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

金门 合成生物学 红酵母 生物 计算生物学 遗传学 计算机科学 工程类 酵母 土木工程 跨度(工程)
作者
Hyun Gi Koh,Payman Tohidifar,Hyunjoon Oh,Quanhui Ye,Suk‐Chae Jung,Christopher V. Rao,Yong‐Su Jin
出处
期刊:ACS Synthetic Biology [American Chemical Society]
标识
DOI:10.1021/acssynbio.4c00848
摘要

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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