Tian Chenfei, Zhang Yixin, Li Jianhua, Wang Yong
CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
University of Chinese Academy of Sciences, Beijing 100039, China.
Biodes Res. 2022 May 26;2022:9834989. doi: 10.34133/2022/9834989. eCollection 2022.
The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expression of firefly/ luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporter protein and mRNA levels. In addition, we investigated the effects of terminator-promoter combinations on gene expression and found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest and weakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterized promoters to engineer the betalain pathway in . Seven selected plant promoters with different expression strengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0-362.4 g/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequences in and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.
新兴的植物合成代谢工程在生产增值代谢产物或治疗性蛋白质方面展现出了巨大的潜力。然而，用于植物途径工程的启动子通常是凭经验选择的。对基于植物的启动子进行定量表征对于在植物底盘中优化基因表达至关重要。在这里，我们使用叶片和BY2悬浮细胞通过萤火虫荧光素酶/荧光素酶的瞬时表达对植物启动子文库进行定量表征。我们通过检查报告蛋白与mRNA水平之间的相关性验证了双荧光素酶报告系统。此外，我们研究了终止子 - 启动子组合对基因表达的影响，发现启动子和终止子的组合导致报告基因表达中最强和最弱表现之间相差326倍。作为概念验证，我们使用定量表征的启动子对甜菜红素途径进行工程改造。七个具有不同表达强度的选定植物启动子被正交使用以表达CYP76AD1和DODA，最终甜菜红素的产量范围为6.0 - 362.4克/克鲜重。我们的系统方法不仅展示了多种启动子序列在叶片和BY2细胞中的不同强度，还为植物工程增加了植物启动子的工具库。
