YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae.
YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae.
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DOI：
10.1093/nar/gkv464
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发表时间：
2015-07-27
影响因子：
14.9
通讯作者：
Dai J
中科院分区：
文献类型：
--
作者：
Guo Y;Dong J;Zhou T;Auxillos J;Li T;Zhang W;Wang L;Shen Y;Luo Y;Zheng Y;Lin J;Chen GQ;Wu Q;Cai Y;Dai J
将多组分途径引入异源宿主以产生代谢物是代谢工程中的一项常规任务。然而，由于缺乏标准化的遗传工具，这个过程有时可能需要数周到数月的时间。在这里，我们提出了一种基于酿酒酵母的天然基因和调控元件设计和构建生物部件的方法。我们开发了高效的方案（称为 YeastFab Assembly），将这些遗传元件合成为标准化的生物部件，可用于在单管反应中组装转录单元。此外，还使用报告构建体开发了标准化表征分析来校准启动子的功能。此外，组装的转录单元可以单独进行测定，也可以用于构建多基因代谢途径，通过简单的体外反应有效地靶向基因组位点或接收质粒。最后，以β-胡萝卜素生物合成途径为例，我们证明我们的方法不仅使我们能够在几天内构建和测试代谢途径，而且还可以通过使用数百个调节生物部件组合组装途径来优化生产。
It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts.