Tianjin Institutes of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China.
Key Laboratories of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China.
Bioprocess Biosyst Eng. 2021 Aug;44(8):1685-1697. doi: 10.1007/s00449-021-02552-4. Epub 2021 Mar 22.
L-tryptophan (L-trp) production in Escherichia coli has been developed by employing random mutagenesis and selection for a long time, but this approach produces an unclear genetic background. Here, we generated the L-trp overproducer TPD5 by combining an intracellular L-trp biosensor and fluorescence-activated cell sorting (FACS) in E. coli, and succeeded in elucidating the genetic basis for L-trp overproduction. The most significant identified positive mutations affected TnaA (deletion), AroG (S211F), TrpE (A63V), and RpoS (nonsense mutation Q33*). The underlying structure-function relationships of the feedback-resistant AroG (S211F) and TrpE (A63V) mutants were uncovered based on protein structure modeling and molecular dynamics simulations, respectively. According to transcriptomic analysis, the global regulator RpoS not only has a great influence on cell growth and morphology, but also on carbon utilization and the direction of carbon flow. Finally, by balancing the concentrations of the L-trp precursors' serine and glutamine based on the above analysis, we further increased the titer of L-trp to 3.18 g/L with a yield of 0.18 g/g. The analysis of the genetic characteristics of an L-trp overproducing E. coli provides valuable information on L-trp synthesis and elucidates the phenotype and complex cellular properties in a high-yielding strain, which opens the possibility to transfer beneficial mutations and reconstruct an overproducer with a clean genetic background.
大肠杆菌中 L-色氨酸（L-Trp）的生产已经通过随机诱变和选择进行了很长时间，但这种方法产生的遗传背景不清晰。在这里，我们通过在大肠杆菌中结合细胞内 L-色氨酸生物传感器和荧光激活细胞分选（FACS），生成了 L-色氨酸过量产生菌 TPD5，并成功阐明了 L-色氨酸过量产生的遗传基础。鉴定出的最显著的阳性突变影响 TnaA（缺失）、AroG（S211F）、TrpE（A63V）和 RpoS（无义突变 Q33*）。基于蛋白质结构建模和分子动力学模拟，分别揭示了反馈抗性 AroG（S211F）和 TrpE（A63V）突变体的潜在结构-功能关系。根据转录组分析，全局调控因子 RpoS 不仅对细胞生长和形态有很大影响，而且对碳利用和碳流方向有很大影响。最后，通过基于上述分析平衡 L-色氨酸前体丝氨酸和谷氨酰胺的浓度，我们进一步将 L-色氨酸的滴度提高到 3.18 g/L，得率为 0.18 g/g。对高产 L-色氨酸大肠杆菌遗传特性的分析为 L-色氨酸合成提供了有价值的信息，并阐明了高产菌株中的表型和复杂细胞特性，为转移有益突变和重建具有清洁遗传背景的高产菌开辟了可能性。
