Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli
Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli
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DOI：
10.1016/j.ymben.2016.05.008
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发表时间：
2016-11-01
影响因子：
8.4
通讯作者：
Klamt, Steffen
中科院分区：
文献类型：
--
作者：
Harder, Bjoern-Johannes;Bettenbrock, Katja;Klamt, Steffen
衣康酸是目前由土曲霉工业化生产的一种高潜力平台化学品。利用大肠杆菌异源生产衣康酸可以克服土曲霉生长缓慢和对供氧高度敏感的局限性。然而，到目前为止，在大肠杆菌中所取得的效果仍然很低。我们将携带衣康酸产生基因的质粒(PCadCS)导入到大肠杆菌中，并应用基于模型的方法构建了高产菌株。基于最小割集的概念，我们确定了在保证衣康酸产量高的同时仍允许增长的干预策略。选择一个割集，反复剔除相应的基因。作为一个概念上的创新，我们采用了一种适应性方法，允许模型中的变化，并初步计算了如果基因修饰导致副产物形成的变化的干预策略。使用这种方法，我们迭代地实施了五种干预措施，导致在以葡萄糖为底物并添加少量谷氨酸的最低限度培养基中高产衣康酸。得到的菌株(ita23：MG1655 Delta ACEA Delta ucCD Delta PYKA Delta pykF Delta PTA Delta Picd：：CaM_BBA_J23115 pCadCS)合成2.27g/L衣康酸，产率为0.77摩尔/(摩尔葡萄糖)。在补料分批培养中，该菌株的衣康酸产量为32g/L，总产率为0.68mol/(摩尔葡萄糖)，最高产量为0.45g/L/h，这是迄今为止异源衣康酸生产中达到的最高值，表明该菌株的实际应用即将到来。(C)2016国际代谢工程学会。爱思唯尔公司出版，版权所有。
Itaconic acid is a high potential platform chemical which is currently industrially produced by Aspergillus terreus. Heterologous production of itaconic acid with Escherichia coli could help to overcome limitations of A. terreus regarding slow growth and high sensitivity to oxygen supply. However, the performance achieved so far with E. coli strains is still low.We introduced a plasmid (pCadCS) carrying genes for itaconic acid production into E. coli and applied a model-based approach to construct a high yield production strain. Based on the concept of minimal cut sets, we identified intervention strategies that guarantee high itaconic acid yield while still allowing growth. One cut set was selected and the corresponding genes were iteratively knocked-out. As a conceptual novelty, we pursued an adaptive approach allowing changes in the model and initially calculated intervention strategy if a genetic modification induces changes in by product formation. Using this approach, we iteratively implemented five interventions leading to high yield itaconic acid production in minimal medium with glucose as substrate supplemented with small amounts of glutamic acid. The derived E. coli strain (ita23: MG1655 Delta aceA Delta sucCD Delta pykA Delta pykF Delta pta Delta Picd::cam_BBa_J23115 pCadCS) synthesized 2.27 g/l itaconic acid with an excellent yield of 0.77 mol/(mol glucose). In a fed-batch cultivation, this strain produced 32 g/l itaconic acid with an overall yield of 0.68 mol/(mol glucose) and a peak productivity of 0.45 g/l/h. These values are by far the highest that have ever been achieved for heterologous itaconic acid production and indicate that realistic applications come into reach. (C) 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.