Hou Sha, Qin Qin, Dai Junbiao
Key Laboratory of Industrial Biocatalysis (Ministry of Education) and Center for Synthetic and Systems Biology, School of Life Sciences , Tsinghua University , Beijing 100084 , China.
Center for Synthetic Genomics, Institute of Synthetic Biology , Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences , Shenzhen 518055 , China.
ACS Synth Biol. 2018 Mar 16;7(3):782-788. doi: 10.1021/acssynbio.7b00391. Epub 2018 Feb 28.
Yeast can be used as a microbial cell factory to produce valuable chemicals. However, introducing an exogenous pathway into particular or different chromosomal locations for stable expression is still a daunting task. To address this issue, we designed a DNA cassette called a "wicket", which can be integrated into the yeast genome at designated loci to accept exogenous DNA upon excision by a nuclease. Using this system, we demonstrated that, in strains with "wickets", we could achieve near 100% efficiency for integration of the β-carotene pathway with no need for selective markers. Furthermore, it allowed independent and simultaneous integration of different genes in a pathway, resulting in a large variety of strains with variable copy numbers of each gene. This system could be a useful tool to modulate the integration of multiple copies of genes within a metabolic pathway and to optimize the yield of the target products.
酵母可作为微生物细胞工厂来生产有价值的化学品。然而，将外源途径引入特定或不同的染色体位置以实现稳定表达仍是一项艰巨的任务。为了解决这个问题，我们设计了一种名为“wicket”的DNA盒，它可以整合到酵母基因组的指定位点，以便在被核酸酶切除后接受外源DNA。使用该系统，我们证明，在带有“wicket”的菌株中，我们可以实现β-胡萝卜素途径整合的近100%效率，而无需选择标记。此外，它允许在一条途径中独立且同时整合不同的基因，从而产生具有每个基因可变拷贝数的多种菌株。该系统可能是一种有用的工具，可用于调节代谢途径内多个基因拷贝的整合，并优化目标产物的产量。
