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Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli
Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cof...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463868/ https://www.ncbi.nlm.nih.gov/pubmed/32781536 http://dx.doi.org/10.3390/molecules25163567 |
Sumario: | Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3′-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that β-galactosidase (lacZ) affects the yield of 3′-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3′-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3′-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3′-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3′-SL. |
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