccharomyces cerevisiae Y5 and genetic immobilization of β-glucosidase1. Bioenerg Res 6:1205–1211CrossRefGoogle Scholar
  24. Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 56:17–34CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Francisco Javier Ríos-Fránquez
    • 1
  • Enrique González-Bautista
    • 1
  • Teresa Ponce-Noyola
    • 1
  • Ana Carmela Ramos-Valdivia
    • 1
  • Héctor Mario Poggi-Varaldo
    • 1
  • Jaime García-Mena
    • 2
  • Alfredo Martinez
    • 3
  1. 1.Departamento de Biotecnología y BioingenieríaCinvestav-IPNMexico CityMexico
  2. 2.Departamento de Genética y Biología MolecularCinvestav-IPNMexico CityMexico
  3. 3.Departamento de Ingeniería Celular y Biocatálisis, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

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