(585an) Engineered CRISPR/Cas9 System for Multiplex Genome Engineering of Industrial Yeast Strains

Compared with laboratory yeast strains, industrial yeast strains have the advantages of higher productivity and robustness under harsh industrial conditions. However, industrial yeast strains are polyploid and the presence of multiple copies of chromosomes makes their genome engineering rather challenging. The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for efficient and multiplex genome engineering in nearly all kingdoms of life by introducing a double strand break followed by DNA repair machinery. Although successful in the engineering of laboratory yeast strains, the application of CRISPR/Cas9 system in polyploid industrial yeast strain engineering is less explored. During CRISPR/Cas9 based genome editing process, the double strand break can be repaired by the provided DNA donor (edited yeast strains) or the other copies of the chromosome (wild-type yeast strains), leading to very low genome editing efficiency. We developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by introducing double strand breaks to all copies of the chromosome simultaneously, which can only be repaired using the provided DNA donors. Using plasmids with increased copy numbers for SgRNA expression, we could knock out four genes in a diploid industrial yeast strain (Ethanol Red, 8 alleles in total) and in a triploid industrial yeast strain (ATCC4124, 12 alleles in total) in a single step with 100% efficiency. The metabolic engineering application was demonstrated by the construction of efficient xylose-fermenting and lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway (XR-XDH-XKS) with URA3 auxotrophic marker and a lactate dehydrogenase gene (LDH) with LEU2 auxotrophic marker. The engineered CRISPR/Cas9 system can be used for the development of industrial yeast strains based microbial cell factories.