Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, 300457, PR China; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, PR China.
Ningxia Eppen Biotech Co., Ltd, Ningxia, 750000, PR China.
Metab Eng. 2023 Mar;76:146-157. doi: 10.1016/j.ymben.2023.02.003. Epub 2023 Feb 8.
L-arginine is a value-added amino acid with promising applications in the pharmaceutical and nutraceutical industries. Further unleashing the potential of microbial cell factories to make L-arginine production more competitive remains challenging due to the sophisticated intracellular interaction networks and the insufficient knowledge of global metabolic regulation. Here, we combined multilevel rational metabolic engineering with biosensor-assisted mutagenesis screening to exploit the L-arginine production potential of Escherichia coli. First, multiple metabolic pathways were systematically reprogrammed to redirect the metabolic flux into L-arginine synthesis, including the L-arginine biosynthesis, TCA cycle, and L-arginine export. Specifically, a toggle switch responding to special cellular physiological conditions was designed to dynamically control the expression of sucA and pull more carbon flux from the TCA cycle toward L-arginine biosynthesis. Subsequently, a biosensor-assisted high-throughput screening platform was designed and applied to further exploit the L-arginine production potential. The best-engineered ARG28 strain produced 132 g/L L-arginine in a 5-L bioreactor with a yield of 0.51 g/g glucose and productivity of 2.75 g/(L ⋅ h), which were the highest values reported so far. Through whole genome sequencing and reverse engineering, Frc frameshift mutant, PqiB A78P mutant, and RpoB P564T mutant were revealed for enhancing the L-arginine biosynthesis. Our study exhibited the power of coupling rational metabolic reprogramming and biosensor-assisted mutagenesis screening to unleash the cellular potential for value-added metabolite production.
L-精氨酸是一种附加值氨基酸，在制药和营养保健品行业具有广阔的应用前景。由于细胞内相互作用网络复杂，以及对全局代谢调控的了解不足，进一步挖掘微生物细胞工厂生产 L-精氨酸的潜力仍然具有挑战性。在这里，我们结合多层次理性代谢工程和生物传感器辅助诱变筛选，来挖掘大肠杆菌生产 L-精氨酸的潜力。首先，我们系统地重新规划了多个代谢途径，将代谢通量重新导向 L-精氨酸合成，包括 L-精氨酸生物合成、三羧酸 (TCA) 循环和 L-精氨酸输出。具体来说，设计了一个响应特殊细胞生理条件的拨动开关，以动态控制 sucA 的表达，从 TCA 循环中拉动更多的碳通量流向 L-精氨酸生物合成。随后，设计并应用了一个生物传感器辅助高通量筛选平台，进一步挖掘 L-精氨酸的生产潜力。经过工程改造的 ARG28 菌株在 5-L 生物反应器中生产 132 g/L 的 L-精氨酸，得率为 0.51 g/g 葡萄糖，产率为 2.75 g/(L ⋅ h)，这是迄今为止报道的最高值。通过全基因组测序和反向工程，揭示了 Frc 移码突变、PqiB A78P 突变和 RpoB P564T 突变增强 L-精氨酸生物合成的作用。我们的研究展示了将理性代谢重编程与生物传感器辅助诱变筛选相结合，释放细胞生产增值代谢产物潜力的强大力量。
