TY - JOUR T1 - Development of a dedicated Golden Gate Assembly platform (RtGGA) for <em>Rhodotorula toruloides</em> JF - bioRxiv DO - 10.1101/2022.03.02.482697 SP - 2022.03.02.482697 AU - Nemailla Bonturi AU - Marina Julio Pinheiro AU - Paola Monteiro de Oliveira AU - Eka Rusadze AU - Tobias Eichinger AU - Gintare Liudžiūtė AU - Juliano Sabedotti De Biaggi AU - Age Brauer AU - Maido Remm AU - Everson Alves Miranda AU - Rodrigo Ledesma-Amaro AU - Petri-Jaan Lahtvee Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/03/02/2022.03.02.482697.abstract N2 - Rhodotorula toruloides is a potential chassis to 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 was adapted to the first basidiomycete, an oleaginous yeast R. toruloides (RtGGA). R. toruloides CCT 0783 was sequenced, and used for the RtGGA 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 were built 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.Competing Interest StatementThe authors have declared no competing interest. ER -