Bonturi Nemailla, Pinheiro Marina Julio, de Oliveira Paola Monteiro, Rusadze Eka, Eichinger Tobias, Liudžiūtė Gintare, De Biaggi Juliano Sabedotti, Brauer Age, Remm Maido, Miranda Everson Alves, Ledesma-Amaro Rodrigo, Lahtvee Petri-Jaan
Institute of Technology, University of Tartu, Tartu, Estonia.
Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia.
Metab Eng Commun. 2022 May 23;15:e00200. doi: 10.1016/j.mec.2022.e00200. eCollection 2022 Dec.
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 . 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 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 , enabling the efficient design of complex metabolic pathways.
作为一种微生物细胞工厂的潜在底盘，这种酵母能够将不同底物代谢为多种天然产物，但缺乏高效的合成生物学工具阻碍了其应用。在本研究中，模块化、通用且高效的金门DNA组装系统（RtGGA）被应用于第一种担子菌，一种产油酵母。对CCT 0783进行了测序，并用于GGA设计。DNA片段通过预先设计的4个核苷酸的突出端进行组装，并创建了一个标准化元件库，其中包含启动子、基因、终止子、插入区域和抗性基因。该文库被组合起来创建用于启动子强度表征和过表达类胡萝卜素生产途径的盒式结构。使用了多种试剂、质粒和策略，结果证明RtGGA是可靠的。通过使用不同的启动子组合，RtGGA被用于构建三种版本的类胡萝卜素过表达盒式结构。这些盒式结构被转化到[具体菌株未给出]中，并对这三种新菌株进行了表征。总类胡萝卜素浓度提高了41%。这个专门的GGA平台填补了[具体物种未给出]先进基因组工程工具包中的一个空白，能够高效设计复杂的代谢途径。
