Engineer P. multocida Heparosan Synthase 2 (PmHS2) for Size-Controlled Synthesis of Longer Heparosan Oligosaccharides.

Pasteurella multocida heparosan synthase 2 (PmHS2) is a dual-function polysaccharide synthase having both α1-4-N-acetylglucosaminyltransferase (α1-4-GlcNAcT) and β1-4-glucuronyltransferase (β1-4-GlcAT) activities located in two separate catalytic domains. We found that removing PmHS2 N-terminal 80-amino acid residues improved enzyme stability and expression level while retaining its substrate promiscuity. We also identified the reverse glycosylation activities of PmHS2 which complicated its application in size-controlled synthesis of oligosaccharides longer than hexasaccharide. Engineered Δ80PmHS2 single-function-glycosyltransferase mutants Δ80PmHS2_D291N (α1-4-GlcNAcT lacking both forward and reverse β1-4-GlcAT activities) and Δ80PmHS2_D569N (β1-4-GlcAT lacking both forward and reverse α1-4-GlcNAcT activities) were designed and showed to minimize side product formation. They were successfully used in a sequential one-pot multienzyme (OPME) platform for size-controlled high-yield production of oligosaccharides up to decasaccharide. The study draws attention to the consideration of reverse glycosylation activities of glycosyltransferases, including polysaccharide synthases, when applying them in the synthesis of oligosaccharides and polysaccharides. The mutagenesis strategy has the potential to be extended to other multifunctional polysaccharide synthases with reverse glycosylation activities to generate catalysts with improved synthetic efficiency.