A genetic toolbox for metabolic engineering of Issatchenkia orientalis
- Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States); University of Illinois at Urbana-Champaign, IL (United States)
- Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States); University of Illinois at Urbana-Champaign, IL (United States); Nankai University, Tianjin (China)
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Iowa State University, Ames, IA (United States)
The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. Yet, 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 evidence of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.
- Research Organization:
- Univ. of Illinois at Urbana-Champaign, IL (United States); University of California, Berkeley, CA (United States); Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); China Scholarship Council; National Science Foundation (NSF)
- Grant/Contract Number:
- SC0018420; AC02-05CH1123; MCB-1716837
- OSTI ID:
- 1599331
- Alternate ID(s):
- OSTI ID: 1599237; OSTI ID: 1991861
- Journal Information:
- Metabolic Engineering, Vol. 59, Issue C; ISSN 1096-7176
- Publisher:
- ElsevierCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
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