Manuel Becerra Fernández inicia su carrera investigadora en 1993, tras finalizar la licenciatura en Biología en la Universidad Autónoma de Madrid, cuando se incorpora como estudiante de tercer ciclo al Dpto. de Bioloxía Celular e Molecular de la Universidade da Coruña (UDC). En 1999, obtiene el grado de Doctor en Ciencias Biológicas por la UDC, siéndole otorgado el premio extraordinario de doctorado por la UDC. Ese mismo año es contratado como profesor ayudante de Bioquímica y Biología Molecular por la UDC, pasando en el 2004 a profesor Contratado Doctor y desde enero del 2012 es profesor titular de Universidad en la UDC.
With their circumneutral pH and their moderate temperature (66 and 68°C, respectively), As Burgas... more With their circumneutral pH and their moderate temperature (66 and 68°C, respectively), As Burgas and Muiño da Veiga are two important human-use hot springs, previously studied with traditional culture methods, but never explored with a metagenomic approach. In the present study, we have performed metagenomic sequence-based analyses to compare the taxonomic composition and functional potential of these hot springs. Proteobacteria, Deinococcus-Thermus, Firmicutes, Nitrospirae, and Aquificae are the dominant phyla in both geothermal springs, but there is a significant difference in the abundance of these phyla between As Burgas and Muiño da Veiga. Phylum Proteobacteria dominates As Burgas ecosystem while Aquificae is the most abundant phylum in Muiño da Veiga. Taxonomic and functional analyses reveal that the variability in water geochemistry might be shaping the differences in the microbial communities inhabiting these geothermal springs. The content in organic compounds of As Burgas...
Contextos universitarios transformadores: a nova normalidade académica. Leccións aprendidas e retos de futuro. V Xornadas de Innovación Docente, 2021
El uso de plataformas virtuales de docencia por los estudiantes genera una gran cantidad de huell... more El uso de plataformas virtuales de docencia por los estudiantes genera una gran cantidad de huellas y el reto consiste en analizar estos datos para sacar conclusiones del proceso de enseñanza-aprendizaje. En el presente trabajo, nos hemos propuesto analizar los registros que dejan los estudiantes en los espacios virtuales de una materia optativa de cuarto curso del grado de Biología con la finalidad de conocer las actividades que han presentado un mayor número de interacciones y que han despertado por tanto un mayor interés por parte del alumnado.
The northwest of Spain has an abundance of non-volcanic hot springs that, until recently, had onl... more The northwest of Spain has an abundance of non-volcanic hot springs that, until recently, had only been used for thermalism activities. One of such hot springs, Muiño da Veiga, has now been explored using metagenomics to study the microbial community that inhabits these high-temperature circumneutral continental waters. Sequencing of the metagenome allowed the characterization of its composition, diversity, metabolic connections and potential as a source for thermozymes, as well as its ability to assemble MAGs. A diverse microbial community dominated by Bacteria domain members was revealed, particularly from the early-branching Aquificales group. The most abundant genus was Sulfurihydrogenibium, known for its implication in sulfur cycling and for forming mats that enable novel niches. The variety of primary producers with autotrophic pathways (and specifically the sulfur oxidizing pathway) expands the range of available nutrients, and the increase in biomass forms thicker mats, resu...
Additional file 9: Table S6. ANOVA for the response surface quadratic model to optimization of Sc... more Additional file 9: Table S6. ANOVA for the response surface quadratic model to optimization of ScAGal production by BJ3505/YEpMEL1His.
Additional file 1: Figure S1. Standard curves (A) and validation of the method 2−ΔΔCt (B) by qPCR... more Additional file 1: Figure S1. Standard curves (A) and validation of the method 2−ΔΔCt (B) by qPCR and SYBGreen detection. Three extractions of genomic DNA of the calibrator strain KGM28 and decimal serial dilutions were performed. MEL1 (full circles), TAF10 (empty circles), E = amplification efficacy. ΔCtc (CtMEL1,c − CtTAF10,c).
Background Endoglucanases from thermophilic microorganisms are a valuable resource as they can be... more Background Endoglucanases from thermophilic microorganisms are a valuable resource as they can be used in a wide variety of biotechnological applications including the valorisation of biomass and the production of biofuels. In the present work we analysed the metagenome from the hot spring Muiño da Veiga, located in the northwest of Spain (in the Galicia region), in search for novel thermostable endoglucanases. Results Sequence analysis of the metagenome revealed a promising enzyme (Cel776). Predictions on protein structure and conserved amino acid sequences were conducted, as well as expression in heterologous systems with Escherichia coli and Saccharomyces cerevisiae as the host. Cel776Ec was correctly expressed and purified by taking advantage of the His-Tag system, with a yield of 0.346 U/mL in the eluted fraction. Cel776Sc was expressed extracellulary and was easily recovered from the supernatant without the need of further purification, requiring only a concentration step by u...
Functional screenings were conducted on two metagenomic libraries from hot springs in order to fi... more Functional screenings were conducted on two metagenomic libraries from hot springs in order to find novel thermozymes with potential biotechnological applications. These included enzymes acting on plant cell walls such as endoglucanases and exoglucanases, β-glucosidases, xylanases, and β-xylosidases, and broad application enzymes such as proteases and lipolytic hydrolases. Of all the enzymes found by this bioprospection, we selected a novel lipolytic enzyme for further characterization. The protein was found to belong to the SGNH/GDSL family of hydrolases. It was purified and its biochemical parameters determined. We found that the enzyme was most active at 60 °C and pH 9 using pNP-laurate as substrate and was highly thermostable. It also showed preference for short-chained substrates and activation with temperature and with certain detergents such as Tween 80. Proteins of this family of hydrolases are relevant for their broad substrate specificity, that coupled with this protein’s ...
Additional file 2: Figure S2. Extracellular α-galactosidase activity produced by the strain KGM21... more Additional file 2: Figure S2. Extracellular α-galactosidase activity produced by the strain KGM21 growing in lactose or cheese whey media, YPL and YPW, respectively. Data shown are average ± SD, N = 3.
Abstract New thermozymes are required by several industries due to their high stability. During t... more Abstract New thermozymes are required by several industries due to their high stability. During the last few years, functional metagenomics from several hot environments has proven to be very useful to find these high temperature-resistant and operating enzymes. In this chapter, the methodological basis of functional metagenomics is reviewed, with a focus on mining themozymes. Conventional and high-throughput methods at present under implementation for library screening are also described. The development of new tools to adapt functional metagenomics to high temperatures, i.e., to the special characteristics of thermophilic microorganisms and their enzymes, is still a challenge.
β-galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or trans... more β-galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or transgalactosylation of β-galactosides. Among them, the Aspergillus niger β-galactosidase (AnβGal) belongs to the glycoside hydrolase family 35 (GH35) and is widely used in the industry due to its high hydrolytic activity degrading lactose. We present here its three-dimensional structure in complex with different oligosaccharides, to illustrate the structural determinants of the broad specificity of the enzyme against different glycoside linkages. Remarkably, the residues Phe264, Tyr304 and Trp806 make a dynamic hydrophobic platform that accommodates the sugar at subsite +1 suggesting a main role on the recognition of structurally different substrates. Moreover, complexes with the trisaccharides show two potential subsites +2 depending on the substrate type. This feature and the peculiar shape of its wide cavity suggest that AnβGal might accommodate branched substrates from the complex net of polysaccharides composing the plant material in its natural environment. Relevant residues were selected and mutagenesis analyses were performed to evaluate their role in the catalytic performance and the hydrolase/transferase ratio of AnβGal. Thus, we generated mutants with improved transgalactosylation activity. In particular, the variant Y304F/Y355H/N357G/W806F displays a higher level of galacto-oligosaccharides (GOS) production than the Aspergillus oryzae β-galactosidase, which is the preferred enzyme in the industry owing to its high transferase activity. Our results provide new knowledge on the determinants modulating specificity and the catalytic performance of fungal GH35 β-galactosidases. In turn, this fundamental background gives novel tools for the future improvement of these enzymes, which represent an interesting target for rational design.
Over the last years, the development of techniques such as metagenomics, that allows the direct a... more Over the last years, the development of techniques such as metagenomics, that allows the direct access to the genetic information of an environment, has enabled us to comprehend the microbial communities as a complex network of interactions between and within cells rather than just as a set of individual microorganisms living together. Therefore, the study of relationships and communication between organisms has become essential to understand how an ecosystem works. In this chapter, we review the main communication pathways previously described in hot spring archaeal populations, and analyze nine published hot spring DNA metagenomes from distant locations, using metagenomics as a tool to increase our insights in this field.
Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respirat... more Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to
With their circumneutral pH and their moderate temperature (66 and 68°C, respectively), As Burgas... more With their circumneutral pH and their moderate temperature (66 and 68°C, respectively), As Burgas and Muiño da Veiga are two important human-use hot springs, previously studied with traditional culture methods, but never explored with a metagenomic approach. In the present study, we have performed metagenomic sequence-based analyses to compare the taxonomic composition and functional potential of these hot springs. Proteobacteria, Deinococcus-Thermus, Firmicutes, Nitrospirae, and Aquificae are the dominant phyla in both geothermal springs, but there is a significant difference in the abundance of these phyla between As Burgas and Muiño da Veiga. Phylum Proteobacteria dominates As Burgas ecosystem while Aquificae is the most abundant phylum in Muiño da Veiga. Taxonomic and functional analyses reveal that the variability in water geochemistry might be shaping the differences in the microbial communities inhabiting these geothermal springs. The content in organic compounds of As Burgas...
Contextos universitarios transformadores: a nova normalidade académica. Leccións aprendidas e retos de futuro. V Xornadas de Innovación Docente, 2021
El uso de plataformas virtuales de docencia por los estudiantes genera una gran cantidad de huell... more El uso de plataformas virtuales de docencia por los estudiantes genera una gran cantidad de huellas y el reto consiste en analizar estos datos para sacar conclusiones del proceso de enseñanza-aprendizaje. En el presente trabajo, nos hemos propuesto analizar los registros que dejan los estudiantes en los espacios virtuales de una materia optativa de cuarto curso del grado de Biología con la finalidad de conocer las actividades que han presentado un mayor número de interacciones y que han despertado por tanto un mayor interés por parte del alumnado.
The northwest of Spain has an abundance of non-volcanic hot springs that, until recently, had onl... more The northwest of Spain has an abundance of non-volcanic hot springs that, until recently, had only been used for thermalism activities. One of such hot springs, Muiño da Veiga, has now been explored using metagenomics to study the microbial community that inhabits these high-temperature circumneutral continental waters. Sequencing of the metagenome allowed the characterization of its composition, diversity, metabolic connections and potential as a source for thermozymes, as well as its ability to assemble MAGs. A diverse microbial community dominated by Bacteria domain members was revealed, particularly from the early-branching Aquificales group. The most abundant genus was Sulfurihydrogenibium, known for its implication in sulfur cycling and for forming mats that enable novel niches. The variety of primary producers with autotrophic pathways (and specifically the sulfur oxidizing pathway) expands the range of available nutrients, and the increase in biomass forms thicker mats, resu...
Additional file 9: Table S6. ANOVA for the response surface quadratic model to optimization of Sc... more Additional file 9: Table S6. ANOVA for the response surface quadratic model to optimization of ScAGal production by BJ3505/YEpMEL1His.
Additional file 1: Figure S1. Standard curves (A) and validation of the method 2−ΔΔCt (B) by qPCR... more Additional file 1: Figure S1. Standard curves (A) and validation of the method 2−ΔΔCt (B) by qPCR and SYBGreen detection. Three extractions of genomic DNA of the calibrator strain KGM28 and decimal serial dilutions were performed. MEL1 (full circles), TAF10 (empty circles), E = amplification efficacy. ΔCtc (CtMEL1,c − CtTAF10,c).
Background Endoglucanases from thermophilic microorganisms are a valuable resource as they can be... more Background Endoglucanases from thermophilic microorganisms are a valuable resource as they can be used in a wide variety of biotechnological applications including the valorisation of biomass and the production of biofuels. In the present work we analysed the metagenome from the hot spring Muiño da Veiga, located in the northwest of Spain (in the Galicia region), in search for novel thermostable endoglucanases. Results Sequence analysis of the metagenome revealed a promising enzyme (Cel776). Predictions on protein structure and conserved amino acid sequences were conducted, as well as expression in heterologous systems with Escherichia coli and Saccharomyces cerevisiae as the host. Cel776Ec was correctly expressed and purified by taking advantage of the His-Tag system, with a yield of 0.346 U/mL in the eluted fraction. Cel776Sc was expressed extracellulary and was easily recovered from the supernatant without the need of further purification, requiring only a concentration step by u...
Functional screenings were conducted on two metagenomic libraries from hot springs in order to fi... more Functional screenings were conducted on two metagenomic libraries from hot springs in order to find novel thermozymes with potential biotechnological applications. These included enzymes acting on plant cell walls such as endoglucanases and exoglucanases, β-glucosidases, xylanases, and β-xylosidases, and broad application enzymes such as proteases and lipolytic hydrolases. Of all the enzymes found by this bioprospection, we selected a novel lipolytic enzyme for further characterization. The protein was found to belong to the SGNH/GDSL family of hydrolases. It was purified and its biochemical parameters determined. We found that the enzyme was most active at 60 °C and pH 9 using pNP-laurate as substrate and was highly thermostable. It also showed preference for short-chained substrates and activation with temperature and with certain detergents such as Tween 80. Proteins of this family of hydrolases are relevant for their broad substrate specificity, that coupled with this protein’s ...
Additional file 2: Figure S2. Extracellular α-galactosidase activity produced by the strain KGM21... more Additional file 2: Figure S2. Extracellular α-galactosidase activity produced by the strain KGM21 growing in lactose or cheese whey media, YPL and YPW, respectively. Data shown are average ± SD, N = 3.
Abstract New thermozymes are required by several industries due to their high stability. During t... more Abstract New thermozymes are required by several industries due to their high stability. During the last few years, functional metagenomics from several hot environments has proven to be very useful to find these high temperature-resistant and operating enzymes. In this chapter, the methodological basis of functional metagenomics is reviewed, with a focus on mining themozymes. Conventional and high-throughput methods at present under implementation for library screening are also described. The development of new tools to adapt functional metagenomics to high temperatures, i.e., to the special characteristics of thermophilic microorganisms and their enzymes, is still a challenge.
β-galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or trans... more β-galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or transgalactosylation of β-galactosides. Among them, the Aspergillus niger β-galactosidase (AnβGal) belongs to the glycoside hydrolase family 35 (GH35) and is widely used in the industry due to its high hydrolytic activity degrading lactose. We present here its three-dimensional structure in complex with different oligosaccharides, to illustrate the structural determinants of the broad specificity of the enzyme against different glycoside linkages. Remarkably, the residues Phe264, Tyr304 and Trp806 make a dynamic hydrophobic platform that accommodates the sugar at subsite +1 suggesting a main role on the recognition of structurally different substrates. Moreover, complexes with the trisaccharides show two potential subsites +2 depending on the substrate type. This feature and the peculiar shape of its wide cavity suggest that AnβGal might accommodate branched substrates from the complex net of polysaccharides composing the plant material in its natural environment. Relevant residues were selected and mutagenesis analyses were performed to evaluate their role in the catalytic performance and the hydrolase/transferase ratio of AnβGal. Thus, we generated mutants with improved transgalactosylation activity. In particular, the variant Y304F/Y355H/N357G/W806F displays a higher level of galacto-oligosaccharides (GOS) production than the Aspergillus oryzae β-galactosidase, which is the preferred enzyme in the industry owing to its high transferase activity. Our results provide new knowledge on the determinants modulating specificity and the catalytic performance of fungal GH35 β-galactosidases. In turn, this fundamental background gives novel tools for the future improvement of these enzymes, which represent an interesting target for rational design.
Over the last years, the development of techniques such as metagenomics, that allows the direct a... more Over the last years, the development of techniques such as metagenomics, that allows the direct access to the genetic information of an environment, has enabled us to comprehend the microbial communities as a complex network of interactions between and within cells rather than just as a set of individual microorganisms living together. Therefore, the study of relationships and communication between organisms has become essential to understand how an ecosystem works. In this chapter, we review the main communication pathways previously described in hot spring archaeal populations, and analyze nine published hot spring DNA metagenomes from distant locations, using metagenomics as a tool to increase our insights in this field.
Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respirat... more Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to
Uploads
Papers by Manuel Becerra