RESUMEN

Filamentous fungus Aspergillus niger has high industrial value due to their lignocellulolytic enzyme activities and ATCC 10864 is one of the few type strains of A. niger which has a unique biofilm forming capability. Here we report the first draft genome sequence of A. niger ATCC 10864 strain. The genome of A. niger ATCC 10864 is 36,172,237 bp long and comprise of 310 scaffolds with 49.5% average GC content. A total of 10,804 protein-coding genes were predicted among which 10,761 genes were with putative functions. A. niger ATCC 10864 genome coded for 709 putative carbohydrate active enzyme families distributed in six functional categories and among them glycoside hydrolases (GHs) represent the most number of families (279). Genes that include pepA, brlA, exgA, LaeA, rodA, GCN have also been identified in this study, which may play a role in biofilm formation. This high-quality draft genome sequence will facilitate our understanding of the mechanisms behind fungal biofilm formation and higher lignocellulolytic enzyme production.

RESUMEN

Here, we report the complete genome sequence of a high alkaline cellulase producing Aspergillus fumigatus strain LMB-35Aa isolated from soil of Peruvian Amazon rainforest. The genome is ∼27.5mb in size, comprises of 228 scaffolds with an average GC content of 50%, and is predicted to contain a total of 8660 protein-coding genes. Of which, 6156 are with known function; it codes for 607 putative CAZymes families potentially involved in carbohydrate metabolism. Several important cellulose degrading genes, such as endoglucanase A, endoglucanase B, endoglucanase D and beta-glucosidase, are also identified. The genome of A. fumigatus strain LMB-35Aa represents the first whole sequenced genome of non-clinical, high cellulase producing A. fumigatus strain isolated from forest soil.

Asunto(s)

Aspergillus fumigatus/genética , Genoma Fúngico , Aspergillus fumigatus/metabolismo , Celulasa/metabolismo , Perú , Filogenia , Bosque Lluvioso , Microbiología del Suelo

RESUMEN

Aguas Calientes (AC) is an isolated geothermal spring located deep into the Amazon rainforest (7°21'12″ S, 75°00'54″ W) of Peru. This geothermal spring is slightly acidic (pH 5.0-7.0) in nature, with temperatures varying from 45 to 90 °C and continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). Pooled water sample was analyzed at 16S rRNA V3-V4 hypervariable region by amplicon metagenome sequencing on Illumina HiSeq platform. A total of 2,976,534 paired ends reads were generated which were assigned into 5434 numbers of OTUs. All the resulting 16S rRNA fragments were then classified into 58 bacterial phyla and 2 archaeal phyla. Proteobacteria (88.06%) was found to be the highest represented phyla followed by Thermi (6.43%), Firmicutes (3.41%) and Aquificae (1.10%), respectively. Crenarchaeota and Euryarchaeota were the only 2 archaeal phyla detected in this study with low abundance. Metagenomic sequences were deposited to SRA database which is available at NCBI with accession number SRX1809286. Functional categorization of the assigned OTUs was performed using PICRUSt tool. In COG analysis "Amino acid transport and metabolism" (8.5%) was found to be the highest represented category whereas among predicted KEGG pathways "Metabolism" (50.6%) was the most abundant. This is the first report of a high resolution microbial phylogenetic profile of an Amazonian hot spring.

RESUMEN

Alkaline cellulase producing fungi were isolated from soils of an undisturbed rain forest of Peru. The soil dilution plate method was used for the enumeration and isolation of fast growing cellulolytic fungi on an enriched selective medium. Eleven out of 50 different morphological colonies were finally selected by using the plate clearing assay with CMC as substrate at different pH values. All 11 strains produced cellulases in liquid culture with activities at alkaline pH values without an apparent decrease of them indicating that they are true alkaline cellulase producers. Aspergillus sp. LM-HP32, Penicillium sp. LM-HP33, and Penicillium sp. LM-HP37 were the best producers of FP cellulase (>3 U mL(-1)) with higher specific productivities (>30 U g(-1) h(-1)). Three strains have been found suitable for developing processes for alkaline cellulase production. Soils from Amazonian rain forests are good sources of industrial fungi with particular characteristics. The results of the present study are of commercial and biological interest. Alkaline cellulases may be used in the polishing and washing of denim processing of the textile industry.

RESUMEN

Industrial enzymes are produced by submerged fermentation (SF) and by solid-state fermentation (SSF) to a lesser extent. Although SSF has several advantages, its scale-up is difficult. The role of physiological and genetic properties of microorganisms growing attached to surfaces could explain the advantages of SSF. Filamentous fungi are naturally adapted to growth on surfaces and in these conditions they show a particular physiological behavior which is different from that in SF; thus, they also form biofilms. Fermentation by filamentous fungal biofilms (FFB) is a homogeneous production system within a liquid environment based on the infrastructure of the SF process with the productive efficiency of the SSF. Enzyme production levels of FFB are much higher than those obtained in SF and they are also amenable of mixed fungal cultivation. Transcriptomic and proteomic tools are used to uncover the fundamental biological issues behind FFB. Several genes encoding cellulolytic enzymes are either differentially expressed or overexpressed in FFB. Moreover, our proteomic studies of Aspergillus niger biofilms compared to SF indicate that many intracellular proteins are either differentially expressed or overexpressed. Clinically important fungi like A. fumigatus also form biofilms when they infect lungs and recent studies demonstrate same gene expression features. These results support our hypothesis of cell adhesion and its role in the new schemes for improved fermentative production of industrial enzymes.

Asunto(s)

Biopelículas , Hongos/fisiología , Industrias , Aspergillus/citología , Aspergillus/genética , Aspergillus/metabolismo , Aspergillus/fisiología , Células Inmovilizadas/metabolismo , Hongos/citología , Hongos/genética , Hongos/metabolismo , Micelio/citología , Micelio/genética , Micelio/metabolismo , Micelio/fisiología

RESUMEN

Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis.

Asunto(s)

Antígenos Helmínticos , Cysticercus/metabolismo , Proteínas del Helminto , Neurocisticercosis/diagnóstico , Taenia solium/metabolismo , Teniasis/diagnóstico , Tripsina , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/genética , Antígenos Helmínticos/metabolismo , Cysticercus/química , Cysticercus/genética , Cysticercus/crecimiento & desarrollo , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Humanos , Datos de Secuencia Molecular , Neurocisticercosis/parasitología , Estructura Terciaria de Proteína , Porcinos , Taenia solium/química , Taenia solium/genética , Taenia solium/crecimiento & desarrollo , Teniasis/parasitología , Tripsina/química , Tripsina/genética , Tripsina/metabolismo

RESUMEN

Las células inmovilizadas tienen aplicación potencial en la producción de biocombustibles posibilitando la reutilización de biomasa, el empleo de diversas configuraciones de reactores y sistemas de cultivo, el manejo de altas densidades celulares alcanzando altas productividades volumétricas, y la simplificación de operaciones de procesamiento de salida. El objetivo del presente estudio fue evaluar la influencia del diámetro de las perlas y la densidad celular en la producción de etanol con Saccharomyces uvarum inmovilizada en alginato al 2% (p/v). Para ello se evaluaron tres diámetros de perlas de 2, 2,5 y 3 mm. Las células inmovilizadas fueron cultivadas en medio con 12% (p/v) de glucosa en biorreactores de columna sin agitación a 28 ºC, y se operaron cuatro lotes consecutivos de 48 horas cada uno. En cada lote se cuantificó el consumo de glucosa y se determinó la cantidad de etanol producido. Los rendimientos máximos de etanol para las esferas de 2, 2,5 y 3 mm de diámetro fueron 81, 83 y 97% del rendimiento teórico. La máxima productividad volumétrica de etanol fue 1,2 g/L-1/h-1 con un consumo de glucosa de 99,8% al término del lote, correspondiente a las columnas con perlas de 3 mm y con una producción de 0,017 g de etanol por esfera. La producción de etanol acumulada en cada sistema fue 178, 189 y 200 g/L-1 para 2, 2,5 y 3 mm respectivamente, encontrándose una relación directa con el diámetro de perla e inversa respecto a la densidad celular. Los rendimientos de etanol obtenidos son superiores a los reportados para la misma especie.

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Immobilized cells have a potential use in biofuel production. They also allow re-using biomass, using diverse reactor configurations and culture systems, handling high cell densities to obtain high volumetric productivities and to simplify the downstream processing. The purpose of this work was to evaluate the influence of bead diameter and cell density on ethanol production using immobilized Saccharomyces uvarum in 2% (w/v) alginate. For that, three bead diameters (2, 2.5 and 3 mm) were evaluated. Immobilized cells were cultured on a 12% (w/v) glucose medium in column bioreactors without agitation at 28 °C for four 48 h–repeated batches. For each batch, both glucose consumption and ethanol produced were measured. Maximum yields for 2, 2.5 and 3 mm bead diameters were 81, 83 and 97% of theoretical yield. Maximum volumetric productivity of ethanol was 1.2 g/L-1/h-1 with 99.8% glucose consumption at the end of the batch, corresponding to the 3 mm bead diameter and the ethanol production per bead was 0.017 g. Accumulated ethanol production for each system was 178, 189 and 200 g/L-1 for 2, 2.5 y 3 mm bead diameter, respectively, being this directly related to bead diameter and inversely related to cell density. Ethanol yields were higher than those reported for the same species.

Asunto(s)

Etanol/aislamiento & purificación , Etanol/análisis , Etanol/síntesis química , Saccharomyces/aislamiento & purificación , Saccharomyces/enzimología , Saccharomyces/química

RESUMEN

Existe un gran interés por el uso de enzimas lignocelulolíticas en varias industrias, y en la biodegradación de biomasa para la producción de biocombustibles y otras aplicaciones. Entre las fuentes microbianas de enzimas, Aspergillus niger es uno de los microorganismos más utilizados en la producción de enzimas industriales, debido a sus niveles altos de secreción de proteína y a su condición GRAS (generally regarded as safe). El objetivo del presente estudio fue evaluar la influencia de la concentración de inóculo en la morfología y producción de celulasas y xilanasas con A. niger en cultivo sumergido. Para ello, fueron inoculados matraces de 250 mL con 40 mL de medio con 3% (v/v) de una suspensión de 104 o 108 esporas por mililitro e incubados a 28 ºC y 175 rpm durante 120 horas. Se utilizaron 10 g*L-1 de lactosa como fuente de carbono. En cada caso se determinó la cantidad de biomasa, la proteína extracelular soluble, lactosa residual, actividad celulasa total y xilanasa cada 24 horas. Aunque no hubo un efecto notorio en la morfología de crecimiento, salvo en el color y el diámetro de pellets obtenidos, sí se afectó la µmax (0,06 y 0,03 h-1 para 104 y 108 esporas*mL-1, respectivamente) y la concentración máxima de biomasa. Además, mientras que las productividades volumétricas de celulasa (ΓFPA) (8,2 y 8,0 UI.*L-1*h-1 para 104 y 108 esporas*mL-1, respectivamente) fueron similares para ambos inóculos, la productividad de xilanasa (ΓXIL) fue mayor para el inóculo más concentrado (29,7 y 33,4 UI¨*L-1*h-1 para 104 y 108 esporas*mL-1, respectivamente). Los resultados indican que la productividad de celulasas y xilanasas está estrechamente relacionada con la concentración de inóculo.

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There is a great interest for the use of lignocellulolytic enzymes in several industries and in biomass degradation for production of biofuels and other applications. Among the microbial sources of enzymes, Aspergillus niger is one of the most used microorganisms in the production of industrial enzymes due to its high levels of protein secretion and its GRAS (generally regarded as safe) condition. The aim of the present study was to evaluate the influence of A. niger inoculum concentration in the morphology and production of cellulases and xylanases in submerged cultures. For this, 250 mL flasks containing 40 mL culture medium were inoculated with a 3% (v/v) of either 104 or 108 spores per milliliter suspension and incubated at 28 º C and 175 rpm during 120 hours. Lactose (10 g*L-1) was used as the carbon source. In each case, the amount of biomass, the extracellular soluble protein, residual lactose, total celullase activity and xylanase activity were determined every 24 hours. Even thought there was not a notorious effect on the growth morphology, except in color and diameter of pellets; µmax was affected (0.06 and 0.03 h-1 for 104 and 108 spores*mL-1, respectively) as well as maximum biomass concentration. In addition, while the volumetric productivity of cellulase (8.2 and 8.0 UI*L-1*h-1 for 104 and 108 spores*mL-1, respectively) were similar for both inocula, the productivity of xylanase was greater for the more concentrated inoculum (29.7 and 33.4 UI*L-1*h-1 for 104 and 108 spores*mL-1, respectively).The results show that cellulase and xylanase productivities are closely related to the inoculum concentration.

Asunto(s)

Celulasa/análisis , Celulasa/biosíntesis , Celulasa/genética , Celulasa/inmunología , Celulasa/química , Celulasa/síntesis química , Aspergillus niger/enzimología , Aspergillus niger/fisiología , Aspergillus niger/genética , Aspergillus niger/inmunología , Aspergillus niger/química

RESUMEN

Se realizó un perfil inicial del proteoma de biopelículas de Aspergillus niger ATCC 10864 desarrolladas sobre tela de poliéster mediante 2D-PAGE y análisis MS-TOF y comparado con el proteoma de cultivos sumergidos convencionales de micelio libre. De ambos tipos de cultivo se analizó un número de muestras proteicas de geles 2D-PAGE mediante MS-TOF y los resultados se compararon con la base de datos NCBI nr disponible para esta especie. Los mapas proteómicos mostraron patrones diferentes de expresión en cada caso. En cultivo de biopelículas, el 19% y el 32% de las muestras seleccionadas fueron sobre-expresadas y diferencialmente expresadas, respectivamente. Por el contrario, en cultivos sumergidos en micelio libre el 44% y el 7% de las muestras seleccionadas fueron sobre-expresadas y diferencialmente expresadas, respectivamente. Aunque preliminares, los resultados presentados en este trabajo muestran que existen diferencias significativas entre los proteomas de biopelículas y micelio libre de A. niger. Parece ser que la adhesión celular es el estímulo más importante para el desarrollo de biopelículas, las cuales son la base de la Fermentación por Adhesión a Superficies.

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An initial profiling of the intracellular proteome of Aspergillus niger ATCC 10864 biofilm cultures developed on polyester cloth was carried out by using 2D-PAGE and MS-TOF analysis and it was compared to the proteome of conventionally grown free-living submerged cultures. A number of 2D-PAGE protein spots from both types of cultures were subjected to MS-TOF analysis and data interrogation of the NCBI nr database available for this species. Proteomic maps showed different expression patterns in both culture systems with differentially expressed proteins in each case. In biofilm cultures, 19% and 32% of the selected protein spots were over-expressed and differentially expressed, respectively. On the contrary, in free-living cultures, 44% and 7% of the selected protein spots were over-expressed and differentially expressed, respectively. Although preliminary, results presented in this paper show that there are significant differences between the proteomes of A. niger biofilm and free-living mycelia. It seems that cell adhesion is the most important stimulus responsible for biofilm development which is the basis of Surface Adhesion Fermentation.

Asunto(s)

Aspergillus niger , Biopelículas , Proteoma

RESUMEN

A preliminary evaluation of transcriptional gene expression in Aspergillus niger ATCC 10864 biofilms developed on polyester cloth was carried out. The expression analysis of genes encoding some lignocellulolytic enzymes and some regulatory genes by means of RT-PCR showed that eng1, eglC, exo, eglA, eglB and xynB genes are differentially expressed in biofilm fermentation either time-related or through the production of more than a transcript as compared to A. niger grown in submerged fermentation. Likewise, the regulatory genes xlnR and creA showed time-related expression patterns that were different in both fermentation systems. Results attained in this work contribute with an initial molecular evidence of differential gene expression as well as differential gene regulation patterns in fungal biofilms that may be related to cell adhesion.

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Se realizó una evaluación génica preliminar a nivel transcripcional de biopelículas de Aspergillus niger ATCC10864 desarrolladas sobre poliéster respecto a algunas enzimas lignocelulolíticas. El análisis de expresión de genes de enzimas lignocelulolíticas y genes reguladores mediante RT-PCR mostró que los genes eng1, eglC, exo y eglA, eglB y xynB son diferencialmente expresados ya sea temporalmente o mediante más de untranscripto en comparación con cultivos sumergidos. Asimismo, los genes reguladores xlnR y creA mostraron patrones temporales de expresión distintos en ambos sistemas. Los resultados obtenidos aportan la evidencia molecular inicial de expresión diferencial de genes en biopelículas así como patrones de regulación diferencial muy probablemente ligada a la adhesión celular.

Asunto(s)

Aspergillus niger/genética , Biopelículas , Celulasas , Enzimas/genética , Expresión Génica

Asunto(s)

Masculino , Femenino , Humanos , Biotecnología , Metabolismo , Plantas Modificadas Genéticamente , Salud Pública

RESUMEN

Lignocellulolytic enzyme production by Aspergillus niger was compared both in submerged fermentation (SF) and biofilm fermentation (BF) at varying water activities. Maximal filter paper activity, endoglucanase and xylanase activities were much higher in BF (2.96, 4.7 and 4.61 IU ml-1, respectively) than in SF cultures (1.71, 1.31 and 2.3 IU ml-1, respectively) but biomass yields were lower in BF than in SF (0.338 g g-1 and 0.431 g g-1, respectively). In the presence of 20 percent ethylene glycol (a w = 0.942) the enzyme activities decreased in both systems but BF still had higher levels (1.0, 1.0 and 2.6 IU ml-1, respectively) than SF cultures (0.6, 0.7 and 1.5 IU ml-1, respectively). An increase in xylanase specific activity of more than 2 fold (from 4.2 to 10.2 IU mg-1 biomass) was observed in the presence of 20 percent ethylene glycol, suggesting differential regulatory mechanisms in biofilm fermentation related to cell adhesion.

RESUMEN

Se resume el conocimiento básico sobre la fermentación en estado sólido y la formación de biopelículas y se relaciona con los procesos de adhesión celular, cubriendo puntos de vista de ingeniería y de biología molecular. Contrariamente a la creencia común, la ventaja de la fermentación en estado sólido esta relacionada a la adhesión de los hongos a partículas sólidas y no al bajo contenido de agua. Por lo tanto, la fermentación en estado sólido y la fermentación en biopelículas (erradamente conocida como inmovilización por adsorción) son variantes técnicas del mismo proceso biológico y deben ser referidas como Fermentación por Adhesión a Superficies.

Asunto(s)

Fermentación , Genes , Moléculas de Adhesión Celular

RESUMEN

Se evaluaron biopelículas de aspergillus niger desarrolladas sobre tela de poliéster en dos aspectos fundamentales inherentes al crecimiento sobre superficies: la estructura y el comportamiento fisiológico específicamente relacionado con la producción de celulasa. La estructura de la biopelícula fue evaluada usando microfotografías de microscopía electrónica de barrido (SEM) desde el momento de la inoculación y adsorción de esporas hasta las 120 horas de crecimiento. Nuestros resultados demuestran que la formación de la biopelícula ocurre en tres fases: la adhesión, la cual es fuertemente favorecida por la hidrofobicidad de las esporas de Aspergillus; la fase de crecimiento inicial y desarrollo, que se inicia con la germinación de la espora entre las 4 y 10 horas y continua hasta las 24 horas cuando ya se percibe la colonización casi total de la superficie; y finalmente la fase de maduración en la cual la densidad de biomasa se incrementa notablemete desde las 48 horas hasta las 120 horas; se observa, además en este momento una organización interna de canales, tambien reportada para biopelículas bacterianas, que aseguran el flujo interno de la biopelícula. Además, la actividad celulolítica de las biopelículas fue evaluada, siendo hasta 40 por ciento mayor que en los cultivos sin soporte de crecimiento (durante el transcurso de la fermentación), lográndose un incremento del 55 por ciento en la productividad. Estos resultados son concordantes con el comportamiento de la gran mayoría de microorganismos que crecen sobre superficies, los cuales muestran por lo general una mayor actividad metabólica resultante de una expresión genética diferencial. Este trabajo es un primer intento por establecer la estructura y fisiología de las biopelículas de hongos filamentosos de interés industrial en respuesta a la escasa información existente, en comparación con el minucioso y vasto estudio de biopelículas bacterianas y de levaduras patógenas.

Asunto(s)

Aspergillus niger , Celulasa , Hongos

RESUMEN

The kinetics of cellulase production of six fungal production of six fungal strain isolated from soils of the Lomas de Lachay (Lima), was studiend and compared with that of the hypercellulolytic mutan T. reesei QM-9414. Submerget batch conditions and lactose (1%) as inducer were used. Increment velocities of enzyme activity were comparable with those of the mutant and a significant correlation between the highest endoglucanase activity and the diameter of hydrolisis on swollen cellulose plates was found. Likewise, all fungal strains overcame the mutant in specific activity (fivefold), productivity (twofold), and B-glucosidase titer (threefold9).

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Seis diferentes hongos aislados de las Lomas de Lachay (Lima, Perú) fueron estudiados en su cinética de producción de celulosas y comparados con el mutante hipercelulotítico T. reesei QM-9414. Trabajando en cultivo sumergido utilizando lactosa al 1% como inductor, se detecto velocidades de incremento de la actividad enzimatica entre otros.

Asunto(s)

Celulasa , Hongos , Trichoderma , Biomasa , Perú

RESUMEN

By induced mutations with UV radiation and caffeine 20 ypercellulolytic strains were obtained from Tichoderma resei ATCC 26921. The cellulotytic ability of the mutants were selected: LM-UC2, LM-UC4, with great increase in their enzyme activities, specific activities and productivity.

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Por mutaciones inducidas con irradiación ultravioleta y cafeína se obtuvieron 20 cepas ipercelulolíticas a partir de Trichodema resei ATCC 26291. La capacidad celulolítica delos mutantes se evaluó en placas de agar celulosa y en cultivos sumergidos. Se seleccionaron 3 mutantes estables LM-UC2, LM-UC4 y LM-UC6 con incrementos notables en las actividades enzimáticas, actividades específicas y productividad.

Asunto(s)

Mutación , Trichoderma , Biomasa , Hidrólisis

RESUMEN

To the ligt of the new findings, started some time ago by Barbara McClinton, biological diversity it is not due only to mulationn and homoloous recombination but also to insertion o "controllin elements" in a specific ocus. These DNA molecules know as iether "transposable genetic elements"or" mobile genetic in a due chromosome produces a recombinant DNA. This process can be done in vitro and it has a very wide scope for Mankind.

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A la luz de los nuevos,descubrimientos, iniciados hace algún tiempo por Bárbara McClintock, la diversidad biológica no sólo es el resultado de la mutación y recombinación homóloga sino tambien la inserción de "elementos controladores" es un locus determinado. Estas moléculas de ADN conocidas como "elementos genéticos transpasables" o "elementos genéticos móviles" presentan terminales repetidos e invertido. La inserción de un elemento móvil es un cromosoma da lugar a un ADN recombinante, tecnología reaizable ya in vitro, cuyas proyecciones para el ser humano son casi infinitas.

Asunto(s)

ADN , ADN Recombinante , Variación Genética