Codon optimization, constitutive expression and antimicrobial characterization of hen egg white lysozyme (HEWL) in <i>Pichia pastoris</i>

Abstract

Fusarium oxysporum (F. oxysporum) and Verticillium dahlia (V. dahlia) causes severe cotton disease in China and other cotton-producing countries. Hen egg white lysozyme (HEWL) has antimicrobial properties. In this study, a codon-optimized HEWL gene was synthesized and cloned into the yeast expression vector, pPIC9K, under the control of the Pichia pastoris (P. pastoris) glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). Results showed that codon-optimized HEWL (oHEWL) was constitutively expressed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the molecular weight of recombinant HEWL (roHEWL) was 14 kDa which corresponds to the standard HEWL. The expression of the roHEWL reached to 54 mg/L. Activity of the roHEWL was 1680 U/mL. The optimum pH for roHEWL was from 6.0 to 7.0, and the optimum temperature was 55°C. In vitro antimicrobial activity assay revealed that roHEWL can lyse cell walls of the gram positive bacteria, Micrococcus lysodeikticus (M. lysodeikticus). In vivo studies showed that it inhibits plant fungi, F. oxysporum and V. dahlia. roHEWL anti-fungal properties might be useful for future genetically engineered cotton plant resistance against pathogenic fungal disease.

Keywords: Hen egg white lysozyme (HEWL), antimicrobial activity, glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, codon optimization, constitutive expression