The oxidative pentose phosphate(OPP)pathway provides metabolic intermediates for the shikimate pathway and directs carbon flow to the biosynthesis of aromatic amino acids(AAAs),which serve as basic protein building bl...The oxidative pentose phosphate(OPP)pathway provides metabolic intermediates for the shikimate pathway and directs carbon flow to the biosynthesis of aromatic amino acids(AAAs),which serve as basic protein building blocks and precursors of numerous metabolites essential for plant growth.However,genetic evidence linking the two pathways is largely unclear.In this study,we identified 6-phosphogluconate dehydrogenase 2(PGD2),the rate-limiting enzyme of the cytosolic OPP pathway,through suppressor screening of arogenate dehydrogenase 2(adh2)in Arabidopsis.Our data indicated that a single amino acid substitution at position 63(glutamic acid to lysine)of PGD2 enhanced its enzyme activity by facilitating the dissociation of products from the active site of PGD2,thus increasing the accumulation of AAAs and partially restoring the defective phenotype of adh2.Phylogenetic analysis indicated that the point mutation occurred in a well-conserved amino acid residue.Plants with different amino acids at this conserved site of PGDs confer diverse catalytic activities,thus exhibiting distinct AAAs producing capability.These findings uncover the genetic link between the OPP pathway and AAAs biosynthesis through PGD2.The gain-of-function point mutation of PGD2 identified here could be considered as a potential engineering target to alter the metabolic flux for the production of AAAs and downstream compounds.展开更多
Programmed cell death(PCD)is essential for both plant development and stress responses including immunity.However,how plants control PCD is not well-understood.The shikimate pathway is one of the most important metabo...Programmed cell death(PCD)is essential for both plant development and stress responses including immunity.However,how plants control PCD is not well-understood.The shikimate pathway is one of the most important metabolic pathways in plants,but its relationship to PCD is unknown.Here,we show that the shikimate pathway promotes PCD in Arabidopsis.We identify a photoperiod-dependent lesion-mimic mutant named Lesion in short-day(lis),which forms spontaneous lesions in short-day conditions.Mapbased cloning and whole-genome resequencing reveal that LIS encodes MEE32,a bifunctional enzyme in the shikimate pathway.Metabolic analysis shows that the level of shikimate is dramatically increased in lis.Through genetic screenings,three suppressors of lis(slis)are identified and the causal genes are cloned.SLISes encode proteins upstream of MEE32 in the shikimate pathway.Furthermore,exogenous shikimate treatment causes PCD.Our study uncovers a link between the shikimate pathway and PCD,and suggests that the accumulation of shikimate is an alternative explanation for the action of glyphosate,the most successful herbicide.展开更多
(6S)-6-Fluoroshikimic acid 2 and (6 R ) 6 hydroxyshikimic acid 3 have been synthesized via an OsO 4 catalysed dihydroxylation of diene 7, which was derived from (-) shikimic acid 1.
The MYB transcription factor genes play important roles in many developmental processes and various defense responses of plants. The shikimate pathway is a major biosynthetic pathway for the production of three aromat...The MYB transcription factor genes play important roles in many developmental processes and various defense responses of plants. The shikimate pathway is a major biosynthetic pathway for the production of three aromatic amino acids and other aromatic compounds that are involved in multiple responses of plants, including protection against UV and defense. Herein, we describe the characterization of the R2R3-MYB gene AtMYB15as an activator of the shikimate pathway in Arabidopsis. The AtMYB15 protein is nuclear localized and a transcriptional activation domain is found in its C-terminal portion. Northern blots showed that AtMYB15 is an early wounding-inducible gene. Resutls of microarray analysis, confirmed using quantitative real-time polymerase chain reaction, showed that overexpression of AtMYB15 in transgenic plants resulted in elevated expression of almost all the genes involved in the shikimate pathway. Bioinformatics analysis showed that one or more AtMYB15-binding AC elements were detected in the promoters of these upregulated genes. Furthermore, these genes in the shikimate pathway were also found to be induced by wounding. These data suggest an important role of AtMYB15as a possible direct regulator of the Arabidopsis shikimate pathway in response to wounding.展开更多
Dried roots and rhizomes of rhubarb are widely used both as a vegetable and a medicinal product around the world.To date,most studies on the drying process of rhubarb have focused primarily on comparing different dryi...Dried roots and rhizomes of rhubarb are widely used both as a vegetable and a medicinal product around the world.To date,most studies on the drying process of rhubarb have focused primarily on comparing different drying techniques and assessing whether the content of functional compounds meets standard requirements.However,few studies have explored the dynamic changes and underlying mechanisms of these functional ingredients during drying process.In this study,HPLC-DAD-based dynamic monitoring of anthraquinones,combined with widely targeted metabolomics,was employed to investigate the impact of moisture loss on the quality of rhubarb and to elucidate potential molecular mechanisms during the drying process.The results indicated that the contents of total and free anthraquinones reached their highest levels when the moisture content was 40%,whereas other moisture levels generally led to a decrease.In contrast to anthraquinones,flavonoids,phenolic acids,and stilbenoids were identified as the most significantly changed functional components throughout the drying process.Further analysis revealed that the key metabolic pathways influencing these functional ingredients were glycolysis,TCA cycle,and pentose phosphate pathway,which are associated with respiratory metabolism.Phosphoenolpyruvate and D-erythrose-4-phosphate were the key precursors of shikimate pathway for the biosynthesis and interconversion between phenolic acids,flavonoids,stilbenoids and anthraquinones in rhubarb.This study provides a comprehensive understanding of how the drying process influences the functional components of rhubarb and offers valuable insights for determining the optimal moisture content in industrial drying processes.展开更多
β-Arbutin is a shikimate-derived phenolic glycoside and a valuable bio-based fine chemical traditionally extracted from plants(e.g.,bearberry).Plant extraction is limited by low yields,high costs,and heavy solvent us...β-Arbutin is a shikimate-derived phenolic glycoside and a valuable bio-based fine chemical traditionally extracted from plants(e.g.,bearberry).Plant extraction is limited by low yields,high costs,and heavy solvent use,motivating microbial production as a sustainable alternative.Most microbial efforts to date have used prokaryotic hosts;eukaryotic systems are comparatively underexplored yet offer distinct engineering opportu-nities and challenges.In this study,we established a biosynthetic pathway forβ-arbutin in Komagataella phaffii.By employing multiple metabolic strategies,including promoter engineering,optimizing the shikimic acid pathway flux,and enhancing precursor supply(PEP,E4P,and UDPG),we increased theβ-arbutin titer from 53.07 mg/L to 960.79 mg/L.Further optimization of glucose concentration in the shake flasks achieved a titer of 5.30 g/L in 144 h,representing a 90-fold increase.In batch replenishment fermentation,strain PC-AMU21 reached 70.87 g/L in a 3 L bioreactor with a productivity of 0.49 g/L/h,which is the highest reported titer for de novoβ-arbutin production from glucose in a eukaryotic microbial system.These results demonstrate the potential of K.phaffii as a eukaryotic chassis for producing shikimate-derived aromatic glycosides and provide generalizable metabolic design principles for improving titers of related compounds.展开更多
基金supported by the National Key Research and Development Program of China(2019YFA0903900)the National Natural Science Foundation of China(32300233)+1 种基金Guangdong Provincial Key Laboratory of Synthetic Genomics(2023B1212060054)Shenzhen Key Laboratory of Synthetic Genomics(ZDSYS201802061806209).
文摘The oxidative pentose phosphate(OPP)pathway provides metabolic intermediates for the shikimate pathway and directs carbon flow to the biosynthesis of aromatic amino acids(AAAs),which serve as basic protein building blocks and precursors of numerous metabolites essential for plant growth.However,genetic evidence linking the two pathways is largely unclear.In this study,we identified 6-phosphogluconate dehydrogenase 2(PGD2),the rate-limiting enzyme of the cytosolic OPP pathway,through suppressor screening of arogenate dehydrogenase 2(adh2)in Arabidopsis.Our data indicated that a single amino acid substitution at position 63(glutamic acid to lysine)of PGD2 enhanced its enzyme activity by facilitating the dissociation of products from the active site of PGD2,thus increasing the accumulation of AAAs and partially restoring the defective phenotype of adh2.Phylogenetic analysis indicated that the point mutation occurred in a well-conserved amino acid residue.Plants with different amino acids at this conserved site of PGDs confer diverse catalytic activities,thus exhibiting distinct AAAs producing capability.These findings uncover the genetic link between the OPP pathway and AAAs biosynthesis through PGD2.The gain-of-function point mutation of PGD2 identified here could be considered as a potential engineering target to alter the metabolic flux for the production of AAAs and downstream compounds.
基金supported by grants from the National Natural Science Foundation of China(31771355,31970311)Thousand Talents Plan of China-Young ProfessionalsHuazhong Agricultural University Scientific&Technological Self-innovation Foundation(2014RC004)。
文摘Programmed cell death(PCD)is essential for both plant development and stress responses including immunity.However,how plants control PCD is not well-understood.The shikimate pathway is one of the most important metabolic pathways in plants,but its relationship to PCD is unknown.Here,we show that the shikimate pathway promotes PCD in Arabidopsis.We identify a photoperiod-dependent lesion-mimic mutant named Lesion in short-day(lis),which forms spontaneous lesions in short-day conditions.Mapbased cloning and whole-genome resequencing reveal that LIS encodes MEE32,a bifunctional enzyme in the shikimate pathway.Metabolic analysis shows that the level of shikimate is dramatically increased in lis.Through genetic screenings,three suppressors of lis(slis)are identified and the causal genes are cloned.SLISes encode proteins upstream of MEE32 in the shikimate pathway.Furthermore,exogenous shikimate treatment causes PCD.Our study uncovers a link between the shikimate pathway and PCD,and suggests that the accumulation of shikimate is an alternative explanation for the action of glyphosate,the most successful herbicide.
文摘(6S)-6-Fluoroshikimic acid 2 and (6 R ) 6 hydroxyshikimic acid 3 have been synthesized via an OsO 4 catalysed dihydroxylation of diene 7, which was derived from (-) shikimic acid 1.
基金Supported by the National Priority Basic Research Programs of People＇s Republic of China： Biosafety Study on GM0s of Agricultural Importance （001CB10902 to L-JQ）, the National Natural Science Foundation of China （30470358）, and the Excellent Young Teachers Program of M0E, China （to L-JQ）. Acknowledgements The authors thank Ms Li Zhang and Professor Meihua Liu （Peking University） for technical assistance.
文摘The MYB transcription factor genes play important roles in many developmental processes and various defense responses of plants. The shikimate pathway is a major biosynthetic pathway for the production of three aromatic amino acids and other aromatic compounds that are involved in multiple responses of plants, including protection against UV and defense. Herein, we describe the characterization of the R2R3-MYB gene AtMYB15as an activator of the shikimate pathway in Arabidopsis. The AtMYB15 protein is nuclear localized and a transcriptional activation domain is found in its C-terminal portion. Northern blots showed that AtMYB15 is an early wounding-inducible gene. Resutls of microarray analysis, confirmed using quantitative real-time polymerase chain reaction, showed that overexpression of AtMYB15 in transgenic plants resulted in elevated expression of almost all the genes involved in the shikimate pathway. Bioinformatics analysis showed that one or more AtMYB15-binding AC elements were detected in the promoters of these upregulated genes. Furthermore, these genes in the shikimate pathway were also found to be induced by wounding. These data suggest an important role of AtMYB15as a possible direct regulator of the Arabidopsis shikimate pathway in response to wounding.
基金supported by National Natural Science Foundation of China(82204762)Youth Innovation Team Program of University in Shandong Province(2023KJ217)Natural science Foundation of Shandong Province(ZR2024MH248).
文摘Dried roots and rhizomes of rhubarb are widely used both as a vegetable and a medicinal product around the world.To date,most studies on the drying process of rhubarb have focused primarily on comparing different drying techniques and assessing whether the content of functional compounds meets standard requirements.However,few studies have explored the dynamic changes and underlying mechanisms of these functional ingredients during drying process.In this study,HPLC-DAD-based dynamic monitoring of anthraquinones,combined with widely targeted metabolomics,was employed to investigate the impact of moisture loss on the quality of rhubarb and to elucidate potential molecular mechanisms during the drying process.The results indicated that the contents of total and free anthraquinones reached their highest levels when the moisture content was 40%,whereas other moisture levels generally led to a decrease.In contrast to anthraquinones,flavonoids,phenolic acids,and stilbenoids were identified as the most significantly changed functional components throughout the drying process.Further analysis revealed that the key metabolic pathways influencing these functional ingredients were glycolysis,TCA cycle,and pentose phosphate pathway,which are associated with respiratory metabolism.Phosphoenolpyruvate and D-erythrose-4-phosphate were the key precursors of shikimate pathway for the biosynthesis and interconversion between phenolic acids,flavonoids,stilbenoids and anthraquinones in rhubarb.This study provides a comprehensive understanding of how the drying process influences the functional components of rhubarb and offers valuable insights for determining the optimal moisture content in industrial drying processes.
基金supported by the National Key Research and Development Program of China[2022YFD2200805].
文摘β-Arbutin is a shikimate-derived phenolic glycoside and a valuable bio-based fine chemical traditionally extracted from plants(e.g.,bearberry).Plant extraction is limited by low yields,high costs,and heavy solvent use,motivating microbial production as a sustainable alternative.Most microbial efforts to date have used prokaryotic hosts;eukaryotic systems are comparatively underexplored yet offer distinct engineering opportu-nities and challenges.In this study,we established a biosynthetic pathway forβ-arbutin in Komagataella phaffii.By employing multiple metabolic strategies,including promoter engineering,optimizing the shikimic acid pathway flux,and enhancing precursor supply(PEP,E4P,and UDPG),we increased theβ-arbutin titer from 53.07 mg/L to 960.79 mg/L.Further optimization of glucose concentration in the shake flasks achieved a titer of 5.30 g/L in 144 h,representing a 90-fold increase.In batch replenishment fermentation,strain PC-AMU21 reached 70.87 g/L in a 3 L bioreactor with a productivity of 0.49 g/L/h,which is the highest reported titer for de novoβ-arbutin production from glucose in a eukaryotic microbial system.These results demonstrate the potential of K.phaffii as a eukaryotic chassis for producing shikimate-derived aromatic glycosides and provide generalizable metabolic design principles for improving titers of related compounds.
