Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis
Clavulanic acid (CA) is a potent inhibitor of class A β-lactamase enzymes produced by Streptomyces clavuligerus (S. clavuligerus) as a defense mechanism. Due to its industrial interest, the process optimization is under continuous investigation. This work aimed at identifying the potential relations...
- Autores:
-
Caicedo Montoya, Carlos Andrés
Yepes García, Jeferyd
Pinilla Mendoza, Laura Inés
Toro Navarro, León Felipe
Ríos Estepa, Rigoberto
- Tipo de recurso:
- Article of investigation
- Fecha de publicación:
- 2020
- Institución:
- Universidad de Antioquia
- Repositorio:
- Repositorio UdeA
- Idioma:
- eng
- OAI Identifier:
- oai:bibliotecadigital.udea.edu.co:10495/45999
- Acceso en línea:
- https://hdl.handle.net/10495/45999
- Palabra clave:
- Cells - Morphology
Células - Morfología
Ácido Clavulánico
Clavulanic Acid
Streptomyces
Tensión cortante
Shear stress
Fermentación
Fermentation
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http://aims.fao.org/aos/agrovoc/c_2855
http://id.loc.gov/authorities/subjects/sh85021688
https://id.nlm.nih.gov/mesh/D019818
https://id.nlm.nih.gov/mesh/D013302
ODS 3: Salud y bienestar. Garantizar una vida sana y promover el bienestar de todos a todas las edades
ODS 9: Industria, innovación e infraestructura. Construir infraestructuras resilientes, promover la industrialización inclusiva y sostenible y fomentar la innovación
ODS 12: Producción y consumo responsables. Garantizar modalidades de consumo y producción sostenibles
- Rights
- openAccess
- License
- http://creativecommons.org/licenses/by/4.0/
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Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis |
| title |
Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis |
| spellingShingle |
Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis Cells - Morphology Células - Morfología Ácido Clavulánico Clavulanic Acid Streptomyces Tensión cortante Shear stress Fermentación Fermentation http://aims.fao.org/aos/agrovoc/c_8b629ee6 http://aims.fao.org/aos/agrovoc/c_2855 http://id.loc.gov/authorities/subjects/sh85021688 https://id.nlm.nih.gov/mesh/D019818 https://id.nlm.nih.gov/mesh/D013302 ODS 3: Salud y bienestar. Garantizar una vida sana y promover el bienestar de todos a todas las edades ODS 9: Industria, innovación e infraestructura. Construir infraestructuras resilientes, promover la industrialización inclusiva y sostenible y fomentar la innovación ODS 12: Producción y consumo responsables. Garantizar modalidades de consumo y producción sostenibles |
| title_short |
Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis |
| title_full |
Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis |
| title_fullStr |
Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis |
| title_full_unstemmed |
Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis |
| title_sort |
Morphological differentiation of streptomyces clavuligerus exposed to diverse environmental conditions and its relationship with clavulanic acid biosynthesis |
| dc.creator.fl_str_mv |
Caicedo Montoya, Carlos Andrés Yepes García, Jeferyd Pinilla Mendoza, Laura Inés Toro Navarro, León Felipe Ríos Estepa, Rigoberto |
| dc.contributor.author.none.fl_str_mv |
Caicedo Montoya, Carlos Andrés Yepes García, Jeferyd Pinilla Mendoza, Laura Inés Toro Navarro, León Felipe Ríos Estepa, Rigoberto |
| dc.contributor.researchgroup.none.fl_str_mv |
Bioprocesos |
| dc.subject.lcsh.none.fl_str_mv |
Cells - Morphology Células - Morfología |
| topic |
Cells - Morphology Células - Morfología Ácido Clavulánico Clavulanic Acid Streptomyces Tensión cortante Shear stress Fermentación Fermentation http://aims.fao.org/aos/agrovoc/c_8b629ee6 http://aims.fao.org/aos/agrovoc/c_2855 http://id.loc.gov/authorities/subjects/sh85021688 https://id.nlm.nih.gov/mesh/D019818 https://id.nlm.nih.gov/mesh/D013302 ODS 3: Salud y bienestar. Garantizar una vida sana y promover el bienestar de todos a todas las edades ODS 9: Industria, innovación e infraestructura. Construir infraestructuras resilientes, promover la industrialización inclusiva y sostenible y fomentar la innovación ODS 12: Producción y consumo responsables. Garantizar modalidades de consumo y producción sostenibles |
| dc.subject.decs.none.fl_str_mv |
Ácido Clavulánico Clavulanic Acid Streptomyces |
| dc.subject.agrovoc.none.fl_str_mv |
Tensión cortante Shear stress Fermentación Fermentation |
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http://aims.fao.org/aos/agrovoc/c_8b629ee6 http://aims.fao.org/aos/agrovoc/c_2855 |
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http://id.loc.gov/authorities/subjects/sh85021688 |
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https://id.nlm.nih.gov/mesh/D019818 https://id.nlm.nih.gov/mesh/D013302 |
| dc.subject.ods.none.fl_str_mv |
ODS 3: Salud y bienestar. Garantizar una vida sana y promover el bienestar de todos a todas las edades ODS 9: Industria, innovación e infraestructura. Construir infraestructuras resilientes, promover la industrialización inclusiva y sostenible y fomentar la innovación ODS 12: Producción y consumo responsables. Garantizar modalidades de consumo y producción sostenibles |
| description |
Clavulanic acid (CA) is a potent inhibitor of class A β-lactamase enzymes produced by Streptomyces clavuligerus (S. clavuligerus) as a defense mechanism. Due to its industrial interest, the process optimization is under continuous investigation. This work aimed at identifying the potential relationship that might exist between S. clavuligerus ATCC 27064 morphology and CA biosynthesis. For this, modified culture conditions such as source, size, and age of inoculum, culture media, and geometry of fermentation flasks were tested. We observed that high density spore suspensions (1 × 107 spores/mL) represent the best inoculum source for S. clavuligerus cell suspension culture. Further, we studied the life cycle of S. clavuligerus in liquid medium, using optic, confocal, and electron microscopy; results allowed us to observe a potential relationship that might exist between the accumulation of CA and the morphology of disperse hyphae. Reactor geometries that increase shear stress promote smaller pellets and a quick disintegration of these in dispersed secondary mycelia, which begins the pseudosporulation process, thus easing CA accumulation. These outcomes greatly contribute to improving the understanding of antibiotic biosynthesis in the Streptomyces genus. |
| publishDate |
2020 |
| dc.date.issued.none.fl_str_mv |
2020 |
| dc.date.accessioned.none.fl_str_mv |
2025-05-19T17:23:58Z |
| dc.type.none.fl_str_mv |
Artículo de investigación |
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info:eu-repo/semantics/publishedVersion |
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Yepes-García, J., Caicedo-Montoya, C., Pinilla, L., Toro, L. F., & Ríos-Estepa, R. (2020). Morphological Differentiation of Streptomyces clavuligerus Exposed to Diverse Environmental Conditions and Its Relationship with Clavulanic Acid Biosynthesis. Processes, 8(9), 1038. https://doi.org/10.3390/pr8091038 |
| dc.identifier.uri.none.fl_str_mv |
https://hdl.handle.net/10495/45999 |
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10.3390/pr8091038 |
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2227-9717 |
| identifier_str_mv |
Yepes-García, J., Caicedo-Montoya, C., Pinilla, L., Toro, L. F., & Ríos-Estepa, R. (2020). Morphological Differentiation of Streptomyces clavuligerus Exposed to Diverse Environmental Conditions and Its Relationship with Clavulanic Acid Biosynthesis. Processes, 8(9), 1038. https://doi.org/10.3390/pr8091038 10.3390/pr8091038 2227-9717 |
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https://hdl.handle.net/10495/45999 |
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eng |
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Processes |
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Basilea, Suiza |
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Caicedo Montoya, Carlos AndrésYepes García, JeferydPinilla Mendoza, Laura InésToro Navarro, León Felipe Ríos Estepa, RigobertoBioprocesos2025-05-19T17:23:58Z2020Yepes-García, J., Caicedo-Montoya, C., Pinilla, L., Toro, L. F., & Ríos-Estepa, R. (2020). Morphological Differentiation of Streptomyces clavuligerus Exposed to Diverse Environmental Conditions and Its Relationship with Clavulanic Acid Biosynthesis. Processes, 8(9), 1038. https://doi.org/10.3390/pr8091038https://hdl.handle.net/10495/4599910.3390/pr80910382227-9717Clavulanic acid (CA) is a potent inhibitor of class A β-lactamase enzymes produced by Streptomyces clavuligerus (S. clavuligerus) as a defense mechanism. Due to its industrial interest, the process optimization is under continuous investigation. This work aimed at identifying the potential relationship that might exist between S. clavuligerus ATCC 27064 morphology and CA biosynthesis. For this, modified culture conditions such as source, size, and age of inoculum, culture media, and geometry of fermentation flasks were tested. We observed that high density spore suspensions (1 × 107 spores/mL) represent the best inoculum source for S. clavuligerus cell suspension culture. Further, we studied the life cycle of S. clavuligerus in liquid medium, using optic, confocal, and electron microscopy; results allowed us to observe a potential relationship that might exist between the accumulation of CA and the morphology of disperse hyphae. Reactor geometries that increase shear stress promote smaller pellets and a quick disintegration of these in dispersed secondary mycelia, which begins the pseudosporulation process, thus easing CA accumulation. These outcomes greatly contribute to improving the understanding of antibiotic biosynthesis in the Streptomyces genus.Colombia. 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