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

For economic and sustainable biomanufacturing, the oleaginous yeast <i>Rhodotorula toruloides</i> has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of <i>R. toruloides</i> 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 (<i>R. toruloides</i> Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline <i>R. toruloides</i> 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 <i>Agrobacterium</i> 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 <i>R. toruloides</i> by coexpressing fatty acid elongases and desaturases. This result underscores the potential of the RT-EZ toolkit to advance synthetic biology in <i>R. toruloides</i>, providing a streamlined method for addressing genetic engineering challenges in the yeast.