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
{"title":"Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides","authors":"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","doi":"10.1016/j.mec.2022.e00200","DOIUrl":null,"url":null,"abstract":"<div><p><em>Rhodotorula toruloides</em> 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 <em>R. toruloides</em>. <em>R. toruloides</em> 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 <em>R. toruloides</em> 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 <em>R. toruloides</em>, enabling the efficient design of complex metabolic pathways.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00200"},"PeriodicalIF":3.7000,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000098/pdfft?md5=a2fe424d543e5912a0eacd9cd6daa4d6&pid=1-s2.0-S2214030122000098-main.pdf","citationCount":"8","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic Engineering Communications","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2214030122000098","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 8
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.
期刊介绍:
Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.