Ultrahigh-throughput discovery and engineering of enzymes for biotechnological applications
Research summary:
Microbial diversity is a vast reservoir of genetic information that can be valorized through industrial application, from biosynthetic gene clusters to novel protein catalysts. The synergy between new experimental discovery tools based on biology and those based on nanotechnologies are instrumental to find relevant genes faster and more efficiently, enabling especially academic labs to undertake screening campaigns until now costly and limited to large enterprises.
En nuestro laboratorio utilizamos selecciones biológicas o métodos de cribado de ultra-alta capacidad basados en microfluídica para encontrar genes y enzimas de interés en la diversidad natural o artificial. Las selecciones biológicas acoplan la mejora en una propiedad de interés a la supervivencia de un huésped bajo presión de selección. Utilizando este y otros métodos de ingeniería de proteínas hemos desarrollado variantes estables y solubles de enzimas para biocatálisis, como esterasas, deshalogenasas, además de proteínas fluorescentes para aplicaciones de localización in vivo a altas temperaturas.
However, the complexity of cellular metabolism limits the application of biological selections. Microfluidics enables the miniaturization of assays with throughput of kHz and a 1000x reduction in volume and costs. Therefore, droplet microfluidics achieves throughputs typical of biological selections with none of their complexity. Within the H2020 project MetaFluidics, in our laboratory, we have implemented a fluorescence-based microfluidic sorting platform and assays to find stable hydrolases in thermal environments as well as other relevant enzymes for biocatalysis. This has entailed developing news hosts and methods for functional expression, compatible with enzymatic assays at high temperatures.
Ultrahigh-thropughput screening using droplet microfluidics. A) Growth of microorganisms and cells in droplets after 24 h incubation. B) Ultrahigh-throughput screening of thermozymes using Thermus thermophilus as host. C) In vitro transcription translation (IVVTT) using T. thermophilus cell-free extracts. D) Digital PCR to detect rare microbes (mcirobial dark matter) and genes). Images by M. Almendros, A. Lopes and M. Sánchez
| Last name | Name | Laboratory | Ext.* | Professional category | |
|---|---|---|---|---|---|
| Blas Muñoz | Laura | 108 | 4525 | laura.blas(at)cbm.csic.es | Titulado Sup.de Actividades Técn. y Profes. GP1 |
| Bosch Reñe | Sandra | 108 | 4525 | sbosch(at)cbm.csic.es | Titulado Superior Grado de Doctor |
| Cecchini | Davide Agostino | 108 | 4525 | davide.cecchini(at)uam.es | Investigador Doctor |
| Dos Santos Pinto Vázquez | Laura | 108 | 4525 | ldossantos(at)cbm.csic.es | Titulado Sup.de Actividades Técn. y Profes. GP1 |
| Hidalgo Huertas | Aurelio | 108 | 4527 | ahidalgo(at)cbm.csic.es | Profesor Titular Universidad, GA |
| Lorente Arévalo | Álvaro | 108 | 4525 | alorente(at)cbm.csic.es | Titulado Sup.de Actividades Técn. y Profes. GP1 |
| Margot | Léa | 108 | 4524 | Titulado Sup.de Actividades Técn. y Profes. GP1 | |
| Martínez Salvador | John | 108 | 4525/4569 | john.martinez(at)cbm.csic.es | Tco. de Investigación y Laboratorio |
| Nejda Hamdoun | Nargisse | 108 | 4524 | n.nejda(at)cbm.csic.es | Titulado Sup.de Actividades Técn. y Profes. GP1 |
| Ortega Plaza | Carmen | 108 | 4525 | cortega(at)cbm.csic.es | Tco. de Investigación y Laboratorio |
| Pérez Arnaiz | Patricia | 108 | 4498 | pparnaiz(at)cbm.csic.es | Profesor Ayudante Doctor Universidad |
Relevant publications:
- Mate DM, Rivera NR, Sanchez-Freire E, Ayala JA, Berenguer J and Hidalgo A (2019) Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus. Biotech. Bioeng. doi: 10.1002/bit.27170
- Verdú C, Sanchez-Freire E, Ortega C, Hidalgo A, Berenguer J* and Mencía M* (2019) A modular vector toolkit with a tailored set of thermosensors to regulate gene expression in Thermus thermophilus. ACS Omega doi: 10.1021/acsomega.9b02107
- Castillo J, Caminata Landriel S, Sánchez Costa M, Taboga OA, Berenguer J, Hidalgo A, Ferrarotti SA, Costa H (2018) A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production. Protein Eng. Des. Sel. 31: 399-407. doi. 10.1093/protein/gzy034
- Consolati T, Bolívar JM, Petrasek Z, Berenguer J, Hidalgo A, Guisán JM, and Nidetzky B (2018) Bio-based pH internally sensitive materials: immobilized yellow fluorescent protein as optical sensor for spatiotemporal mapping of pH inside porous matrices. ACS Appl. Mater. Inter. 10: 6858-6868. doi: 10.1021/acsami.7b16639
- Ribeiro AL, Mencía M and Hidalgo A (2018) A brief guide to the high-throughput expression of directed evolution libraries. Methods Mol. Biol. 1685: 131-143. doi: 10.1007/978-1-4939-7366-8_7
- Ribeiro AL, Sánchez M, Hidalgo A, and Berenguer J (2017) Stabilization of enzymes by using thermophiles. Methods Mol. Biol. 1465: 297-312. doi: 10.1007/978-1-4939-7183-1_21
- Berenguer J, Mencía M, and Hidalgo A (2017) Are in vivo selections on the path to extinction? Microb. Biotechnol. 10: 46-49. doi: 10.1111/1751-7915.12490
- Vettone A, Serpe M, Hidalgo A, Berenguer J, Del Monaco G, Valenti A, Rossi M, Ciaramella M and Perugino G (2016) A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA-alkyl-transferase by protein engineering. Extremophiles 2016, 20: 1-13. Doi: 10.1007/s00792-015-0791-9
- Hidalgo A, Schließmann A and Bornscheuer UT (2014) One-pot Simple methodology for CAssette Randomization and Recombination for focused directed evolution (OSCARR). Methods. Mol. Biol. 1179: 207-212. doi: 10.1007/978-1-4939-1053-3_14
- Hidalgo A and Berenguer J (2013) Biotechnological applications of Thermus thermophilus as host. Curr. Biotechnol. 2: 304-312. doi: 10.2174/18722083113076660030
- Torres LL, Cantero A, Del Valle M, Marina A, Lopez-Gallego F, Guisan JM, Berenguer J and Hidalgo A (2013) Engineering the substrate specificity of a thermophilic penicillin acylase from Thermus thermophilus. Appl. Environ. Microbiol. 79: 1555-1562. doi: 10.1128/AEM.03215-12
- Torres LL, Ferreras ER, Cantero A, Hidalgo A and Berenguer J (2012) Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli. Microb. Cell Fact. 11: 105. doi: 10.1186/1475-2859-11-105.
- Torres LL, Schließmann A, Schmidt M, Silva-Martin N, Hermoso JA, Berenguer J, Bornscheuer UT and Hidalgo A (2012) Promiscuous enantioselective (-)-γ-lactamase activity in the Pseudomonas fluorescens esterase I. Org. Biomol. Chem. 10: 3388-3392. doi:
10.1039/C2OB06887G.
Last doctoral theses:
- Yamal Al-ramahi González (2013) Fluorescent protein engineering and applications in thermophile microorganisms. Universidad Autónoma de Madrid. Supervisors: Aurelio Hidalgo y José Berenguer.
- Noé R. Rivera (2013) Thermal stabilization of biotechnologically relevant proteins. Universidad Autónoma de Madrid. Supervisors: Aurelio Hidalgo y José Berenguer.
Ongoing doctoral theses:
- Mercedes Sánchez Costa. Discovery and characterization of thermostable enzymes for biocatalysis. Supervisors: Aurelio Hidalgo y José Berenguer.
- Sandra Bosch Reñé. Selection of thermostable biocatalysts for green chemistry using thermophile microorganisms. Supervisor: Aurelio Hidalgo.
- Jorge Bravo Villanueva. Ultrahigh-throughput screening methods based on components from thermophile microorganisms. Supervisor: Aurelio Hidalgo.
Funding:
- Desarrollo de sistemas para la selección de proteínas termoestables mediante expresión in vivo e in vitro a alta temperatura. Entidad financiadora: UAM. UAM/89. Período: 01/02/2017-31/12/2021. Tipo de participación: IP.
- Advanced toolbox for rapid and cost-effective functional metagenomic screening: microbiology meets microfluidics (METAFLUIDICS). Entidad financiadora: Unión Europea H2020. GA 685474. Período: 01/06/2016-30/05/2020. Tipo de participación: Coordinación.
- Sustainable industrial processes based on a C-C bond-forming enzyme platform (CarbaZymes). Entidad financiadora: Unión Europea H2020. GA 635595. Período: 01/04/2015-31/03/2019. Tipo de participación: Socio.
- Ultrahigh-throughput platform for the screening of thermostable proteins by thermophilic in vitro transcription-translation and microfluidics (HOTDROPS). Entidad financiadora: Unión Europea FP7. GA 324439. Período: 01/06/2013-30/05/2017. Tipo de participación: Socio.
- Selección de alta eficiencia de biocatalizadores termoestables para química verde empleando microorganismos termófilos modificados. Entidad financiadora: Ministerio de Economía y Competitividad. Período: 01/01/2013-31/12/2016. Tipo de participación: Co-IP.
