Evolución dirigida de penicilina V acilasa de "Streptomyces lavendulae" y aculeacina A acilasa de "Actinoplanes utahensis"

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Microbiología II, leída el 14-07-2016Actualmente 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...Depto. de Microbiología y ParasitologíaFac. de FarmaciaTRUEunpu

Novel Bifunctional Acylase from Actinoplanes utahensis: A Versatile Enzyme to Synthesize Antimicrobial Compounds and Use in Quorum Quenching Processes

Many intercellular communication processes, known as quorum sensing (QS), are regulated by the autoinducers N-acyl-L-homoserine lactones (AHLs) in Gram-negative bacteria. The inactivation of these QS processes using different quorum quenching (QQ) strategies, such as enzymatic degradation of the autoinducers or the receptor blocking with non-active analogs, could be the basis for the development of new antimicrobials. This study details the heterologous expression, purification, and characterization of a novel N-acylhomoserine lactone acylase from Actinoplanes utahensis NRRL 12052 (AuAHLA), which can hydrolyze different natural penicillins and N-acyl-homoserine lactones (with or without 3-oxo substitution), as well as synthesize them. Kinetic parameters for the hydrolysis of a broad range of substrates have shown that AuAHLA prefers penicillin V, followed by C12-HSL. In addition, AuAHLA inhibits the production of violacein by Chromobacterium violaceum CV026, confirming its potential use as a QQ agent. Noteworthy, AuAHLA is also able to efficiently synthesize penicillin V, besides natural AHLs and phenoxyacetyl-homoserine lactone (POHL), a nonnatural analog of AHLs that could be used to block QS receptors and inhibit signal of autoinducers, being the first reported AHL acylase capable of synthesizing AHLs

Engineering of Bacillus megaterium for improving PHA production from glycerol

9 p.-4 fig.There are a few PHA-producer bacteria that can uptake glycerol to produce this biopolymer. Among them, Bacillus megaterium LVN01 has demonstrated to be able to grow up using glycerol as a carbon source. Glycerol dehydrogenase (GD) plays a key role in the synthesis of PHA from glycerol. In this study, the improvement of glycerol uptake by a recombinant strain of B. megaterium carrying pHT01-bmgd was evaluated in order to enhance PHA production. The biomass and PHA production were evaluated and compared to wild-type. It was determined that the PHA produced by both strains was PHB and the highest improvement in PHB yield was 226% at 30 h.This study was funded by Departamento Administrativo de Ciencia, Tecnología e Innovación de Colombia (COLCIENCIAS) (Called 647 of 2014). The authors would like to thank COLCIENCIAS for funding. This work is framed at the “Contrato de acceso a recursos genéticos y productos derivados del Ministerio del Medio Ambiente y Desarrollo Sostenible de Colombia Nº95 and Nº159”.Peer reviewe

Draft genome sequence of actinoplanes utahensis NRRL 12052, a microorganism involved in industrial production of pharmaceutical intermediates

Here, we describe the draft genome sequence of Actinoplanes utahensis NRRL 12052, a filamentous bacterium that encodes an aculeacin A acylase and a putative N-acyl-homoserine lactone acylase of biotechnological interest. Moreover, several nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) clusters and antibiotic resistance genes have been identified.No data (2015)UE

Fed-batch production and characterization of polyhydroxybutyrate by Bacillus megaterium LVN01 from residual glycerol

The operating conditions of polyhydroxybutyrate (PHB) production processes are among the factors that most influence yields. In this study, we evaluated PHB production synthesized by Bacillus megaterium LVN01. Batch and fed-batch cultures were used to produce PHB from residual glycerol. For this, dry cell weight (DCW) and PHB productivity were analyzed at a preliminary stage by central composite design using batch systems under different temperature, C/N ratio, and fermentation time conditions. The maximum PHB productivity occurred at 30.8 °C, 44.9 mol mol-1, and 39.9 h. The same conditions were tested for studies in fed-batch culture. Fed-batch experiments were comparable to each other, where the DCW was around 1.9 g L-1, with PHB productivities of 29.5 mg L-1 h-1 and 35.6 mg L-1 h-1 for bioreactors of 5 L and 14 L, respectively. The PHB was characterized by NMR, FTIR, DSC, TGA, and DTG analyses.Las condiciones de operación de los procesos de polihidroxibutirato (PHB) se encuentran entre los factores que más influyen en los rendimientos. En este estudio se evaluó la producción de PHB sintetizado por Bacillus megaterium LVN01. Se implementaron cultivos en lotes y lotes alimentados para producir PHB, a partir de glicerol residual. Para esto, en una etapa preliminar, se analizó el peso de las células secas (PSC) y la productividad de PHB mediante un diseño central compuesto, utilizando sistemas de lotes bajo diferentes condiciones de temperatura, relación molar C/N y tiempo de incubación. La productividad máxima de PHB ocurrió a 30.8°C, 44.9 mol mol-1 y 39.9 h. Las mismas condiciones se utilizaron para estudios en cultivos por lotes alimentados. Los experimentos en modo lote alimentado fueron comparables entre sí, donde el PSC osciló alrededor de 1.9 g L-1, con productividades de PHB de 29.5 mg L-1 h-1 y 35.6 mg L-1 h-1 para biorreactores de 5 L y 14 L, respectivamente. El PHB se caracterizó mediante análisis de RMN, FTIR, DSC, TGA y DT

Penicillin Acylase from Streptomyces lavendulae and Aculeacin A Acylase from Actinoplanes utahensis: Two Versatile Enzymes as Useful Tools for Quorum Quenching Processes

Many Gram-negative bacteria produceN-acyl-homoserine lactones (AHLs), quorum sensing(QS) molecules that can be enzymatically inactivated by quorum quenching (QQ) processes; this approachis considered an emerging antimicrobial alternative. In this study, kinetic parameters of several AHLshydrolyzed by penicillin acylase fromStreptomyces lavendulae(SlPA) and aculeacin A acylase fromActinoplanes utahensis(AuAAC) have been determined. Both enzymes catalyze efficiently the amide bondhydrolysis in AHLs with different acyl chain moieties (with or without 3-oxo modification) and exhibit aclear preference for AHLs with long acyl chains (C12-HSL>C14-HSL>C10-HSL>C8-HSL forSlPA,whereas C14-HSL>C12-HSL>C10-HSL>C8-HSL forAuAAC). Involvement ofSlPA andAuAAC inQQ processes was demonstrated byChromobacterium violaceumCV026-based bioassays and inhibitionof biofilm formation byPseudomonas aeruginosa, a process controlled by QS molecules, suggesting theapplication of these multifunctional enzymes as quorum quenching agents, this being the first time thatquorum quenching activity was shown by an aculeacin A acylase. In addition, a phylogenetic studysuggests thatSlPA andAuAAC could be part of a new family of actinomycete acylases, with a preferencefor substrates with long aliphatic acyl chains, and likely involved in QQ processes.Sin financiación4.146 JCR (2020) Q2, 67/162 Chemistry, Physical0.800 SJR (2020) Q2, 27/60No data IDR 2020UE