Publication: Directed evolution of penicillin V acylase from "Streptomyces lavendulae" and aculeacin A acylase from "Actinoplanes utahensis"
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2017-05-24
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Universidad Complutense de Madrid
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Abstract
Actualmente la secuenciación y el consecuente depósito en bases de datos públicas de genomas bacterianos han incrementado de manera exponencial y constituye una herramienta inevitable en la investigación básica y aplicada. Sin embargo, la elucidación de la información encriptada en sus secuencias codificantes aunado a las particularidades de cada microorganismo, constituyen las barreras a ser superadas por parte de los investigadores, para lo cual estudios bioinformáticos integrados con evidencias experimentales son ineludibles de abordar en el laboratorio. En particular, es menester reconocer la versatilidad que ostentan las bacterias Gram-positivas y sus implicaciones que trascienden los entornos naturales y se inmiscuyen cada vez más en procesos biotecnológicos. Por tal motivo, en el presente estudio se secuenciaron los genomas de las cepas bacterianas Streptomyces lavendulae ATCC 13664 y Actinoplanes utahensis NRRL 12052, gracias a lo cual se logró determinar múltiples características de dichos microorganismos. En este sentido, el presente estudio logró determinar con base en la secuencia del 16S rRNA, al igual que con fundamento en una comparación de todo el genoma frente a una base de datos local de genomas, que la cepa de S. lavendulae se encuentra mal asignada y por lo tanto debería ser reasignada como una nueva especie, dado que fue detectada filogenéticamente cerca a otras especies de S. lavendulae, y en contraste dicha cepa se localiza aún más cerca de otras especies de S. griseus. Igualmente, dentro del genoma de A. utahensis resalta la detección de una acil-homoserin lactona acilasa (AuAHLA) putativa, la cual es documentada por primera vez en este estudio. Los análisis bioinformáticos desarrollados destacaron que dicha enzima presenta características similares a la aculeacin A acilasa (AuAAC) de A. utahensis y a la penicilina V acilasa (SlPVA) de S. lavendulae. Igualmente, cabe mencionar que no fue detectada la equinocandina B (ECB) deacilasa transmembrana dentro del genoma de A. utahensis, la cual se había descrito previamente por otros autores y que solo difiere ligeramente en su secuencia con respecto a AuAAC (aunque no se ha depositado la secuencia completa de la ECB deacilasa, si se ha informado sobre fragmentos del amino-terminal de cada subunidad), lo cual permite proponer que la ECB deacilasa debe ser reasignada. Asimismo, es de resaltar que en los dos microorganismos secuenciados fueron detectados clúster relacionados con la biosíntesis de NRPS (de su sigla en inglés non-ribosomal peptide-synthase) y PKS (de su sigla en inglés polyketide synthase). Específicamente, tanto AuAAC como AuAHLA fueron localizadas dentro de clústeres relacionados con la biosíntesis de sideróforos (i.e. gobichelina y laspartomicina, respectivamente según la predicción realizada), moléculas que son empleadas por las bacterias como compuestos quelantes del hierro, y que los seres humanos aprovechan gracias a su actividad biológica. En contraste, a pesar de que la plataforma empleada no predijo ningún clúster que contenga SlPVA, estudios adicionales permitieron que el presente estudio no descarte que SlPVA esté implicada en la biosíntesis de algún sideróforo, tal y como fue el caso de las acilasas de A. utahensis...
Nowadays sequencing and consequent deposit in public data bases of bacterial genomes have been increased exponentially and constitutes an inevitable tool in basic and applied research. However, the elucidation of the encrypted information along their coding sequences and the particularities of each microorganism are barriers to be overcome by the researcher. Thus, bioinformatic studies integrated with experimental evidences are inescapably addressed in the laboratory. In particular, it is important to mention the versatility that holds the Gram-positive bacteria and its implications that transcends natural environments and interferes time after time in biotechnological processes. For this reason, in this study the genomes of the bacterial strains Streptomyces lavendulae ATCC 13664 and Actinoplanes utahensis NRRL 12052 were sequenced, and thanks to this information, it was possible to determine several features from those microorganisms. In this sense, the analysis of the 16S rRNA sequence as well as the comparison of the whole genome against a local database of genomes suggests that the strain of S. lavendulae is misassigned and should be assigned as a new specie, because despite the fact that it was detected phylogenetically close to other strains of S. lavendulae, it was located closer to other S. griseus species. Likewise, within the genome of A. utahensis highlights the presence of acyl-homoserine lactone acylase (AuAHLA), which is reported here for the first time. The bioinformatic analyses developed emphasizes that this enzyme had similar characteristics with respect to aculeacin A acylase (AuAAC) from A. utahensis and penicillin V acylase (SlPVA) from S. lavendulae. Surprisingly, it is noteworthy to mention that the transmembrane echinocandin B (ECB) deacylase was not detected within the genome of A. utahensis. Information about ECB deacylase reported by other authors and its sequence differs slightly with respect to AuAAC. Although the sequence of ECB deacylase has not been deposited, the authors reported the amino-terminus of each subunit. Thus, the present study suggests that this ECB deacylase should be reassigned. Likewise, it is important to mention that in both genomes clusters related with the biosynthesis of NRPS (non-ribosomal peptide-synthase) and PKS (polyketide synthase) were detected. Specifically, both AuAAC as AuAHLA were located within a cluster associated with the biosynthesis of siderophores (i.e. predicted gobichelin and laspartomycin, respectively). These molecules are employed by the bacteria as iron chelating compounds, and humans use their biological activity. In contrast, although the platform employed did not predict any cluster containing SlPVA, further studies might indicate that SlPVA could be implicated in the biosynthesis of some siderophore, similarly to that exposed with the acylases from A. utahensis...
Nowadays sequencing and consequent deposit in public data bases of bacterial genomes have been increased exponentially and constitutes an inevitable tool in basic and applied research. However, the elucidation of the encrypted information along their coding sequences and the particularities of each microorganism are barriers to be overcome by the researcher. Thus, bioinformatic studies integrated with experimental evidences are inescapably addressed in the laboratory. In particular, it is important to mention the versatility that holds the Gram-positive bacteria and its implications that transcends natural environments and interferes time after time in biotechnological processes. For this reason, in this study the genomes of the bacterial strains Streptomyces lavendulae ATCC 13664 and Actinoplanes utahensis NRRL 12052 were sequenced, and thanks to this information, it was possible to determine several features from those microorganisms. In this sense, the analysis of the 16S rRNA sequence as well as the comparison of the whole genome against a local database of genomes suggests that the strain of S. lavendulae is misassigned and should be assigned as a new specie, because despite the fact that it was detected phylogenetically close to other strains of S. lavendulae, it was located closer to other S. griseus species. Likewise, within the genome of A. utahensis highlights the presence of acyl-homoserine lactone acylase (AuAHLA), which is reported here for the first time. The bioinformatic analyses developed emphasizes that this enzyme had similar characteristics with respect to aculeacin A acylase (AuAAC) from A. utahensis and penicillin V acylase (SlPVA) from S. lavendulae. Surprisingly, it is noteworthy to mention that the transmembrane echinocandin B (ECB) deacylase was not detected within the genome of A. utahensis. Information about ECB deacylase reported by other authors and its sequence differs slightly with respect to AuAAC. Although the sequence of ECB deacylase has not been deposited, the authors reported the amino-terminus of each subunit. Thus, the present study suggests that this ECB deacylase should be reassigned. Likewise, it is important to mention that in both genomes clusters related with the biosynthesis of NRPS (non-ribosomal peptide-synthase) and PKS (polyketide synthase) were detected. Specifically, both AuAAC as AuAHLA were located within a cluster associated with the biosynthesis of siderophores (i.e. predicted gobichelin and laspartomycin, respectively). These molecules are employed by the bacteria as iron chelating compounds, and humans use their biological activity. In contrast, although the platform employed did not predict any cluster containing SlPVA, further studies might indicate that SlPVA could be implicated in the biosynthesis of some siderophore, similarly to that exposed with the acylases from A. utahensis...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Microbiología II, leída el 14-07-2016