Toward a Serine Integrase Mediated Integration System in Yarrowia lipolytica

• Caleb Lawson, Chemical Engineering, University of Delaware

• Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware

Monoterpene indole alkaloids (MIAs) constitute a class of vital medicines that are difficult to manufacture. The low concentrations of these chemicals from their native species make MIAs incredibly expensive to extract. A better way to produce MIAs is with a heterologous host. Yarrowia lipolytica is a yeast with a high natural flux of Acetyl-CoA, a precursor for all MIAs. However, there is a need to integrate large DNA fragments to produce MIAs, and Y. lipolytica lacks the genetic tools to do so. Serine integrases have been proven to deliver large fragments of DNA, catalyzing the recombination of recognition sites in the genome. Using this method of integration in Y. lipolytica requires a “donor vector” and a “landing pad”. The landing pad is a linear piece of DNA that we assembled via Golden Gate reaction and includes a selection marker and a fluorescent reporter flanked by recognition sites. The landing pad was integrated randomly into Y. lipolytica leveraging the native non-homologous end joining (NHEJ) system. The fluorescence of integrated cultures is used to find high expression loci. Fluorescence behavior of cultures was investigated under multiple culture conditions. High fluorescence strains were identified, and the locus of integration will be elucidated by inverse PCR.