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Published on: June 23, 2023
Development, optimization, and application of an episomal plasmid system for Rhodotorula toruloides
Longyuan Shi1, Hao Xu1, Huimin Zhao2
1Department of Chemical and Biomolecular Engineering, University of Illinois Urbana-Champaign, Urbana, IL, 61801, United States; DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois Urbana-Champaign, Urbana, IL, 61801, United States.
Researchers developed the first episomal plasmid system for Rhodotorula toruloides, enabling robust genetic engineering. This breakthrough facilitates the production of valuable bioproducts and advances synthetic biology applications in this oleaginous yeast.
Area of Science:
- Biotechnology
- Synthetic Biology
- Microbial Engineering
Background:
- Rhodotorula toruloides is a promising oleaginous yeast for producing acetyl-CoA derived bioproducts.
- Engineering R. toruloides has been hindered by the lack of a functional episomal plasmid system for genetic manipulation.
Purpose of the Study:
- To establish the first functional episomal plasmid system for Rhodotorula toruloides.
- To develop tools for enhanced genetic engineering and synthetic biology applications in R. toruloides.
Main Methods:
- Systematic screening of autonomously replicating sequences (ARSs) to identify functional elements.
- Development of a Cre-loxP-mediated in vivo re-circularization strategy to improve transformation efficiency.
- Demonstration of episomal expression of metabolic genes and pathways, and establishment of a CRISPR system.
Main Results:
- Identified and validated C63F4 as a stable ARS element for episomal plasmid construction (pC63F4).
- Achieved stable episomal maintenance, functional reporter gene expression, and efficient transformation.
- Successfully demonstrated episomal expression of pathways for triacetic acid lactone, fatty alcohols, and limonene production.
- Established a novel CRISPR system for seamless genome editing and marker recycling in R. toruloides.
Conclusions:
- The developed episomal plasmid system and CRISPR toolkit significantly advance genetic engineering capabilities in R. toruloides.
- This platform will accelerate metabolic engineering, synthetic biology research, and bioproduct development using R. toruloides.
- The findings provide foundational tools for unlocking the full potential of R. toruloides as a microbial cell factory.
