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Efficient yeast cell-surface display of an endoglucanase of Aspergillus flavus and functional characterization of the whole-cell enzyme

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Abstract

The endoglucanase gene endo753 from Aspergillus flavus NRRL3357 strains was cloned, and the recombinant Endo753 was displayed on the cell surface of Saccharomyces cerevisiae EBY100 strain by the C-terminal fusion using Aga2p protein as anchor attachment tag. The results of indirect immunofluorescence and Western blot confirmed the expression and localization of Endo753 on the yeast cell surface. The hydrolytic activity test of the whole-cell enzyme revealed that Endo753 immobilized on the yeast cell surface had high endoglucanase activity. The functional characterization of the whole-cell enzyme was investigated, and the whole-cell enzyme displayed the maximum activity at pH 8 and 50 °C. The enzyme was stable in a pH range of 7.0–10.0. Furthermore, the whole-cell enzyme displayed high thermostability below 50 °C and moderate stability between 50 and 70 °C. These properties make endo753 a good candidate in bioethanol production from lignocellulosic materials after displaying on the yeast cell surface.

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References

  • Alvira P, Tomás-Pejó E, Ballesteros M, Negro MJ (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 101:4851–4861

    Article  CAS  Google Scholar 

  • Bakare MK, Adewale IO, Ajayi A et al (2005) Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens. Afr J Biotechnol 4:898–904

    CAS  Google Scholar 

  • Becker Onofre S, Silva GC, Mattiello SP et al (2013) Production of cellulolytic enzymes by Aspergillus flavus using solid state fermentation based on sugarcane bagasse. Am J Biochem 3:25–28

    Article  Google Scholar 

  • Berlin A, Gilkes N, Kilburn D et al (2006) Evaluation of cellulase preparations for hydrolysis of hardwood substrates. Appl Biochem Biotechnol 130:528–545

    Article  Google Scholar 

  • Boonvitthya N, Bozonnet S, Burapatana V et al (2013) Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica. Mol Biotechnol 54:158–169

    Article  CAS  Google Scholar 

  • Cardona CA, Sánchez OJ (2007) Fuel ethanol production: process design trends and integration opportunities. Bioresour Technol 98:2415–2457

    Article  CAS  Google Scholar 

  • Cleveland TE, Yu JJ, Fedorova N et al (2009) Potential of Aspergillus flavus genomics for applications in biotechnology. Trends Biotechnol 27:151–157

    Article  CAS  Google Scholar 

  • Duan CJ, Feng JX (2010) Mining metagenomes for novel cellulase genes. Biotechnol Lett 32:1765–1775

    Article  CAS  Google Scholar 

  • Foreman PK, Brown D, Dankmeyer L et al (2003) Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. J Biol Chem 278:31988–31997

    Article  Google Scholar 

  • Fujii T, Fang X, Inoue H et al (2009) Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials. Biotechnol Biofuels 2:24

    Article  Google Scholar 

  • Fujita Y, Ito J, Ueda M, Fukuda H, Kondo A (2004) Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Appl Environ Microbiol 70:1207–1212

    Article  CAS  Google Scholar 

  • Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268

    CAS  Google Scholar 

  • Gomathi D, Muthulakshmi C, Kumar DG et al (2012) Production of bio-ethanol from pretreated agricultural byproduct using enzymatic hydrolysis and simultaneous saccharification. Microbiology 81:201–207

    Article  CAS  Google Scholar 

  • Gusakov AV (2011) Alternatives to Trichoderma reesei in biofuel production. Trends Biotechnol 29:419–425

    Article  CAS  Google Scholar 

  • Hasunuma T, Kondo A (2012) Development of yeast cell factories for consolidated bioprocessing of lignocellulose to bioethanol through cell surface engineering. Biotechnol Adv 30:1207–1218

    Article  CAS  Google Scholar 

  • Hendriks ATWM, Zeeman G (2009) Pretreatment to enhance the digestability of lignocellulosic biomass. Bioresour Technol 100:10–18

    Article  CAS  Google Scholar 

  • Huang GL, Anderson TD, Clubb RT (2014) Engineering microbial surfaces to degrade lignocellulosic biomass. Bioengineered 5:96–106

    Article  Google Scholar 

  • Ikeda Y, Hayashi H, Okuda N et al (2007) Efficient cellulase production by the filamentous fungus Acremonium cellulolyticus. Biotechnol Prog 23:333–338

    Article  CAS  Google Scholar 

  • Inokuma K, Hasunuma T, Kondo A (2014) Efficient yeast cell-surface display of exo- and endo-cellulase using the SED1 anchoring region and its original promoter. Biotechnol Biofuels 7:8

    Article  Google Scholar 

  • Jorgensen H, Morkeberg A, Krogh KBR et al (2005) Production of cellulases and hemicellulases by three Penicillium species: effect of substrate and evaluation of cellulase adsorption by capillary electrophoresis. Enzyme Microb Technol 36:42–48

    Article  CAS  Google Scholar 

  • Kondo A, Shigechi H, Abe M et al (2002) High-level ethanol production from starch by a flocculent Saccharomyces cerevisiae strain displaying cell-surface glucoamylase. Appl Microbiol Biotechnol 58:291–296

    Article  CAS  Google Scholar 

  • Kotaka A, Bando H, Kaya M et al (2008) Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase. J Biosci Bioeng 105:622–627

    Article  CAS  Google Scholar 

  • Kubicek CP (2013) Systems biological approaches towards understanding cellulase production by Trichoderma reesei. J Biotechnol 163:133–142

    Article  CAS  Google Scholar 

  • Kumar R, Wyman CE (2014) Strong cellulase inhibition by Mannan polysaccharides in cellulose conversion to sugars. Biotechnol Bioeng 111:1341–1353

    Article  CAS  Google Scholar 

  • Kuroda K, Ueda M (2011) Cell surface engineering of yeast for applications in white biotechnology. Biotechnol Lett 33:1–9

    Article  CAS  Google Scholar 

  • Lau MW, Gunawan C, Balan V, Dale BE (2010) Comparing the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A (LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production. Biotechnol Biofuels 3:11

    Article  Google Scholar 

  • Lee YJ, Kim BK, Lee BH et al (2008) Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresour Technol 99:378–386

    Article  CAS  Google Scholar 

  • Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:739

    Article  Google Scholar 

  • Machida M, Asai K, Sano M et al (2005) Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161

    Article  Google Scholar 

  • Nierman WC, Pain A, Anderson MJ et al (2005) Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151–1156

    Article  CAS  Google Scholar 

  • Pandey A (2009) Handbook of plant based biofuels. CRC Press, Boca Raton, pp 159–173

    Google Scholar 

  • Pel HJ, de Winde JH, Archer DB et al (2007) Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol 25:221–231

    Article  Google Scholar 

  • Phitsuwan P, Laohakunjit N, Kerdchoechuen O, Kyu KL, Ratanakhanokchai K (2013) Present and potential applications of cellulases in agriculture, biotechnology, and bioenergy. Folia Microbiol (Praha) 58:163–176

    Article  CAS  Google Scholar 

  • Sajith S, Sreedevi S, Priji P et al (2014) Production and partial purification of cellulase from a novel fungus, Aspergillus flavus BS1. Ann Microbiol 64:763–771

    Article  CAS  Google Scholar 

  • Segato F, Damasio ARL, de Lucas RC et al (2014) Genomics review of holocellulose deconstruction by aspergilli. Microbiol Mol Biol Rev 78:588–613

    Article  Google Scholar 

  • Strakowska J, Blaszczyk L, Chelkowski J (2014) The significance of cellulolytic enzymes produced by Trichoderma in opportunistic lifestyle of this fungus. J Basic Microbiol 54:S2–S13

    Article  CAS  Google Scholar 

  • Tanaka T, Yamada R, Ogino C et al (2012) Recent developments in yeast cell surface display toward extended applications in biotechnology. Appl Microbiol Biotechnol 95:577–591

    Article  CAS  Google Scholar 

  • Tsai SL, Goyal G, Chen W (2010) Surface display of a functional minicellulosome by intracellular complementation using a synthetic yeast consortium and its application to cellulose hydrolysis and ethanol production. Appl Environ Microbiol 76:7514–7520

    Article  CAS  Google Scholar 

  • Ueda M, Tanaka A (2000) Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv 18:121–140

    Article  CAS  Google Scholar 

  • Xu Q, Singh A, Himmel ME (2009) Perspectives and new directions for the production of bioethanol using consolidated biopreocessing of lignocellulose. Curr Opin Biotechnol 20:364–371

    Article  CAS  Google Scholar 

  • Yan PA, Su LQ, Chen J et al (2013) Heterologous expression and biochemical characterization of an endo-1,4-glucanase from Thermobifida fusca. Biotechnol Appl Biochem 60:348–355

    Article  CAS  Google Scholar 

  • Yanase G, Hasunuma T, Yamada R et al (2010) Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes. Appl Microbiol Biotechnol 88:381–388

    Article  CAS  Google Scholar 

  • Yin YR, Zhang F, Hu QW et al (2015) Heterologous expression and characterization of a novel halotolerant, thermostable, and alkali-stable GH6 endoglucanase from Thermobifida halotolerans. Biotechnol Lett 37:857–862

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the grant of National Science Foundation of China (31270156), National marine research funds for public welfare projects of China (20120520), the Research programs of Guangdong Province (2012B061800091, 2013A061402006).

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Correspondence to Run-Qian Mao or Gang Li.

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Gao, G., Mao, RQ., Xiao, Y. et al. Efficient yeast cell-surface display of an endoglucanase of Aspergillus flavus and functional characterization of the whole-cell enzyme. World J Microbiol Biotechnol 33, 114 (2017). https://doi.org/10.1007/s11274-016-2182-5

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