World Journal of Microbiology and Biotechnology

, Volume 30, Issue 11, pp 2985–2993 | Cite as

Development of a cellulolytic Saccharomyces cerevisiae strain with enhanced cellobiohydrolase activity

  • Jiefang Hong
  • Huajun Yang
  • Kun Zhang
  • Cheng Liu
  • Shaolan Zou
  • Minhua Zhang
Original Paper

Abstract

Consolidated bioprocessing (CBP) is a promising technology for lignocellulosic ethanol production, and the key is the engineering of a microorganism that can efficiently utilize cellulose. Development of Saccharomyces cerevisiae for CBP requires high level expression of cellulases, particularly cellobiohydrolases (CBH). In this study, to construct a CBP-enabling yeast with enhanced CBH activity, three cassettes containing constitutively expressed CBH-encoding genes (cbh1 from Aspergillus aculeatus, cbh1 and cbh2 from Trichoderma reesei) were constructed. T. reesei eg2, A. aculeatus bgl1, and the three CBH-encoding genes were then sequentially integrated into the S. cerevisiae W303-1A chromosome via δ-sequence-mediated integration. The resultant strains W1, W2, and W3, expressing uni-, bi-, and trifunctional cellulases, respectively, exhibited corresponding cellulase activities. Furthermore, both the activities and glucose producing activity ascended. The growth test on cellulose containing plates indicated that CBH was a necessary component for successful utilization of crystalline cellulose. The three recombinant strains and the control strains W303-1A and AADY were evaluated in acid- and alkali-pretreated corncob containing media with 5 FPU exogenous cellulase/g biomass loading. The highest ethanol titer (g/l) within 7 days was 5.92 ± 0.51, 18.60 ± 0.81, 28.20 ± 0.84, 1.40 ± 0.12, and 2.12 ± 0.35, respectively. Compared with the control strains, W3 efficiently fermented pretreated corncob to ethanol. To our knowledge, this is the first study aimed at creating cellulolytic yeast with enhanced CBH activity by integrating three types of CBH-encoding gene with a strong constitutive promoter Ptpi.

Keywords

Endoglucanase β-Glucosidase Cellobiohydrolase Consolidated bioprocessing Yeast 

Supplementary material

11274_2014_1726_MOESM1_ESM.docx (42 kb)
Supplementary material 1 (DOCX 41 kb)

References

  1. den Haan R, McBride JE, la Grange DC, Lynd LR, van Zyl WH (2007) Functional expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. Enzyme Microb Technol 40:1291–1299CrossRefGoogle Scholar
  2. den Haan R, Kroukamp H, van Zyl JHD, van Zyl WH (2013) Cellobiohydrolase secretion by yeast: current state and prospects for improvement. Process Biochem 48:1–12CrossRefGoogle Scholar
  3. Ekino K, Hayashi H, Moriyama M, Matsuda M, Goto M, Yoshino S, Furukawa K (2002) Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase. Appl Environ Microbiol 68:5693–5697CrossRefGoogle Scholar
  4. Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11(4):355–360CrossRefGoogle Scholar
  5. Hahn-Hägerdal B, Galbe M, Gorwa-Grauslund M-F, Lidén G, Zacchi G (2006) Bio-ethanol—the fuel of tomorrow from the residues of today. Trends Biotechnol 24:549–556CrossRefGoogle Scholar
  6. Hasunuma T, Kondo A (2012) Development of yeast cell factories for consolidated bioprocessing of lignocellulose to bioethanol through cell surface engineering. Biotechnol Adv 30:1207–1218CrossRefGoogle Scholar
  7. Henrissat B, Driguez H, Viet C, SchlÜein M (1985) Synergism of cellulases from Trichoderma reesei in the degradation of cellulose. Bio/Technology 3:722–726CrossRefGoogle Scholar
  8. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315:804–807CrossRefGoogle Scholar
  9. Ilmén M, den Haan R, Brevnova E, McBride J, Wiswall E, Froehlich A, Koivula A, Voutilainen SP, Siika-Aho M, la Grange DC, Thorngren N, Ahlgren S, Mellon M, Deleault K, Rajgarhia V, van Zyl WH, Penttilä M (2011) High level secretion of cellobiohydrolases by Saccharomyces cerevisiae. Biotechnol Biofuels 4:30CrossRefGoogle Scholar
  10. Khramtsov N, McDade L, Amerik A, Yu E, Divatia K, Tikhonov A, Minto M, Kabongo-Mubalamate G, Markovic Z, Ruiz-Martinez M, Henck S (2011) Industrial yeast strain engineered to ferment ethanol from lignocellulosic biomass. Bioresour Technol 102(17):8310–8313CrossRefGoogle Scholar
  11. la Grange DC, den Haan R, van Zyl WH (2010) Engineering cellulolytic ability into bioprocessing organisms. Appl Microbiol Biotechnol 87:1195–1208CrossRefGoogle Scholar
  12. Liu L, Liu C, Zou S, Yang H, Hong J, Ma Y, Zhang M (2013) Expression of cellulase genes in Saccharomyces cerevisiae via δ-integration subject to auxotrophic markers. Biotechnol Lett 35:1303–1307CrossRefGoogle Scholar
  13. Lynd LR, van Zyl WH, McBride JE, Laser M (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 16:577–583CrossRefGoogle Scholar
  14. Medve J, Ståhlberg J, Tjerneld F (1994) Adsorption and synergism of cellobiohydrolase I and cellobiohydrolase II of Trichoderma reesei during hydrolysis of microcrystalline cellulose. Biotechnol Bioeng 44:1064–1073CrossRefGoogle Scholar
  15. Nidetzky B, Steiner W, Hayn M, Claeyssens M (1994) Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interactions. Biochem J 298:705–710Google Scholar
  16. Oliveira GS, Ulhoa CJ, Silveira MH, Andreaus J, Silva-Pereira I, Poças-Fonseca MJ, Faria FP (2013) An **alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates. World J Microbiol Biotechnol 29(1):19–26CrossRefGoogle Scholar
  17. Olson DG, McBride JE, Shaw AJ, Lynd LR (2012) Recent progress in consolidated bioprocessing. Curr Opin Biotechnol 23:396–405CrossRefGoogle Scholar
  18. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  19. Takada G, Kawaguchi T, Sumitani J, Arai M (1998) Expression of Aspergillus aculeatus NO. F-50 cellobiohydrolase I (cbh1) and β-glucosidase 1 (bgl1) genes by Saccharomyces cerevisiae. Biosci Biotechnol Biochem 62:1615–1618CrossRefGoogle Scholar
  20. van Zyl WH, Lynd LR, den Haan R, McBride JE (2007) Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae. Adv Biochem Eng Biotechnol 108:205–235Google Scholar
  21. Voutilainen SP, Murray PG, Tuohy MG, Koivula A (2010) Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity. Protein Eng Des Sel 23:69–79CrossRefGoogle Scholar
  22. Wang G, Liu C, Hong J, Ma Y, Zhang K, Huang X, Zou S, Zhang M (2013) Comparison of process configurations for ethanol production from acid- and alkali-pretreated corncob by Saccharomyces cerevisiae strains with and without β-glucosidase expression. Bioresour Technol 142:154–161CrossRefGoogle Scholar
  23. Wood TM (1992) Fungal cellulases. Biochem Soc Trans 20:46Google Scholar
  24. Xu L, Shen Y, Hou J, Peng B, Tang H, Bao X (2014) Secretory pathway engineering enhances secretion of cellobiohydrolase I from Trichoderma reesei in Saccharomyces cerevisiae. J Biosci Bioeng 117(1):45–52CrossRefGoogle Scholar
  25. Yamada R, Tanaka T, Ogino C, Fukuda H, Kondo A (2010a) Novel strategy for yeast construction using delta-integration and cell fusion to efficiently produce ethanol from raw starch. Appl Microbiol Biotechnol 85(5):1491–1498CrossRefGoogle Scholar
  26. Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A (2010b) Cocktail delta-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains. Microb Cell Fact 9:32CrossRefGoogle Scholar
  27. 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 4:8CrossRefGoogle Scholar
  28. Yang H, Liu C, Zou S, Ma Y, Hong J, Zhang M (2014) Improving bgl1 gene expression in Saccharomyces cerevisiae through meiosis in an isogenic triploid. Biotechnol Lett. doi:10.1007/s10529-014-1471-z Google Scholar
  29. Zhang GC (2011) Metabolic engineering, cofactor engineering and enzymatic engineering for construction of recombinant xylose-utilizing Saccharomyces cerevisiae strains to produce ethanol. Dissertation, Tianjin University, Tianjin, ChinaGoogle Scholar
  30. Zhang YH, Lynd LR (2004) Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng 88:797–824CrossRefGoogle Scholar
  31. Zhang WN, Liu C, Wang GC, Ma YY, Zhang K, Zou SL, Zhang MH (2012) Comparison of the expression in Saccharomyces cerevisiae of endoglucanase II from Trichoderma reesei and Endoglucanase I from Aspergillus aculeatus. BioResources 7:4031–4045Google Scholar
  32. Zou SL (2011) Construction and evaluation of recombinant strains for ethanol production from lignocellulosic hydrolysate. Dissertation, Tianjin University, Tianjin, ChinaGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Jiefang Hong
    • 1
    • 2
  • Huajun Yang
    • 1
    • 2
  • Kun Zhang
    • 1
  • Cheng Liu
    • 1
  • Shaolan Zou
    • 1
  • Minhua Zhang
    • 1
  1. 1.Tianjin R&D Center for Petrochemical TechnologyTianjin UniversityTianjinChina
  2. 2.School of Chemical Engineering and TechnologyTianjin UniversityTianjinChina

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