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
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引用次数: 8
摘要
toruloides红酵母是微生物细胞工厂的潜在基础,因为这种酵母可以将不同的底物代谢成多种天然产物,但缺乏有效的合成生物学工具阻碍了其适用性。本研究将模块化、多用途和高效的金门DNA组装系统(Golden Gate DNA assembly system, RtGGA)应用于第一担子菌——产油酵母toruloides。对toruloides CCT 0783进行测序,并用于GGA设计。将DNA片段与预先设计的4-nt悬垂进行组装,并创建一个包含启动子、基因、终止子、插入区和抗性基因的标准化部分文库。该文库被组合成盒式磁带,用于表征启动子强度和过表达类胡萝卜素的产生途径。使用了各种试剂、质粒和策略,RtGGA被证明是健壮的。RtGGA通过使用不同的启动子组合构建了三个版本的类胡萝卜素过表达盒。将这些盒式菌转化为toruloides,并对3个新菌株进行了鉴定。总类胡萝卜素浓度增加41%。专用的GGA平台填补了toruloides先进基因组工程工具包的空白,使复杂代谢途径的有效设计成为可能。
Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides
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.