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 (pPICZαA) and constitutive (pGAPZαA) 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 μg/ml) was much lower than that from inducible vector (880 μg/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 μg/ml, respectively, while those of constitutive plectasin were 4, 4, and 16 μg/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 (pGAPZαA-Plectasin-GS115) had lower expression than P. pastoris GS115 (pPICZαA-plectasin-GS115), it reached the plateau phase faster, had steadier yields and showed superiority in antimicrobial activities. Therefore, pGAPZαA might be more suitable for expression of plectasin in GS115 compared with pPICZαA.
Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China.
Xin
X
Chen
Biosafety Level-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China.
Yong-hao
YH
Hu
Biosafety Level-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China.
Wei-di
WD
Chen
Biosafety Level-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China.
Wen-di
WD
Li
Biosafety Level-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China.
Zi-chao
ZC
Huang
Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China.
Yun
Y
Li
Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China.
Yu-wen
YW
Luo
Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China.
Yu-xia
YX
Huang
Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China.
Yi-tai
YT
Chen
Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China.
Kai
K
Wang
Biosafety Level-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. Electronic address: liling@smu.edu.cn.
Ling
L
Li
D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D010843:Pichia / Q000145:classification / Q000235:genetics* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D010455:Peptides; C504857:plectasin
Antimicrobial activity; Constitutive expression; High level expression; Inducible expression; Plectasin