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...

Full description

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
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
Rights
openAccess
License
Atribución-NoComercial-SinDerivadas 2.5 Colombia (CC BY-NC-ND 2.5 CO)
id UDEA2_2daa8b6c0bb9911c46912e9b886a525b
oai_identifier_str oai:bibliotecadigital.udea.edu.co:10495/16422
network_acronym_str UDEA2
network_name_str Repositorio UdeA
repository_id_str
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
dc.rights.uri.*.fl_str_mv 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/
rights_invalid_str_mv 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 http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/1/GomezDavid_2020_DynamicsClavulanalateProduction.pdf
http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/2/GomezDavid_2020_DynamicsClavulanalateProduction.docx
http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/3/Aval%20CENDOI.pdf
http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/4/Autorizaci%c3%b3n.pdf
http://bibliotecadigital.udea.edu.co/bitstream/10495/16422/5/license.txt
bitstream.checksum.fl_str_mv 48892096c0669b2c65f82ea09d1d5703
d88da5464a9ba863e6200fb1b12579b2
d351ea8e7ebc18f4f148abca4f60139d
514169099a1c55b93db573fb7eb9e91a
8a4605be74aa9ea9d79846c1fba20a33
bitstream.checksumAlgorithm.fl_str_mv MD5
MD5
MD5
MD5
MD5
repository.name.fl_str_mv Repositorio Institucional Universidad de Antioquia
repository.mail.fl_str_mv andres.perez@udea.edu.co
_version_ 1812173257235759104
spelling 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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