Breeding of Brevibacillin Producing Strain by Genome Shuffling and Optimization of Culture Conditions

doi:10.13523/j.cb.2103022

China Biotechnology

2021, Vol. 41

Issue (8): 42-51 DOI: 10.13523/j.cb.2103022

Breeding of Brevibacillin Producing Strain by Genome Shuffling and Optimization of Culture Conditions

WANG Xiao-jie,MENG Fan-qiang,ZHOU Li-bang,LV Feng-xia,BIE Xiao-mei,ZHAO Hai-zhen,LU Zhao-xin(

)

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

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Abstract

Brevibacillin, a broad-spectrum antimicrobial peptide, has shown strong antibacterial effect on bacteria and fungi, which can be regard as a kind of potential substitute for antibiotics. The wild strain Brevibacillus laterosporus was carried out the conventional mutagenesis techniques including UV mutagenesis, atmospheric and room temperature plasma mutagenesis and nitrosoguanidine mutagenesis to obtain four mutant strains. After two rounds of genome shuffling, strain F₂-24 was obtained, and the yield of brevibacillin reached (340.5±16.35) μg/mL, which was 1.92-fold than that of wild strain fmb70. Furthermore, the fusion strain F₂-24 possessed great stability after five generations. Finally, the fermentation process of fusion strain F₂-24 was optimized. The results of the best carbon source, nitrogen source and inorganic salt ion was sucrose, beef extract and Mg ²⁺, respectively and the addition dose was 4%, 2% and 0.5%, respectively. The yield of brevibacillin was significantly enhanced via fermentation at 30℃ pH6.0 for 24 h, and meanwhile, the results suggested that the yield of brevibacillin was remarkably improved by a series of optimization and the yield reached (442.45±9.58) μg/mL, which was 2.50-fold than that produced by wild strain fmb70.

Key words： Brevibacillin Brevibacillus laterosporus Genome shuffling Medium optimization

Received: 15 March 2021 Published: 31 August 2021

ZTFLH:

Q933

Corresponding Authors: Zhao-xin LU E-mail: fmb@njau.edu.cn

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	Xiao-jie WANG
	Fan-qiang MENG
	Li-bang ZHOU
	Feng-xia LV
	Xiao-mei BIE
	Hai-zhen ZHAO
	Zhao-xin LU

Cite this article:

WANG Xiao-jie,MENG Fan-qiang,ZHOU Li-bang,LV Feng-xia,BIE Xiao-mei,ZHAO Hai-zhen,LU Zhao-xin. Breeding of Brevibacillin Producing Strain by Genome Shuffling and Optimization of Culture Conditions. China Biotechnology, 2021, 41(8): 42-51.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2103022 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I8/42

Table 1 HPLC detection program of brevibacillin

Table 2 Factors and levels of medium main ingredient in the orthogonal experiment

Table 3 Conditions and positive mutation rates of physical and chemical mutagenesis

Fig.1 Effects of different conditions on protoplast formation and regeneration (a) Lysozyme concentration (b) Treatment temperature (c) Treatment time

Fig.2 Effect of UV irradiation and heat treatment on the survival rate of protoplasts

Fig.3 Effects of different conditions on protoplast fusion rate (a) Molecular weight of PEG (b) PEG6000 concentration (c) pH (d) Ca²⁺ concentration (e) Fusion time Different lowercase letters express significant difference under the condition of P<0.05

Fig.4 Selection of fusants in the first round of genome shuffling

Table 4 Analysis of fusant yield stability in the second round of genome shuffling

Fig.5 Effect of different carbon sources on brevibacillin production (a) Types of carbon sources (b) Addition of sucrose Different lowercase letters express significant difference under the condition of P<0.05

Fig.6 Effect of different nitrogen sources on brevibacillin production (a) Types of nitrogen sources (b) Addition of beef extract Different lowercase letters express significant difference under the condition of P<0.05

Fig.7 Effect of different inorganic salt ions on brevibacillin production (a) Types of inorganic salt ions (b) Addition of Mg²⁺ Different lowercase letters express significant difference under the condition of P<0.05

Table 5 The results of medium main ingredient in the orthogonal experiment

Fig. 8 Effect of different fermentation conditions on brevibacillin production (a) Types of inorganic salt ions (b) Addition of Mg²⁺ Different lowercase letters express significant difference under the condition of P<0.05

Fig. 9 Fermentation validation of wild strain and fusant F₂-24 (a) Verification of fermentation yield by HPLC (b) Bacteriostatic circle of fermentation broth Different lowercase letters express significant difference under the condition of P<0.05


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