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 geranyl-geranyl 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 enhanc-ing 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.

Chen Dong;Ge Qu;Jinggong Guo;Fang Wei;Shuwen Gao;Zhoutong Sun;Lifeng Jin;Xuwu Sun;Jean-David Rochaix;Yuchen Miao;Ran Wang

Zhengzhou Tobacco Research Institute,Zhengzhou 450001,China;Key Laboratory of Plant Stress Biology,State Key Laboratory of Cotton Biology,School of Life Sciences,Henan University,Kaifeng 475001,China;College of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;School of Agricultural Sciences,Zhengzhou University,Zhengzhou 450001,China;Departments of Molecular Biology and Plant Biology,University of Geneva,Geneva 1211,Switzerland

科学通报（英文版）

[1]Chen Dong;Ge Qu;Jinggong Guo;Fang Wei;Shuwen Gao;Zhoutong Sun;Lifeng Jin;Xuwu Sun;Jean-David Rochaix;Yuchen Miao;Ran Wang-.Rational design of geranylgeranyl diphosphate synthase enhances carotenoid production and improves photosynthetic efficiency in Nicotiana tabacum)[J].科学通报（英文版）,2022(03):315-327

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