Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides

Nemailla Bonturi; Marina Julio Pinheiro; Paola Monteiro de Oliveira; Eka Rusadze; Tobias Eichinger; Gintare Liudžiūtė; Juliano Sabedotti De Biaggi; Age Brauer; Maido Remm; Everson Alves Miranda; Rodrigo Ledesma-Amaro; Petri-Jaan Lahtvee

Metabolic Engineering Communications (Dec 2022)

Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides

Nemailla Bonturi,
Marina Julio Pinheiro,
Paola Monteiro de Oliveira,
Eka Rusadze,
Tobias Eichinger,
Gintare Liudžiūtė,
Juliano Sabedotti De Biaggi,
Age Brauer,
Maido Remm,
Everson Alves Miranda,
Rodrigo Ledesma-Amaro,
Petri-Jaan Lahtvee

Affiliations

Nemailla Bonturi: Institute of Technology, University of Tartu, Tartu, Estonia; Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia
Marina Julio Pinheiro: Institute of Technology, University of Tartu, Tartu, Estonia; Department of Materials and Bioprocess Engineering, School of Chemical Engineering, University of Campinas, Campinas, Brazil
Paola Monteiro de Oliveira: Institute of Technology, University of Tartu, Tartu, Estonia
Eka Rusadze: Institute of Technology, University of Tartu, Tartu, Estonia
Tobias Eichinger: Institute of Technology, University of Tartu, Tartu, Estonia
Gintare Liudžiūtė: Institute of Technology, University of Tartu, Tartu, Estonia; Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia
Juliano Sabedotti De Biaggi: Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia; Department of Materials and Bioprocess Engineering, School of Chemical Engineering, University of Campinas, Campinas, Brazil
Age Brauer: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
Maido Remm: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
Everson Alves Miranda: Department of Materials and Bioprocess Engineering, School of Chemical Engineering, University of Campinas, Campinas, Brazil
Rodrigo Ledesma-Amaro: Imperial College Centre for Synthetic Biology, Imperial College London, London, UK; Department of Bioengineering, Imperial College London, London, UK
Petri-Jaan Lahtvee: Institute of Technology, University of Tartu, Tartu, Estonia; Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia; Corresponding author. Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia.

Journal volume & issue: Vol. 15
p. e00200

Abstract

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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.

Published in Metabolic Engineering Communications

ISSN: 2214-0301 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Technology: Chemical technology: Biotechnology; Science: Biology (General)
Website: http://www.journals.elsevier.com/metabolic-engineering-communications/

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