The methylerythritol phosphate pathway is responsible for the biosynthesis of terpenoids, the largest class of secondary metabolites. Although the structures and functions of the proteins involved in this pathway have...The methylerythritol phosphate pathway is responsible for the biosynthesis of terpenoids, the largest class of secondary metabolites. Although the structures and functions of the proteins involved in this pathway have been well studied in Bacillus subtilis, only a few studies have reported the transcriptional profile of the genes involved. Therefore, we analyzed methylerythritol phosphate pathway genes in the genome of B. subtilis 916, which has been developed as a biological control agent against some rice diseases in China. Our results showed that methylerythritol phosphate pathway genes were distributed throughout the genome of this strain. These genes were transcribed during both the exponential and stationary phases. We further confirmed the transcription units of dxs, dxr, ispD, ispF, ipK, ispG, ispH, idi, and ispA in B. subtilis 916 through reverse transcription-PCR analyses; the results showed that these nine genes were located in seven different operons. The transcript start sites of the seven different operons were determined by 5′-rapid amplification of cDNA ends-PCR. Thus, our study provides a molecular basis at the transcriptional level for investigating homoterpene synthesis in the methylerythritol phosphate pathway of B. subtilis 916.展开更多
Biosynthesis of paclitaxel(Taxol™)is a hot topic with extensive and durable interests for decades.However,it is severely hindered due to the very low titers of intermediates.In this study,Escherichia coli was employed...Biosynthesis of paclitaxel(Taxol™)is a hot topic with extensive and durable interests for decades.However,it is severely hindered due to the very low titers of intermediates.In this study,Escherichia coli was employed to de novo synthesize a key intermediate of paclitaxel,taxadien-5α-yl-acetate(T5OAc).Plasmid-based pathway reconstruction and optimization were conducted for T5OAc production.The endogenous methylerythritol phosphate pathway was enhanced to increase the precursor supply.Three taxadien-5α-ol O-acetyltransferases were tested to obtain the best enzyme for the acetylation step.Metabolic burden was relieved to restore cell growth and promote production through optimizing the plasmid production system.In order to achieve metabolic balance,the biosynthesis pathway was regulated precisely by multivariate-modular metabolic engineering.Finally,in a 5-L bioreactor,the T5OAc titer was enhanced to reach 10.9 mg/L.This represents an approximately 272-fold increase in production compared to the original strain,marking the highest yield of T5OAc ever documented in E.coli,which is believed to be helpful for promoting the progress of paclitaxel biosynthesis.展开更多
1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-g...1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.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supported by the National Natural Science Foundation of China (31530095)
文摘The methylerythritol phosphate pathway is responsible for the biosynthesis of terpenoids, the largest class of secondary metabolites. Although the structures and functions of the proteins involved in this pathway have been well studied in Bacillus subtilis, only a few studies have reported the transcriptional profile of the genes involved. Therefore, we analyzed methylerythritol phosphate pathway genes in the genome of B. subtilis 916, which has been developed as a biological control agent against some rice diseases in China. Our results showed that methylerythritol phosphate pathway genes were distributed throughout the genome of this strain. These genes were transcribed during both the exponential and stationary phases. We further confirmed the transcription units of dxs, dxr, ispD, ispF, ipK, ispG, ispH, idi, and ispA in B. subtilis 916 through reverse transcription-PCR analyses; the results showed that these nine genes were located in seven different operons. The transcript start sites of the seven different operons were determined by 5′-rapid amplification of cDNA ends-PCR. Thus, our study provides a molecular basis at the transcriptional level for investigating homoterpene synthesis in the methylerythritol phosphate pathway of B. subtilis 916.
基金Funding for this research was graciously provided by several key sources:the National Key Research and Development Program of China under grant number 2019YFA0905000the Fundamental Research Funds for the Central Universities,grant number 222201714026the National Natural Science Foundation of China with grant numbers 21871085 and 31971380.
文摘Biosynthesis of paclitaxel(Taxol™)is a hot topic with extensive and durable interests for decades.However,it is severely hindered due to the very low titers of intermediates.In this study,Escherichia coli was employed to de novo synthesize a key intermediate of paclitaxel,taxadien-5α-yl-acetate(T5OAc).Plasmid-based pathway reconstruction and optimization were conducted for T5OAc production.The endogenous methylerythritol phosphate pathway was enhanced to increase the precursor supply.Three taxadien-5α-ol O-acetyltransferases were tested to obtain the best enzyme for the acetylation step.Metabolic burden was relieved to restore cell growth and promote production through optimizing the plasmid production system.In order to achieve metabolic balance,the biosynthesis pathway was regulated precisely by multivariate-modular metabolic engineering.Finally,in a 5-L bioreactor,the T5OAc titer was enhanced to reach 10.9 mg/L.This represents an approximately 272-fold increase in production compared to the original strain,marking the highest yield of T5OAc ever documented in E.coli,which is believed to be helpful for promoting the progress of paclitaxel biosynthesis.
文摘1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.
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。
