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Transcriptional profile of gene clusters involved in the methylerythritol phosphate pathway in Bacillus subtilis 916 认领 引用
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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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A metabolic optimization strategy based on modular genomic integration of the MVA pathway to enhance lycopene production in Escherichia coli 认领 引用
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作者 Wanpeng Xia Wenqian Li +1 位作者 Qiang Yao Jingyu Chen 《Food Innovation and Advances》 2026年第2期251-260,共10页
Lycopene,a bioactive tetraterpenoid antioxidant valued in food,pharmaceutical,and cosmetic sectors,remains constrained by low plant extractability and expensive chemical synthesis;microbial engineering now presents a ... Lycopene,a bioactive tetraterpenoid antioxidant valued in food,pharmaceutical,and cosmetic sectors,remains constrained by low plant extractability and expensive chemical synthesis;microbial engineering now presents a cost-effective alternative.In this study,key genes from the methylerythritol phosphate(MEP)pathway—dxs,idi,and ispDF—were integrated into the genome of the chassis strain Escherichia coli MG1655 using a CRISPR-Cpf1-based system,resulting in MEP pathway-overexpressed strains.Additionally,the downstream module(MBot)of the mevalonate(MVA)pathway was optimized by introducing T7 RNA polymerase,mvaE,and mvaS from different species,including Saccharomyces cerevisiae,Streptococcus pneumoniae,and Staphylococcus aureus.Integration of the MEP genes improved lycopene production by 2 mg/L compared with the initial strain.Notably,fermentation performance varied significantly depending on the source of the downstream MBot module.The optimal combination—erg12 from Saccharomyces cerevisiae,mvaK and mvd1 from Streptococcus pneumoniae,and idi from Escherichia coli—achieved a lycopene titer of 86 mg/L in shake-flask cultures,representing a 21-fold increase compared to the parental strain.Paradoxically,dxr deletion to eliminate endogenous MEP flux precipitated a 7-fold drop in lycopene titre,whereas MEP overexpression failed to enhance production—revealing that MEP–MVA pathway synergy,rather than simple precursor supply,governs efficient carotenoid biosynthesis. 展开更多
关键词 lycopene production mva pathway methylerythritol phosphate mep pathway dxsidiand integrated genome metabolic optimization modular genomic integration chemical synthesismicrobial engineering escherichia coli mg
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穿心莲内酯抑制刚地弓形虫增殖作用的研究 认领 引用 被引量：1
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作者 柳润春 邹伟浩 +2 位作者 郑书雨 吴蔚玲 彭鸿娟 《中国寄生虫学与寄生虫病杂志》 CSCD 北大核心 2025年第3期329-334,共6页
目的探讨穿心莲内酯(AG)对刚地弓形虫的抑制作用及作用靶点。方法在人包皮成纤维细胞(HFF)中加入0、5、10、20、40、80、120、160、320μmol/L AG和10μl细胞计数试剂盒‑8(CCK‑8)溶液,检测各组细胞的吸光度(A450值),绘制增殖曲线,筛... 目的探讨穿心莲内酯(AG)对刚地弓形虫的抑制作用及作用靶点。方法在人包皮成纤维细胞(HFF)中加入0、5、10、20、40、80、120、160、320μmol/L AG和10μl细胞计数试剂盒‑8(CCK‑8)溶液,检测各组细胞的吸光度(A450值),绘制增殖曲线,筛选对细胞无明显毒性的浓度用于后续研究。将弓形虫感染的HFF细胞分为二甲基亚砜(DMSO)组、AG组和乙胺嘧啶(PYR)组,分别加入兔源抗弓形虫表面抗原1抗体,绿色荧光标记的羊抗兔IgG抗体(1∶1000),红色荧光标记的羊抗兔IgG抗体(1∶1000),光学显微镜下观察弓形虫入侵情况,并计算入侵率。将HFF细胞分为DMSO组和AG组,加入绿色荧光标记的羊抗鼠IgG抗体(1∶1000),光学显微镜下观察弓形虫增殖情况。将HFF细胞分为DMSO组、AG组,分别加入100μl DMSO、40μmol/L AG,显微镜下观察HFF细胞形成的空斑面积。采用表面等离子体共振成像(SPRi)技术筛选互作蛋白,药物亲和响应靶标稳定性实验验证异戊烯基焦磷酸合成酶(IspG)蛋白与AG的互作,蛋白质免疫印迹(Western blotting)检测IspG蛋白变化情况。实时荧光定量PCR(qPCR)检测互作蛋白基因mRNA相对转录水平。使用GraphPad Prism 8.0.2软件进行统计学分析。结果增殖实验结果显示,在0、5、10、20、40μmol/L AG中HFF细胞相对活力分别为100.00%、107.45%、100.66%、109.21%和90.94%,细胞活力维持在较高水平,无明显毒性;在80、120、160、320μmol/L AG中相对活力分别为57.83%、34.16%、48.25%和30.75%,毒性明显(F=14.96,P<0.01)。选择40μmol/L浓度用于后续研究。间接免疫荧光结果显示,AG组和PYR组弓形虫入侵率分别为(8.06±2.40)%和(6.36±1.79)%,均低于DMSO组的(42.49±9.75)%(F=35.88,P<0.01)。DMSO组平均每个纳虫泡的弓形虫数量约为(5.78±0.94)个,高于AG组的(1.40±0.12)个(t=7.98,P<0.01)。空斑实验结果显示,AG组和DMSO组的空斑形成面积分别为0μm²和(3210±1840)μm²,差异有统计学意义(t=19.03,P<0.01)。SPRi鉴定AG与弓形虫的互作蛋白结果显示,蛋白质谱评分较高的弓形虫蛋白分子为核糖体RNA加工蛋白、ATP合酶α亚基、IspG蛋白和START结构域蛋白,分别为2.19、4.01、4.01和2.12分,分子间具有高度的相关性。药物亲和响应靶标稳定性实验结果显示,10μmol/L AG组的IspG蛋白相对表达水平为0.25±0.01,高于对照组的0.12±0.01(F=294.2,P<0.01)。qPCR结果显示,40μmol/L AG组IspG mRNA的相对转录水平为4.903±1.546,高于DMSO组的1.19±0.20(t=4.123,P<0.05)。Western blotting检测结果显示,IspG蛋白在0、10、20和40μmol/L AG组的相对表达水平分别为0.57±0.01、0.52±0.02、0.24±0.05和0.03±0.01,呈现明显的浓度依赖性下降趋势(F=313.4,P<0.01)。结论AG通过靶向IspG蛋白,抑制弓形虫的入侵和增殖,具有显著的抗弓形虫活性。 展开更多
关键词 刚地弓形虫 穿心莲内酯 异戊烯基焦磷酸合成酶 甲基赤藓醇磷酸途径
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Enhancing the biosynthesis of taxadien-5α- yl-acetate in Escherichia coli by combinatorial metabolic engineering approaches 认领 引用 被引量：1
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作者 Wen-Liang Xie Mei-Fang Zhang +3 位作者 Zheng-Yu Huang Man Xu Chun-Xiu Li Jian-He Xu 《Bioresources and Bioprocessing》 SCIE EI 2024年第1期675-687,共13页
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
关键词 Escherichia coli Methylerythritol phosphate pathway Taxadien-5α-yl-acetate Taxadien-5α-ol O-acetyltransferase Multivariate-modular metabolic engineering
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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 认领 引用 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7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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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 认领 引用 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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Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase 认领 引用
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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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Rational design of geranylgeranyl diphosphate synthase enhances carotenoid production and improves photosynthetic efficiency in Nicotiana tabacum 认领 引用 被引量：23
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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 CAS 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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