Data from A Genetic Toolbox for Metabolic Engineering of Issatchenkia orientalis

Dataset · Fri Jan 31 00:00:00 EST 2020

DOI:https://doi.org/10.13012/B2IDB-8429399_V1· OSTI ID:3013818

Cao, Mingfeng ^[1]; Fatma, Zia ^[1]; Song, Xiaofei ^[2]; Hsieh, Ping-Hung ^[3]; Tran, Vinh G. ^[1]; Lyon, William L. ^[4]; Sayadi, Maryam ^[5]; Shao, Zengyi ^[6]; Yoshikuni, Yasuo ^[7]; Zhao, Huimin ^[8]

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1. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
2. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Department of Microbiology, Nankai University, Tianjin, China
3. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
4. Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States
5. Genome Informatics Facility, Office of Biotechnology, Iowa State University, Ames, IA, 50011, United States
6. Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, 50011, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
7. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States; U.S. Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States; Biological Systems Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
8. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States); Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States

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 multiplexed 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.

• Research Organization: Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States); University of Illinois Urbana-Champaign
• Sponsoring Organization: U.S. Department of Energy (DOE)
• DOE Contract Number: SC0018420
• OSTI ID: 3013818
• Country of Publication: United States
• Language: English

Related Subjects

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