Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli.
Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli.
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DOI：
10.1007/s00253-010-2749-2
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发表时间：
2010-09
影响因子：
5
通讯作者：
Stephanopoulos, Gregory
中科院分区：
文献类型：
--
作者：
Fischer, Curt R.;Tseng, Hsien-Chung;Tai, Mitchell;Prather, Kristala L. J.;Stephanopoulos, Gregory
据报道，在梭状芽胞杆菌中，碳水化合物生产正丁醇的产率高达理论最大值的 76%，滴度高达 13 g/L。然而，在大肠杆菌中，一些研究小组报道了通过梭菌基因的重组表达产生丁酸或丁醇，但滴度和产量要低得多。为了查明重组途径中的缺陷步骤，我们开发了一种分析程序来测定关键途径中间体的细胞内库，并将该技术应用于分析三组表达不同丁酸生物合成基因组合的大肠杆菌菌株。 hbd 编码的 S-3-羟基丁酰-CoA 脱氢酶的低表达水平不足以将乙酰-CoA 转化为 3-羟基丁酰-CoA，表明 hbd 是丁酰-CoA 生产中的限速步骤。增加 hbd 表达缓解了这一瓶颈，但在所得菌株中，我们的库大小测量和热力学分析表明，由 bcd 编码的丁酰辅酶 A 脱氢酶催化的反应步骤是限速的。表达 hbd 和 ptb-buk 的大肠杆菌菌株产生副产物巴豆酸，但在非梭菌生物的相关基因表达中未观察到这种副产物。我们对池大小测量的热力学解释适用于其他代谢途径的分析。本文的在线版本 (doi:10.1007/s00253-010-2749-2) 包含补充材料，可供授权用户使用。
In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways. The online version of this article (doi:10.1007/s00253-010-2749-2) contains supplementary material, which is available to authorized users.
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影响因子：
2.9
作者：
Liu, Xiaoguang;Zhu, Ying;Yang, Shang-Tian
通讯作者：
Yang, Shang-Tian
影响因子：
8.4
作者：
Nielsen, David R.;Leonard, Effendi;Prather, Kristala L. Jones
通讯作者：
Prather, Kristala L. Jones
影响因子：
2.3
作者：
Park, Je Won;Jung, Won Seok;Yoon, Yeo Joon
通讯作者：
Yoon, Yeo Joon
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
8.4
作者：
Atsumi, Shota;Cann, Anthony F.;Liao, James C.
通讯作者：
Liao, James C.