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Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars

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Abstract

The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially improved prebiotic properties. The Xd-INV gene comprises an open reading frame of 1995 bp, which encodes a 665-amino acid protein. Initial N-terminal sequencing of Xd-INV pointed to a majority extracellular protein of 595 amino acids lacking the first 70 residues (potential signal peptide). Functionality of the last 1785 bp of Xd-INV gene was previously proved in Saccharomyces cerevisiae but only weak β-fructofuranosidase activity was quantified. In this study, different strategies to improve this enzyme level in a heterologous system have been used. Curiously, best results were obtained by increasing the protein N-terminus sequence in 39 amino acids, protein of 634 residues. The higher β-fructofuranosidase activity detected in this study, about 15 U/mL, was obtained using Pichia pastoris and represents an improvement of about 1500 times the level previously obtained in a heterologous organism and doubles the best level of activity obtained by the natural producer. Heterologously expressed protein was purified and characterized biochemically and kinetically. Except by its glycosylation degree (10 % lower) and thermal stability (4–5 °C lower in the 60–85 °C range), the properties of the heterologous enzyme, including ability to produce neo-FOS, remained unchanged. Interestingly, besides the neo-FOS referred before blastose was also detected (8–22 g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date.

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References

  • Alvaro-Benito M, de Abreu M, Fernandez-Arrojo L, Plou FJ, Jimenez-Barbero J, Ballesteros A, Polaina J, Fernandez-Lobato M (2007) Characterization of a β-fructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose. J Biotechnol 132:75–81. doi:10.1016/j.jbiotec.2007.07.939

    Article  CAS  PubMed  Google Scholar 

  • Alvaro-Benito M, Polo A, Gonzalez B, Fernandez-Lobato M, Sanz-Aparicio J (2010a) Structural and kinetic analysis of Schwanniomyces occidentalis invertase reveals a new oligomerization pattern and the role of its supplementary domain in substrate binding. J Biol Chem 285:13930–13941. doi:10.1074/jbc.M109.095430

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Alvaro-Benito M, de Abreu M, Portillo F, Sanz-Aparicio J, Fernandez-Lobato M (2010b) New insights into the fructosyltransferase activity of Schwanniomyces occidentalis β-fructofuranosidase, emerging from nonconventional codon usage and directed mutation. Appl Environ Microbiol 76:7491–7499. doi:10.1128/AEM. 01614-10

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, Fernandes GR, Tap J, Bruls T, Batto JM, Bertalan M, Borruel N, Casellas F, Fernandez L, Gautier L, Hansen T, Hattori M, Hayashi T, Kleerebezem M, Kurokawa K, Leclerc M, Levenez F, Manichanh C, Nielsen HB, Nielsen T, Pons N, Poulain J, Qin J, Sicheritz-Ponten T, Tims S, Torrents D, Ugarte E, Zoetendal EG, Wang J, Guarner F, Pedersen O, de Vos WM, Brunak S, Dore J, Antolin M, Artiguenave F, Blottiere HM, Almeida M, Brechot C, Cara C, Chervaux C, Cultrone A, Delorme C, Denariaz G, Dervyn R, Foerstner KU, Friss C, van de Guchte M, Guedon E, Haimet F, Huber W, van Hylckama-Vlieg J, Jamet A, Juste C, Kaci G, Knol J, Lakhdari O, Layec S, Le Roux K, Maguin E, Merieux A, Melo Minardi R, M’Rini C, Muller J, Oozeer R, Parkhill J, Renault P, Rescigno M, Sanchez N, Sunagawa S, Torrejon A, Turner K, Vandemeulebrouck G, Varela E, Winogradsky Y, Zeller G, Weissenbach J, Ehrlich SD, Bork P (2011) Enterotypes of the human gut microbiome. Nature 473:174–180. doi:10.1038/nature09944

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bang ML, Villadsen I, Sandal T (1999) Cloning and characterization of an endo-β-1,3(4) glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938. Appl Microbiol Biotechnol 51:215–222. doi:10.1007/s002530051384

    Article  CAS  PubMed  Google Scholar 

  • Bekers M, Laukevics J, Upite D, Kaminska E, Vigants A, Viesturs U, Pankova L, Danilevics A (2002) Fructooligosaccharides and levan producing activity of Zymomonas mobilis extracellular levansucrase. Process Biochem 38:701–706. doi:10.1016/S0032-9592(02)00189-9

    Article  CAS  Google Scholar 

  • Capone S, Pletzenauer R, Maresch D, Metzger K, Altmann F, Herwig C, Spadiut O (2014) Glyco-variant library of the versatile enzyme horseradish peroxidase. Glycobiology 24:852–863. doi:10.1093/glycob/cwu047

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Çelik E, Çalik P (2012) Production of recombinant proteins by yeast cells. Biotechnol Adv 30:1108–1118. doi:10.1016/j.biotechadv.2011.09.011

    Article  PubMed  Google Scholar 

  • Chen J, Chen X, Xu X, Ning Y, Jin Z, Tian Y (2011) Biochemical characterization of an intracellular 6G-fructofuranosidase from Xanthophyllomyces dendrorhous and its use in production of neo-fructooligosaccharides (neo-FOSs). Bioresour Technol 102:1715–1721. doi:10.1016/j.biortech.2010.08.033

    Article  CAS  PubMed  Google Scholar 

  • Farine S, Versluis C, Bonnici PJ, Heck A, L’Homme C, Puigserver A, Biagini A (2001) Application of high performance anion exchange chromatography to study invertase-catalysed hydrolysis of sucrose and formation of intermediate fructan products. Appl Microbiol Biotechnol 55:55–60. doi:10.1007/s002530000493

    Article  CAS  PubMed  Google Scholar 

  • Flieger M, Kantorova M, Halada P, Kuzma M, Pazoutova S, Stodulkova E, Kolinska R (2005) Oligosaccharides produced by submerged cultures of Claviceps africana and Claviceps sorghi. Folia Microbiol (Praha) 50:198–204. doi:10.1007/BF02931566

    Article  CAS  Google Scholar 

  • Gimeno-Perez M, Santos-Moriano P, Fernandez-Arrojo L, Poveda A, Jimenez-Barbero J, Ballesteros AO, Fernandez-Lobato M, Plou FJ (2014) Regioselective synthesis of neo-erlose by the β-fructofuranosidase from Xanthophyllomyces dendrorhous. Process Biochem 49:423–429. doi:10.1016/j.procbio.2013.12.018

    Article  CAS  Google Scholar 

  • Gutierrez-Alonso P, Fernandez-Arrojo L, Plou FJ, Fernandez-Lobato M (2009) Biochemical characterization of a β-fructofuranosidase from Rhodotorula dairenensis with transfructosylating activity. FEMS Yeast Res 9:768–773. doi:10.1111/j.1567-1364.2009.00526.x

    Article  CAS  PubMed  Google Scholar 

  • Han M, Wang X, Ding H, Jin M, Yu L, Wang J, Yu X (2014) The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris. Enzym Microb Technol 54:32–37. doi:10.1016/j.enzmictec.2013.09.014

    Article  CAS  Google Scholar 

  • Homann A, Biedendieck R, Gotze S, Jahn D, Seibel J (2007) Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium. Biochem J 407:189–198. doi:10.1042/BJ20070600

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Huang CJ, Damasceno LM, Anderson KA, Zhang S, Old LJ, Batt CA (2011) A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures. Appl Microbiol Biotechnol 90:235–247. doi:10.1007/s00253-011-3118-5

    Article  CAS  PubMed  Google Scholar 

  • Kaur N, Gupta AK (2002) Applications of inulin and oligofructose in health and nutrition. J Biosci 27:703–714. doi:10.1007/BF02708379

    Article  CAS  PubMed  Google Scholar 

  • Kilian SG, Kritzinger SM, Rycroft C, du Gibson GR, Preez JC (2002) The effects of the novel bifidogenic trisaccharide, neokestose, on the human colonic microbiota. World J Microbiol Biotechnol 18:637–644. doi:10.1023/A:1016808015630

    Article  CAS  Google Scholar 

  • Kritzinger SM, Kilian SG, Potgieter MA, du Preez JC (2003) The effect of production parameters on the synthesis of the prebiotic trisaccharide, neokestose, by Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Enzym Microb Technol 32:728–737. doi:10.1016/S0141-0229(03)00035-8

    Article  CAS  Google Scholar 

  • Lim JS, Lee JH, Kang SW, Park SW, Kim SW (2007) Studies on production and physical properties of neo-FOS produced by co-immobilized Penicillium citrinum and neo-fructosyltransferase. Eur Food Res Technol 225:457–462. doi:10.1007/s00217-006-0440-8

    Article  CAS  Google Scholar 

  • Linde D, Macias I, Fernandez-Arrojo L, Plou FJ, Jimenez A, Fernandez-Lobato M (2009) Molecular and biochemical characterization of a β-fructofuranosidase from Xanthophyllomyces dendrorhous. Appl Environ Microbiol 75:1065–1073. doi:10.1128/AEM. 02061-08

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Linde D, Rodriguez-Colinas B, Estevez M, Poveda A, Plou FJ, Fernandez Lobato M (2012) Analysis of neofructooligosaccharides production mediated by the extracellular β-fructofuranosidase from Xanthophyllomyces dendrorhous. Bioresour Technol 109:123–130. doi:10.1016/j.biortech.2012.01.023

    Article  CAS  PubMed  Google Scholar 

  • Lu L, Wang TN, Xu TF, Wang JY, Wang CL, Zhao M (2013) Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization. Bioresour Technol 134:81–86. doi:10.1016/j.biortech.2013.02.015

    Article  CAS  PubMed  Google Scholar 

  • Piirainen MA, de Ruijter JC, Koskela EV, Frey AD (2014) Glycoengineering of yeasts from the perspective of glycosylation efficiency. N Biotechnol 31:532–537. doi:10.1016/j.nbt.2014.03.001

    Article  CAS  PubMed  Google Scholar 

  • Sabater-Molina M, Larque E, Torrella F, Plaza J, Ramis G, Zamora S (2011) Effects of fructooligosaccharides on cecum polyamine concentration and gut maturation in early-weaned piglets. J Clin Biochem Nutr 48:230–236. doi:10.3164/jcbn. 10-100

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sangeetha PT, Ramesh MN, Prapulla SG (2005) Recent trends in the microbial production, analysis and application of fructooligosaccharides. Trends Food Sci Tech 16:442–457. doi:10.1016/j.tifs.2005.05.003

    Article  CAS  Google Scholar 

  • Sears IB, O’Connor J, Rossanese OW, Glick BS (1998) A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris. Yeast 14:783–790. doi:10.1002/(SICI)1097-0061(19980615)14:8<783::AID-YEA272>3.0.CO;2-Y

    Article  CAS  PubMed  Google Scholar 

  • Skropeta D (2009) The effect of individual N-glycans on enzyme activity. Bioorg Med Chem 17:2645–2653. doi:10.1016/j.bmc.2009.02.037

    Article  CAS  PubMed  Google Scholar 

  • Swennen K, Courtin CM, Delcour JA (2006) Non-digestible oligosaccharides with prebiotic properties. Crit Rev Food Sci Nutr 46:459–471. doi:10.1080/10408390500215746

    Article  CAS  PubMed  Google Scholar 

  • Tang SJ, Shaw JF, Sun KH, Sun GH, Chang TY, Lin CK, Lo YC, Lee GC (2001) Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability. Arch Biochem Biophys 387:93–98. doi:10.1006/abbi.2000.2235

    Article  CAS  PubMed  Google Scholar 

  • Tian B, Chen Y, Ding S (2012) A combined approach for improving alkaline acetyl xylan esterase production in Pichia pastoris, and effects of glycosylation on enzyme secretion, activity and stability. Protein Expr Purif 85:44–50. doi:10.1016/j.pep.2012.06.008

    Article  CAS  PubMed  Google Scholar 

  • Tschopp JF, Sverlow G, Kosson R, Craig W, Grinna L (1987) High-level secretion of glycosylated invertase in the methylotrophic yeast, Pichia pastoris. Nat Biotechnol 5:1305–1308. doi:10.1038/nbt1287-1305

    Article  CAS  Google Scholar 

  • Vogl T, Glieder A (2013) Regulation of Pichia pastoris promoters and its consequences for protein production. N Biotechnol 30:385–404. doi:10.1016/j.nbt.2012.11.010

    Article  CAS  PubMed  Google Scholar 

  • Zambelli P, Fernández-Arrojo L, Romano D, Santos-Moriano P, Gimeno-Pérez M, Poveda A, Gandolfi R, Fernández-Lobato M, Molinari F, Plou FJ (2014) Production of fructooligosaccharides by mycelium-bound transfructosylation activity present in Cladosporium cladosporioides and Penicillium sizovae. Process Biochem. doi:10.1016/j.procbio.2014.09.021

  • Zou S, Huang S, Kaleem I, Li C (2013) N-Glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris. J Biotechnol 164:75–81. doi:10.1016/j.jbiotec.2012.12.015

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

Projects BIO2010-20508-C4-1/-4 and BIO2013-48779-C4-1/-4 from the Spanish Ministry of Economy and Competitiveness supported this research. We thank Fundación Ramón Areces for the institutional grant to the Centro de Biologia Molecular Severo Ochoa. M.G.P. was supported by a Spanish FPU fellowship from the Ministry of Education, Culture and Sports.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, 28049, Madrid, Spain

    María Gimeno-Pérez, Dolores Linde & María Fernández-Lobato

  2. Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049, Madrid, Spain

    Lucía Fernández-Arrojo & Francisco J. Plou

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  1. María Gimeno-Pérez
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  2. Dolores Linde
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Correspondence to María Fernández-Lobato.

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Gimeno-Pérez, M., Linde, D., Fernández-Arrojo, L. et al. Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars. Appl Microbiol Biotechnol 99, 3459–3467 (2015). https://doi.org/10.1007/s00253-014-6145-1

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