Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.
Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.
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DOI：
10.1186/1475-2859-9-21
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发表时间：
2010-04-12
影响因子：
6.4
通讯作者：
Bolívar F
中科院分区：
文献类型：
--
作者：
Escalante A;Calderón R;Valdivia A;de Anda R;Hernández G;Ramírez OT;Gosset G;Bolívar F
莽草酸（SA）用于合成抗流感药物磷酸奥司他韦。在这项工作中，利用代谢工程方法在大肠杆菌菌株（PB12）中产生SA，该菌株源自进化菌株（PB12），该菌株缺乏磷酸烯醇丙酮酸：碳水化合物磷酸转移酶系统（PTS-），但具有在葡萄糖上生长的能力。构建PB12菌株衍生物，测定灭活aroK、aroL、pykF或pykA以及质粒编码基因aroGfbr、tktA、aroB和aroE的表达对SA合成的影响。进行了批量培养，以评估遗传修饰对生长，葡萄糖消耗和芳香中间体生产的影响。所有衍生物均表现为两相生长行为，初始特定生长率（μ）和特定葡萄糖消耗率（qs）较高，但芳香中间体产量较低。在第二生长期，芳香族中间体的产量达到最大值，而μ下降。表达质粒编码基因的双aroK- aroL突变株（菌株PB12）。SA22在葡萄糖上的SA产率为0.29 mol/mol，总芳香化合物产率（TACY）为0.38 mol/mol，累积SA达7 g/L。在PB12中进行了pykF或pykA的单次失活。SA22压力。pykF失活导致μ、qs、SA产量和产量下降；而TACY升高33% （0.5 mol/mol）。研究了碳代谢物有效性的增加、它们进入芳香中间体合成的通道以及SA途径的破坏对SA生产的影响。aroK和aroL失活后，质粒编码基因转化后，SA积累量可达7 g/L，葡萄糖产量为0.29 mol/mol PB12。SA22，代表最高的报告产量。菌株PB12的pykF和pykA基因失活。SA22增加PTS背景下芳香族化合物的生成。结果表明，Pyk同工酶在植物生长和芳香族化合物生产中的作用是不同的。本研究首次证明了PTS和pykF的同时失活是提高大肠杆菌SA及其芳香前体产量的策略的一部分，从而使葡萄糖的芳香化合物产量达到0.5 mol/mol。
Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis. Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (μ) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the μ decreased, whereas aromatic intermediate production reached its maximum. The double aroK- aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in μ, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol). The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.
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影响因子：
6.4
作者：
Báez-Viveros JL;Flores N;Juárez K;Castillo-España P;Bolivar F;Gosset G
通讯作者：
Gosset G
影响因子：
120.7
作者：
Dharan, Nila J.;Gubareva, Larisa V.;Fry, Alicia M.
通讯作者：
Fry, Alicia M.
影响因子：
2.9
作者：
Chandran, SS;Yi, J;Frost, JW
通讯作者：
Frost, JW
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：
1.2
作者：
Flores, N;de Anda, R;Bolivar, F
通讯作者：
Bolivar, F