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Transcriptional profile of gene clusters involved in the methylerythritol phosphate pathway in Bacillus subtilis 916
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作者 XIAO Ya-jing GAO Tan-tan +3 位作者 PENG Qi ZHANG Jie SUN Dong-mei SONG Fu-ping 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2019年第3期644-655,共12页
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
关键词 methylerythritol PHOSPHATE pathway TRANSCRIPTION BACILLUS SUBTILIS biological control
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巴西橡胶树胶乳和悬浮细胞中MVA和MEP代谢途径基因的表达分析 被引量:4
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作者 邓小敏 吴绍华 +1 位作者 戴雪梅 田维敏 《广西植物》 CAS CSCD 北大核心 2016年第4期449-455,396,共8页
MVA和MEP代谢途径是植物类异戊二烯代谢途径的两条重要次生代谢途径。该研究利用荧光定量PCR技术,分析了橡胶树胶乳和橡胶树花药愈伤组织来源的悬浮细胞中MVA代谢途径和MEP代谢途径中关键基因的表达水平,同时分析了茉莉酸的结构类似物... MVA和MEP代谢途径是植物类异戊二烯代谢途径的两条重要次生代谢途径。该研究利用荧光定量PCR技术,分析了橡胶树胶乳和橡胶树花药愈伤组织来源的悬浮细胞中MVA代谢途径和MEP代谢途径中关键基因的表达水平,同时分析了茉莉酸的结构类似物冠菌素(coronatine,COR)对悬浮细胞中Hb AACT3,Hb HMGR4,Hb HMGR5,Hb DXS2,Hb DXR和Hb SQS1基因表达的调节作用。结果表明:在MVA代谢途径中,基因Hb AACT1,Hb AACT2,Hb HMGS1,Hb HMGS2,Hb HMGR1,Hb HMGR3,Hb MVK,Hb PMK,Hb MVD1,Hb MVD2和IPP下游代谢基因Hb IPPI1和Hb FDPS1在胶乳中的表达量要相对高于其在悬浮细胞中的表达量,然而橡胶树悬浮细胞中MEP代谢途径基因Hb DXS1,Hb DXS2,Hb DXR,Hb CMS1,Hb CMS2,Hb CMK,Hb MCS1,Hb MCS2,Hb HDS,Hb HDR和鲨烯合酶基因Hb SQS1的表达水平要相对高于胶乳。而且COR能不同程度地上调Hb HMGR5,Hb HMGR4,Hb SQS1,Hb DXS2和Hb DXR基因的表达水平。该研究结果为探索利用橡胶树悬浮细胞体系研究次生代谢合成调控以及生产活性次生代谢产物奠定了基础。 展开更多
关键词 巴西橡胶树 甲羟戊酸 脱氧木酮糖-5-磷酸 冠菌素 鲨烯合酶基因
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Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase
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作者 Jordi Querol-Audí Albert Boronat +1 位作者 Josep J. Centelles Santiago Imperial 《Journal of Biosciences and Medicines》 2014年第4期30-35,共6页
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
关键词 Active Site 1-Deoxy-D-Xylulose 5-Phosphate SYNTHASE ISOPRENOID Biosynthesis Kinetic Parameters MEP Pathway methylerythritol Phosphate MUTAGENESIS
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掌叶大黄转录组测序及蒽醌类合成关键酶基因差异表达分析 被引量:2
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作者 邹建珍 胡晓晨 +3 位作者 李依民 郝紫彤 张岗 刘蒙蒙 《中草药》 CAS CSCD 北大核心 2023年第7期2217-2225,共9页
目的研究掌叶大黄Rheum palmatum蒽醌合成关键基因。方法利用RNA-seq技术对掌叶大黄根、根茎、叶进行转录组测序和生物信息学分析。结果经Trinity软件组装后获得140224条Unigenes,全部Unigenes能被非冗余蛋白库、核酸序列数据库、京都... 目的研究掌叶大黄Rheum palmatum蒽醌合成关键基因。方法利用RNA-seq技术对掌叶大黄根、根茎、叶进行转录组测序和生物信息学分析。结果经Trinity软件组装后获得140224条Unigenes,全部Unigenes能被非冗余蛋白库、核酸序列数据库、京都基因组百科全书数据库、蛋白质序列数据库、蛋白家族数据库、基因本体联合数据库、同源蛋白簇数据库等公共数据库注释。差异基因分析结果显示,掌叶大黄的根与叶相比,差异表达基因总数为4175个,根与根茎相比,差异表达基因总数为992个,叶与根茎相比,差异表达基因总数为4469个。差异表达基因代谢途径分析分析发现,苯丙烷生物合成通路在叶比根中被显著富集。蒽醌类化合物合成相关关键差异表达基因分析发现,关键差异表达基因主要涉及甲羟戊酸(mevalonic acid,MVA)途径、甲基赤藓糖醇(methylerythritol 4-phosphate,MEP)、莽草酸及聚酮途径的13个基因。结论为进一步挖掘大黄有效成分生物合成过程中的关键基因,为解析调控其有效成分生物合成途径提供研究基础。 展开更多
关键词 掌叶大黄 蒽醌 转录组 基因表达 甲羟戊酸途径 甲基赤藓糖醇途径
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Rational design of geranylgeranyl diphosphate synthase enhances carotenoid production and improves photosynthetic efficiency in Nicotiana tabacum 被引量:5
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作者 Chen Dong Ge Qu +8 位作者 Jinggong Guo Fang Wei Shuwen Gao Zhoutong Sun Lifeng Jin Xuwu Sun Jean-David Rochaix Yuchen Miao Ran Wang 《Science Bulletin》 SCIE EI CSCD 2022年第3期315-327,共13页
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
关键词 Rational design CAROTENOID methylerythritol phosphate pathway(MEP) Geranylgeranyl diphosphate synthase(GGPPS) Nicotiana tabacum
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