Advances in Genome Engineering Techniques Enable Fast and Efficient Construction of Microbial Cell Factories

One of the key components for successful construction of microbial cell factories is the speed and efficiency by which genome can be manipulated to produce desired compounds. In recent years, advances in synthetic biology and genome-engineering techniques, enabled much faster and efficient integration or removal of key components from the genome, hence allowing for rapid construction and optimization of metabolic pathways in host organisms. Here, we present various techniques based on CRISPR/Cas9 for yeast Saccharomyces cerevisiae genome engineering. Developed techniques, allow rapidly and in multiplexed manner integrate, delete, in vivo assemble/integrate components required for production of high value chemicals. Further, we developed and validated a set of vectors capable of carrying multiple gRNA expression cassettes and allowing easy exchange of them for targeting up to five desired genomic sites simultaneously. In addition, we employed CRISPR/Cas9 for protein engineering, which allows screening for more robust or higher specificity enzymes. All together, these genome manipulation techniques, allow for building and testing of various metabolic pathways in a faster, marker-free, multiplex, cost-effective manner and advances the design of yeast cell factories further.