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Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid
Author(s) -
Mandawe John,
Infanzon Belen,
Eisele Anna,
Zaun Henning,
Kuballa Jürgen,
Davari Mehdi D.,
Jakob Felix,
Elling Lothar,
Schwaneberg Ulrich
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201800093
Subject(s) - pasteurella multocida , hyaluronic acid , glycosyltransferase , hyaluronan synthase , biochemistry , atp synthase , chemistry , protein engineering , enzyme , biology , bacteria , genetics
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.