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

N-Acetylneuraminic acid (NeuAc) is widely used as a supplement to promote brain health and enhance immunity. However, the low efficiency of de novo 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 Bacillus subtilis; 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.