Redirection of sialic acid metabolism in genetically engineered Escherichia coli.

Most microorganisms do not produce sialic acid (sialate), and those that do appear to use a biosynthetic mechanism distinct from mammals. Genetic hybrids of nonpathogenic, sialate-negative laboratory Escherichia coli K-12 strains designed for the de novo synthesis of the polysialic acid capsule from E. coli K1 proved useful in elucidating the genetics and biochemistry of capsule biosynthesis. In this article we propose a dynamic model of sialometabolism to investigate the effects of biosynthetic neu (N-acetylneuraminic acid) and catabolic nan (N-acylneuraminate) mutations on the flux of intermediates through the sialate synthetic pathway. Intracellular sialate concentrations were determined by high pH anion exchange chromatography with pulsed amperometric detection. The results indicated that a strain carrying a null defect in the gene encoding polysialyltransferase (neuS) accumulated > 50 times more CMP-sialic acid than the wild type when strains were grown in a minimal medium supplemented with glucose and casamino acids. Metabolic accumulation of CMP-sialic acid depended on a functional sialic acid synthase (neuB), as shown by the inability of a strain lacking this enzyme to accumulate a detectable endogenous sialate pool. The neuB mutant concentrated trace sialate from the medium, indicating its potential value for quantitative analysis of free sialic acids in complex biological samples. The function of the sialate aldolase (encoded by nanA) in limiting intermediate flux through the synthetic pathway was determined by analyzing free sialate accumulation in neuA (CMP-sialic acid synthetase) nanA double mutants. The combined results demonstrate how E. coli avoids a futile cycle in which biosynthetic sialate induces the system for its own degradation and indicate the feasibility of generating sialooligosaccharide precursors through targeted manipulation of sialate metabolism.