Observatorio de I+D+i UPM

Memorias de investigación
Communications at congresses:
Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of mitochondrial proteins in yeast.
Year:2017
Research Areas
  • Microbiology,
  • Biotechnology
Information
Abstract
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.
International
Si
Congress
20th International Congress on Nitrogen Fixation
970
Place
Granada
Reviewers
Si
ISBN/ISSN
00000000
Start Date
03/09/2017
End Date
09/09/2017
From page
26
To page
26
Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of mitochondrial proteins in yeast.
Participants
  • Autor: Ana Perez Gonzalez (UPM)
  • Autor: Marcel Veldhuizen . (UPM)
  • Autor: Mónica Navarro Rodríguez (UPM)
  • Autor: Luis Manuel Rubio Herrero (UPM)
  • Autor: Elena Caro Bernat (UPM)
Research Group, Departaments and Institutes related
  • Creador: Grupo de Investigación: BIOLOGÍA MOLECULAR Y COMPUTACIONAL
  • Centro o Instituto I+D+i: Centro de Biotecnología y Genómica de Plantas, CBGP
  • Departamento: Biotecnología - Biología Vegetal
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