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 d...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。展开更多
基金the Natural Science Foundation of Henan Province(182300410053)the China Postdoctoral Science Foundation(2020M672308)+3 种基金Henan Postdoctoral Science Foundation(227462)Science Project(902019AA0140)the National Key Research and Development Program of China(2019YFA0905100)the National Natural Science Foundation of China(U2004143)。
文摘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。
