Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid

Abstract

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

Key Factors for a One-Pot Enzyme Cascade Synthesis of High Molecular Weight Hyaluronic Acid

“…For example, sucrose enhances the stability of some enzymes significantly [31]. Protein engineering could also help to improve stability or other characteristics as it was recently shown that a PmHAS variant with better activity and chain length specificity was created [57].…”

KnowVolution of a Fungal Laccase toward Alkaline pH

“…As a result of the balance between screening efforts and gained molecular understanding, KnowVolution enables the tailoring of protein properties with minimal experimental efforts through the integration of directed evolution and computational analysis in four phases. More than ten KnowVolution success stories have been reported for diverse protein properties, for instance, to increase cellobiose activity of an aryl sulfotransferase, to improve the polypropylene binding of a polymer‐binding peptide, or to improve the chain‐length specificity of a hyaluronic acid synthase . Herein, we report the first laccase KnowVolution campaign toward increased tolerance at alkaline pH (pH≥9.0).…”

The beauty of biocatalysis: sustainable synthesis of ingredients in cosmetics

“…474 Hyaluranon synthase has also be engineered for improved turnover and an increase in the molecular weight of the acid produced. 475…”