Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products:: Homoacetate production
Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products:: Homoacetate production
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DOI：
10.1073/pnas.0337684100
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发表时间：
2003-02-04
影响因子：
11.1
通讯作者：
Ingram, LO
中科院分区：
文献类型：
--
作者：
Causey, TB;Zhou, S;Ingram, LO
用于商品化学品的微生物工艺集中于还原产物和厌氧条件，其中基质损失到细胞质量和CO2最小并且产物产率高。为了促进扩展到更多的氧化化学品，大肠杆菌W3110通过使用将发酵和氧化代谢（快速生长，外部电子受体）的属性结合到单个生物催化剂中的方法进行遗传工程化以用于乙酸盐生产。所得菌株（TC 36）在18小时内将333 mM葡萄糖转化为572 mM乙酸盐，乙酸盐是等效氧化态的产物。在过量葡萄糖的情况下，产生最多878 mM乙酸盐。通过顺序组装缺失来构建菌株TC 36，所述缺失使氧化磷酸化失活（Δ atpFH）、破坏三羧酸途径的循环功能（Δ tasucA）并消除天然发酵途径（Δ focA-pflB Δ frdBC Δ dhA Δ adhE）。这些突变使底物碳的损失和氧化还原平衡的氧需求最小化。尽管TC 36每单位葡萄糖仅产生4个ATP，但该菌株在矿物盐培养基中生长良好，并且没有营养缺陷型要求。TC 36（0.3 μ mol·min（-1）·mg（-1）protein）的糖酵解通量是亲本的2倍。较高的通量归因于（F1 F0）H+-ATP合酶中的膜偶联亚基的缺失，其在保持细胞质F-1-ATP酶活性的同时使ATP合成失活。这种缺失在刺激流量方面的有效性为ATP供给和需求在中枢代谢调节中的重要性提供了进一步的证据。TC 36的衍生物可证明可用于多种商品化学品的商业生产。
Microbial processes for commodity chemicals have focused on reduced products and anaerobic conditions where substrate loss to cell mass and CO2 are minimal and product yields are high. To facilitate expansion into more oxidized chemicals, Escherichia coli W3110 was genetically engineered for acetate production by using an approach that combines attributes of fermentative and oxidative metabolism (rapid growth, external electron acceptor) into a single biocatalyst. The resulting strain (TC36) converted 333 mM glucose into 572 mM acetate, a product of equivalent oxidation state, in 18 h. With excess glucose, a maximum of 878 mM acetate was produced. Strain TC36 was constructed by sequentially assembling deletions that inactivated oxidative phosphorylation (DeltaatpFH), disrupted the cyclic function of the tricarboxylic acid pathway (DeltasucA), and eliminated native fermentation pathways (DeltafocA-pflB DeltafrdBC DeltaldhA DeltaadhE). These mutations minimized the loss of substrate carbon and the oxygen requirement for redox balance. Although TC36 produces only four ATPs per glucose, this strain grows well in mineral salts medium and has no auxotrophic requirement. Glycolytic flux in TC36 (0.3,mumol.min(-1).mg(-1) protein) was twice that of the parent. Higher flux was attributed to a deletion of membrane-coupling subunits in (F1F0)H+-ATP synthase that inactivated ATP synthesis while retaining cytoplasmic F-1-ATPase activity. The effectiveness of this deletion in stimulating flux provides further evidence for the importance of ATP supply and demand in the regulation of central metabolism. Derivatives of TC36 may prove useful for the commercial production of a variety of commodity chemicals.