Study of N-acetylneuraminic acid.
N-acetylneuraminic acid (NEUAC) is one of the most common and important sialic acids (SA). Neuac has anti-adhesion, antibacterial and antiviral properties, which can improve brain development, improve cognitive performance, in addition, play a role in immune regulation, cardiovascular protection, anticancer and antioxidant. Neuac has a certain whitening effect. As a result, NEUAC has great commercial potential in the pharmaceutical, cosmetics and food industries.
Recently, a research team from the State Key Laboratory of Food Science and Technology of Jiangnan University published a paper entitled "Efficient Production of N-Acetylneuraminic Acid in Escherichia Coli Based on The" in the Journal of Agricultural and Food Chemistry udp-n-acetylglucosamine biosynthetic pathway**.
In this study, the researchers used Escherichia coli BL21 (DE3) to biosynthesize NeuAC through the UDP-GLCNAC pathway, and used a variety of strategies to improve the synthesis of NEUAC, such as increasing the ** of UDP-GLCNAC, removing the ineffective pathway, replacing NEUAC synthase (NEUB) and UDP-N-acetylglucosamine 2-epimerase (NEUC), and optimizing the carbon source**, and finally obtained the engineered strain EM19. Through fed-batch fermentation, the titer of the engineered bacteria EM19 for the production of neuac was increased to 4692g l with a productivity of 082 g l h, is the highest reported concentration of microbial production of neuac to date.
Specific lines of study for NEUAC.
Enzymatic synthesis of Neuac is often dependent on the efficient expression and purification of the desired enzymes, with GLCNAC and pyruvate as starting materials, both of which are not inexpensive to produce on a large scale. Therefore, the synthesis of neuAC by cells using metabolic engineering strategies not only allows cheaper glycerol and glucose as starting materials, but also makes it easier to scale up production. Microbial synthesis of NeuAC generally uses N-acetylmannoseamine (MANNAC) as a direct precursor, and then uses phosphoenolpyruvate (PEP) or pyruvate as auxiliary substrates to convert to NeuAC through Neuac synthase (NeuB) or Neuac aldolase (Nana). MANNAC can be synthesized from three pathways, including the UDP-GLCNAC, GLCNAC, and MANNAC-6-phosphate pathways.
Both the GLCNAC and MANNAC-6-phosphate pathways use GLCNAC6P as a common metabolic intermediate, which is endogenously converted to GLCNAC and then accumulated in the culture medium, resulting in an insufficient carbon source for the synthesis of the key precursor MANNAC. In contrast, in the UDP-GLCNAC pathway, all intermediates except Mannac cannot be released into the medium, reducing the accumulation of by-products.
The researchers used Escherichia coli BL21 (DE3) to biosynthesize NeuAC via the UDP-GLCNAC pathway. The neuac synthesis pathway was constructed in Escherichia coli BL21 (DE3) by deleting the competitive pathway genes and introducing two genes encoding Neub and Neuc. Genes overexpressing the UDP-GLCNAC pathway (GLMS, GLMM, and GLMU) were then introduced, which strengthened the precursors** and promoted neuAC synthesis. Finally, the microorganisms** of neuc and neub were optimized, and the engineered strain EM19 was obtained. In addition, glycerol was found to have a better effect on neuac synthesis than glucose as a carbon source.
The seed culture of the engineered bacteria EM19 was added to a 5L bioreactor and incubated at 37 until the OD600 reached 13, the temperature was reduced to 25, and the final concentration was 02 mM iPTG induces expression of Neuac, growth11After 4 hours, the OD600 reached 176. Add a final concentration of 0 to the bioreactor1 mM IPTG, the neuac titer continued to increase throughout the process. When the glycerol concentration is less than 6 g l, 600 g l glycerol, 20 g l mgso4·7h2o, 02 g l vitamin bug ml ampicillin and 50 ml kanamycin to maintain glycerol concentration between 6 and 10 g l. NH3·H2O (14%, W V) is automatically added to maintain a pH at 68。By adjusting the stirring speed and aeration, the dissolved oxygen is controlled to 30%. Eventually, the engineering bacterium EM19 produced 4692 g L of extracellular neuac with a productivity of 082 g l h, which is the highest concentration reported so far.
References: Mingli Zhao, Yingying Zhu, Hao Wang, Jiameng Zhang, Wei Xu, and Wanmeng Mu efficient production of n-acetylneuraminic acid in escherichia coli based on the udp-n-acetylglucosamine biosynthetic pathway. journal of agricultural and food chemistry. 2023，71 (28), 10701-10709.