Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods.
Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods.
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使用体外和体内合成基因组 SCRaMbLE-in 方法进行快速途径原型设计和工程设计
DOI：
10.1038/s41467-018-04254-0
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发表时间：
2018-05-22
影响因子：
16.6
通讯作者：
Cai Y
中科院分区：
文献类型：
--
作者：
Liu W;Luo Z;Wang Y;Pham NT;Tuck L;Pérez-Pi I;Liu L;Shen Y;French C;Auer M;Marles-Wright J;Dai J;Cai Y
外源途径优化和底盘工程是外源途径表达的两种重要方法。这两种方法通常是逐步进行，并以试错的方式。在这里，我们报告了一种基于重组酶的组合方法（称为“SCRaMbLE-in”），以同时应对这两个挑战。SCRaMbLE-in包括一个体外重组酶工具包，用于在途径水平上快速原型化和多样化基因表达，以及一个体内基因组重排系统，用于将组装的途径整合到合成酵母基因组中，同时组合地在宿主底盘中引起大规模基因组重排。鉴定了一组loxP突变体对以使体外多样化的效率最大化。使用该SCRaMbLE-in方法成功组装、多样化和整合了β-胡萝卜素和紫霉素的示例性途径。对选定的工程菌株进行高通量测序，以揭示所得基因型与表型的关系。SCRaMbLE-in方法被证明是快速跟踪工程生物学周期的快速、有效和通用的方法。
Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. The two methods are normally carried out step-wise and in a trial-and-error manner. Here we report a recombinase-based combinatorial method (termed “SCRaMbLE-in”) to tackle both challenges simultaneously. SCRaMbLE-in includes an in vitro recombinase toolkit to rapidly prototype and diversify gene expression at the pathway level and an in vivo genome reshuffling system to integrate assembled pathways into the synthetic yeast genome while combinatorially causing massive genome rearrangements in the host chassis. A set of loxP mutant pairs was identified to maximize the efficiency of the in vitro diversification. Exemplar pathways of β-carotene and violacein were successfully assembled, diversified, and integrated using this SCRaMbLE-in method. High-throughput sequencing was performed on selected engineered strains to reveal the resulting genotype-to-phenotype relationships. The SCRaMbLE-in method proves to be a rapid, efficient, and universal method to fast track the cycle of engineering biology.
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影响因子：
4.6
作者：
Jouhten P;Boruta T;Andrejev S;Pereira F;Rocha I;Patil KR
通讯作者：
Patil KR
影响因子：
5.6
作者：
BERNARD, P;KEZDY, KE;COUTURIER, M
通讯作者：
COUTURIER, M
影响因子：
7
作者：
Ay F;Bunnik EM;Varoquaux N;Bol SM;Prudhomme J;Vert JP;Noble WS;Le Roch KG
通讯作者：
Le Roch KG
影响因子：
4.8
作者：
Ghosh, K;Van Duyne, GD
通讯作者：
Van Duyne, GD
影响因子：
64.8
作者：
Dymond, Jessica S.;Richardson, Sarah M.;Coombes, Candice E.;Babatz, Timothy;Muller, Heloise;Annaluru, Narayana;Blake, William J.;Schwerzmann, Joy W.;Dai, Junbiao;Lindstrom, Derek L.;Boeke, Annabel C.;Gottschling, Daniel E.;Chandrasegaran, Srinivasan;Bader, Joel S.;Boeke, Jef D.
通讯作者：
Boeke, Jef D.