Zahoor Ahmed, Küttner Felix T F, Blank Lars M, Ebert Birgitta E
Institute of Applied Microbiology - iAMB Aachen Biology and Biotechnology - ABBt RWTH Aachen University Aachen Germany.
Eng Life Sci. 2019 Aug 29;19(10):711-720. doi: 10.1002/elsc.201900080. eCollection 2019 Oct.
Dicarboxylic acids are important bio-based building blocks, and is postulated to be an advantageous host for their fermentative production. Here, we engineered a pyruvate decarboxylase-negative strain for succinic acid production to exploit its promising properties, that is, lack of ethanol production and accumulation of the precursor pyruvate. The metabolic engineering steps included genomic integration of a biosynthesis pathway based on the reductive branch of the tricarboxylic acid cycle and a dicarboxylic acid transporter. Further modifications were the combined deletion of and and multi-copy integration of the native gene, encoding a pyruvate carboxylase required to drain pyruvate into the synthesis pathway. The effect of increased redox cofactor supply was tested by modulating oxygen limitation and supplementing formate. The physiologic analysis of the differently engineered strains focused on elucidating metabolic bottlenecks. The data not only highlight the importance of a balanced activity of pathway enzymes and selective export systems but also shows the importance to find an optimal trade-off between redox cofactor supply and energy availability in the form of ATP.
二羧酸是重要的生物基构建模块，据推测是其发酵生产的有利宿主。在此，我们构建了一株丙酮酸脱羧酶阴性的琥珀酸生产菌株，以利用其有前景的特性，即不产生乙醇且积累前体丙酮酸。代谢工程步骤包括基于三羧酸循环还原分支的生物合成途径和二羧酸转运体的基因组整合。进一步的修饰是联合缺失[具体基因]和多拷贝整合天然的[具体基因]，该基因编码将丙酮酸引流到合成途径所需的丙酮酸羧化酶。通过调节氧限制和补充甲酸来测试增加氧化还原辅因子供应的效果。对不同工程改造菌株的生理分析着重于阐明代谢瓶颈。这些数据不仅突出了途径酶和选择性输出系统平衡活性的重要性，还表明了在氧化还原辅因子供应和以ATP形式存在的能量可用性之间找到最佳权衡的重要性。
