Publicação
Engineering a biosynthetic pathway for high value glycosaminoglycans production in Saccharomyces cerevisiae
| Resumo: | Glycosaminoglycans (GAGs), such as hyaluronic acid, heparosan and chondroitin, have emerged as key substances with diverse applications in both the medical and cosmetic industries. Particularly, chondroitin is a crucial polysaccharide with anti-inflammatory properties, mostly used in the treatment of osteoarthritis. Historically, chondroitin has been extracted from animal sources, including shark fins and animal cartilage. However, rising concerns regarding the risks associated with the use of animal-derived products, coupled with the ecological repercussions of overfishing, have been driving the search for sustainable, alternative methodologies for chondroitin production. Inspired by the inherent biosynthetic capabilities of pathogenic strains of Pasteurella multocida and Escherichia coli to produce GAG-like polysaccharides, several studies have sought to use non-pathogenic hosts for the biosynthetic production of chondroitin. The aim of this thesis was to design and engineer an artificial pathway to produce chondroitin in Saccharomyces cerevisiae, due to its well-established status as industrial host organism, its genetic manipulability, and its robust fermentation capabilities. Alternative key genes were carefully curated and evaluated using literature mining and kinetic assays to streamline and optimize the production of chondroitin. The methodologies for bioinformatic metabolic flux prediction and optimization, as well as for the analysis of chondroitin production and quantification have been firstly evaluated and validated in a simpler organism, E. coli. Several targets were identified for under and overexpression that were able to enhance up to 1.9-fold the in vivo chondroitin production in E. coli. Further bioprocess scale-up with a mutant overexpressing lytic murein transglycosylase (mltB) led to 535 mg/L of chondroitin. Subsequently, S. cerevisiae harboring chondroitin production pathways was designed and engineered leading to up to 125 mg/L of chondroitin in flask. These titers might be improved in the future with several under and overexpressions enhancing the precursors availability predicted by the computational approach. This thesis represents a step forward toward a sustainable, cost-effective, and scalable platform for the large-scale production of chondroitin, thereby circumventing the challenges posed by conventional extraction methods and alleviating the environmental and availability concerns associated with animal-sourced chondroitin. Also, the methods herein used can be further applied to other GAG production processes. |
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| Autores principais: | Couto, Márcia Regina Linhares |
| Assunto: | Chondroitin Glycosaminoglycans Metabolic engineering Nutraceuticals Synthetic biology Biologia sintética Condroitina Engenharia metabólica Glicosaminoglicanos Nutracêuticos |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Glycosaminoglycans (GAGs), such as hyaluronic acid, heparosan and chondroitin, have emerged as key substances with diverse applications in both the medical and cosmetic industries. Particularly, chondroitin is a crucial polysaccharide with anti-inflammatory properties, mostly used in the treatment of osteoarthritis. Historically, chondroitin has been extracted from animal sources, including shark fins and animal cartilage. However, rising concerns regarding the risks associated with the use of animal-derived products, coupled with the ecological repercussions of overfishing, have been driving the search for sustainable, alternative methodologies for chondroitin production. Inspired by the inherent biosynthetic capabilities of pathogenic strains of Pasteurella multocida and Escherichia coli to produce GAG-like polysaccharides, several studies have sought to use non-pathogenic hosts for the biosynthetic production of chondroitin. The aim of this thesis was to design and engineer an artificial pathway to produce chondroitin in Saccharomyces cerevisiae, due to its well-established status as industrial host organism, its genetic manipulability, and its robust fermentation capabilities. Alternative key genes were carefully curated and evaluated using literature mining and kinetic assays to streamline and optimize the production of chondroitin. The methodologies for bioinformatic metabolic flux prediction and optimization, as well as for the analysis of chondroitin production and quantification have been firstly evaluated and validated in a simpler organism, E. coli. Several targets were identified for under and overexpression that were able to enhance up to 1.9-fold the in vivo chondroitin production in E. coli. Further bioprocess scale-up with a mutant overexpressing lytic murein transglycosylase (mltB) led to 535 mg/L of chondroitin. Subsequently, S. cerevisiae harboring chondroitin production pathways was designed and engineered leading to up to 125 mg/L of chondroitin in flask. These titers might be improved in the future with several under and overexpressions enhancing the precursors availability predicted by the computational approach. This thesis represents a step forward toward a sustainable, cost-effective, and scalable platform for the large-scale production of chondroitin, thereby circumventing the challenges posed by conventional extraction methods and alleviating the environmental and availability concerns associated with animal-sourced chondroitin. Also, the methods herein used can be further applied to other GAG production processes. |
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