The sialic acids Neu5Ac and Neu5Gc are common components of mammalian glycoproteins, glycolipids and milk oligosaccharides. Of the two, humans only synthesize Neu5Ac, but they can incorporate Neu5Gc from dietary sources such as red meats and dairy into tissues, eliciting an immune response which leads to inflammation and increased risk of carcinomas. The disease relevance and biological importance of sialic acids and sialoglycans make them important targets for research, but they are often expensive to purchase or difficult and time-consuming to synthesize. Adding further complexity is the diversity of linkages (a2-3, a2-6, a2-8) and underlying asialoglycans (Galβ, GalNAcβ, Lewis X, etc.) which modulate the biological impact of both Neu5Ac and Neu5Gc sialoglycans. Purified bacterial enzymes combined in a one-pot multi-enzyme (OPME) system are an efficient way to synthesize sialic acids and sialoglycans with specific desired linkages and structures. Our collaborators and other researchers have previously developed and characterized recombinant enzymes for sialic acid synthesis and sugar activation and numerous sialyltransferases and glycosyltransferases capable of generating a wide variety of complex structures. Here, we demonstrate multi-gram-scale production and purification of a bacterial sialic acid aldolase in <i>E. coli</i> and its application in OPME systems for efficient chemoenzymatic sialoglycan synthesis. All chemicals were purchased from MilliporeSigma, Carbosynth, ThermoFisher or Cayman Chemicals. Recombinant enzymes were expressed with polyhistidine tag and purified by IMAC on an Akta FPLC (Cytiva). Enzymes were assayed using HPAEC-UV on a Vanquish UPLC (Thermo). Recombinant expression of the bacterial sialic acid aldolase by fed-batch fermentation improved expression yields to over 7 g/L. By combining the aldolase with a CMP-sialic acid synthetase and a pyrophosphatase, we demonstrate the production of CMP-Neu5Ac and CMP-Neu5Gc. Using stereo- and regioselective bacterial sialyltransferases, we additionally demonstrate the OPME synthesis of sialosides bearing Neu5Ac and Neu5Gc using the substrate analog LacβMU. We expect that these enzymes, alongside others currently in development, will provide cost-effective means for the research community to synthesize a wide array of rare and complex sialoglycans.

Commercial production of recombinant sialic acid aldolase for one-pot multi-enzyme (OPME) synthesis of Neu5Gc-containing sialosides

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