Improved N‐acetylneuraminic acid bioproduction by optimizing pathway for reducing intermediate accumulation

Abstract

Abstract N‐acetylneuraminic acid (NeuAc), which has been widely used as a nutraceutical and pharmaceutical intermediate, plays an important role in improving brain development and cognition while enhancing immunity. Bacillus subtilis, generally regarded as a food‐safe microorganism, is suitable for developing as a chassis cell for efficient NeuAc synthesis. However, accumulated intermediates can lead to metabolic bottlenecks for NeuAc synthesis. To eliminate the accumulated byproduct N‐acetylglucosamine (GlcNAc), the UDP‐GlcNAc epimerase pathway without GlcNAc production was first reconstructed and optimized in B. subtilis, resulting in the NeuAc titer increase of 5.9 g/L with GlcNAc elimination. In addition, to reduce another accumulated byproduct N‐acetylmannosamine (ManNAc), the directed evolution of N‐acetylneuraminic acid synthase and the enhancement of phosphoenolpyruvate supply was implemented. Using this strategy, ManNAc decreased by 46.3%, and the NeuAc titer increased by 54.9%, reaching 7.9 g/L. Finally, the maximum titer of NeuAc in a 3‐L fermenter reached 21.8 g/L with a productivity of 0.34 g/L/h.

Keywords

• Bacillus subtilis
• N‐acetylneuraminic acid
• reducing intermediate accumulation