Multi-enzyme cascades starting from a cost-effective substrate are regarded as valuable biotechnological production systems for complex oligosaccharides. Enzyme cascades have been described in literature for decades, however, there has been little attempt to optimize flux through the cascade based on systematic evaluation of process efficiency. Ideally kinetic modelling can guide cascade optimization and minimize experimental effort.
Here we focused on systematic optimization of 3'-sialylactose production by multi-enzyme cascades in a one pot approach. 3'-sialyllactose is a major human milk oligosaccharide and the simplest target for biotechnological sialoside synthesis . Sialoside syntheses with sialyltransferases depend on high cost nucleotide donor substrates and therefore are ideally integrated in multi-enzyme cascades enabling donor synthesis from a cost-efficient substrate. In the here used cascade 3'-sialyllactose production started with sialic acid production from N-acetyl mannosamine. Sialic acid was then converted to the activated sialyl donor CMP-N-acetylneuraminic acid and in a final step N-acetylneuraminic acid was transferred to the acceptor substrate by the sialyltransferase. Previously, a sialic acid aldolase was used in the initial step for sialic acid production . Sialic acid aldolases use cost efficient pyruvate as a co-substrate, however, an unfavorable thermodynamic equilibrium on the substrate side decreases the reactant flux through the cascade.
In an alternative approach we replaced the sialic acid aldolase by a sialic acid synthase for sialic acid formation from N-acetyl mannosamine. Sialic acid synthases rely on the more cost intensive phosphoenolpyruvate as co-substrate, however the thermodynamic equilibrium of the catalyzed reaction is clearly on the product side. A comparison of alternative application of synthase or aldolase in a one-pot approach for 3'-sialyllactose synthesis showed that the production rate with the synthase significantly increased compared to the aldolase with high 3'-sialyllactose production yields for both enzymes.
In the here optimized multi enzyme 3'-sialyllactose synthesis a strong focus was put on the quantitative analysis of reactants. Analysis results supported development of a Michaelis-Menten kinetics based model. The kinetic model will guide fine-tuning of enzyme-ratio and minimizing substrate usage in order to optimize productivity of the cascade
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