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 ;

【DOI】

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

Download this article

(Translated by LU R)

    References

    [1] Silva NC, Sarmento B, Pintado M. The importance of antimicrobial peptides and their potential for therapeutic use in ophthalmology [J]. International Journal of Antimicrobial Agents, 2013, 41 (1): 5–10

    [2] Guaní-Guerra E, Santos-Mendoza T, Lugo-Reyes SO, et al. Antimicrobial peptides: general overview and clinical implications in human health and disease [J]. Clinical Immunology, 2010, 135 (1): 1–11

    [3] Yong D, Toleman MA, Giske CG, et al. Characterization of a new metallo-β-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India [J]. Antimicrobial Agents and Chemotherapy, 2009, 53 (12): 5046–5054

    [4] Zhao DH, Dai ZY, Zhou KY. The research advances in function, mechanism and molecular biology of insect antibacterial peptides [J]. Progress in Biotechnology, 1999, 19 (5): 14–18 (in Chinese)

    [5] Boman HG, Nilsson I, Rasmuson B. Inducible antibacterial defence system in Drosophila [J]. Nature, 1972, 237 (5352): 232–235

    [6] Jin LL, Geng YJ, Wang QY. Overview of the membrane-associated methods of antibacterial mechanism of antimicrobial peptides [J]. Journal of Liaoning University (Natural Science Edition), 2014, 41 (4): 356–361 (in Chinese)

    [7] Jiang X, Ma G, Xu ZC, et al. Secreted expression and activity detection of two hybrid antimicrobial peptides in Pichia pastoris [J]. Journal of Sichuan University (Natural Science Edition), 2007, 44 (3): 673–678 (in Chinese)

    [8] Ding YC, Zhang SC. Antimicrobial peptides from marine invertebrates and fish [J]. Chinese Journal of Marine Drugs, 2013, 32 (6): 87–96 (in Chinese)

    [9] Yang LL, Wang B, Zhu HH, et al. Bacteriostatic effect of antibacterial peptide from Cipangopaludina cathayensis and the determination of physical and chemical properties and tumor-suppressor activity [J]. Food Research and Development, 2013, 34 (20): 5–8 (in Chinese)

    [10] Yin J. Primary studies on antibacterial activity of recombinant antibacterial peptide MgJ [J]. Journal of Jilin Institute of Chemical Technology, 2013, 30 (11): 7–9 (in Chinese)

    [11] Miao JY, Ke C, Guo HX, et al. Extraction, isolation and antibacterial mechanism of antibacterial peptides [J]. Modern Food Science & Technology, 2014, 30 (1): 233–240 (in Chinese)

    [12] Li GN, Xia XJ, Long YH, et al. Research progresses and applications of antimicrobial peptides [J]. Chinese Journal of Animal Nutrition, 2014, 26 (1): 17–25 (in Chinese)

    [13] Hancock REW, Diamond G. The role of cationic antimicrobial peptides in innate host defences [J]. Trends in Microbiology, 2000, 8 (9): 402–410

    [14] Yu SL, Wang YY, Xu ZY, et al. Research advance in application of antimicrobial peptide in aquaculture feed [J]. China Feed, 2014 (8): 31–34 (in Chinese)

    [15] Merrifield RB, Vizioli LD, Boman HG. Synthesis of the antibacterial peptide cecropin A (1-33) [J]. Biochemistry, 1982, 21 (20): 5020–5031

    [16] Chen XM, Li J, Sun HL, et al. High-level heterologous production and functional secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin [J]. Scientific Reports, 2017, 7: 14543

    [17] Yin N. Expression, purification and characterization in vitro of antibacterial peptide Cecropin D [D]. Beijing: Master’s Thesis of Union Medical College of China, 2008 (in Chinese)

    [18] Wang Z, Jiang JY, Zhu FM, et al. Constitutive expression of antibacterial peptide Cecropin AD gene in Pichia pastoris [J]. Chinese Journal of Animal Science, 2010, 46 (5): 51–54 (in Chinese)

    [19] Guo CH, Huang YM, Zheng HY, et al. Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris [J]. Experimental and Therapeutic Medicine, 2012, 4 (6): 1063–1068

    [20] Meng DM, Zhao JF, Ling X, et al. Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastoris [J]. Protein Expression and Purification, 2017, 130: 90–99

    [21] Wan J, Li Y, Chen DW, et al. Expression of a tandemly arrayed plectasin gene from Pseudoplectania nigrella in Pichia pastoris and its antimicrobial activity [J]. Journal of Microbiology and Biotechnology, 2015, 26 (3): 461–468

    [22] Chen X, Hu YH, Chen WD, et al. Comparison of inducible versus constitutive expression of plectasin on yields and antimicrobial activities in Pichia pastoris [J]. Protein Expression and Purification, 2016, 118: 70–76

    [23] Ma P, Xue LG, Shang H, et al. High-level expression in Pichia pastoris SMD1168 of misgurin and its antimicrobial activity [J]. Microbiology China, 2018, 45 (5): 970–980 (in Chinese)

    [24] Zhang QH, Wang Q, Shang TT, et al. Studies on antibacterial activity and hemolysis of novel hemoglobin antimicrobial peptide [J]. Progress in Veterinary Medicine, 2014, 35 (9): 54–57 (in Chinese)

    [25] Macauley-Patrick S, Fazenda ML, Mc Neil B, et al. Heterologous protein production using the Pichia pastoris expression system [J]. Yeast, 2005, 22 (4): 249–270

    [26] Chen X, Zhu FM, Cao YH, et al. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity [J]. Antimicrobial Agents and Chemotherapy, 2009, 53 (9): 3683–3689

    [27] Huang YD, Zheng Q, Li XK, et al. Modification of cecropin AD gene and its expression in Pichia pastoris [J]. Journal of South China University of Technology (Natural Science Edition), 2002, 30 (2): 13–16 (in Chinese)

This Article

ISSN:0253-2654

CN: 11-1996/Q

Vol 47, No. 05, Pages 1441-1451

May 2020

Downloads:2

Share
Article Outline

Abstract

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