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Descripción
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| Synthetic biology methods and tools need to be developed to facilitate rapid and efficient engineering of yeast that accommodates the necessities of specific biotechnology projects. We have adapted a modular cloning system, the GoldenBraid2.0 (1), for the integration of transcriptional units in two different sites of the yeast chromosome characterized by a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, the toolkit includes an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. The manipulation of the mitochondrial proteome has interesting potential applications. One of these advantages reside in the fact that metalation occurs after protein import into the organelle, which contains pools of iron, zinc, copper, and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins since their high respiration rate creates a low oxygen environment within the matrix (2). As a proof of concept, we present the integration and expression of fourteen nitrogen fixation (nif) genes, some of which are known to require different metalloclusters to accumulate and to be highly sensitive to the presence of oxygen. For one of these genes, nifU, we show how the utilization of the Su9 pre-sequence and the use of glycerol as carbon source to sustain aerobic respiration exhibits best results in protein accumulation. | |
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Internacional
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Si |
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Nombre congreso
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20th International Congress on Nitrogen Fixation |
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Tipo de participación
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970 |
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Lugar del congreso
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Granada |
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Revisores
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Si |
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ISBN o ISSN
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00000000 |
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DOI
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Fecha inicio congreso
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03/09/2017 |
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Fecha fin congreso
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09/09/2017 |
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Desde la página
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26 |
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Hasta la página
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26 |
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Título de las actas
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Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of mitochondrial proteins in yeast. |