Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110.
Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110.
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DOI：
10.1186/1475-2859-7-30
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发表时间：
2008-10-21
影响因子：
6.4
通讯作者：
Neubauer P
中科院分区：
文献类型：
--
作者：
Soini J;Falschlehner C;Liedert C;Bernhardt J;Vuoristo J;Neubauer P
Norvaline是一种罕见的非蛋白性支链氨基酸，在粘质沙雷氏菌支链氨基酸途径调节突变体的早期酶学研究中引起了人们的兴趣。直到最近，去甲戊氨酸和其他支链氨基酸合成途径的修饰氨基酸才再次引起人们的注意，因为它们被发现少量结合在亮氨酸或蛋氨酸含量较高的异源蛋白中。早期的实验已经令人信服地表明，去甲戊酸和去甲亮氨酸是由丙酮酸作为α-异丙基苹果酸合成酶的替代底物而形成的，然而到目前为止，还没有发现去甲伐他汀在非重组菌株中发生积累。在这里，我们证明了氧气限制导致诺瓦林积累在大肠杆菌K-12W3110在葡萄糖为基础的矿物盐培养基中。诺伐他林在高葡萄糖浓度下转换到氧气限制后立即积累。相反，在好氧培养中，大肠杆菌W3110中不积累游离去甲伐他汀。在转录学研究的支持下，对介质成分的分析提出了一种从丙酮酸到支链氨基酸合成途径的纯粹代谢溢出机制，这进一步得到了丙酮酸在氧气降档后的显著积累的支持。结果表明，丙酮酸的溢出代谢是必要和充分的，但支链氨基酸途径的解除调控可能是一个额外的调节参数。到目前为止，诺瓦林的合成主要与合成支链氨基酸的不平衡有关，在丙酮酸水平较高的条件下。在这里，我们表明，在高糖浓度下，由于丙酮酸的积累及其在α-酮丁酸酯和α-酮戊酸酯上的直接链延长，简单的氧气下移就足以导致去甲戊酸的积累。尽管去甲伐他汀的通量很低，但在发酵液中积累了毫摩尔浓度，这远远高于已经讨论过的与去甲伐他汀错误结合到重组蛋白中有关的水平。因此，我们认为我们的发现与重组蛋白的生产有关，但也可能对一般氧气限制下的大肠杆菌的生理学产生影响。
Norvaline is an unusual non-proteinogenic branched-chain amino acid which has been of interest especially during the early enzymological studies on regulatory mutants of the branched-chain amino acid pathway in Serratia marcescens. Only recently norvaline and other modified amino acids of the branched-chain amino acid synthesis pathway got attention again when they were found to be incorporated in minor amounts in heterologous proteins with a high leucine or methionine content. Earlier experiments have convincingly shown that norvaline and norleucine are formed from pyruvate being an alternative substrate of α-isopropylmalate synthase, however so far norvaline accumulation was not shown to occur in non-recombinant strains of E. coli. Here we show that oxygen limitation causes norvaline accumulation in E. coli K-12 W3110 during grow in glucose-based mineral salt medium. Norvaline accumulates immediately after a shift to oxygen limitation at high glucose concentration. On the contrary free norvaline is not accumulated in E. coli W3110 in aerobic cultures. The analysis of medium components, supported by transcriptomic studies proposes a purely metabolic overflow mechanism from pyruvate into the branched chain amino acid synthesis pathway, which is further supported by the significant accumulation of pyruvate after the oxygen downshift. The results indicate overflow metabolism from pyruvate as necessary and sufficient, but deregulation of the branched chain amino acid pathway may be an additional modulating parameter. Norvaline synthesis has been so far mainly related to an imbalance of the synthesis of the branched chain amino acids under conditions were pyruvate level is high. Here we show that simply a downshift of oxygen is sufficient to cause norvaline accumulation at a high glucose concentration as a consequence of the accumulation of pyruvate and its direct chain elongation over α-ketobutyrate and α-ketovalerate. Although the flux to norvaline is low, millimolar concentrations are accumulated in the cultivation broth, which is far above the level which has been discussed for being relevant for misincorporation of norvaline into recombinant proteins. Therefore we believe that our finding is relevant for recombinant protein production but also may even have implications for the physiology of E. coli under oxygen limitation in general.
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影响因子：
2.9
作者：
RANDHAWA, ZI;WITKOWSKA, HE;SHACKLETON, CHL
通讯作者：
SHACKLETON, CHL
影响因子：
4.8
作者：
Apostol, I;Levine, J;Blackmore, R
通讯作者：
Blackmore, R
影响因子：
4.1
作者：
Enfors, SO;Jahic, M;Manelius, Å
通讯作者：
Manelius, Å
DOI：
10.1016/s0006-291x(88)80916-1
发表时间：
1988-10-31
影响因子：
3.1
作者：
LU, HS;TSAI, LB;LAI, PH
通讯作者：
LAI, PH
影响因子：
4.1
作者：
Muramatsu, R;Negishi, T;Hayashi, H
通讯作者：
Hayashi, H