Chemical and Biological Engineering, Microbiology, Ames Laboratory, NSF Engineering Research Center for Biorenewable Chemicals, Genome Informatics Facility, Office of Biotechnology
Journal or Book Title
The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplex gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ∼100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.
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This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
International Metabolic Engineering Society. Published by Elsevier Inc.
Cao, Mingfeng; Fatma, Zia; Song, Xiaofei; Hsieh, Ping-Hung; Tran, Vinh G.; Lyon, William L.; Sayadi, Maryam; Shao, Zengyi; Yoshikuni, Yasuo; and Zhao, Huimin, "A genetic toolbox for metabolic engineering of Issatchenkia orientalis" (2020). Chemical and Biological Engineering Publications. 406.