Publication
Exploitation of Ashbya gossypii for the production of high-value products from glycerol feedstocks
| Summary: | The implementation of biorefineries has been proposed as a mean to increase the economic viability of the biobased industries. In its “conventional” form, a biorefinery makes use of by-products (e.g. crude glycerol) generated during the production of biodiesel to coproduce high-value products. For this, it is necessary to develop robust microorganisms, easily metabolically and genetically manipulated in order to develop tailor made cell factories. Ashbya gossypii is considered an example of the sustainable white biotechnology business model in what concerns the industrial production of riboflavin. Its biotechnological relevance has allowed the development of directed and based-knowledge methodologies for strain optimization for riboflavin production. The metabolic engineering strategies tested to create overproducing strains have focused on pathways strongly connected with riboflavin production. However, the A. gossypii pyrimidine pathway and its connection with the riboflavin biosynthetic pathway had not yet been assessed. We report that the blockage of the de novo pyrimidine biosynthetic pathway in the recently generated A. gossypii Agura3 uridine/uracil auxotrophic strain led to improved riboflavin production. Considering that the riboflavin and the pyrimidine pathways share the same precursors and that riboflavin overproduction may be triggered by stress, we suggest that overproduction of riboflavin by A. gossypii Agura3 may occur as an outcome of a nutritional stress response and/or of an increased availability in precursors for riboflavin biosynthesis, due to their reduced consumption by the pyrimidine pathway. Despite the recognized capabilities of A. gossypii to use industrial wastes as substrate for the production of riboflavin, glycerol had not yet been exploited. In this study we explore riboflavin production by A. gossypii from glycerol. Additionally, we designed a strategy to improve its glycerol consumption profile by overexpressing glycerol uptake proteins (GUP1) genes. A. gossypii strains overexpressing the native GUP1 gene (pRSAG) or GUP1 gene from Saccharomyces cerevisiae (pRSSG) under the control of the AgTEF promoter displayed significantly improved glycerol-dependent hyperosmotic stress tolerance and glycerol consumption profiles, indicating that our strategy successfully led to the improvement of glycerol utilization. These results contribute to the further development of A. gossypii as an environmentalfriendly cell factory organism, contributing to its establishment in the biorefinery concept. |
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| Main Authors: | Silva, Rui Miguel Correia da |
| Subject: | Engenharia e Tecnologia::Biotecnologia Industrial |
| Year: | 2014 |
| Country: | Portugal |
| Document type: | master thesis |
| Access type: | open access |
| Associated institution: | Universidade do Minho |
| Language: | English |
| Origin: | RepositóriUM - Universidade do Minho |
| Summary: | The implementation of biorefineries has been proposed as a mean to increase the economic viability of the biobased industries. In its “conventional” form, a biorefinery makes use of by-products (e.g. crude glycerol) generated during the production of biodiesel to coproduce high-value products. For this, it is necessary to develop robust microorganisms, easily metabolically and genetically manipulated in order to develop tailor made cell factories. Ashbya gossypii is considered an example of the sustainable white biotechnology business model in what concerns the industrial production of riboflavin. Its biotechnological relevance has allowed the development of directed and based-knowledge methodologies for strain optimization for riboflavin production. The metabolic engineering strategies tested to create overproducing strains have focused on pathways strongly connected with riboflavin production. However, the A. gossypii pyrimidine pathway and its connection with the riboflavin biosynthetic pathway had not yet been assessed. We report that the blockage of the de novo pyrimidine biosynthetic pathway in the recently generated A. gossypii Agura3 uridine/uracil auxotrophic strain led to improved riboflavin production. Considering that the riboflavin and the pyrimidine pathways share the same precursors and that riboflavin overproduction may be triggered by stress, we suggest that overproduction of riboflavin by A. gossypii Agura3 may occur as an outcome of a nutritional stress response and/or of an increased availability in precursors for riboflavin biosynthesis, due to their reduced consumption by the pyrimidine pathway. Despite the recognized capabilities of A. gossypii to use industrial wastes as substrate for the production of riboflavin, glycerol had not yet been exploited. In this study we explore riboflavin production by A. gossypii from glycerol. Additionally, we designed a strategy to improve its glycerol consumption profile by overexpressing glycerol uptake proteins (GUP1) genes. A. gossypii strains overexpressing the native GUP1 gene (pRSAG) or GUP1 gene from Saccharomyces cerevisiae (pRSSG) under the control of the AgTEF promoter displayed significantly improved glycerol-dependent hyperosmotic stress tolerance and glycerol consumption profiles, indicating that our strategy successfully led to the improvement of glycerol utilization. These results contribute to the further development of A. gossypii as an environmentalfriendly cell factory organism, contributing to its establishment in the biorefinery concept. |
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