Optimization of taxadiene synthesis in tobacco via fine-tuning of plant promoter strength

Aims: Quantitative and appropriate introduction of complex synthetic pathways is pivotal for heterologous synthesis of natural products in a plant-based chassis. Transient transformation enables multi-gene co-expression but it often suffers from inadequate regulation of the introduced pathways, limiting the synthesis efficiency of target products. Promoters are core regulatory elements mediating gene expression, yet the effect of orthogonal combinations of different-strength promoters on product synthesis efficiency is rarely studied. This study aims to reconstruct taxadiene pathway in Nicotiana benthamiana and optimize its yield via orthogonal promoter combinations. Methods: Taxus-derived DXS, GGPPS and taxadiene synthase were introduced into N. benthamiana to reconstruct the pathway. Golden Gate cloning combined with yeast homologous recombination was used to rapidly construct orthogonal vectors carrying different-strength promoters for pathway optimization. Results: The facile and rapid construction of orthogonal vectors with promoters of distinct strengths enabled fine-tuning of the taxadiene biosynthetic pathway in N. benthamiana. The orthogonal vectors precisely modulated the coordinated expression of heterologous Taxus DXS, GGPPS and TS genes, achieving a maximum taxadiene yield of (36.0±6.6) μg·g-1 (FW) in N. benthamiana leaves. Conclusion: These results lay a foundation for paclitaxel heterologous synthesis in N. benthamiana and provide insights for metabolic engineering optimization of heterologous pathways via orthogonal promoter regulation.