Background:Limonene is an important monoterpene used as a chemical commodity and precursor for producing biofuels,flavor and medicinal compounds.Results:In this paper,we engineered Escherichia coli by embedding two ex...Background:Limonene is an important monoterpene used as a chemical commodity and precursor for producing biofuels,flavor and medicinal compounds.Results:In this paper,we engineered Escherichia coli by embedding two exogenous genes encoding a limonene synthase(LS)and a geranyl diphosphate synthase(GPPS)for production of limonene.Out of 12 E.coli strains transformed with various plasmids,the best one with p15T7-ls-gpps produced limonene with a titer of 4.87 mg/L.In order to enhance the limonene production,two rate-limiting enzymes in the endogenous MEP pathway of E.coli,1-deoxy-xylulose-5-phosphate synthase(DXS)and isopentenyl diphosphate isomerase(IDI),were overexpressed consecutively on vector pET21a+,resulting in a production of 17.4 mg_(limonene)/L at 48 h.Conclusions:After the preliminary optimization of the medium in a two-phase culture system composed of n-hexadecane(1/50,V_(org)/V_(aq)),the final production of limonene was raised up to 35.8 mg/L,representing approximately a 7-fold improvement compared to the initial titer.展开更多
1-Deoxy-D-xylulose-5-phosphate reductoisomerase （DXR） is an important enzyme involved in the 2-C-methyi-D- erythritol-4-phosphate （MEP） pathway which provides the basic five-carbon units for isoprenoid biosynthesi...1-Deoxy-D-xylulose-5-phosphate reductoisomerase （DXR） is an important enzyme involved in the 2-C-methyi-D- erythritol-4-phosphate （MEP） pathway which provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the role of the MEP pathway in plant development and metabolism, we carried out detailed analyses on a dxr mutant （GK_215C01） and two DXR transgenic co-suppression fines, OX-DXR-L2 and OX-DXR-L7. We found that the dxr mutant was albino and dwarf. It never bolted, had significantly reduced number of trichomes and most of the stomata could not close normally in the leaves. The two co-suppression lines produced more yellow inflorescences and albino sepals with no trichomes. The transcription levels of genes involved in tricbome initiation were found to be strongly affected, including GLABRA1, TRANSPARENT TESTA GLABROUS 1, TRIPTYCHON and SPINDLY, expression of which is regulated by gibberellic acids （GAs）. Exogenous application of GA3 could partially rescue the dwarf phenotype and the trichome initiation of dxr, whereas exogenous application of abscisic acid （ABA） could rescue the stomata closure defect, suggesting that lower levels of both GA and ABA contribute to the phenotype in the dxr mutants. We further found that genes involved in the biosynthetic pathways of GA and ABA were coordinately regulated. These results indicate that disruption of the plastidial MEP pathway leads to biosynthetic deficiency of photosynthetic pigments, GAs and ABA, and thus the developmental abnormalities, and that the flux from the cytoplasmic mevalonate pathway is not sufficient to rescue the deficiency caused by the blockage of the plastidial MEP pathway. These results reveal a critical role for the MEP biosynthetic pathway in controlling the biosynthesis of isoprenoids.展开更多
Ulva prolifera is a green alga that plays an important role in green tides.Carotenoid biosynthesis is a basic terpenoid metabolism that is very important for maintaining normal life activities in algae.In this study,w...Ulva prolifera is a green alga that plays an important role in green tides.Carotenoid biosynthesis is a basic terpenoid metabolism that is very important for maintaining normal life activities in algae.In this study,we first reported the complete sequences of all genes in the 2-C-methyl-D-erythritol 4-phosphate(MEP)pathway,which is the only carotenoid synthesis pathway in U.prolifera.Then,we compared these genes with those of other species.Additionally,by detecting the carotenoid contents and expression levels of key genes participating in carotenoid biosynthesis in U.prolifera under three different light(1000 lx,5000 lx and 12000 lx)and salinity(12,24 and 40)regimes,we found that carotenoid synthesis could be influenced by light and salinity,such that low light and high salinity could promote the synthesis of carotenoids.The results showed that the expression levels of genes involved in the MEP and the downstream pathway could affect the biosynthesis of carotenoids at the molecular level.This study contributes to a better understanding of the roles of genes participating in carotenoid biosynthesis in U.prolifera and the environmental regulation of these genes.展开更多
Isoprenoids are among the largest and most chemically diverse classes of organic compounds in nature and are involved in the processes of photosynthesis, respiration, growth, development,and plant responses to stress....Isoprenoids are among the largest and most chemically diverse classes of organic compounds in nature and are involved in the processes of photosynthesis, respiration, growth, development,and plant responses to stress. The basic building block units for isoprenoid synthesis-isopentenyl diphosphate and its isomer dimethylallyl diphosphate-are generated by the mevalonate (MVA) and methylerythritol phosphate(MEP) pathways. Here, we summarize recent advances on the roles of the MEP and MVA pathways in plant growth, development and stress responses, and attempt to define the underlying gene networks that orchestrate the MEP and MVA pathways in response to developmental or environmental cues.Through phylogenomic analysis, we also provide a new perspective on the evolution of the plant isoprenoid pathway. We conclude that the presence of the MVA pathway in plants may be associated with the transition from aquatic to subaerial and terrestrial environments, as lineages for its core components are absent in green algae. The emergence of the MVA pathway has acted as a key evolutionary event in plants that facilitated land colonization and subsequent embryo development, as well as adaptation to new and varied environments.展开更多
Plant isoprenoids are formed from precursors synthesized by the mevalonate （MVA） pathway in the cytosol or by the methyl-D-erythritol 4-phosphate （MEP） pathway in plastids. Although some exchange of precursors occ...Plant isoprenoids are formed from precursors synthesized by the mevalonate （MVA） pathway in the cytosol or by the methyl-D-erythritol 4-phosphate （MEP） pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms （DXS1, DXS2）. In tomato （Solanum lycopersicum）, the SIDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SIDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SIDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SIDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ^13C to ^12C natural isotope ratios. A compensatory up-regulation of SIDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.展开更多
Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub（s） involved have remained an enigma. We have previously established t...Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub（s） involved have remained an enigma. We have previously established that methylerythritol cyclodiphosphate （MEcPP）, a precursor of plastidial isoprenoids and a stress- specific retrograde signaling metabolite, enables cellular readjustments for high-order adaptive functions. Here, we specifically show that MEcPP promotes two Brassicaceae-specific traits, namely endoplasmic reticulum （ER） body formation and induction of indole glucosinolate （IGs） metabolism selectively, via tran- scriptional regulation of key regulators NAIl for ER body formation and MYB51/122 for IGs biosynthesis）. The specificity of MEcPP is further confirmed by the lack of induction of wound-inducible ER body genes as well as IGs by other altered methylerythritol phosphate pathway enzymes. Genetic analyses revealed MEcPP-mediated COil-dependent induction of these traits. Moreover, MEcPP signaling integrates the biosynthesis and hydrolysis of IGs through induction of nitrile-specifier protein1 and reduction of the sup- pressor, ESM1, and production of simple nitriles as the bioactive end product. The findings position the plastidial metabolite, MEcPP, as the initiation hub, transducing signals to adjust the activity of hard- wired gene circuitry to expand phytochemical diversity and alter the associated subcellular structure required for functionality of the secondary metabolites, thereby tailoring plant stress responses.展开更多
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financially supported by the National Natural Science Foundation of China(No.21276082)Ministry of Science and Technology,P.R.China(Nos.2011CB710800)Shanghai Commission of Science and Technology(No.11431921600).
文摘Background:Limonene is an important monoterpene used as a chemical commodity and precursor for producing biofuels,flavor and medicinal compounds.Results:In this paper,we engineered Escherichia coli by embedding two exogenous genes encoding a limonene synthase(LS)and a geranyl diphosphate synthase(GPPS)for production of limonene.Out of 12 E.coli strains transformed with various plasmids,the best one with p15T7-ls-gpps produced limonene with a titer of 4.87 mg/L.In order to enhance the limonene production,two rate-limiting enzymes in the endogenous MEP pathway of E.coli,1-deoxy-xylulose-5-phosphate synthase(DXS)and isopentenyl diphosphate isomerase(IDI),were overexpressed consecutively on vector pET21a+,resulting in a production of 17.4 mg_(limonene)/L at 48 h.Conclusions:After the preliminary optimization of the medium in a two-phase culture system composed of n-hexadecane(1/50,V_(org)/V_(aq)),the final production of limonene was raised up to 35.8 mg/L,representing approximately a 7-fold improvement compared to the initial titer.
基金Acknowledgments This work was supported by the National Natural Science Foundation of China （NSFC Grant 90717003 to L-J Qu）.
文摘1-Deoxy-D-xylulose-5-phosphate reductoisomerase （DXR） is an important enzyme involved in the 2-C-methyi-D- erythritol-4-phosphate （MEP） pathway which provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the role of the MEP pathway in plant development and metabolism, we carried out detailed analyses on a dxr mutant （GK_215C01） and two DXR transgenic co-suppression fines, OX-DXR-L2 and OX-DXR-L7. We found that the dxr mutant was albino and dwarf. It never bolted, had significantly reduced number of trichomes and most of the stomata could not close normally in the leaves. The two co-suppression lines produced more yellow inflorescences and albino sepals with no trichomes. The transcription levels of genes involved in tricbome initiation were found to be strongly affected, including GLABRA1, TRANSPARENT TESTA GLABROUS 1, TRIPTYCHON and SPINDLY, expression of which is regulated by gibberellic acids （GAs）. Exogenous application of GA3 could partially rescue the dwarf phenotype and the trichome initiation of dxr, whereas exogenous application of abscisic acid （ABA） could rescue the stomata closure defect, suggesting that lower levels of both GA and ABA contribute to the phenotype in the dxr mutants. We further found that genes involved in the biosynthetic pathways of GA and ABA were coordinately regulated. These results indicate that disruption of the plastidial MEP pathway leads to biosynthetic deficiency of photosynthetic pigments, GAs and ABA, and thus the developmental abnormalities, and that the flux from the cytoplasmic mevalonate pathway is not sufficient to rescue the deficiency caused by the blockage of the plastidial MEP pathway. These results reveal a critical role for the MEP biosynthetic pathway in controlling the biosynthesis of isoprenoids.
基金Foundation item:The National Key R&D Program of China under contract No.2016YFC1402102the MNR Key Laboratory of Eco-Environmental Science and Technology,China under contract No.MEEST-2020-2+1 种基金the Jiangsu Planned Projects for Postdoctoral Research Fundsthe Priority Academic Program Development of Jiangsu Higher Education Institutions。
文摘Ulva prolifera is a green alga that plays an important role in green tides.Carotenoid biosynthesis is a basic terpenoid metabolism that is very important for maintaining normal life activities in algae.In this study,we first reported the complete sequences of all genes in the 2-C-methyl-D-erythritol 4-phosphate(MEP)pathway,which is the only carotenoid synthesis pathway in U.prolifera.Then,we compared these genes with those of other species.Additionally,by detecting the carotenoid contents and expression levels of key genes participating in carotenoid biosynthesis in U.prolifera under three different light(1000 lx,5000 lx and 12000 lx)and salinity(12,24 and 40)regimes,we found that carotenoid synthesis could be influenced by light and salinity,such that low light and high salinity could promote the synthesis of carotenoids.The results showed that the expression levels of genes involved in the MEP and the downstream pathway could affect the biosynthesis of carotenoids at the molecular level.This study contributes to a better understanding of the roles of genes participating in carotenoid biosynthesis in U.prolifera and the environmental regulation of these genes.
基金supported by National Natural Science Foundation of China (31971410)the Postdoctoral Fund of Yunnan Province (Y835981261 and Y732681261)Postdoctoral Science Foundation of China (X.P., 2018M633434)
文摘Isoprenoids are among the largest and most chemically diverse classes of organic compounds in nature and are involved in the processes of photosynthesis, respiration, growth, development,and plant responses to stress. The basic building block units for isoprenoid synthesis-isopentenyl diphosphate and its isomer dimethylallyl diphosphate-are generated by the mevalonate (MVA) and methylerythritol phosphate(MEP) pathways. Here, we summarize recent advances on the roles of the MEP and MVA pathways in plant growth, development and stress responses, and attempt to define the underlying gene networks that orchestrate the MEP and MVA pathways in response to developmental or environmental cues.Through phylogenomic analysis, we also provide a new perspective on the evolution of the plant isoprenoid pathway. We conclude that the presence of the MVA pathway in plants may be associated with the transition from aquatic to subaerial and terrestrial environments, as lineages for its core components are absent in green algae. The emergence of the MVA pathway has acted as a key evolutionary event in plants that facilitated land colonization and subsequent embryo development, as well as adaptation to new and varied environments.
文摘Plant isoprenoids are formed from precursors synthesized by the mevalonate （MVA） pathway in the cytosol or by the methyl-D-erythritol 4-phosphate （MEP） pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms （DXS1, DXS2）. In tomato （Solanum lycopersicum）, the SIDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SIDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SIDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SIDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ^13C to ^12C natural isotope ratios. A compensatory up-regulation of SIDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.
文摘Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub（s） involved have remained an enigma. We have previously established that methylerythritol cyclodiphosphate （MEcPP）, a precursor of plastidial isoprenoids and a stress- specific retrograde signaling metabolite, enables cellular readjustments for high-order adaptive functions. Here, we specifically show that MEcPP promotes two Brassicaceae-specific traits, namely endoplasmic reticulum （ER） body formation and induction of indole glucosinolate （IGs） metabolism selectively, via tran- scriptional regulation of key regulators NAIl for ER body formation and MYB51/122 for IGs biosynthesis）. The specificity of MEcPP is further confirmed by the lack of induction of wound-inducible ER body genes as well as IGs by other altered methylerythritol phosphate pathway enzymes. Genetic analyses revealed MEcPP-mediated COil-dependent induction of these traits. Moreover, MEcPP signaling integrates the biosynthesis and hydrolysis of IGs through induction of nitrile-specifier protein1 and reduction of the sup- pressor, ESM1, and production of simple nitriles as the bioactive end product. The findings position the plastidial metabolite, MEcPP, as the initiation hub, transducing signals to adjust the activity of hard- wired gene circuitry to expand phytochemical diversity and alter the associated subcellular structure required for functionality of the secondary metabolites, thereby tailoring plant stress responses.
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。
