Biosensor-Assisted Multitarget Gene Fine-Tuning for N-Acetylneuraminic Acid Production in Escherichia coli with Sole...

N-Acetylneuraminic acid (NeuAc) is widely used in the food and medical industries. Microbial fermentation has become one of the most important approaches for NeuAc production. However, current research on NeuAc is confronted with challenges, including high production costs, interference from competitive pathways, and low conversion efficiency, all of which impede its efficient production. In this study, an engineered Escherichia coli capable of utilizing glucose as the sole carbon source for NeuAc production was constructed by optimizing the glucose utilization pathway, competitive pathways, and redox balance of NADH/NAD⁺. Subsequently, pathway genes were systematically upregulated to identify key target genes for improving NeuAc biosynthesis. The gene cluster glmS-glmM-SeglmU was identified as the key engineering target. To achieve multitarget coordinated optimization of this gene cluster in vivo, a highly responsive biosensor for NeuAc was developed, exhibiting a maximum response ratio of 10.62-fold. By the construction of random mutation libraries and integration of the NeuAc-responsive biosensor with high-throughput screening using flow cytometry, the expression levels of three key genes were synergistically optimized. As a result, highly efficient NeuAc-producing strain A39 was successfully obtained. In a 3-L bioreactor, the strain achieved a NeuAc titer of 58.26 g·L^-1 with a productivity of 0.83 g·L^-1·h^-1, representing the highest reported production of NeuAc using glucose as the sole carbon source.