Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production
ABSTRACT: Clavulanic acid (CA) is a β-lactam antibiotic with potent inhibitory activity against β-lactamase enzymes, which are responsible for the antibiotic resistance phenomenon in several pathogenic bacteria. CA is a secondary metabolite of pharmaceutical and industrial interest naturally produce...
- Autores:
-
Gómez Ríos, David Andrés
- Tipo de recurso:
- Doctoral thesis
- Fecha de publicación:
- 2020
- Institución:
- Universidad de Antioquia
- Repositorio:
- Repositorio UdeA
- Idioma:
- spa
- OAI Identifier:
- oai:bibliotecadigital.udea.edu.co:10495/16422
- Acceso en línea:
- http://hdl.handle.net/10495/16422
- Palabra clave:
- Clavulanic acid
Enzymes
Enzima
Scientific information systems
Sistema de información científica
Metabolism
Metabolismo
Pharmaceutical technology
Tecnología farmacéutica
Bacteria
Bacteria
Hydrodynamics
Hidrodinámica
Resistance to antibiotics
Resistencia a los antibióticos
Dynamic flux balance analysis
Fed-batch
Flux balance analysis
Genome-scale model
Single-use bioreactor
Streptomyces clavuligerus
http://aims.fao.org/aos/agrovoc/c_8848
http://id.loc.gov/authorities/subjects/sh85026861
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http://vocabularies.unesco.org/thesaurus/concept1950
http://vocabularies.unesco.org/thesaurus/concept8575
- Rights
- openAccess
- License
- Atribución-NoComercial-SinDerivadas 2.5 Colombia (CC BY-NC-ND 2.5 CO)
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dc.title.spa.fl_str_mv |
Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production |
title |
Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production |
spellingShingle |
Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production Clavulanic acid Enzymes Enzima Scientific information systems Sistema de información científica Metabolism Metabolismo Pharmaceutical technology Tecnología farmacéutica Bacteria Bacteria Hydrodynamics Hidrodinámica Resistance to antibiotics Resistencia a los antibióticos Dynamic flux balance analysis Fed-batch Flux balance analysis Genome-scale model Single-use bioreactor Streptomyces clavuligerus http://aims.fao.org/aos/agrovoc/c_8848 http://id.loc.gov/authorities/subjects/sh85026861 http://vocabularies.unesco.org/thesaurus/concept3945 http://vocabularies.unesco.org/thesaurus/concept3777 http://vocabularies.unesco.org/thesaurus/concept3954 http://vocabularies.unesco.org/thesaurus/concept4099 http://vocabularies.unesco.org/thesaurus/concept1950 http://vocabularies.unesco.org/thesaurus/concept8575 |
title_short |
Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production |
title_full |
Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production |
title_fullStr |
Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production |
title_full_unstemmed |
Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production |
title_sort |
Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid production |
dc.creator.fl_str_mv |
Gómez Ríos, David Andrés |
dc.contributor.advisor.none.fl_str_mv |
Ríos Estepa, Rigoberto Ochoa Cáceres, Silvia Mercedes Ramírez Malule, Howard Diego |
dc.contributor.author.none.fl_str_mv |
Gómez Ríos, David Andrés |
dc.subject.lcsh.none.fl_str_mv |
Clavulanic acid |
topic |
Clavulanic acid Enzymes Enzima Scientific information systems Sistema de información científica Metabolism Metabolismo Pharmaceutical technology Tecnología farmacéutica Bacteria Bacteria Hydrodynamics Hidrodinámica Resistance to antibiotics Resistencia a los antibióticos Dynamic flux balance analysis Fed-batch Flux balance analysis Genome-scale model Single-use bioreactor Streptomyces clavuligerus http://aims.fao.org/aos/agrovoc/c_8848 http://id.loc.gov/authorities/subjects/sh85026861 http://vocabularies.unesco.org/thesaurus/concept3945 http://vocabularies.unesco.org/thesaurus/concept3777 http://vocabularies.unesco.org/thesaurus/concept3954 http://vocabularies.unesco.org/thesaurus/concept4099 http://vocabularies.unesco.org/thesaurus/concept1950 http://vocabularies.unesco.org/thesaurus/concept8575 |
dc.subject.unesco.none.fl_str_mv |
Enzymes Enzima Scientific information systems Sistema de información científica Metabolism Metabolismo Pharmaceutical technology Tecnología farmacéutica Bacteria Bacteria Hydrodynamics Hidrodinámica |
dc.subject.agrovoc.none.fl_str_mv |
Resistance to antibiotics Resistencia a los antibióticos |
dc.subject.proposal.spa.fl_str_mv |
Dynamic flux balance analysis Fed-batch Flux balance analysis Genome-scale model Single-use bioreactor Streptomyces clavuligerus |
dc.subject.agrovocuri.none.fl_str_mv |
http://aims.fao.org/aos/agrovoc/c_8848 |
dc.subject.lcshuri.none.fl_str_mv |
http://id.loc.gov/authorities/subjects/sh85026861 |
dc.subject.unescouri.none.fl_str_mv |
http://vocabularies.unesco.org/thesaurus/concept3945 http://vocabularies.unesco.org/thesaurus/concept3777 http://vocabularies.unesco.org/thesaurus/concept3954 http://vocabularies.unesco.org/thesaurus/concept4099 http://vocabularies.unesco.org/thesaurus/concept1950 http://vocabularies.unesco.org/thesaurus/concept8575 |
description |
ABSTRACT: Clavulanic acid (CA) is a β-lactam antibiotic with potent inhibitory activity against β-lactamase enzymes, which are responsible for the antibiotic resistance phenomenon in several pathogenic bacteria. CA is a secondary metabolite of pharmaceutical and industrial interest naturally produced by the filamentous Gram-positive bacterium Streptomyces clavuligerus (S. clavuligerus) under limited nutritional conditions. CA has limited availability in the market, and its high cost is a consequence of the complexity of the production process. It is determined mainly by the low titers of CA obtained in submerged cultivations of S. clavuligerus and the difficulties associated with the down-stream process. Several authors have applied experimental approaches to study the influence of some variables, especially those of nutritional nature, on CA accumulation. Nevertheless, the effect of variables relevant to the bioprocess operation, as the reactor hydrodynamics and shear stress conditions, has not been well explored. Fluxomic approaches have been recently applied to S. clavuligerus aimed to improve the understanding of its metabolism. Such approaches were mostly developed under steady-state assumptions, resulting in a limited comprehension of the metabolism under the dynamic conditions of batch and fed-batch processes. In this thesis, the reconstruction of a new and enhanced genome-scale model of S. clavuligerus, the successful combination of experimental studies in the shake flask and bioreactor scales coupled with constraint-based modeling in pseudo-steady (flux balance analysis) and dynamic (dynamic flux balance analysis) conditions were used as strategies for studying the metabolic response of S. clavuligerus to environmental and nutritional perturbations in connection with CA biosynthesis. Experimental studies in stirred tank and 2-D rocking-motion bioreactors provided valuable information on the strain's metabolic response to environmental conditions, especially regarding the effect of shear forces. Moreover, the experimental data obtained allowed to test different in silico scenarios by using constraint-based modeling with a new and enhanced reconstruction of a genome-scale metabolic network of S. clavuligerus, aimed to understand the carbon fluxes distribution during the different environmental conditions attained during the cultivations. The use of constrained-based modeling under pseudo-steady state conditions (Flux Balance Analysis, FBA) and dynamic conditions (Dynamic Flux Balance Analysis, DFBA) allowed to explain the role of primary metabolism and revealed the dynamics of intracellular carbon fluxes distribution during CA biosynthesis. Furthermore, the in silico simulation of metabolic scenarios and experimental testing showed that fed-batch operation with glutamate supplementation is a favorable condition for increasing the CA production. Potential genetic engineering targets were identified and evaluated in silico, aiming to improve the CA titers in S. clavuligerus cultures. This is the first work considering the cultivation dynamics on experimental and in silico studies of S. clavuligerus metabolism. |
publishDate |
2020 |
dc.date.accessioned.none.fl_str_mv |
2020-08-24T17:33:41Z |
dc.date.available.none.fl_str_mv |
2020-08-24T17:33:41Z |
dc.date.issued.none.fl_str_mv |
2020 |
dc.type.spa.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
dc.type.coarversion.fl_str_mv |
http://purl.org/coar/version/c_b1a7d7d4d402bcce |
dc.type.hasversion.spa.fl_str_mv |
info:eu-repo/semantics/draft |
dc.type.coar.spa.fl_str_mv |
http://purl.org/coar/resource_type/c_db06 |
dc.type.redcol.spa.fl_str_mv |
https://purl.org/redcol/resource_type/TD |
dc.type.local.spa.fl_str_mv |
Tesis/Trabajo de grado - Monografía - Doctorado |
format |
http://purl.org/coar/resource_type/c_db06 |
status_str |
draft |
dc.identifier.uri.none.fl_str_mv |
http://hdl.handle.net/10495/16422 |
url |
http://hdl.handle.net/10495/16422 |
dc.language.iso.spa.fl_str_mv |
spa |
language |
spa |
dc.rights.*.fl_str_mv |
Atribución-NoComercial-SinDerivadas 2.5 Colombia (CC BY-NC-ND 2.5 CO) |
dc.rights.spa.fl_str_mv |
info:eu-repo/semantics/openAccess |
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http://creativecommons.org/licenses/by-nc-nd/2.5/co/ |
dc.rights.accessrights.spa.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
dc.rights.creativecommons.spa.fl_str_mv |
https://creativecommons.org/licenses/by-nc-nd/4.0/ |
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Atribución-NoComercial-SinDerivadas 2.5 Colombia (CC BY-NC-ND 2.5 CO) http://creativecommons.org/licenses/by-nc-nd/2.5/co/ http://purl.org/coar/access_right/c_abf2 https://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.extent.spa.fl_str_mv |
115 |
dc.format.mimetype.spa.fl_str_mv |
application/pdf |
dc.publisher.group.spa.fl_str_mv |
Bioprocesos Simulación, Diseño, Control y Optimización de Procesos (SIDCOP) |
dc.publisher.place.spa.fl_str_mv |
Medellín, Colombia |
institution |
Universidad de Antioquia |
bitstream.url.fl_str_mv |
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Ríos Estepa, RigobertoOchoa Cáceres, Silvia MercedesRamírez Malule, Howard DiegoGómez Ríos, David Andrés2020-08-24T17:33:41Z2020-08-24T17:33:41Z2020http://hdl.handle.net/10495/16422ABSTRACT: Clavulanic acid (CA) is a β-lactam antibiotic with potent inhibitory activity against β-lactamase enzymes, which are responsible for the antibiotic resistance phenomenon in several pathogenic bacteria. CA is a secondary metabolite of pharmaceutical and industrial interest naturally produced by the filamentous Gram-positive bacterium Streptomyces clavuligerus (S. clavuligerus) under limited nutritional conditions. CA has limited availability in the market, and its high cost is a consequence of the complexity of the production process. It is determined mainly by the low titers of CA obtained in submerged cultivations of S. clavuligerus and the difficulties associated with the down-stream process. Several authors have applied experimental approaches to study the influence of some variables, especially those of nutritional nature, on CA accumulation. Nevertheless, the effect of variables relevant to the bioprocess operation, as the reactor hydrodynamics and shear stress conditions, has not been well explored. Fluxomic approaches have been recently applied to S. clavuligerus aimed to improve the understanding of its metabolism. Such approaches were mostly developed under steady-state assumptions, resulting in a limited comprehension of the metabolism under the dynamic conditions of batch and fed-batch processes. In this thesis, the reconstruction of a new and enhanced genome-scale model of S. clavuligerus, the successful combination of experimental studies in the shake flask and bioreactor scales coupled with constraint-based modeling in pseudo-steady (flux balance analysis) and dynamic (dynamic flux balance analysis) conditions were used as strategies for studying the metabolic response of S. clavuligerus to environmental and nutritional perturbations in connection with CA biosynthesis. Experimental studies in stirred tank and 2-D rocking-motion bioreactors provided valuable information on the strain's metabolic response to environmental conditions, especially regarding the effect of shear forces. Moreover, the experimental data obtained allowed to test different in silico scenarios by using constraint-based modeling with a new and enhanced reconstruction of a genome-scale metabolic network of S. clavuligerus, aimed to understand the carbon fluxes distribution during the different environmental conditions attained during the cultivations. The use of constrained-based modeling under pseudo-steady state conditions (Flux Balance Analysis, FBA) and dynamic conditions (Dynamic Flux Balance Analysis, DFBA) allowed to explain the role of primary metabolism and revealed the dynamics of intracellular carbon fluxes distribution during CA biosynthesis. Furthermore, the in silico simulation of metabolic scenarios and experimental testing showed that fed-batch operation with glutamate supplementation is a favorable condition for increasing the CA production. Potential genetic engineering targets were identified and evaluated in silico, aiming to improve the CA titers in S. clavuligerus cultures. This is the first work considering the cultivation dynamics on experimental and in silico studies of S. clavuligerus metabolism.115application/pdfspainfo:eu-repo/semantics/draftinfo:eu-repo/semantics/doctoralThesishttp://purl.org/coar/resource_type/c_db06https://purl.org/redcol/resource_type/TDTesis/Trabajo de grado - Monografía - Doctoradohttp://purl.org/coar/version/c_b1a7d7d4d402bcceAtribución-NoComercial-SinDerivadas 2.5 Colombia (CC BY-NC-ND 2.5 CO)info:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-nd/2.5/co/http://purl.org/coar/access_right/c_abf2https://creativecommons.org/licenses/by-nc-nd/4.0/Clavulanic acidEnzymesEnzimaScientific information systemsSistema de información científicaMetabolismMetabolismoPharmaceutical technologyTecnología farmacéuticaBacteriaBacteriaHydrodynamicsHidrodinámicaResistance to antibioticsResistencia a los antibióticosDynamic flux balance analysisFed-batchFlux balance analysisGenome-scale modelSingle-use bioreactorStreptomyces clavuligerushttp://aims.fao.org/aos/agrovoc/c_8848http://id.loc.gov/authorities/subjects/sh85026861http://vocabularies.unesco.org/thesaurus/concept3945http://vocabularies.unesco.org/thesaurus/concept3777http://vocabularies.unesco.org/thesaurus/concept3954http://vocabularies.unesco.org/thesaurus/concept4099http://vocabularies.unesco.org/thesaurus/concept1950http://vocabularies.unesco.org/thesaurus/concept8575Dynamic quantitative description of the metabolic capabilities of Streptomyces clavuligerus for clavulanic acid productionBioprocesosSimulación, Diseño, Control y Optimización de Procesos (SIDCOP)Medellín, ColombiaDoctor en Ingeniería QuímicaDoctoradoFacultad de Ingeniería. Doctorado en Ingeniería QuímicaUniversidad de AntioquiaORIGINALGomezDavid_2020_DynamicsClavulanalateProduction.pdfGomezDavid_2020_DynamicsClavulanalateProduction.pdfTesis doctoralapplication/pdf6303365http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/1/GomezDavid_2020_DynamicsClavulanalateProduction.pdf48892096c0669b2c65f82ea09d1d5703MD51GomezDavid_2020_DynamicsClavulanalateProduction.docxGomezDavid_2020_DynamicsClavulanalateProduction.docxTesis doctoralapplication/vnd.openxmlformats-officedocument.wordprocessingml.document8627398http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/2/GomezDavid_2020_DynamicsClavulanalateProduction.docxd88da5464a9ba863e6200fb1b12579b2MD52Aval CENDOI.pdfAval CENDOI.pdfAval archivo finalapplication/pdf351690http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/3/Aval%20CENDOI.pdfd351ea8e7ebc18f4f148abca4f60139dMD53Autorización.pdfAutorización.pdfAutorizaciónapplication/pdf514919http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/4/Autorizaci%c3%b3n.pdf514169099a1c55b93db573fb7eb9e91aMD54LICENSElicense.txtlicense.txttext/plain; charset=utf-81748http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/5/license.txt8a4605be74aa9ea9d79846c1fba20a33MD5510495/16422oai:bibliotecadigital.udea.edu.co:10495/164222021-05-21 11:44:34.902Repositorio Institucional Universidad de Antioquiaandres.perez@udea.edu.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 |