Harvesting novel biocatalysts from the metagenome

Bacteriën spelen in de biotechnologie een bijzondere rol. Naast schimmels en gisten vormen ze de basis van sommige van de oudste biotechnologische processen zoals de productie van azijn, gefermenteerde zuivelproducten, zuurkool en bij het pekelen. Niet alleen de intacte (levende) bacteriecel kan worden ingezet om bepaalde chemische reacties te doen, maar ook de uit bacteriecellen geïsoleerde afzonderlijke enzymen. Enzymen zijn katalysatoren, dat wil zeggen dat ze chemische reacties versnellen en ze behoren, op een paar uitzonderingen na, allen tot de eiwitklasse. In de industrie worden enzymen bijvoorbeeld op grote schaal toegepast in de productie van “high fructose corn sirup” (glucose isomerase), de vorming van acrylamide (nitril hydratase) en de isolatie van aminopenicillaanzuur (penicilline acylase). ... Zie: Samenvatting

Simultaneous clarification and purification of recombinant penicillin G acylase using tangential flow filtration anion-exchange membrane chromatography

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained

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

Identification of Factors Limiting Heterologous Lipase Expression in the Cytoplasm and the Periplasm as well as Display on Cell Surface of Escherichia coli

Lipase B from Pseudozyma antarctica (PalB), had been expressed in several recombinant protein hosts and showed very good transesterification activity for biodiesel production. However, the functional expression could not be demonstrated until recently in the most popular recombinant protein expression system, e.g. Escherichia coli, and the expression performance stands improvement. The probable reason is that PalB is a lipase with more hydrophobic surface and three disulfide bonds and thus may not be easily expressed in E. coli functionally. This thesis focuses on the identification of factors limiting heterologous expression of PalB in E. coli through a systematic study by using several strategies, including the different expression compartments, fusion tags, folding factors, and host strains. Functional expression of PalB in the cytoplasm of E. coli was explored using BL21(DE3) and its mutant derivative Origami B(DE3) as the host. Bioactive PalB was obtained in the reduced cytoplasm of BL21(DE3), implying that the formation of disulfide bond was not strictly required for functional expression. However, the expression was ineffective and was primarily limited by formation of PalB inclusion bodies and growth arrest, both of which were associated with PalB misfolding and deteriorated physiology. The culture performance in terms of cell growth and PalB expression level could be significantly improved by simultaneous coexpression of multiple chaperones of trigger factor and GroEL/ES, but not individual coexpression of either one of them. It was proposed that the two chaperones mediate the early stage and late stage of cytoplasmic PalB folding and would be required simultaneously for boosting both the overall PalB synthesis rate and the cytoplasmic folding efficiency. On the other hand, a much higher bioactive PalB was produced in Origami B(DE3) harboring the same PalB expression vector. Furthermore, the significant high bioactive PalB was produced by coexpression of periplasmic folding factor without a signal peptide (e.g., coexpression of DsbA, and DsbC). Coexpression of DsbA was found to be effective in enhancing PalB expression and such an improvement was more pronounced in Origami B(DE3), suggesting that both folding and disulfide bond formation could be the major factors limiting PalB expression. The fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity possibly via both enhanced solubility and correct disulfide bond formation. Our results suggest that solubilization of PalB fusions did not necessarily result in the development of PalB activity which could be closely associated with correct disulfide bond formation. While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC, but not cytoplasmic chaperones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. This is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable. On the other hand, functional expression of PalB in the periplasm was explored using four fusion tags, e.g., DsbC, DsbA, maltose binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags for functional expression of PalB, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of gene products, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegPS210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance for functional expression of PalB was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/liter. Without extracting protein from cells the whole cell can be directly used as a platform for the immobilized enzyme. Proof-of-concept experimentation was conducted by PalB display on the E. coli cell surface. By fusing the palB gene in between the signal peptide phoA and an autotransporter Protein EstA’s gene under the lac promoter, PalB was successfully displayed on the E. coli cell surface. However, cells encountered a severe physiological stress. Coexpression of various periplasmic folding factors, e.g., DegP, SurA, DsbA and DsbC could erease the physiological stress, but only DsbA was demonstrated to be effective to restore cell physiology and increase PalB expression level. Key words: enzyme, Escherichia coli, chaperone, folding factor, fusion tag, gene expression, Pseudozyma antarctica, lipase B, recombinant protein productio

Nanomaterials for controlling bacterial pathogens and resistance occurrence

lnfectious diseases are the leading cause of death worldwide while the constantly raising antimicrobial resistance (AMR) is a major concern for the public health. During the infection establishment bacteria! pathogens communicate via expression of signaling molecules, controlled through a phenomenon called quorum sensing (QS). As a result of this, bacteria produce virulence factors and form resistant biofilms on living and non-living surfaces causing persistent infections. The infection complexity, especially in chronic diseases, requires the use of broad-spectrum antibiotics responsive for the appearance and the spread of drug resistant species. lnfections caused by antibiotic-resistant pathogens are associated with high morbidity, mortality, and huge economic burden. Unlike the decrease over the past three decades of the number of novel marketed antimicrobial drugs, the number of antibiotic resistant bacteria! strains steadily increases. Thus, there is an urgent need for development of alternative strategies to manage difficult-to-treat infections. This thesis aims at the engineering of advanced nano-enabled materials and nanostructured coatings for controlling bacteria! pathogenesis and resistance occurrence. To achieve this, biopolymers, antibiofilm and anti-infective enzymes. and inorganic compounds were nano-hybridized as altemative modalities to the conventional antibiotics. The nanoform was able to provide enhanced interaction with bacteria! cell membranes and easier penetration into biofilms, increasing the antimicrobial efficacy at lower dosages, while preventing from development of antimicrobial resistance. Additionally, specific targeting moieties increased the nanomaterial's interaction with the pathogens, avoiding the drug resistance appearance and cytotoxicity. The first part ofthe thesis describes the functionalization of biologically inert nanoparticles (NPs) with membrane disturbing antimicrobial aminocellulose (AM) and biocompatible hyaluronic acid (HA) in an Lbl fashion for elimination of medically relevant pathogens. The generated nanoentities demonstrated high potential to inhibit the biofilm formation, without affecting the human cell viability. Further, the Lbl technique was applied to decorate antimicrobial, but potentially toxic silver (Ag) nano-templates with biocompatible AM and quorum quenching (QQ) acylase in order to obtain safe antibacterial and antibiofilm nanomaterials. The deposition of acylase and AM on the Ag core interfered with the QS signaling and bacteria! pathogenesis, and enhanced the NPs interaction with the bacteria! membrane. The integration of a triple mechanisms of action in the hybrid nanoentities resulted in complete bacteria and biofilm eradication and improved biocompatibility ofthe AgNPs. The thesís also describes the development of targeted nanocapsules (NCs) for selective elimination of Staphylococcus aureus. Herein, self-assembling nanoencapsulation technology using the biocompatible and biodegradable proteín zein was applied for the generation of zein NCs loaded with bactericida! oregano essential oil (EO). An antibody specifically targeting S. aureus was covalently grafted on the NCs surface. The obtained targeted NCs demonstrated antibacterial selectivity in a mixed bacteria! inoculum, and the treatment efficacy was validated in an in vitro coculture model of bacteria and mammalian cells. Finally, high intensity ultrasonochemistry (US) process was employed for engineering of durable antibacterial/antibiofilm coating on urinary catheters. The simultaneous deposition of zinc oxide (ZnO) NPs anda matrix-degrading amylase enzyme improved the NPs adhesion on the silicone material, and prevented its bacteria! colonization and biofilm formation in vitro. The hybrid nanostructured coating delayed the occurrence of early onset urinary tract infections (UTls) and showed excellent biosafety in an in vivo animal model.Las enfermedades infecciosas son la principal causa de muerte en todo el mundo. Mientras que la resistencia a los antimicrobianos es una preocupación importante para la salud pública. Durante el establecimiento de la infección los patógenos bacterianos se comunican mediante la expresión de moléculas de señalización controladas mediante un fenómeno llamado detección de quórum (QS). Como resultado, las bacterias producen factores de virulencia y forman biopelículas resistentes que causan infecciones persistentes. Las infecciones causadas por patógenos resistentes a los antibióticos se asocian con una alta morbilidad mortalidad y una enorme carga económica. A diferencia de la disminución en las últimas tres décadas del número de nuevos medicamentos antimicrobianos comercializados el número de cepas bacterianas resistentes a los antibióticos aumenta constantemente. Por lo tanto existe la necesidad urgente de desarrollar estrategias alternativas para manejar infecciones diflciles de tratar. Esta tesis tiene como objeto de trabajo la ingenieria de materiales y recubrimientos avanzados nano estructurados para controlar la patogénesis bacteriana y la aparición de resistencias. Para lograrlo se combina polímeros anti biopelícula, enzimas anti infecciosas y compuestos inorgánicos como estrategias alternativas a los antibióticos convencionales. La nanoforma puede proporcionar una interacción mejorada con las membranas celulares bacterianas y una penetración más fácil en las biopelículas, aumentando la eficacia antimicrobiana en dosis más bajas al mismo tiempo que previene el desarrollo de resistencia antimicrobiana. Además las fracciones de orientación especificas aumentan la interacción del nano material con los patógenos evitando la aparición de resistencia al fármaco y la citotoxicidad.La primera parte de la tesis describe la funcionalización de nano partículas (NP) biológicamente inertes con aminocelulosa antimicrobiana (AM) perturbadora de la membrana y ácido hialurónico (HA) biocompatible en forma de LbL para la eliminación de patógenos médicamente relevantes. Las nanoentidades generadas demuestran un alto potencial para inhibir la formación de biopelículas sin afectar la viabilidad en las células humanas. Además, la técnica L.bL se aplica para decorar nanoplantillas de plata (Ag) antimicrobianas,pero potencialmente tóxicas con PNt biocompatible y acilasa de extinción de quórum (QQ) para obtener nanomateriales antibacterianos y anti biopelícula seguros. La deposición de acilasa y PNt en el núcleo de Ag interfiere con la señalización de QS y la patogénesis bacteriana y mejora la interacción de las NP con la membrana bacteriana. La integración de un triple mecanismo de acción en las nanoentidades híbridas da como resultado la erradicación completa de bacterias y biopelículas y una mejor biocompatibilidad de los AgNP. La tesis también describe el desarrollo de nanocápsulas dirigidas (NC) para la eliminación selectiva de Staphylococcus aureus. En este trabajo se aplica la tecnología de nanoencapsulación de autoensamblaje que utiliza la proteína Zeína biocompatible y biodegradable para la generación de NC de Zelna cargadas con aceite esencial de orégano bactericida.Un anticuerpo dirigido específicamente a S.aureus se injerta covalentemente en la superficie de las NC. Las NC dirigidos obtenidos demuestran selectividad antibacteriana en un inóculo bacteriano mixto.y la eficacia del tratamiento se valida en un modelo de cocultivo in vitro de bacterias y células de mamíferos.Finalmente, se emplea un proceso de ultrasonoqufmica de alta intensidad. Para la ingeniería de un recubrimiento antibacterlano/anti biopelícula duradero en catéteres urinarios. La deposición de NP de Óxido de Zinc y enzima amilasa que degrada la matriz.mejora la adhesión de las NP en el material de silicona evitando su colonización bacteriana y la formación de biopeliculas in vitroPostprint (published version

Applied Biocatalysis in Europe: A Sustainable Tool for Improving Life Quality

Applied biocatalysis and biotransformation, that is, the use of enzymes and whole-cell systems in manufacturing processes for synthetic purposes, has been experiencing a clear boom in recent years, which has led to the start of the so-called “fourth wave”. In fact, the latest advances in bioinformatics, system biology, process intensification, and, in particular, enzyme-directed evolution (encouraged by the 2018 Nobel Prize awarded to F. Arnold), are widening the range of the efficacy of biocatalysts and accelerating the rate at which new enzymes are becoming available, even for activities not previously known. European scientists have been very actively involved in different aspects of this field. Nine contributions dealing with different aspects of applied biocatalysis developed by European researchers are gathered in this Special Issu