Presentation Title

Development of a synthetic biology tool for standardized gene integration in Yarrowia Lipolytica

Start Date

November 2016

End Date

November 2016

Location

HUB 302-#31

Type of Presentation

Poster

Abstract

The nonconventional yeast Yarrowia lipolytica has been the subject of a number of studies due to its potential for lipid based chemical production. To facilitate more rapid and standardized strain development for metabolic engineering applications of Y. lipolytica, advanced synthetic biology tools are needed. In this work, we used a CRISPR-Cas9 system to induce homologous recombination to integrate heterologous genes into the genome of the yeast. A total of 17 genomic loci were tested for the rate of integration of an hrGFP expression cassette. Five sites were identified as having high rates of integration, and were characterized further. Expression from each site was quantified using flow cytometry under a range of growth conditions to enable rational selection of sites for pathway integration. To demonstrate the application of the tool, two heterologous genes enabling production of the carotenoid, lycopene, were integrated (phytoene synthase and phytoene desaturase from Pantoea ananatis). Lycopene production was then enhanced by integrating overexpression cassettes of native Hmg-CoA reductase and heterologous GGPP synthase, resulting in a strain producing 3.38 mg lycopene/g DCW in shake flasks. This new genome integration tool provides synthetic biologists and metabolic engineers the capability to efficiently integrate metabolic pathways in Y. lipolytica.

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Development of a synthetic biology tool for standardized gene integration in Yarrowia Lipolytica

HUB 302-#31

The nonconventional yeast Yarrowia lipolytica has been the subject of a number of studies due to its potential for lipid based chemical production. To facilitate more rapid and standardized strain development for metabolic engineering applications of Y. lipolytica, advanced synthetic biology tools are needed. In this work, we used a CRISPR-Cas9 system to induce homologous recombination to integrate heterologous genes into the genome of the yeast. A total of 17 genomic loci were tested for the rate of integration of an hrGFP expression cassette. Five sites were identified as having high rates of integration, and were characterized further. Expression from each site was quantified using flow cytometry under a range of growth conditions to enable rational selection of sites for pathway integration. To demonstrate the application of the tool, two heterologous genes enabling production of the carotenoid, lycopene, were integrated (phytoene synthase and phytoene desaturase from Pantoea ananatis). Lycopene production was then enhanced by integrating overexpression cassettes of native Hmg-CoA reductase and heterologous GGPP synthase, resulting in a strain producing 3.38 mg lycopene/g DCW in shake flasks. This new genome integration tool provides synthetic biologists and metabolic engineers the capability to efficiently integrate metabolic pathways in Y. lipolytica.