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

Koh, H.G., Tohidifar, P., Oh, H., Ye, Q., Jung, S.C., Rao, C.V., Jin, Y.S. April 15, 2025. “RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides.” ACS Synthetic Biology. DOI: 10.1021/acssynbio.4c00848.

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.

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• mRNA expression levels
• Self cleavage 2A peptides
• Estimated litigation frequency