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Development of a multigene expression system using 2A peptides in Rhodosporidium toruloides
Xiao Guo1, Zhenzhen Bai1, Huimin Zhao2
1Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Engineering, Beijing, China.
Biotechnology and Bioengineering
|September 17, 2024
Summary
Scientists developed a new multigene expression system for Rhodosporidium toruloides using 2A peptides. This system efficiently expresses multiple genes simultaneously, enhancing the production of valuable compounds like alpha-linolenic acid.
Area of Science:
- Biotechnology
- Molecular Biology
- Synthetic Biology
Background:
- Eukaryotic gene expression typically requires individual promoters and terminators, complicating the simultaneous expression of multiple genes, particularly in nonmodel organisms like Rhodosporidium toruloides.
- Polycistronic expression offers a more streamlined approach for multigene expression, providing benefits such as reduced construct size and simplified cloning procedures.
Purpose of the Study:
- To develop and validate a novel polycistronic gene expression system for the nonmodel yeast Rhodosporidium toruloides.
- To identify highly efficient 2A peptides for mediating simultaneous protein expression in R. toruloides.
- To apply the developed system for metabolic pathway optimization and enhance the production of alpha-linolenic acid.
Main Methods:
- Screening of twenty-two different 2A peptides for their cleavage efficiencies in R. toruloides.
- Selection of the most efficient 2A peptide (ERBV-1) for constructing a polycistronic expression system.
- Engineering the alpha-linolenic acid biosynthetic pathway using the ERBV-1 mediated polycistronic expression system.
Main Results:
- Cleavage efficiencies of the evaluated 2A peptides varied significantly, ranging from 33.65% to 93.32%.
- The ERBV-1 2A peptide demonstrated the highest cleavage efficiency and was successfully used to achieve simultaneous expression of four proteins.
- Polycistronic expression mediated by the ERBV-1 peptide led to a 104.72% increase in alpha-linolenic acid production through pathway optimization.
Conclusions:
- The ERBV-1 peptide-mediated polycistronic expression system is an effective tool for multigene expression in Rhodosporidium toruloides.
- This system significantly enhances the production of target compounds, such as alpha-linolenic acid, by enabling efficient metabolic engineering.
- The developed system holds promise for advancing genetic engineering applications in underdeveloped nonmodel organisms.
