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Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis

热1 dechang 添加于 2012-11-5 19:16 | 4351 次阅读 | 0 个评论

作者
Hoefnagel MHN, Starrenburg MJC, Martens DE, Hugenholtz J, Kleerebezem M, Van Swam II, Bongers R, Westerhoff HV, Snoep JL
摘要
Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches \'bottle-necks\' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected \'rate-limiting\' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.
详细资料
- 关键词: Base Sequence; DNA Primers; Fermentation; Kinetics; L-Lactate Dehydrogenase/genetics/*metabolism; Lactococcus lactis/genetics/growth & development/*metabolism; Models, Biological; Molecular Sequence Data; Mutagenesis; Polymerase Chain Reaction; Templates, Genetic
- 文献种类:期刊
- 期刊名称: Microbiology (Reading, England)
- 期刊缩写: Microbiology
- 期卷页: 2002年第148卷第Pt 4期 1003-1013页
- 地址: Wageningen Centre for Food Sciences and Food and Bioprocess Engineering Group, Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands. marcel.hoefnagel@algemeen.pk.wau.nl
- ISBN: 1350-0872
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Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis

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乳酸菌代谢工程

每个曾经试图从根本上改变代谢通量的人都知道通常确定哪些酶必须修改比实际实现这些变化更困难。在更传统的遗传工程方法 \'瓶颈 \'在于使用定性,直观的方法,但是减轻敏感的 \'限速 \'步骤并不总是成功的。本文作者说明了基于酶动力学结合代谢控制分析的丙酮酸在Lactococcus lactis的分布模型清楚地说明关键控制点的通量在于乙偶姻和双乙酰,这是重要的风味化合物。本文介绍的模型(http://jjj.biochem.sun.ac.za/wcfs.html)表明,酶对该通量的最大效果在于乙酰乳酸合成酶分支本身。实验证实了模型的预测,即敲除乳酸脱氢酶和过度表达NADH氧化酶可以增加乙酰乳酸合成酶分支通量从0到75%。

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