Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering
Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering
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DOI：
10.1093/nar/gky1182
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发表时间：
2019-02-20
影响因子：
14.9
通讯作者：
Heap, John T.
中科院分区：
文献类型：
--
作者：
Taylor, George M.;Mordaka, Pawel M.;Heap, John T.
DNA组装允许快速、可靠地组装单个DNA构建物或文库。大多数方法要么是模块化的，易于扩展的，适合组合组装，但会留下不需要的SCAR‘序列；要么是定制的(非模块化)，无疤痕，但不太适合组合文库的构建。两者在代谢工程方面都有局限性。为了克服这种权衡，我们设计了一种起止组装，这是一种多部分的模块化DNA组装方法，既没有功能上的疤痕，又适合组合组装。重要的是，与起始和终止密码子相对应的3个核苷酸悬垂被用来将编码序列组装成表达单位，从而避免在敏感的编码序列边界上留下疤痕。在这一概念的基础上，设计并实施了一个完整的DNA组装框架，允许从多达60个部分(或混合物)组装多达15个基因；单顺反子、操纵子或混合配置；以及新的精简组装层次结构，最大限度地减少载体数量。每个生物体只需要一个目的载体，这反映了我们对不同生物体中代谢工程系统的优化。通过在大肠杆菌中组合组装类胡萝卜素途径，证明了使用起止组装的代谢工程，从而产生了广泛的类胡萝卜素生产和菌落大小表型，表明了对设计空间的有意探索。
DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable scar' sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimizing the number of vectors. Only one destination vector is required per organism, reflecting our optimization of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in Escherichia coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.