Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates

Abstract

Issatchenkia orientalis, 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. orientalis 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. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis 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. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.

Enhanced protein degradation by black soldier fly larvae (Hermetia illucens L.) and its gut microbes

“…were dominated by Issatchenkia orientalis ( Figure 3F ). I. orientalis is also known as Pichia kudriavzevii ( Kurtzman et al, 2008 ; Lee et al, 2022 ). I. orientalis is often genetically engineered to produce organic acids ( Cao et al, 2020 ).…”