(281d) Building a Comprehensive Set of Genetic Toolkit for Metabolic Engineering of Issatchenkia Orientalis

The nonconventional yeast Issatchenkia orientalis is a potential platform microorganism for production of organic acids thanks to its unusual ability to grow in highly acidic conditions. However, lack of efficient genetic tools, including stable episomal plasmid and precise genome editing tool, remains a major bottleneck in metabolic engineering of this organism. Here we isolated one functional 0.8 kb centromere-like (CEN-L) sequence through in silico centromere prediction and library-based screening from I. orientalis genome. The identified CEN-L sequence was only about 2.5% size of a previously reported centromere, and it greatly improved the stability of the episomal plasmid, resulting in enhanced biochemical production and CRISPR/Cas9-mediated genome editing efficiency. The optimized CRISPR/Cas9 system consisting of an improved Cas9 and single guide RNA (sgRNA) expressed by a fusion RPR1’-tRNA promoter could achieve efficiencies of 95%, 72% and 27% for single, double and triple gene disruptions. Additionally, we developed an in vivo assembly method in I. orientalis, which could achieve ~100% fidelity for assembly of an 8 kb-plasmid by 7 DNA fragments ranging from 0.7 kb to 1.7 kb. These genetic tools should be generally applicable for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals.