Comparison of Inducible Versus Constitutive Expression of Plectasin on Yields and Antimicrobial Activities in Pichia Pastoris

Background: Plectasin might serve as a substitute for traditional antibiotics, but its yields and antimicrobial activities warrant further investigation.Objective: To identify the influence of inducible versus constitutive expression of plectasin on yields and antimicrobial activities.Methods: Through SOE-PCR, a recombinant plectasin gene was generated and inserted into inducible (pPICZ alpha A) and constitutive (pGAPZ alpha A) vectors in order to create Pichia pastoris GS115 strains. After 120 h of fermentation, supernatants were purified by an AKTA purifier using nickel columns. Minimal inhibitory concentration (MIC) and inhibition zone assays were performed after Tricine-SDS-PAGE.Results: After 120 h of fermentation, the yield of constitutive plectasin (370 mu g/ml) was much lower than that from inducible vector (880 mu g/ml) (P < 0.05). However, constitutive strain reached its plateau phase faster and keep more consistent yield (P < 0.05). The MICs of inducible plectasin against Methicillin-resistant Staphylococcus aureus (MRSA) 15471118, vancomycin-resistant Enterococcus feces (VREF), and penicillin-resistant Streptococcus pneumonia (PRSP) 31355 were 64, 32, and 64 mu g/ml, respectively, while those of constitutive plectasin were 4, 4, and 16 mu g/ml. No significant differences were observed in antimicrobial activities between inducible and constitutive plectasin for MRSA 15471118, VREF and PRSP 31355 (all P > 0.05). However, constitutive plectasin had a larger inhibition zone than inducible plectasin with the same mass.Conclusions: Although P. pastoris GS115 (pGAPZ alpha A-Plectasin-GS115) had lower expression than P. pastoris GS115 (pPICZ alpha A-plectasin-GS115), it reached the plateau phase faster, had steadier yields and showed superiority in antimicrobial activities. Therefore, pGAPZ alpha A might be more suitable for expression of plectasin in GS115 compared with pPICZ alpha A. (C) 2015 Elsevier Inc. All rights reserved.