Modular pathway engineering of key carbon‐precursor supply‐pathways for improved N‐acetylneuraminic acid production in Bacillus subtilis

Abstract

N-acetylneuraminic acid (NeuAc) is widely used as a nutraceutical for facilitating infant brain development, maintaining brain health, and enhancing immunity. Currently, NeuAc is mainly produced by extraction from egg yolk and milk, or via chemical synthesis. However, its low concentration in natural resources and its non-ecofriendly chemical synthesis result in insufficient NeuAc production and environmental pollution, respectively. In this study, improved NeuAc production was attained via modular pathway engineering of the supply pathways of two key precursors-N-acetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP)-and by balancing NeuAc biosynthesis and cell growth in engineered Bacillus subtilis. Specifically, we used a previously constructed GlcNAc-producing B. subtilis as the initial host for NeuAc biosynthesis. First, we constructed a de novo NeuAc biosynthetic pathway utilizing glucose by coexpressing glucosamine-6-phosphate acetyl-transferase (GNA1), N-acetylglucosamine 2-epimerase (AGE), and N-acetylneuraminic acid synthase (NeuB), resulting in 0.33 g/l NeuAc production. Next, to balance the supply of the two key precursors for NeuAc biosynthesis, modular pathway engineering was performed. The optimal strategy for balancing the GlcNAc module and PEP supply module involved the use of an engineered, unique glucose and malate coutilization pathway in B. subtilis, supplied with both glucose (for the GlcNAc moiety) and malate (for the PEP moiety) at high strength. This led to 1.65 g/L NeuAc production, representing a 5.0-fold improvement over the existing methods. Furthermore, to enhance the NeuAc yield on cell, glucose and malate coutilization pathways were engineered to balance NeuAc biosynthesis and cell growth via the blocking of glycolysis, the introduction of the Entner-Doudoroff pathway, and the overexpression of the malic enzyme YtsJ. NeuAc titer reached 2.18 g/L, with 0.38 g/g dry cell weight NeuAc yield on cell, which represented a 1.32-fold and 2.64-fold improvement over the existing methods, respectively. The strategy of modular pathway engineering of key carbon precursor supply pathways via engineering of the unique glucose-malate coutilization pathway in B. subtilis should be generically applicable for engineering of B. subtilis for the production of other important biomolecules. Our study also provides a good starting point for further metabolic engineering to achieve industrial production of NeuAc by a Generally Regarded As Safe bacterial strain.

Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis

“…The precursors UDP-Gal and UDP-GlcNAc are derived from glucose 6-phosphate and fructose 6-phosphate of Embden–Meyerhof–Parnas pathway (EMP), respectively. Modular pathway engineering divides complex synthetic networks into modules, systematically strengthens and balances the various modules to optimize the entire metabolic network via assembling different strengths between modules [24, 25]. Therefore, modular pathway engineering can be used as an effective strategy to balance the UDP-GlcNAc and UDP-Gal supply for LNnT synthesis.…”

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

“…Plasmids and strains. Gibson assembly was used to construct all plasmids used in this study with pET28a, pHT01, and p43NMK as backbones [54][55][56][57] . For genome editing of E. coli Δ321AM, λ-Red recombination was used, and pCP20 served to eliminate resistance selection markers on the E. coli genome.…”

Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production

“…The concentrations of these stock solutions were 1× in the final media. The bioproduction of acetoin and proteins (GFP and ovalbumin) was carried out in the media containing 12 ​g/L yeast extract, 6 ​g/L tryptone, 2.5 ​g/L KH 2 PO 4 , 12 ​g/L K 2 HPO 4 ∙3H 2 O, 6 ​g/L (NH 4 ) 2 SO 4 , 3 ​g/L MgSO 4 ∙7H 2 O, and 60 ​g/L glucose ( Zhang et al., 2018 ). All strains were grown at 37 ​°C and 220 ​rpm.…”