Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose

Rodrigo-Frutos, David; Piedrabuena, David; Sanz-Aparicio, Julia; Fernández-Lobato, María

doi:10.1007/s00253-018-9446-y

Biotechnologically relevant enzymes and proteins
Published: 24 October 2018

Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose

David Rodrigo-Frutos¹,
David Piedrabuena¹,
Julia Sanz-Aparicio² &
María Fernández-Lobato ORCID: orcid.org/0000-0003-0778-7443¹

Applied Microbiology and Biotechnology volume 103, pages 279–289 (2019)Cite this article

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Abstract

The β-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and a mutated Ffase variant including the Ser196Leu substitution (Ffase-Leu196). Best results were obtained using yeast cultures supplemented with sucrose and expressing the Ffase-Leu196, which after only 4 h produced ~ 116 g/L of 6-kestose, twice the amount obtained with the corresponding purified enzyme. 6-Kestose represented ~ 70% of the products synthesized. In addition, a small amount of 1-kestose and the neofructoligosaccharides neokestose and blastose were also produced. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~ 2.2- and 1.5-fold, respectively, without affecting production of 1-kestose. Supplementing yeast cultures with glucose clearly showed that blastose originates from direct fructosylation of glucose, a property that has not been described for other similar proteins from yeasts. Modeling neokestose and blastose into the Ffase-active site revealed the molecular basis explaining the peculiar specificity of this enzyme.

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References

Adamberg S, Tomson K, Vija H, Puurand M, Kabanova N, Visnapuu T, Jõgi E, Alamäe T, Adamberg K (2014) Degradation of fructans and production of propionic acid by Bacteroides thetaiotaomicron are enhanced by the shortage of amino acids. Front Nutr 5:1–21. https://doi.org/10.3389/fnut.2014.00021
Álvaro-Benito M, de Abreu M, Fernández-Arrojo L, Plou FJ, Jiménez-Barbero J, Ballesteros A, Polaina J, Fernández-Lobato M (2007) Characterization of a β-fructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose. J Biotechnol 132:75–81. https://doi.org/10.1016/j.jbiotec.2007.07.939
CAS Article PubMed Google Scholar
Álvaro-Benito M, de Abreu M, Portillo F, Sánz-Aparicio J, Fernández-Lobato M (2010a) New insights into the fructosyltransferase activity of Schwanniomyces occidentalis β-fructofuranosidase emerging from a non-conventional codon usage and directed mutation. Appl Environ Microbiol 76:7491–7499. https://doi.org/10.1128/AEM.01614-10
CAS Article PubMed PubMed Central Google Scholar
Álvaro-Benito M, Polo A, González B, Fernández-Lobato M, Sánz-Aparicio J (2010b) 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. https://doi.org/10.1074/jbc.M109.095430
CAS Article PubMed PubMed Central Google Scholar
Álvaro-Benito M, Sainz-Polo MA, González-Pérez D, González B, Plou FJ, Fernández-Lobato M, Sanz-Aparicio J (2012) Structural and kinetic insights reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, an enzyme that produces prebiotics. J Biol Chem 287:19674–19686. https://doi.org/10.1074/jbc.M112.355503
CAS Article PubMed PubMed Central Google Scholar
Bali V, Panesar PS, Bera MB, Panesar R (2015) Fructo-oligosaccharides: production, purification and potential applications. Crit Rev Food Sci 55:1475–1490. https://doi.org/10.1080/10408398
CAS Article Google Scholar
Bekers M, Laukevics J, Upite D, Kaminska E, Vigants A, Viesturs U, Pankova L, Danilevics A (2002) Fructooligosaccharide and levan producing activity of Zymomonas mobilis extracellular levansucrase. Process Biochem 38:701–706. https://doi.org/10.1016/S0032-9592(02)00189-9
CAS Article Google Scholar
Caputi L, Nepogodiev SA, Malnoy M, Rejzek M, Field RA, Benini S (2013) Biomolecular characterization of the levansucrase of Erwinia amylovora a promising biocatalyst for the synthesis of fructooligosaccharides. J Agric Food Chem 61:12265–12273. https://doi.org/10.1021/jf4023178
de Abreu MA, Álvaro-Benito M, Plou FJ, Fernández-Lobato M, Alcalde M (2013) Synthesis of 6-kestose using a highly efficient β-fructofuranosidase engineered by directed evolution. Adv Synth Catal 355:1698–1702. https://doi.org/10.1002/adsc.201200769
CAS Article Google Scholar
DeLano WL (2002) The PyMOL Molecular Graphics System, version 1.6.0.0, DeLano Scientific, San Carlos, CA
Dominguez A, Nobre C, Rodrigues LR, Peres AM, Torres D, Rocha I, Lima N, Teixeira J (2012) New improved method for fructooligosaccharides production by Aureobasidium pullulans. Carbohydr Polym 89:1174–1179. https://doi.org/10.1016/j.carbpol.2012.03.091
CAS Article PubMed Google Scholar
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132. https://doi.org/10.1107/S0907444904019158
CAS Article PubMed Google Scholar
Farine S, Versluis C, Bonnici PJ, Heck A, L’homme C, Puicserver 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 55:75–81. https://doi.org/10.1007/s002530000493
Fialho MB, Simoes K, Barros CD, Possoni RAB, Braga MR, Figueiredo-Ribeiro RDL (2013) Production of 6-kestose by the filamentous fungus Gliocladium virens as affected by sucrose concentration. Mycoscience 54:198–204. https://doi.org/10.1016/j.myc.2012.09.012
CAS Article Google Scholar
Franck AS, De Leenheer L (2005) Inulin. In Biopolymers Online, Wiley Online Library https://doi.org/10.1002/3527600035.bpol6014
Gimeno-Pérez M, Linde D, Fernández-Arrojo L, Plou FJ, Fernández-Lobato M (2015) Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars. Appl Microbiol Biotechnol 99:3459–3467. https://doi.org/10.1007/s00253-014-6145-1
CAS Article PubMed Google Scholar
Gutiérrez-Alonso P, Fernández-Arrojo L, Plou FJ, Fernández-Lobato M (2009) Biochemical characterization of a β-fructofuranosidase from Rhodotorula dairenensis with transfructosylating activity. FEMS Yeast Res 9:768–773. https://doi.org/10.1111/j.1567-1364.2009.00526
Article PubMed Google Scholar
He C, Yang Y, Zhao R, Qu J, Jin LL, Xiao M (2018) Rational designed mutagenesis of levansucrase from Bacillus licheniformis 8-37-0-1 for product specificity study. Appl Microbiol Biotechnol 102:3217–3228. https://doi.org/10.1007/s00253-018-8854-3
Jõgi, E, Metsla K, Visnapuu T, Aasamets A, Vija H, Alamäe T (2015) Synthesis and purification of functional fructans. Proceedings of the 11th International Conference on Polysaccharides-Glycoscience: 5–9
Katapodis P, Kalogeris E, Kekos D, Macris BJ, Chistakopoulos P (2014) Biosynthesis of fructo-oligosaccharides by Sporotrichum thermophile during submerged batch cultivation in high sucrose media. Appl Microbiol Biotechnol 63:378–382. https://doi.org/10.1007/s11274-004-0684-z
Article Google Scholar
Kilian SG, Kritzinger SM, Rycroft C, Gibson GR, du Preez JC (2002) The effects of the novel bifidogenic trisaccharide, neokestose, on the human colonic microbiota World J Microbiol Biotechnol 18:637–644. https://doi.org/10.1023/A:1016808015630
Kirschner KN, Yongye AB, Tschampel SM, González-Outeiriño J, Daniels CR, Foley BL, Woods RJ (2008) GLYCAM06: a generalizable biomolecular force field. Carbohydrates J Comput Chem 29:622–655. https://doi.org/10.1002/jcc.20820
CAS Article PubMed Google Scholar
Koga Y, Tokunaga S, Nagano J, Sato F, Konishi K, Tochio T, Murakami Y, Masumoto N, Tezuka J, Sudo N, Kubo C, Shibata R (2016) Age-associated effect of kestose on Faecalibacterium prausnitzii and symptoms in the atopic dermatitis infants. Pediatr Res 80:844–851. https://doi.org/10.1038/pr.2016.167
CAS Article PubMed PubMed Central Google Scholar
Lafraya A, Sanz-Aparicio J, Polaina J, Marín-Navarro J (2011) Fructooligosaccharide synthesis by mutant versions of Saccharomyces cerevisiae invertase. Appl Environ Microbiol 77:6148–6157. https://doi.org/10.1128/AEM.05032-11
CAS Article PubMed PubMed Central Google Scholar
Lee SM, Chang JY, Wu JS, Sheu DC (2015) Antineoplastic effect of a novel chemopreventive agent, neokestose, on the Caco-2 cell line via inhibition of expression of nuclear factor-kappa B and cyclooxygenase-2. Mol Med Rep 12:1114–1118. https://doi.org/10.3892/mmr.2015.3507
CAS Article PubMed Google Scholar
Lim JS, Lee JH, Kang SW, Park SW, Kim SW (2007) Studies on production and physical properties of neo-FOS by co-inmovilized Penicillium and neo-fructosyltransferase. Eur Food Res Technol 225:457–462. https://doi.org/10.1016/j.biortech.2013.01.061
CAS Article Google Scholar
Linde D, Macías I, Fernández-Arrojo L, Plou FJ, Jiménez A, Fernández-Lobato M (2009) Molecular and biochemical characterization of a β-fructofuranosidase from Xanthophyllomyces dendrorhous. Appl Environ Microbiol 75:1065–1073. https://doi.org/10.1128/AEM.02061-08
CAS Article PubMed Google Scholar
Mardo K, Visnapuu T, Vija H, Aasamens A, Viigand K, Alamäe T (2017) A highly active endo-levanase BT1760 of a dominant mammalian gut commensal Bacteroides thetaiotaomicron cleaves not only various bacterial levans, but also levan of timothy grass. PLoS One e0169989:12. https://doi.org/10.1371/journal.pone.0169989
Marín-Navarro J, Talens-Perales D, Polaina J (2015) One-pot production of fructooligosaccharides by a Saccharomyces cerevisiae strain expressing an engineered invertase. Appl Microbiol Biotechnol 99:2549–2555. https://doi.org/10.1007/s00253-014-6312-4
CAS Article PubMed Google Scholar
Marx SP, Winkler S, Hartmaier W (2000) Metabolization of β-(2,6) linked fructose-oligosaccharides by different bifidobacteria. FEMS Microbiol Lett 182:163–169. https://doi.org/10.1111/j.1574-6968.2000.tb08891.x
CAS Article PubMed Google Scholar
Miranda-Molina A, Castillo E, Lopez-Munguia A (2017) A novel two-step enzymatic synthesis of blastose, a β-D-fructofuranosyl-(2↔6)-D-glucopyranose sucrose analogue. Food Chem 227:202–210. https://doi.org/10.1016/j.foodchem.2017.01.094
Ning Y, Wang J, Chen J, Yang N, Jin Z, Xu X (2010) Production of neo-fructooligosaccharides using free-whole-cells biotransformation by Xanthophyllomyces dendrorhous. Bioresour Technol 101:7472–7478. https://doi.org/10.1016/j.biortech.2010.04.026
CAS Article PubMed Google Scholar
Nobre C, Teixeira JA, Rodrigues LR (2015) New trends and technological challenges in the industrial production and purification of fructo-oligosaccharides. Crit Rev Food Sci Nutri 55:1444–1455. https://doi.org/10.1080/10408398.2012.697082
CAS Article Google Scholar
Nobre C, Castro CC, Hantson AL, Teixeira JA, de Weireld G, Rodrigues LR (2016) Strategies for the production of high-content fructo-oligosaccharides through the removal of small saccharides by co-culture or successive fermentation with yeast. Carbohydr Polym 136:274–281. https://doi.org/10.1016/j.carbpol.201508.088
CAS Article PubMed Google Scholar
Piedrabuena D, Míguez N, Poveda A, Plou FJ (2016) Exploring the transferase activity of Ffase from Schwanniomyces occidentalis, a β-fructofuranosidase showing high fructosyl-acceptor promiscuity. Appl Microbiol Biotechnol 100:8769–8778. https://doi.org/10.1007/s00253-016-7628-z
CAS Article PubMed Google Scholar
Porras-Domínguez JR, Ávila-Fernández A, Rodriguez-Alegría ME, Miranda-Molina A, Escalante A, González-Cervantes R, Olvera C, López-Munguía A (2014) Levan-type FOS production using a Bacillus licheniformis endolevanase. Process Biochem 49:783–790. https://doi.org/10.1016/j.procbio.2014.02.005
CAS Article Google Scholar
Ramírez-Escudero M, Gimeno-Pérez M, Gonzalez B, Linde D, Merdzo Z, Fernández-Lobato M, Sanz-Aparicio J (2016) Structural analysis of β-fructofuranosidase from Xanthophyllomyces dendrorhous reveals unique features and the crucial role of N-glycosylation in oligomerization and activity. J Biol Chem 291:6843–6857. https://doi.org/10.1074/jbc.M115.708495
Ramirez-Farias C, Slezak K, Fuller Z, Duncan A, Holtrop G, Louis P (2009) Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii. Br J Nutr 101:541–550. https://doi.org/10.1017/S0007114508019880
Roberfroid M (2007) Prebiotics: the concept revisited. J Nutr 137:830S–837S
CAS Article Google Scholar
Santos-Moriano P, Fernandez-Arrojo L, Poveda A, Jimenez-Barbero J, Ballesteros AO, Plou FJ (2015) Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: effect of reaction conditions. J Mol Catal B Enzym 119:18–25. https://doi.org/10.1016/j.molcatb.2015.05.011
CAS Article Google Scholar
Scott KP, Martin JC, Duncan SH, Flint HJ (2014) Prebiotic stimulation of human colonic butyrate-producing bacteria and bifidobacteria in vitro. FEMS Microbiol Ecol 87:30–40. https://doi.org/10.1111/1574-6941.12186
CAS Article PubMed Google Scholar
Semjonovs SP, Marauska M, Linde R, Grube M, Zikmanis P, Bekers M (2004) Development of Bifidobacterium lactis Bb 12 on β-(2,6)-linked fructan-containing substrate. Eng Life Sci 4:433–437. https://doi.org/10.1002/elsc.200420043
CAS Article Google Scholar
Sheu DC, Chang JY, Chen YJ, Lee CW (2013) Production of high-purity neofructooligosaccharides by culture of Xanthophyllomyces dendrorhous. Bioresour Technol 132:432–435. https://doi.org/10.1016/j.biortech.2013.01.061
CAS Article PubMed Google Scholar
Stewart ML, Tim DA, Slavin JL (2008) Fructooligosaccharides exhibit more rapid fermentation than long-chain inulin in an in vitro fermentation system. Nutr Res 28:329–334. https://doi.org/10.1016/j.nutres.2008.02.014
Swennen K, Courtin CM, Delcour JA (2006) Non-digestible oligosaccharides with prebiotic properties. Crit Rev Food Sci Nutr 46:459–471. https://doi.org/10.1080/10408390500215746
CAS Article PubMed Google Scholar
Wu JS, Chang JY, Chen CW, Lin MT, Sheu DC, Lee SM (2017) Neokestose suppresses the growth of human melanoma A2058 cells via inhibition of the nuclear factor-kappa B signalling pathway. Mol Med Rep 16:295–300. https://doi.org/10.3892/mmr.2017.6594
CAS Article PubMed Google Scholar
Yun JW (1996) Fructooligosaccharides-occurrence, preparation and application. Enzyme Microbiol Technol 19:107–117. https://doi.org/10.1016/0141-0229(95)00188-3
CAS Article 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 49:2174–2180. https://doi.org/10.1016/j.procbio.2014.09.021
CAS Article Google Scholar
Zambelli P, Tamborini L, Cazzamalli S, Pinto A, Arioli S, Balzarettio S, Plou FJ, Fernández-Arrojo L, Molinari F, Conti P, Romano D (2016) An efficient continuous flow process for the synthesis of a non-conventional mixture of fructoligosaccharides. Food Chem 190:607–613. https://doi.org/10.1016/j.foodchem.2015.06.002
CAS Article PubMed Google Scholar
Zhang F, Hang XM, Fan XB, Li GJ, Yang H (2007) Selection and optimization procedure of symbiotic for cholesterol removal. Anaerobe 13:185–192. https://doi.org/10.5897/ABJ11.3703
CAS Article PubMed Google Scholar
Zhang L, An J, Li L, Wang H, Liu D, Li N, Cheng H, Deng Z (2016) Highly efficient fructooligosaccharides production by an erythritol producing yeast Yarrowia lipolytica displaying fructosyltransferase. J Agric Food Chem 64:3828–3837. https://doi.org/10.1021/acs.jafc.6b00115
CAS Article PubMed Google Scholar

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Acknowledgements

We thank Mrs. Asunción Martín-Redondo and María Gimeno-Pérez for their technical support and Mr. Tom Halmos for reading and correcting this manuscript.

Funding

This work was supported by the Spanish Ministry of Economy and Competitiveness: BIO2016-76601-C3-2/-3, and by institutional grants from Fundación Ramón Areces and Banco de Santander to the Centro de Biología Molecular Severo Ochoa. Besides, funding has been received from the European Union’s Horizon 2020 research and innovation program [Blue Growth: Unlocking the potential of Seas and Oceans] under grant agreement No [634486; INMARE]. D.P. was supported by the Spanish Ministry of Education’s University Personnel Training Plan ref. FPU014/01004.

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Department of Molecular Biology, Center of Molecular Biology Severo Ochoa (CBMSO; CSIC-UAM), University Autónoma from Madrid, 28049, Madrid, Spain
David Rodrigo-Frutos, David Piedrabuena & María Fernández-Lobato
Department of Crystallography and Structural Biology, Institute of Physical Chemistry-Rocasolano (CSIC), 28006, Madrid, Spain
Julia Sanz-Aparicio

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David Rodrigo-Frutos
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David Piedrabuena
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Julia Sanz-Aparicio
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María Fernández-Lobato
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Correspondence to María Fernández-Lobato.

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Rodrigo-Frutos, D., Piedrabuena, D., Sanz-Aparicio, J. et al. Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose. Appl Microbiol Biotechnol 103, 279–289 (2019). https://doi.org/10.1007/s00253-018-9446-y

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Received: 20 July 2018
Revised: 02 October 2018
Accepted: 09 October 2018
Published: 24 October 2018
Issue Date: January 2019
DOI: https://doi.org/10.1007/s00253-018-9446-y

Keywords

Schwanniomyces occidentalis
β-Fructofuranosidase
Prebiotic sugars
6-Kestose
Blastose
Yeast cultures