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Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Published on: February 5, 2021
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Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides.
Nemailla Bonturi1,2, Marina Julio Pinheiro1,3, Paola Monteiro de Oliveira1
1Institute of Technology, University of Tartu, Tartu, Estonia.
Metabolic Engineering Communications
|June 6, 2022
Summary
A new Golden Gate DNA assembly system (RtGGA) was developed for Rhodotorula toruloides, a yeast valuable for producing natural products. This tool enhances microbial cell factory applications by enabling efficient genetic engineering, leading to a 41% increase in carotenoid production.
Area of Science:
- Synthetic biology
- Metabolic engineering
- Yeast genetics
Background:
- Rhodotorula toruloides is a promising yeast for microbial cell factories due to its metabolic capabilities.
- Limited synthetic biology tools restrict the full potential of R. toruloides for producing diverse natural products.
- Efficient genome engineering is crucial for advancing R. toruloides as a viable microbial chassis.
Purpose of the Study:
- To adapt and validate the Golden Gate DNA assembly system (RtGGA) for the basidiomycete yeast R. toruloides.
- To create a standardized library of genetic parts for R. toruloides.
- To demonstrate the utility of RtGGA for pathway engineering, specifically for carotenoid overproduction.
Main Methods:
- Sequencing of R. toruloides CCT 0783 to inform RtGGA design.
- Assembly of DNA fragments using predesigned 4-nt overhangs.
- Creation of a standardized library of promoters, genes, terminators, and resistance genes.
- Construction and transformation of carotenoid overexpression cassettes using RtGGA.
- Characterization of engineered R. toruloides strains for carotenoid production.
Main Results:
- The modular and efficient RtGGA system was successfully adapted for R. toruloides.
- A versatile library of standardized genetic parts was established for R. toruloides.
- Three distinct carotenoid overexpression cassettes were constructed using RtGGA with varying promoter combinations.
- Engineered R. toruloides strains exhibited a significant 41% increase in total carotenoid concentration.
- The RtGGA system proved robust across various reagents, plasmids, and strategies.
Conclusions:
- The developed RtGGA platform significantly enhances the genome engineering toolkit for R. toruloides.
- This system facilitates the efficient design and construction of complex metabolic pathways in R. toruloides.
- RtGGA empowers R. toruloides as a more applicable microbial cell factory for natural product biosynthesis.
