Liu Tingting, Zhao Qianru, Li Yang, Zhu Liying, Jiang Ling, Huang He
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China.
College of Food Science and Light Industry, Nanjing Tech University, Nanjing, China.
Front Microbiol. 2020 Aug 13;11:1564. doi: 10.3389/fmicb.2020.01564. eCollection 2020.
We first performed a combination of metabolic engineering (deletion of and and overexpression of ) with evolutionary engineering (selection under oxygen stress, acid stress and osmotic stress) in . The results indicated that the mutants had superior physiological activity, especially the mutant III obtained from Δ-Δ+ by evolutionary engineering, with 1.5-3.5 times higher growth rates, as well as a 37.1% increase of propionic acid (PA) titer and a 37.8% increase PA productivity compared to the wild type. Moreover, the integrative transcriptomics and proteomics analyses revealed that the differentially expressed genes (DEGs) and proteins (DEPs) in the mutant III were involved in energy metabolism, including the glycolysis pathway and tricarboxylic acid cycle (TCA cycle). These genes were up-regulated to supply increased amounts of energy and precursors for PA synthesis compared to the wild type. In addition, the down-regulation of fatty acid biosynthesis and fatty acid metabolism may indicate that the repressed metabolic flux was related to the production of PA. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to verify the differential expression levels of 16 selected key genes. The results offer deep insights into the mechanism of high PA production, which provides the theoretical foundation for the construction of advanced microbial cell factories.
我们首先在大肠杆菌中进行了代谢工程（敲除 和 并过表达 ）与进化工程（在氧胁迫、酸胁迫和渗透胁迫下进行筛选）的组合操作。结果表明，这些突变体具有优异的生理活性，特别是通过进化工程从Δ - Δ +获得的突变体III，其生长速率比野生型高1.5 - 3.5倍，丙酸（PA）滴度提高了37.1%，PA生产率提高了37.8%。此外，综合转录组学和蛋白质组学分析表明，突变体III中差异表达的基因（DEGs）和蛋白质（DEPs）参与能量代谢，包括糖酵解途径和三羧酸循环（TCA循环）。与野生型相比，这些基因上调以供应更多能量和前体用于PA合成。此外，脂肪酸生物合成和脂肪酸代谢的下调可能表明受抑制的代谢通量与PA的产生有关。进行了定量逆转录聚合酶链反应（qRT-PCR）以验证16个选定关键基因的差异表达水平。这些结果为高PA产量的机制提供了深入见解，为构建先进的微生物细胞工厂提供了理论基础。
