Multicopy integrative expression and expression optimization of cecropin AD in Pichia pastoris

NIU Jun-Bo1,2 XUE Lin-Gui1 WU Juan-Li1 CHEN Xi-Ming2 LI Juan2,3 WANG Xia1 LIU Yang2,4 Emaneghemi Brown1

(1.School of Chemical and Biological Engineering, Lanzhou Jiaotong University, Lanzhou, Gansu, China 730070)
(2.Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou, Gansu, China 730000)
(3.University of Chinese Academy of Sciences, Beijing, China 100049)
(4.Northwest Normal University, Lanzhou, Gansu, China 730070)

【Abstract】[Background] Cecropin is an extensively studied effective antimicrobial peptide, which has the potential of commercial production for the application in aquaculture and agriculture. [Objective] To obtain a genetically engineered strain for efficient production of cecropin AD. [Methods] The recombinant vector pGAPZαA-CAD was firstly constructed and then transformed to P. pastoris X33 strain by electric shock. Cecropin AD gene was successfully expressed and X33/GCAD strain was obtained. Secondly, the recombinant vector pUCGAP-CAD was constructed and transformed into X33/GCAD strain. The pGAPZαA-CAD plasmid was integrated into the GAPDH promoter region of P. pastoris X33 with Zeocin as resistance screening label, while pUCGAP-CAD plasmid was integrated into the non-translated rDNA region of P. pastoris X33 with geneticin as resistance screening label. Finally, a recombinant X33/GUCAD strain with an efficiently expressed cecropin AD gene was obtained. [Results] The antimicrobial compound of X33/GUCAD was cecropin AD which was identified by mass spectrometry. Afterward optimizing its fermentation conditions, we found that the X33/GUCAD strain had a higher potential to express cecropin AD by consuming glycerol as a sole carbon source while peptone and yeast extract as organic nitrogen source. [Conclusion] The higher copy number is more beneficial to the increase of cecropin AD titer, and the engineered strain is more stable in the later fermentation process and suitable for industrial production.

【Keywords】 Cecropin AD; Genetically engineered bacteria; Biological value ;


【Funds】 National Natural Science Foundation of China (31860163) Project of Key Laboratory of Extreme Environmental Microbial Resources and Engineering of Gansu Province (EEMRE201603)

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(Translated by LU R)


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This Article


CN: 11-1996/Q

Vol 47, No. 05, Pages 1441-1451

May 2020


Article Outline


  • 1 Materials and methods
  • 2 Results
  • 3 Discussion and conclusion
  • References