Riassunto analitico
In recent years, the focus on more sustainable and eco-friendly production processes has gained greater importance, especially in the field of industrial biotechnology. The use of microorganisms as platforms for the biosynthesis of high-value products appears to be among the most feasible approaches to mitigate environmental issues and enhance the efficiency of production processes. Among the yeasts widely used in industrial applications, Komagataella phaffii (formerly Pichia pastoris) stands out for its ability to produce high amounts of heterologous proteins, making it an established industrial platform to produce pharmaceutical and biotechnological proteins. However, like many other microorganisms, K. phaffii is often auxotrophic for several essential metabolites, particularly biotin, a vital vitamin for cellular growth and development.
Traditional selection of production strains relies on the use of antibiotic resistance markers, which raises both economic and environmental concerns. Antibiotics not only represent a significant expense in industrial processes but also contribute to the growing issue of antimicrobial resistance and the release of pollutants into the environment. In this context, genetic engineering emerges as a viable alternative. When applied to production strains, it can address the issue of auxotrophy, enabling selection and, eventually, growth on nutrient-poor media, such as those lacking biotin.
In this study, K. phaffii was engineered to become a biotin prototroph through the insertion of four essential genes involved in the biosynthesis of the vitamin. The aim was to evaluate the possibility of selection using biotin-free media, thereby eliminating the need for antibiotics.
The results showed that the prototrophic strains obtained were capable of growing in biotin-free media, with good protein production capabilities when compared to antibiotic resistance-based selection. However, protein production decreased drastically when selection was conducted on biotin-free media, which proved suboptimal for production purposes.
In conclusion, this study demonstrates the feasibility of using non-antibiotic-based selection approaches, such as biotin absence, for K. phaffii. However, from a production perspective, this method is not recommended due to the low yield in protein production, highlighting the need for further optimizations for industrial applications
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Abstract
In recent years, the focus on more sustainable and eco-friendly production processes has gained greater importance, especially in the field of industrial biotechnology. The use of microorganisms as platforms for the biosynthesis of high-value products appears to be among the most feasible approaches to mitigate environmental issues and enhance the efficiency of production processes. Among the yeasts widely used in industrial applications, Komagataella phaffii (formerly Pichia pastoris) stands out for its ability to produce high amounts of heterologous proteins, making it an established industrial platform to produce pharmaceutical and biotechnological proteins. However, like many other microorganisms, K. phaffii is often auxotrophic for several essential metabolites, particularly biotin, a vital vitamin for cellular growth and development.
Traditional selection of production strains relies on the use of antibiotic resistance markers, which raises both economic and environmental concerns. Antibiotics not only represent a significant expense in industrial processes but also contribute to the growing issue of antimicrobial resistance and the release of pollutants into the environment. In this context, genetic engineering emerges as a viable alternative. When applied to production strains, it can address the issue of auxotrophy, enabling selection and, eventually, growth on nutrient-poor media, such as those lacking biotin.
In this study, K. phaffii was engineered to become a biotin prototroph through the insertion of four essential genes involved in the biosynthesis of the vitamin. The aim was to evaluate the possibility of selection using biotin-free media, thereby eliminating the need for antibiotics.
The results showed that the prototrophic strains obtained were capable of growing in biotin-free media, with good protein production capabilities when compared to antibiotic resistance-based selection. However, protein production decreased drastically when selection was conducted on biotin-free media, which proved suboptimal for production purposes.
In conclusion, this study demonstrates the feasibility of using non-antibiotic-based selection approaches, such as biotin absence, for K. phaffii. However, from a production perspective, this method is not recommended due to the low yield in protein production, highlighting the need for further optimizations for industrial applications
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