Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate.
Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate.
复制标题
莽草酸靶向扰动L-色氨酸产生菌的代谢控制分析。
DOI：
10.1007/s00449-021-02630-7
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发表时间：
2021-12
影响因子：
3.8
通讯作者：
Weuster-Botz D
中科院分区：
文献类型：
--
作者：
Schoppel K;Trachtmann N;Mittermeier F;Sprenger GA;Weuster-Botz D
采用摄动实验和代谢控制分析方法，对大肠杆菌发酵甘油生产L色氨酸进行了研究。将非天然的莽草酸转运体插入到大肠杆菌L-色氨酸生产菌株的基因组中，使得在对L 15小时补料分批生产过程中的细胞进行并行短期扰动实验期间，能够使用莽草酸在产物途径内进行靶向扰动。谷氨酸棒杆菌的莽草酸/H+-转运蛋白基因(ShiA)的表达在估计误差范围内没有改变工艺性能。根据平行分析反应堆和生产过程的数据，进行了代谢分析和随后的广泛数据评估。由于莽草酸的干扰，胞外速率和胞内代谢物浓度在细胞代谢，特别是分支酸的生物合成方面表现出明显的偏离。使用基于热力学的通量分析方法估计细胞内的通量分布，该方法结合热力学约束和细胞内代谢物浓度来约束溶液空间。对于全基因组代谢模型，同时计算了可行的通量分布、吉布斯反应能和浓度范围，与代谢反应的方向相关的偏差最小。代谢控制分析被用来估计弹性和通量控制系数，预测L-色氨酸生物合成的控制位点。莽草酸的加入增加了分支酸生物合成的偏差，揭示了迄今未观察到的3-脱氢奎尼酸合酶对L-色氨酸形成的控制。用RT-qPCR法分析鉴定的目的基因的相对表达。转录组分析揭示了基因表达的差异和目标基因的定位，从而通过代谢工程进一步提高微生物L-色氨酸的产量。网上版载有补充材料，可在10.1007/s00449-021-02630-7查阅。
L-tryptophan production from glycerol with Escherichia coli was analysed by perturbation studies and metabolic control analysis. The insertion of a non-natural shikimate transporter into the genome of an Escherichia coli L-tryptophan production strain enabled targeted perturbation within the product pathway with shikimate during parallelised short-term perturbation experiments with cells withdrawn from a 15 L fed-batch production process. Expression of the shikimate/H+-symporter gene (shiA) from Corynebacterium glutamicum did not alter process performance within the estimation error. Metabolic analyses and subsequent extensive data evaluation were performed based on the data of the parallel analysis reactors and the production process. Extracellular rates and intracellular metabolite concentrations displayed evident deflections in cell metabolism and particularly in chorismate biosynthesis due to the perturbations with shikimate. Intracellular flux distributions were estimated using a thermodynamics-based flux analysis method, which integrates thermodynamic constraints and intracellular metabolite concentrations to restrain the solution space. Feasible flux distributions, Gibbs reaction energies and concentration ranges were computed simultaneously for the genome-wide metabolic model, with minimum bias in relation to the direction of metabolic reactions. Metabolic control analysis was applied to estimate elasticities and flux control coefficients, predicting controlling sites for L-tryptophan biosynthesis. The addition of shikimate led to enhanced deviations in chorismate biosynthesis, revealing a so far not observed control of 3-dehydroquinate synthase on L-tryptophan formation. The relative expression of the identified target genes was analysed with RT-qPCR. Transcriptome analysis revealed disparities in gene expression and the localisation of target genes to further improve the microbial L-tryptophan producer by metabolic engineering. The online version contains supplementary material available at 10.1007/s00449-021-02630-7.
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影响因子：
4.4
作者：
Jiang, Yu;Chen, Biao;Yang, Sheng
通讯作者：
Yang, Sheng
DOI：
10.1016/s1389-0344(02)00003-5
发表时间：
2002-06-01
期刊：
BIOMOLECULAR ENGINEERING
影响因子：
--
作者：
Buchholz, A;Hurlebaus, J;Takors, R
通讯作者：
Takors, R
DOI：
10.1007/s10295-018-2020-x
发表时间：
2018-05-01
影响因子：
3.4
作者：
Chen, Yuanye;Liu, Yongfei;Zhang, Dawei
通讯作者：
Zhang, Dawei
影响因子：
3.8
作者：
Cintolesi, Angela;Clomburg, James M.;Gonzalez, Ramon
通讯作者：
Gonzalez, Ramon
影响因子：
3.8
作者：
Aboka, Fredrick O.;Yang, Huiling;Heijnen, Joseph J.
通讯作者：
Heijnen, Joseph J.