Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli.
Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli.
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DOI：
10.1186/1475-2859-11-127
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发表时间：
2012-09-13
影响因子：
6.4
通讯作者：
Wittmann C
中科院分区：
文献类型：
--
作者：
Meza E;Becker J;Bolivar F;Gosset G;Wittmann C
磷酸烯醇式丙酮酸（PEP）是大肠杆菌中参与葡萄糖转运的关键代谢中间体，是多种生物合成途径的前体，参与糖酵解酶的变构调节。在这项工作中，我们产生了W3110衍生菌株，缺乏主要的PEP消费者PEP：糖磷酸转移酶系统（PTS-）和丙酮酸激酶同工酶PykA和PykF（PTS-pykA-和PTS-pykF-）。为了表征这些修饰对细胞生理学的影响，测定了碳通量分布和芳烃生产能力。与参考菌株W3110相比，菌株VH 33（PTS-）显示出较低的生长、葡萄糖消耗和乙酸盐产生的比速率以及较高的葡萄糖生物量产量。通过PykA或PykF的额外失活，这些表型效应甚至更加明显。碳通量分析表明，PTS失活导致重定向的代谢通量对生物质的形成。PEP羧化酶（Ppc）和PEP羧激酶（Pck）的循环在所有菌株中检测到。在菌株W3110，VH 33（PTS-）和VH 35（PTS-，pykF-），在这个循环中的净流量与葡萄糖消耗和Pck在这些菌株中的失活的特定速率负相关导致生长速率的降低。在PTS背景下，PykA的失活导致Ppc和Pck循环的减少以及到TCA的通量的减少，而PykF的失活导致从PEP到OAA的回补通量的增加以及到TCA的通量的增加。对野生型和突变株进行修饰以过量产生L-苯丙氨酸。在静息细胞实验中，与参考菌株相比，由于PTS、PTS PykA和PTS PykF失活，观察到葡萄糖的芳烃产率高10、4和7倍。对缺乏从PEP产生丙酮酸的主要活性的菌株进行的代谢通量分析揭示了E.杆菌观察到的葡萄糖摄取减少和PEP转化为丙酮酸的速度所造成的PTS，PykA和PykF失活的反应包括通量改道在几个中央代谢节点的合成代谢生物合成反应，从而补偿这些突变株的碳限制。发现检测到的涉及Ppc和Pck的循环是维持所有研究菌株的特定生长和葡萄糖消耗速率所需的。菌株VH 33（PTS-）、VH 34（PTS-pykA-）和VH 35（PTS-pykF-）具有用于生物技术方法的有用性质，例如增加的PEP可用性和来自葡萄糖的高生物质产率，使得它们可用于生产芳香族化合物或重组蛋白。
In Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS-) and pyruvate kinase isozymes PykA and PykF (PTS-pykA- and PTS-pykF-). To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined. When compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc) and PEP carboxykinase (Pck) was detected in all strains. In strains W3110, VH33 (PTS-) and VH35 (PTS-, pykF-), the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation. Metabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high degree of flexibility to perturbations of the central metabolic network in E. coli. The observed responses to reduced glucose uptake and PEP to pyruvate rate of conversion caused by PTS, PykA and PykF inactivation included flux rerouting in several central metabolism nodes towards anabolic biosynthetic reactions, thus compensating for carbon limitation in these mutant strains. The detected cycle involving Ppc and Pck was found to be required for maintaining the specific growth and glucose consumption rates in all studied strains. Strains VH33 (PTS-), VH34 (PTS-pykA-) and VH35 (PTS-pykF-) have useful properties for biotechnological processes, such as increased PEP availability and high biomass yields from glucose, making them useful for the production of aromatic compounds or recombinant proteins.
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DOI：
10.1007/bf01570148
发表时间：
1996-07-01
期刊：
JOURNAL OF INDUSTRIAL MICROBIOLOGY
影响因子：
--
作者：
Gosset, G;YongXiao, J;Berry, A
通讯作者：
Berry, A
影响因子：
3.5
作者：
CHEREPANOV, PP;WACKERNAGEL, W
通讯作者：
WACKERNAGEL, W
影响因子：
1.2
作者：
Carlos Sigala, Juan;Flores, Salvador;Bolivar, Francisco
通讯作者：
Bolivar, Francisco
影响因子：
4.1
作者：
HOLMSEN, H;STORM, E
通讯作者：
STORM, E
影响因子：
6.4
作者：
Escalante A;Calderón R;Valdivia A;de Anda R;Hernández G;Ramírez OT;Gosset G;Bolívar F
通讯作者：
Bolívar F