Engineering of Synthetic Multiplexed Pathways for High-Level -Acetylneuraminic Acid Bioproduction.

-Acetylneuraminic acid (NeuAc) is widely used as a supplement to promote brain health and enhance immunity. However, the low efficiency of NeuAc synthesis limits its cost-efficient bioproduction. Herein, a synthetic multiplexed pathway engineering (SMPE) strategy is proposed to improve NeuAc synthesis. First, we compare the key enzyme sources and optimize the expression levels of three NeuAc synthesis pathways in ; the AGE, NeuC, and NanE pathways, for which NeuAc production reached 3.94, 5.67, and 0.19 g/L, respectively. Next, these synthesis pathways were combined and modularly optimized via the SMPE strategy, with production reaching 7.87 g/L. Finally, fed-batch fermentation in a 5 L fermenter reached 30.10 g/L NeuAc production, the highest reported production using glucose as the sole carbon source. Using a generally regarded as safe strain as a production host, the developed NeuAc-producing approach should be favorable for efficient bioproduction, without the need for plasmids, antibiotics, or chemical inducers.