Easyclone-Markerfree: A Vector Toolkit for Marker-Less Integration of Genes into Saccharomyces Cerevisiae

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of vectors into previously described loci is facilitated by Cas9 endonuclease introducing double strand break at the particular sites in the genome. Cas9 is recruited to the chromosomal location by specific gRNA molecules expressed from a set of gRNA helper vectors. Single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency correspondingly. We demonstrate application of the vector toolkit by constructing a haploid laboratory (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each.