High-Efficiency, Multi-Copy, Markerless Integration of Large Biochemical Pathways in Saccharomyces Cerevisiae 

Saccharomyces cerevisiae has many advantages as a production host due to its robustness, tolerance towards low pH, genetic tractability and long history of industrial use. However, despite recent advances in genome editing capabilities, the chromosomal integration of large multi-gene biochemical pathways for stable industrial production using S. cerevisiae remains challenging. To address this limitation, we developed a simple strategy for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in S. cerevisiae. In this so-called Di-CRISPR (Delta integration CRISPR-Cas) strategy, we specifically designed guide RNA sequences to direct Cas9 cleavage at delta sites that are distributed throughout the yeast genome. The generation of double stranded breaks at the delta sites greatly enhanced the efficiency of homologous recombination at these loci and allowed simultaneous integration of multiple large DNA fragments. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways ranging from 8 kb to 24 kb, achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, and the stable production of BDO directly from xylose with our engineered strain. The simplicity and high efficiency of the Di-CRISPR strategy would provide a superior and more industrially-relevant alternative to high copy plasmids and would render this strategy an invaluable tool for genome editing and metabolic engineering in S. cerevisiae.