Skip to main content
SpringerLink
Log in

Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The development of a yeast that converts raw starch to ethanol in one step (called consolidated bioprocessing) could yield large cost reductions in the bioethanol industry. The aim of this study was to develop an efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production. A native and codon-optimized variant of the Aspergillus awamori glucoamylase gene were expressed in the S. cerevisiae Y294 laboratory strain. Codon optimization resulted to be effective and the synthetic sequence sGAI was then δ-integrated into a S. cerevisiae strain with promising industrial fermentative traits. The mitotically stable recombinant strains showed high enzymatic capabilities both on soluble and raw starch (2425 and 1140 nkat/g dry cell weight, respectively). On raw corn starch, the engineered yeasts exhibited improved fermentative performance with an ethanol yield of 0.42 (g/g), corresponding to 75 % of the theoretical maximum yield.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aggarwal P, Dollimore D (1998) A thermal analysis investigation of partially hydrolyzed starch. Thermochim Acta 319:17–25

    Article  CAS  Google Scholar 

  • Albers E, Larsson C (2009) A comparison of stress tolerance in YPD and industrial lignocellulose-based medium among industrial and laboratory yeast strains. J Ind Microbiol Biotechnol 36:1085–1091

    Article  CAS  Google Scholar 

  • Allen MJ, Coutinho PM, Ford CF (1998) Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations. Protein Eng 11:783–788

    Article  CAS  Google Scholar 

  • Bothast RJ, Schlicher MA (2005) Biotechnological processes for conversion of corn into ethanol. Appl Microbiol Biotechnol 67:19–25

    Article  CAS  Google Scholar 

  • Chen HM, Ford C, Reilly PJ (1995) Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase. Protein Eng 8:575–582

    Article  CAS  Google Scholar 

  • Cho KM, Yoo YJ, Kang 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 

  • de Moraes LMP, Astolfi-Filho S, Oliver SG (1995) Development of yeast strains for the efficient utilization of starch: evaluation of constructs that express alpha-amylase and glucoamylase separately or as bifunctional fusion proteins. Appl Microbiol Biotechnol 43:1067–1076

    Article  Google Scholar 

  • 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 40:1291–1299

    Article  Google Scholar 

  • Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

    Article  CAS  Google Scholar 

  • Favaro L, Basaglia M, Saayman M, Rose SH, van Zyl WH, Casella S (2010a) Engineering amylolytic yeasts for industrial bioethanol production. Chemical Engineering Transactions 20:97–102. doi:10.3303/CET1020017

    Google Scholar 

  • Favaro L, Basaglia M, Trento A, Saayman M, Rose SH, van Zyl WH, Casella S (2010b) Development of raw starch hydrolysing yeasts for industrial bioethanol production. J Biotechnol 150(1):142. doi:10.1016/j.jbiotec.2010.08.371

    Article  Google Scholar 

  • Fierobe HP, Mirgorodskaya E, Frandsen TP, Roepstorff P, Svensson B (1997) Overexpression and characterization of Aspergillus awamori wild-type and mutant glucoamylase secreted by the methylotrophic yeast Pichia pastoris: comparison with wild-type recombinant glucoamylase produced using Saccharomyces cerevisiae and Aspergillus niger as hosts. Protein Expression Purif 9:159–170

    Article  CAS  Google Scholar 

  • Garay-Arroyo A, Covarrubias AA, Clark I, Niño I, Gosset G, Martinez A (2004) Response to different environmental stress conditions of industrial and laboratory Saccharomyces cerevisiae strains. Appl Microbiol Biotechnol 63:734–741

    Article  CAS  Google Scholar 

  • Goto M, Ekino K, Furukawa K (1997) Expression and functional analysis of a hyperglycosylated glucoamylase in a parental host. Aspergillus awamori var. kawachi. Appl Environ Microbiol 63:2940–2943

    CAS  Google Scholar 

  • Hayashida S, Kuroda K, Ohta K, Kuhara S, Fukuda K, Sakaki Y (1989) Molecular cloning of the glucoamylase I gene of Aspergillus awamori var. kawachi for localization of the raw-starch-affinity site. RL Agric Biol Chem 53:923–929

    Article  CAS  Google Scholar 

  • Hahn-Hägerdal B, Karhumaa K, Larsson CU, Gorwa-Grauslund M, Görgens J, van Zyl WH (2005) Role of cultivation media in the development of yeast strains for large scale industrial use. Microb Cells Fact 4:31

    Article  Google Scholar 

  • Hill J, Donald KA, Griffiths DE (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res 19:5791

    Article  CAS  Google Scholar 

  • Kang NY, Park JN, Chin JE, Blaise LH, Im SY, Bai S (2003) Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis α-amylase gene. Biotechnol Lett 25:1847–1851

    Article  CAS  Google Scholar 

  • Kern L, de Montigny J, Jund R, Lacroute F (1990) The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene 88:149–157

    Article  CAS  Google Scholar 

  • Khaw TS, Katakura Y, Koh J, Kondo A, Ueda M, Shioya S (2005) Evaluation of performance of different surface-engineered yeast strains for direct ethanol production from raw starch. Appl Microbiol Biotechnol 70:573–579

    Article  Google Scholar 

  • Kim MD, Rhee SK, Seo JH (2001) Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal delta-integration. J Biotechnol 85:41–48

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Lee FWF, Silva NA (1997) Improved efficiency and stability of multiple cloned gene insertions at the δ sequences of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 48:339–345

    Article  CAS  Google Scholar 

  • Lee J, Choi SI, Jang JS, Jang K, Moon JW, Bae CS, Yang DS, Seong BL (1999) Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer. Biotechnol Prog 15:884–890

    Article  CAS  Google Scholar 

  • Martin C, Jönsson LJ (2003) Comparison of the resistance of industrial and laboratory strains of Saccharomyces and Zygosaccharomyces to lignocellulose-derived fermentation inhibitors. Enzyme Microb Technol 32:386–395

    Article  CAS  Google Scholar 

  • McBride JEE, Deleault KM, Lynd LR, Pronk JT (2008) Recombinant yeast strains expressing tethered cellulase enzymes. Patent PCT/US2007/085390.

  • Murai T, Yoshino T, Ueda M, Haranoya I, Ashikari T, Yoshizumi H, Tanaka A (1998) Evaluation of the function of arming yeast displaying glucoamylase on its cell surface by direct fermentation of corn to ethanol. J Ferment Bioeng 86:569–572

    Article  CAS  Google Scholar 

  • Nakamura Y, Kobayashi F, Ohnaga M, Sawada T (1997) Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity. Biotechnol Bioeng 53:21–25

    Article  CAS  Google Scholar 

  • Plüddemann A, van Zyl WH (2003) Evaluation of Aspergillus niger as host for virus-like particle production, using the hepatitis B surface antigen as a model. Curr Genet 43:439–446

    Article  Google Scholar 

  • Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8:423–488

    Article  CAS  Google Scholar 

  • Sambrook J, Russel DB (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Sharp PM, Cowe E (1991) Synonymous codon usage in Saccharomyces cerevisiae. Yeast 7:657–678

    Article  CAS  Google Scholar 

  • Shibuya I, Gomi K, Iimura Y, Takahashi K, Tamura G, Hara S (1990) Molecular cloning of the glucoamylase gene of Aspergillus shirousami and its expression in Aspergillus oryzae. RL Agric Biol Chem 54:1905–1914

    Article  CAS  Google Scholar 

  • van Rooyen R, Hahn-Hägerdal B, La Grange DC, van Zyl WH (2005) Construction of cellobiose-growing and fermenting Saccharomyces cerevisiae strains. J Biotechnol 120:284–295

    Article  Google Scholar 

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

    Google Scholar 

  • Wiedemann B, Boles E (2008) Codon-optimized bacterial genes improve L-arabinose fermentation in recombinant Saccharomyces cerevisiae. Appl Environ Microbiol 74:2043–2050

    Article  CAS  Google Scholar 

  • Yamada R, Bito Y, Adachi T, Tanaka T, Ogino C, Fukuda H, Kondo A (2009) Efficient production of ethanol from raw starch by a mated diploid Saccharomyces cerevisiae with integrated α-amylase and glucoamylase genes. Enzyme Microb Technol 44:344–349

    Article  CAS  Google Scholar 

  • Zhang J, Reddy J, Buckland B, Greasham R (2003) Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process. Biotechnol Bioeng 82:640–652

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was partially financed by the BiotechIIbis and BiotechIII projects (Regione Veneto, Italy) and the National Research Foundation (South Africa). Dr. Lorenzo Favaro is recipient of “Assegno di ricerca Junior” grant (University of Padova). Stefania Zannoni (University of Padova) is acknowledged for HPLC analysis. Authors are grateful to Claudio Furlan (CUGAS, University of Padova) for performing SEM.

Author information

Authors and Affiliations

  1. Department of Agronomy Food Natural Resources Animals and Environment (DAFNAE), University of Padova, Agripolis, Viale dell’Università 16, 35020, Legnaro PADOVA, Italy

    Lorenzo Favaro, Marina Basaglia & Sergio Casella

  2. Department of Microbiology, Stellenbosch University, Private Bag X1, 7602, Matieland, Stellenbosch, South Africa

    Tania Jooste, Shaunita H. Rose, Maryna Saayman & Willem H. van Zyl

  3. Department of Process Engineering, University of Stellenbosch, Private Bag X1, 7602, Matieland, Stellenbosch, South Africa

    Johann F. Görgens

Authors
  1. Lorenzo Favaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tania Jooste
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marina Basaglia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shaunita H. Rose
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maryna Saayman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Johann F. Görgens
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sergio Casella
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Willem H. van Zyl
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marina Basaglia.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Favaro, L., Jooste, T., Basaglia, M. et al. Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast. Appl Microbiol Biotechnol 95, 957–968 (2012). https://doi.org/10.1007/s00253-012-4001-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4001-8

Keywords

Search

Navigation