Model-driven intracellular redox status modulation for increasing isobutanol production in Escherichia coli.
Model-driven intracellular redox status modulation for increasing isobutanol production in Escherichia coli.
复制标题
模型驱动的细胞内氧化还原状态调节以增加大肠杆菌中的异丁醇产量
DOI：
10.1186/s13068-015-0291-2
复制
发表时间：
2015
影响因子：
6.3
通讯作者：
Wen J
中科院分区：
文献类型：
--
作者：
Liu J;Qi H;Wang C;Wen J
背景：几乎没有发现天然产生异丁醇的菌株。为了构建高效的异丁醇产生菌，通过对氧化还原辅因子代谢的系统研究，重新平衡细胞的氧化还原状态是非常关键的。结果：首先，构建了一株异丁醇产生菌LA02，其异丁醇产量仅为2.7g/L。然后，结合通量平衡分析和最小化代谢调节，对菌株LA02的氧化还原辅因子代谢进行了基因组水平的代谢模拟，预测了3-磷酸甘油醛脱氢酶催化的GAPD反应是氧化还原状态改善的关键靶点。在代谢模型预测的指导下，构建了编码NADP(+)依赖的GapN依赖的甘油醛-3-磷酸脱氢酶途径，并用5个构成启动子对其进行了微调。其中启动子最强的菌株LA09的启动子最强。菌株LA09的NADPH/NADP+比值在对数期达到0.67，在稳定期达到0.64。氧化还原调节导致乙醇和乳酸的产量分别下降17.5%和51.7%，分别降至1.32g/L和6.08g/L。因此，异丁醇效价提高了221%，达到8.68g/L。结论：在人工合成启动子的指导下，异丁醇产生菌基因组代谢模型的预测和建模实现了异丁醇产生菌氧化还原状态的合理改善。因此，异丁醇的产量从2.7g/L大幅提高到8.68g/L，提高了2.21倍。此外，所开发的氧化还原辅因子代谢模型驱动方法对其他生物产品的氧化还原状态调节非常有帮助。
Background:Few strains have been found to produce isobutanol naturally. For building a high performance isobutanol-producing strain, rebalancing redox status of the cell was very crucial through systematic investigation of redox cofactors metabolism. Then, the metabolic model provided a powerful tool for the rational modulation of the redox status.Results:Firstly, a starting isobutanol-producing E. coli strain LA02 was engineered with only 2.7 g/L isobutanol produced. Then, the genome-scale metabolic modeling was specially carried out for the redox cofactor metabolism of the strain LA02 by combining flux balance analysis and minimization of metabolic adjustment, and the GAPD reaction catalyzed by the glyceraldehyde-3-phosphate dehydrogenase was predicted as the key target for redox status improvement. Under guidance of the metabolic model prediction, a gapN-encoding NADP(+) dependent glyceraldehyde-3-phosphate dehydrogenase pathway was constructed and then fine-tuned using five constitutive promoters. The best strain LA09 was obtained with the strongest promoter BBa_J23100. The NADPH/NADP + ratios of strain LA09 reached 0.67 at exponential phase and 0.64 at stationary phase. The redox modulations resulted in the decrease production of ethanol and lactate by 17.5 and 51.7% to 1.32 and 6.08 g/L, respectively. Therefore, the isobutanol titer was increased by 221% to 8.68 g/L.Conclusions:This research has achieved rational redox status improvement of isobutanol-producing strain under guidance of the prediction and modeling of the genome-scale metabolic model of isobutanol-producing E. coli strain with the aid of synthetic promoters. Therefore, the production of isobutanol was dramatically increased by 2.21-fold from 2.7 to 8.68 g/L. Moreover, the developed model-driven method special for redox cofactor metabolism was of very helpful to the redox status modulation of other bio-products.