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

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

doi:10.1021/acs.jafc.2c04251

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

Journal Article · Wed Sep 14 00:00:00 EDT 2022 · Journal of Agricultural and Food Chemistry

DOI:https://doi.org/10.1021/acs.jafc.2c04251· OSTI ID:1902741

Lee, Ye-Gi ^[1]; Kim, Chanwoo ^[2]; Kuanyshev, Nurzhan ^[2]; ^[3]; Fatma, Zia ^[2]; Wu, Zong-Yen ^[4]; Cheng, Ming-Hsun ^[2]; ^[2]; ^[4]; ^[2]; ^[2]

Univ. of Illinois at Urbana-Champaign, IL (United States); Kookmin Univ., Seoul (South Korea); University of Illinois
Univ. of Illinois at Urbana-Champaign, IL (United States)
Kyung Hee Univ., Gyeonggi-do (South Korea)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

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. Furthermore, 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.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Illinois at Urbana-Champaign, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; SC0018420

OSTI ID:: 1902741

Journal Information:: Journal of Agricultural and Food Chemistry, Journal Name: Journal of Agricultural and Food Chemistry Journal Issue: 38 Vol. 70; ISSN 0021-8561

Publisher:: American Chemical SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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