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

Ye Gi Lee, Chanwoo Kim, Nurzhan Kuanyshev, Nam Kyu Kang, Zia Fatma, Zong Yen Wu, Ming Hsun Cheng, Vijay Singh, Yasuo Yoshikuni, Huimin Zhao, Yong Su Jin

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)12085-12094
Number of pages10
JournalJournal of Agricultural and Food Chemistry
Issue number38
StatePublished - Sep 28 2022


  • Cas9 genome-editing strategies
  • Issatchenkia orientalis
  • bioenergy sorghum
  • no pH adjustment
  • nonconventional yeast
  • xylose fermentation

ASJC Scopus subject areas

  • General Chemistry
  • General Agricultural and Biological Sciences


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