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

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Oleaginous yeast Rhodotorula toruloides is a key platform for sustainable biomanufacturing of biofuels and chemicals.
• Genetic manipulation of R. toruloides is challenging due to high GC content and lack of plasmids, requiring genome integration.

Purpose of the Study:

• To develop a streamlined and efficient genetic engineering toolkit for Rhodotorula toruloides.
• To overcome limitations in R. toruloides genetic manipulation for enhanced biomanufacturing applications.

Main Methods:

• Developed the RT-EZ (Rhodotorula toruloides Efficient Zipper) toolkit utilizing Golden Gate assembly.
• Incorporated features like bidirectional promoters, 2A peptides, RFP screening, and optimized sequences for Agrobacterium tumefaciens-mediated transformation (ATMT).
• Demonstrated utility by constructing a four-gene expression cassette and synthesizing arachidonic acid.

Main Results:

• The RT-EZ toolkit significantly improves vector construction speed and efficiency in R. toruloides.
• Successfully engineered R. toruloides for arachidonic acid synthesis by coexpressing key enzymes.
• The toolkit enables straightforward selection and transformation, overcoming previous genetic engineering hurdles.

Conclusions:

• The RT-EZ toolkit provides a versatile and efficient solution for genetic engineering in R. toruloides.
• This advancement facilitates synthetic biology applications and sustainable biomanufacturing using R. toruloides.
• The toolkit accelerates the development of R. toruloides as a cell factory for valuable compounds.