Yeast enzymes bioengineering to generate bioactive compounds

Research summary:

We work with microorganisms of biotechnological interest, mainly fungi and yeasts, producers of bioactive compounds. We try to connect the generation of knowledge to the development of biotechnological applications. Basically we focus on the characterization of new enzymes producing bioactive compounds, the analysis of their structural-functional determinants, the operational improvement using molecular biology tools and in obtaining and characterization of new molecules with potential industrial utility. We have patented in different countries the industrial applicability of most proteins characterized and designed methods for their attachment to solid supports.

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Figure 1. The Xd-INV from Xanthophyllomyces dendrorhous 3D-Structure, a fructofuranosidase producing the prebiotic neokestose. Close-up view of active site including neo-erlose (fructosil maltose).

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Figure 2. The conformation of the substrates at the Ffase active site. A. Six units of inulin (left) and fructosylnystose (right) molecules represented as spheres. B. Inulin (brown) and fructosylnystose (lime green) moieties in stick representation are superimposed.

During the last years we have been characterizing and studying several fungi and non-conventional yeast proteins (from genera Xanthophyllomyces, Schwanniomyces, Rhodotorula, etc.) showing glycosyltransferase activity, and applicable in the production of sugars with prebiotic properties. All are glycosylhydrolases (GH) structurally included in family GH32, 31, 13 or 18. Indeed, we have resolved the 3-D structure of the first yeast protein including in family GH32, assigned a function to the beta-sandwich domain that is present in all members of this family and proved that the oligomerization is directly involved in the substrate recognition and specificity. We have obtained numerous enzymatic variants that increase or alter the biosynthetic product patterns. Recently we have found that some of the characterized enzymes can glycosylate compounds with aromatic rings such as the hydroxytyrosol or pterostilbene (both antioxidants), which confers them a special biotechnological interest. We intend to extend our study to hydrolases including in other structural families, to increase and modify the transferase/biosynthetic activity of the enzymes studied, to scale up to industrial level the enzyme production and the products generated, as well as to validate the biological activity or give new uses to the molecules obtained. Objectives included in those of the consortia Glicoenz, Fish4Fish and a project founded by the Fundación Ramon Areces (XIX Concurso Nacional-Ciencias de la Vida y la Materia).

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Last nameNameLaboratoryExt.*e-mailProfessional category
Fernández LobatoMaría1024492mfernandez(at)cbm.csic.esCatedrático Universidad, GA
García GonzálezDiego Martín1024521diegom.garcia(at)cbm.csic.esTit.Sup.Activ.Técn.y Profes. GP1
Kidibule Peter Elias1024521pkidibule(at)cbm.csic.esTit.Sup.Activ.Técn.y Profes. GP1 66%
Martínez RanzMaría1024521Estudiante TFM
Merdzo KunovacZoran1024521zoran(at)cbm.csic.esTco. de Investigación y Laboratorio
Míguez RodríguezNoa1024492Titulado Sup. Actividades Tecn. y Prof.GP1 66%
Minguet LobatoMarina1024521m.minguet(at)cbm.csic.esAyudante Investigación
Narmontaite Egle4521Tco. de Investigación y Laboratorio
Remacha MorenoMiguel1024521mremacha(at)cbm.csic.esProfesor Titular Universidad, GA

Relevant publications:

  • Garcia-Gonzalez, M., Plou, F.J., Cervantes, F.V., Remacha, M., Poveda, A., Jiménez-Barbero3, J., Fernandez-Lobato, M. (2019) Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts. Microbial Biotechnol. 12: 1274-1285.  Open Access. DOI: 10.1111/1751-7915.13490 
  • Ramírez-Escudero, M., Miguez, N., Gimeno-Perez, M., Ballesteros, A.O., Fernández-Lobato, M., Plou, F.J., and Sanz- Aparicio, J. (2019)  Deciphering the molecular specificity of phenolic compounds as  inhibitors or glycosyl acceptors of β-fructofuranosidase from Xanthophyllomyces dendrorhous.  Scientific Reports 9, 17441. Open Access. https://www.nature.com/articles/s41598-019-53948-y
  • Kidibule, P.E., Santos-Moriano, P., Jiménez-Ortega, E., Ramírez-Escudero, M., Limón, M.C., Remacha, M., Plou, F.J., Sanz-Aparicio, J., Fernández-Lobato, M.  (2018) Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity. Microb. Cell. Fact. 17, 47. Open Access. doi:10.1186/s12934-018-0895-x
  • Míguez, N., Ramírez-Escudero, M., Gimeno-Pérez, M., Poveda, A., Jiménez-Barbero, J. Ballesteros, A. O., Fernández-Lobato, M., Sanz-Aparicio, J. and Plou F. J. (2018) Fructosylation of hydroxytyrosol by the β-fructofuranosidase from Xanthophyllomyces dendrorhous: Insights into the molecular basis of the enzyme specificity. ChemCatChem. 10(21). doi.org/10.1002/cctc.201801171
  • Ramírez-Escudero, M., M. Gimeno-Pérez, B. González, D. Linde, Z. Merdzo, M. Fernández-Lobato* and J. Sanz-Aparicio* (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. 29 (13): 6843-6857 (doi/10.1074/jbc.M115.708495). *Both corresponding authors.
  • Gimeno-Pérez,M., D. Linde, L. Fernández-Arrojo, F. J. Plou, and M. Fernández Lobato (2015) Heterologous overproduction of β-fructofuranosidase from Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars. Appl. Microbiol. Biotechnol. 98 (8) 3459-3467 (doi: 10.1007/s00253-014-6145-1).
  • de Abreu, M.A., Alvaro-Benito, M., Plou, F.J., Fernández Lobato, M.* and Alcalde, M.* (2013) Synthesis of 6-kestose using a highly efficient β-fructofuranosidase engineered by directed evolution. Adv. Synth. Catal. 355 (9), 1698-1702. * Both corresponding authors. (doi: 10.1002/adsc.201200769).
  • Linde, D., Estévez, M., Plou, F. J. and Fernández Lobato, M. (2012) Analysis of the neofructooligosaccharides production mediated by the extracellular β-fructofuranosidase Xd-INV from Xanthophyllomyces dendrorhous. Bioresour. Technol. 109, 123-130 (doi: 10.1016/j.biortech.2012.01.023)
  • Alvaro-Benito, M., Sainz-Polo, M.A., González, B., Plou, F.J., Fernández-Lobato*, M. and Sanz-Aparicio*, J. (2012) Structural and kinetic insight reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, and enzyme that produces prebiotics. J. Biol. Chem. 287, 19674-19686. *Both corresponding authors. (doi:10.1074/jbc.M112.355503)

Doctoral theses:

  • María Gimeno-Pérez (2019) Estudio estructural de la β-fructofuranosidase de Xanthophyllomyces dendrorhous y su empleo para la producción de oligosacáridos prebióticos y otros dereivados fructosilados. UAM
  • Patricia Gutiérrez Alonso (2013) Caracterización de dos glicosiltransferasas de oligosacáridos prebióticos de las levaduras Phaffia rhodozyma y Rhodotorula dairenensis. UAM
  • Miguel Antonio de Abreu Felipe (2011) Studies aimed at improving the funtionality of non-conventional yeast enzymes able to synthesize prebiotic oligosaccarides. UAM. European Program
  • Miguel Álvaro Benito (2011) The study of β-fructofuranosidase from Schwanniomyces occidentalis reveals new functional elements in the family GH32 of glycosyltransferases and an unconventional genetic code use in this yeast. UAM. European Program
  • María Dolores Linde López (2010) Caracterización Bioquímica, molecular y estructural de una b-fructofuranosidasa con capacidad transferasa de la levadura Phaffia rhodozyma aplicable a la producción de oligosacáridos prebióticos. UAM

Patents:

  • P200402994. New enzyme for the prebiotic production: WO 2006/064078 A1; PCT-ES2005/070177; Europa No 05825223.0. Primer premio a la mejor patente Madrid+d 2008.
  • P200501875. New fructofuranosydase for prebiotic oligosaccharides obtaining: PCT-ES2006/000435. Europa Nº 06807883.1; USA No.  11/997.233
  • P200503195. “New fructofuranosydase for the 6-kestose production: PCT-ES2006/000693; Europea No. 06841745-0; USA No. 12/159.164; Japón No. 2008-547993.
  • P200930001. Biocatalizador inmovilizado basado en alginato para la biotransformación de carbohidratos: PCT-ES2010/070104

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