Metabolic regulation of Escherichia coli and its gdhA, glnL, gltB, D mutants under different carbon and nitrogen limitations in the continuous culture.
Metabolic regulation of Escherichia coli and its gdhA, glnL, gltB, D mutants under different carbon and nitrogen limitations in the continuous culture.
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DOI：
10.1186/1475-2859-9-8
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发表时间：
2010-01-27
影响因子：
6.4
通讯作者：
Shimizu K
中科院分区：
文献类型：
--
作者：
Kumar R;Shimizu K
了解在氮(N)-限制和碳(C)-限制下中枢代谢是如何调节的是非常重要的。特别是碳氮比对代谢的影响对异源蛋白生产、PHB生产等具有实际意义。虽然碳氮代谢相互关联，整体机制复杂，但从实际应用的角度阐明培养环境对代谢的影响是非常必要的。以发酵数据、转录RNA水平和酶活性数据为基础，在0.2 h-1稀释率的好氧连续培养条件下，研究C/N比对大肠杆菌代谢的影响。葡萄糖浓度为10 g/l，硫酸铵浓度为5.94 ~ 0.594 g/l。得到的C/N比值分别为1.68（100%）、2.81（60%）、4.21（40%）、8.42（20%）和16.84(10%)，括号内的百分比值表示与5.94 g/l硫酸铵情况下N浓度的比值。随着C/N比的升高，crp和mlc mRNA水平降低，导致ptsG转录物表达上调。随着C/N比值的升高，cra转录物表达降低，导致ptsH、pfkA、pykF上调。高C/N时，soxR/S转录mRNA水平升高，这可能与呼吸链激活有关，表现为cyoA、cydB、ndh等基因表达上调，特定CO2产率升高。rpoN转录本的表达量随着C/N比的增加而增加，这导致glnA、L、G和gltD转录本的表达也以类似的方式变化。nac转录本表达趋势与rpoN相似，而gdhA转录本表达方向相反。编码PII、glnD和glnK的glnB转录mRNA水平随着碳氮比的升高而升高。结果表明，富氮条件下gdhA突变体的GS-GOGAT通路被激活。在氮素限制下，glnL突变体的GOGAT酶活性比野生型降低。在gltB， D突变体中，GDH和GS酶在富氮和限氮条件下均被使用。在这种情况下，限氮条件下的gdhA转录mRNA水平和相应的GDH酶活性高于富氮条件。根据发酵、转录mRNA水平和酶活性，阐明了大肠杆菌在碳(C)限制和氮(N)限制下的代谢调节。提出了总体调控机制。同时阐明了敲除N同化途径基因的作用。
It is quite important to understand how the central metabolism is regulated under nitrogen (N)- limitation as well as carbon (C)- limitation. In particular, the effect of C/N ratio on the metabolism is of practical interest for the heterologous protein production, PHB production, etc. Although the carbon and nitrogen metabolisms are interconnected and the overall mechanism is complicated, it is strongly desirable to clarify the effects of culture environment on the metabolism from the practical application point of view. The effect of C/N ratio on the metabolism in Escherichia coli was investigated in the aerobic continuous culture at the dilution rate of 0.2 h-1 based on fermentation data, transcriptional RNA level, and enzyme activity data. The glucose concentration was kept at 10 g/l, while ammonium sulfate concentration was varied from 5.94 to 0.594 g/l. The resultant C/N ratios were 1.68 (100%), 2.81(60%), 4.21(40%), 8.42(20%), and 16.84(10%), where the percentage values in brackets indicate the ratio of N- concentration as compared to the case of 5.94 g/l of ammonium sulfate. The mRNA levels of crp and mlc decreased, which caused ptsG transcript expression to be up-regulated as C/N ratio increased. As C/N ratio increased cra transcript expression decreased, which caused ptsH, pfkA, and pykF to be up-regulated. At high C/N ratio, transcriptional mRNA level of soxR/S increased, which may be due to the activated respiratory chain as indicated by up-regulations of such genes as cyoA, cydB, ndh as well as the increase in the specific CO2 production rate. The rpoN transcript expression increased with the increase in C/N ratio, which led glnA, L, G and gltD transcript expression to change in similar fashion. The nac transcript expression showed similar trend as rpoN, while gdhA transcript expression changed in reverse direction. The transcriptional mRNA level of glnB, which codes for PII, glnD and glnK increased as C/N ratio increases. It was shown that GS-GOGAT pathway was activated for gdhA mutant under N- rich condition. In the case of glnL mutant, GOGAT enzyme activity was reduced as compared to the wild type under N- limitation. In the case of gltB, D mutants, GDH and GS enzymes were utilized under both N- rich and N- limited conditions. In this case, the transcriptional mRNA level of gdhA and corresponding GDH enzyme activity was higher under N- limitation as compared to N- rich condition. The metabolic regulation of E.coli was clarified under both carbon (C)- limitation and nitrogen (N)- limitation based on fermentation, transcriptional mRNA level and enzyme activities. The overall regulation mechanism was proposed. The effects of knocking out N- assimilation pathway genes were also clarified.
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影响因子：
2.9
作者：
Jiang, Peng;Ninfa, Alexander J.
通讯作者：
Ninfa, Alexander J.
DOI：
10.1073/pnas.88.5.1631
发表时间：
1991-03-01
影响因子：
11.1
作者：
CLAVERIEMARTIN, F;MAGASANIK, B
通讯作者：
MAGASANIK, B
大肠杆菌K-12的构造框架，单基因敲除突变体：Keio Collection。
影响因子：
9.9
作者：
通讯作者：
--
CRP训练营和PII-NTR系统之间的相互作用形成了大肠杆菌中碳代谢和氮同化之间的新调节网络。
影响因子：
14.9
作者：
Mao, Xian-Jun;Huo, Yi-Xin;Buck, Martin;Kolb, Annie;Wang, Yi-Ping
通讯作者：
Wang, Yi-Ping
影响因子：
11.4
作者：
Jaggi, R;vanHeeswijk, WC;Vasudevan, SG
通讯作者：
Vasudevan, SG