摘要
The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolitesor therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitativecharacterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used N.benthamiana leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expressionof firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporterprotein and mRNA levels. In addition, we investigated the effects of terminator–promoter combinations on gene expressionand found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest andweakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterizedpromoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expressionstrengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0–362.4 μg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequencesin N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.
基金
This work was supported by the National Key Research and Development Program of China(2018YFA0900600)
the Strategic Priority Research Program“Molecular Mech-anisms of Plant Growth and Development”of the Chinese Academy of Sciences(CAS)(XDB27020202)
the National Natural Science Foundation of China(22077129,32070328,and 41876084)
the Natural Science Foundation of Shanghai Municipal Science and Technology Committee(21ZR1470900)
the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-002-15),and the Program of Shanghai Academic Research Leader(20XD1404400)
This work was also financially supported by the Construction of the Registry and Data-base of Bioparts for Synthetic Biology of the CAS(ZSYS-016)
the International Partnership Program of the CAS(153D31KYSB20170121)
the National Key Laboratory of Plant Molecular Genetics,Shanghai Institute of Plant Physiology and Ecology,CAS.
