Kinetic characterization of two neuraminic acid synthases and evaluation of their application potential.

Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH5, obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co2+, Mn2+ and Mg2+, was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH5, some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids. KEY POINTS: • A novel neuraminic acid synthase was characterized. • The effect of cofactors on NeuS activity was elucidated. • Kinetic and stability characterization of two neuraminic acid synthases was performed.

Engineering new-to-nature biochemical conversions by combining fermentative metabolism with respiratory modules.

Anaerobic microbial fermentations provide high product yields and are a cornerstone of industrial bio-based processes. However, the need for redox balancing limits the array of fermentable substrate-product combinations. To overcome this limitation, here we design an aerobic fermentative metabolism that allows the introduction of selected respiratory modules. These can use oxygen to re-balance otherwise unbalanced fermentations, hence achieving controlled respiro-fermentative growth. Following this design, we engineer and characterize an obligate fermentative Escherichia coli strain that aerobically ferments glucose to stoichiometric amounts of lactate. We then re-integrate the quinone-dependent glycerol 3-phosphate dehydrogenase and demonstrate glycerol fermentation to lactate while selectively transferring the surplus of electrons to the respiratory chain. To showcase the potential of this fermentation mode, we direct fermentative flux from glycerol towards isobutanol production. In summary, our design permits using oxygen to selectively re-balance fermentations. This concept is an advance freeing highly efficient microbial fermentation from the limitations imposed by traditional redox balancing.