Abstract

To accelerate the construction of superior yeast strains producing high-value chemicals, we developed a modular CRISPR/Cas9 integration platform in S. cerevisiae that accommodates marker-less, multi-copy gene integration. Our engineering strategy introduced a series of synthetic DNA parts, called Landing Pads (LP), into the S. cerevisiae genome to act as modular anchors for heterologous gene integrations. The LPs have been designed to accommodate the CRISPR/Cas9 genome editing system and to facilitate multi-copy gene integration of one, two, three and four copies in a single step. First we designed ten synthetic gRNA targeting sequences and evaluated their targeting specificity, integration efficiency, and possible off-target effects. We also surveyed 16 genomic loci for landing pad integration by evaluating the integration efficiency and gene expression profiles at each site. The results gleaned from our preliminary tests informed the final configuration of the LP platform strain. To demonstrate the utility of our LP integration system, we used the platform to screen ten variants of norcoclaurine synthase (NCS), a notoriously inefficient enzyme that catalyzes the first committed step in the production of high-value benzylisoquinoline alkaloids (BIA). The platform enabled rapid integration of each NCS variant in one, two, three and four copies in parallel, yielding 40 strains total. LC/MS analysis identified two variants, NdNCS and ScNCS that produce higher concentrations of the BIA scaffold (S)-norcoclaurine by increasing copy number, suggesting that the proposed strategy may help alleviate enzyme inefficiencies in pathway engineering.