RT-EZ: A golden gate assembly toolkit for streamlined genetic engineering of rhodotorula toruloides

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels and other valuable chemicals. However, its genetic manipulation has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration.

To address these challenges, researchers at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a Department of Energy-funded Bioenergy Research Center, developed the RT-EZ toolkit (R. toruloides Efficient Zipper). This versatile tool, based on Golden Gate assembly, is 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. A series of vectors were tested in R. toruloides to validate the Golden Gate assembly, promoter functionality, and fluorescent protein expression. The multigene expression was validated using 2A peptides.

Validation experiments demonstrated the toolkit’s functionality through successful fluorescent protein expression using monodirectional and bidirectional promoters, as well as effective multigene expression using 2A peptides. Notably, it can construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The toolkit’s capability was further showcased by the biosynthesis of arachidonic acid, achieved through the efficient coexpression of four pathway enzymes.

This toolkit provides a streamlined method for addressing genetic engineering challenges in R. toruloides, reducing the time and effort required for complex metabolic engineering projects and unlocking the full biotechnological potential of this oleaginous yeast.