Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides.
Author(s)
Bonturi, Nemailla
Pinheiro, Marina Julio
de Oliveira, Paola Monteiro
Rusadze, Eka
Eichinger, Tobias
Type
Journal Article
Abstract
Rhodotorula toruloides is a potential chassis for microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast R. toruloides. R. toruloides CCT 0783 was sequenced, and used for the GGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways.
Date Issued
2022-12
Date Acceptance
2022-05-15
Citation
Metabolic Engineering Communications, 2022, 15, pp.1-10
ISSN
2214-0301
Publisher
Elsevier
Start Page
1
End Page
10
Journal / Book Title
Metabolic Engineering Communications
Volume
15
Copyright Statement
© 2022 Published by Elsevier B.V. on behalf of International Metabolic Engineering Society. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/35662893
PII: S2214-0301(22)00009-8
Subjects
Golden gate assembly
Metabolic engineering
Non-conventional yeast
Oleaginous yeast
Rhodotorula toruloides
Synthetic biology
Publication Status
Published
Coverage Spatial
Netherlands
Date Publish Online
2022-05-23