Metabolic flux analysis using isotope distribution together with gene and protein expression analyses

使用同位素分布以及基因和蛋白质表达分析进行代谢通量分析

• 批准号: 14350438
• 负责人: SHIMIZU Kazuyuki
• 金额: $ 7.49万
• 依托单位: Kyushu Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14350438/
• 关键词: Metbolic engineering; Protein expression; gene expression; Isotope distribution; NMR; GC-MS; Metabolic flux distribution; Metabolic regulation; 代謝解析; 微生物

In order to develop a quantitative model for the main metabolic pathway of Escherichia coli, we cultivated E.coliK12 and W3110, as well as E.coli BW25113 developed in Keio University, and its several single-gene-knockout mutants (zwf^-, pykF^-, gnd^-, ppc^-, pck^-, pgi^- etc.) in batch and continuous modes. We also cultivated several gene knockout mutants (pflA, B, C, D-, pta-, ppc-, adhA- etc.) in microaerobic condition for lactate production. Then we made the metabolic flux analysis based on ^<13>C labeling experiments, measured enzyme activities and the intracellular metabolite concentrations as well as gene expressions using RT-PCR to understand the metabolic regulation quantitatively for the modeling.

为了建立大肠杆菌主要代谢途径的定量模型，我们培养了E.coliK12和W3110，以及庆应大学培育的E.coliBW25113及其几个单基因敲除突变体(zwf^-、pykF^-、GND^-、ppc^-、pck^-、pgi^-等)。以批处理和连续模式。我们还培育了几个基因敲除突变体(pflA、B、C、D-、PTA-、PPC-、Adha-等)。在微氧条件下生产乳酸盐。在此基础上进行代谢通量分析，利用RT-PCR技术检测酶活性、细胞内代谢物浓度和基因表达，定量了解代谢调控规律，为建模提供依据。

项目成果

Metabolic flux analysis of S, cerevisiae grown on glucose, glycerol or acetate by ^<13>C-labeling experiments

通过^ 13 C标记实验对在葡萄糖、甘油或乙酸盐上生长的酿酒酵母进行代谢通量分析

• 发表时间: 2003
• 期刊: Biochemical Engineering Journal 16
• 作者: Zhang,H.;K.Shimizu;S.Yao
• 通讯作者: S.Yao

M.Kabir, K.Shimizu: "Metabolic regulation analysis of icd-gene knockout E.coli based on 2DE with TOF-MASS and enzyme activity"Applied Microbiology and Biotechnology. (in press). (2004)

M.Kabir、K.Shimizu：“基于 2DE、TOF-MASS 和酶活性的 icd 基因敲除大肠杆菌的代谢调控分析”应用微生物学和生物技术。

H.Zhang, K.Shimizu, S.Yano: "Metabolic flux analysis of S.cerevisiae grown ox glucose, glycerol, or acetate by ^<13>C-labeling experiments"Biochemical Engineering Journal. (in press). 1-9 (2003)

H.Zhang、K.Shimizu、S.Yano：“通过^ 13 C标记实验对酿酒酵母生长的葡萄糖、甘油或乙酸盐进行代谢通量分析”《生物化学工程杂志》。

An improved method for statistical analysis of metabolic flux analysis using isotopomer mapping matrices with analytical expressions

• DOI: 10.1016/s0168-1656(03)00169-x
• 发表时间: 2003-10-09
• 期刊: JOURNAL OF BIOTECHNOLOGY
• 影响因子: 4.1
• 作者: Araúzo-Bravo, MJ;Shimizu, K
• 通讯作者: Shimizu, K

Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements

• DOI: 10.1007/s00253-004-1627-1
• 发表时间: 2004-07-01
• 期刊: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
• 影响因子: 5
• 作者: Kabir, MM;Shimizu, K
• 通讯作者: Shimizu, K