Cas9-Based Metabolic Engineering of <i>Issatchenkia orientalis</i> for Enhanced Utilization of Cellulosic Hydrolysates

Abstract

<i>Issatchenkia orientalis</i>, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for <i>I. orientalis</i> exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any <i>I. orientalis</i> strains and engineered <i>I. orientalis</i> strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from <i>Scheffersomyces stipitis</i> were integrated into an intended chromosomal locus in four <i>I. orientalis</i> strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. The resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host <i>I. orientalis</i> strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.