Data for "RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides"

Dataset · Tue Apr 15 00:00:00 EDT 2025

DOI:https://doi.org/10.13012/B2IDB-3167257_V1· OSTI ID:3015308

Jin, Yong-Su ^[1]; Rao, Christopher ^[2]; Ye, Quanhui ^[3]; Oh, Hyunjoon ^[4]; Tohidifar, Payman ^[5]; Koh, Hyun Gi ^[6]

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1. Department of Food Science and Human Nutrition, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana−Champaign, Urbana, Illinois 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, Illinois 61801, United States; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
3. Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
4. Department of Food Science and Human Nutrition, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
5. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
6. Department of Biological and Chemical Engineering, Hongik University, Sejong 30016, Republic of Korea

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ (R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. This result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides, providing a streamlined method for addressing genetic engineering challenges in the yeast.

• 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: 3015308
• Country of Publication: United States
• Language: English

Related Subjects

gene editing
genome engineering