Restricted genetic diversity can supply only a limited number of elite genes for modern plant cultivation and transgenesis. In this study, we demonstrate that rational design enables the engineering of geranylgeranyl diphosphate synthase (NtGGPPS), an enzyme of the methylerythritol phosphate pathway (MEP) in the model plant Nicotiana tabacum. As the crucial bottleneck in carotenoid biosynthesis, NtGGPPS1 interacts with phytoene synthase (NtPSY1) to channel GGPP into the production of carotenoids. Loss of this enzyme in the ntggpps1 mutant leads to decreased carotenoid accumulation. With the aim of enhancing NtGGPPS1 activity, we undertook structure-guided rational redesign of its substrate binding pocket in combination with sequence alignment. The activity of the designed NtGGPPS1 (a pentuple mutant of five sites V154A/I161L/F218Y/I209S/V233E, d-NtGGPPS1) was measured by a high-throughput colorimetric assay. d-NtGGPPS1 exhibited significantly higher conversion of IPP and each co-substrate (DMAPP ~1995.5-fold, GPP ~25.9-fold, and FPP ~16.7-fold) for GGPP synthesis compared with wild-type NtGGPPS1. Importantly, the transient and stable expression of d-NtGGPPS1 in the ntggpps1 mutant increased carotenoid levels in leaves, improved photosynthetic efficiency, and increased biomass relative to NtGGPPS1. These findings provide a firm basis for the engineering of GGPPS and will facilitate the development of quality and yield traits. Our results open the door for the structure-guided rational design of elite genes in higher plants.

有限的遗传多样性只能为现代植物栽培和转基因提供有限数量的优良基因。在这项研究中，我们证明合理的设计使得香叶基香叶基二磷酸合酶（NtGGPPS）的工程化成为可能，NtGGPPS是模型植物烟草中甲基赤藓糖醇磷酸途径（MEP）的一种酶。作为类胡萝卜素生物合成的关键瓶颈，NtGGPPS1 与八氢番茄红素合酶 (NtPSY1) 相互作用，引导 GGPP 参与类胡萝卜素的生产。 ntggpps1突变体中这种酶的缺失会导致类胡萝卜素积累减少。为了增强NtGGPPS1活性，我们结合序列比对对其底物结合口袋进行了结构引导的合理重新设计。通过高通量比色测定法测量了设计的 NtGGPPS1（五个位点 V154A/I161L/F218Y/I209S/V233E，d-NtGGPPS1 的五重突变体）的活性。与野生型 NtGGPPS1 相比，d-NtGGPPS1 在 GGPP 合成中表现出显着更高的 IPP 和每种共底物转化率（DMAPP 约 1995.5 倍，GPP 约 25.9 倍，FPP 约 16.7 倍）。重要的是，相对于 NtGGPPS1，d-NtGGPPS1 在ntggpps1突变体中的瞬时和稳定表达增加了叶片中的类胡萝卜素水平，提高了光合效率并增加了生物量。这些发现为 GGPPS 的工程设计提供了坚实的基础，并将促进品质和产量性状的开发。我们的结果为高等植物中精英基因的结构引导合理设计打开了大门。