Next-generation synthetic biology tools for rapid metabolic engineering of yeast

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Anastasia Krivoruchko, Verena Siewers, Jens Nielsen

Chalmers University, Sweden

The yeast Saccharomyces cerevisiae possesses characteristics that make it an attractive cell factory for production of biotechnology products. It is a robust industrial organism and is able to grow under a variety of industrial conditions including low pH, high temperature, and less stringent nutritional requirements compared to bacteria. In addition, S. cerevisiae is genetically tractable and well-characterized. Much information is available on S. cerevisiae, including a complete genome sequence, as well as characterization of its metabolic pathways and various ‘omics’ data. However, despite these resources, introduction of entire pathways into yeast is still relatively slow when compared to E. coli and it can take a significantly longer time to obtain industrially-usable strains. The goal of this project is, therefore, to develop a synthetic biology platform to facilitate the rapid introduction of entire pathways into yeast. This includes a library of parts, including constitutive and inducible promoters of various strengths, terminators and markers. A series of improved episomal plasmids for rapid pathway screening has also been developed. In addition, this platform includes a variety of integration vectors for single and multi-copy integration. These vectors contain different auxotrophic and dominant markers that allow a single-step introduction of entire pathways into both laboratory and industrial strains. In addition, novel marker recycling systems that facilitate excision of all the markers without additional transformation steps are presented.