利用基因组改组技术提高短杆菌素产量及其培养条件优化<sup>*</sup>

doi:10.13523/j.cb.2103022

中国生物工程杂志

2021, Vol. 41

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

技术与方法

利用基因组改组技术提高短杆菌素产量及其培养条件优化^*

王晓洁,孟凡强,周立邦,吕凤霞,别小妹,赵海珍,陆兆新(

)

南京农业大学食品科学技术学院南京 210095

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

全文: PDF(1959 KB) HTML

摘要：

短杆菌素是一种广谱抗菌肽,对细菌和真菌均有较好的抑制作用,具有潜在的抗生素替代价值。通过对侧孢短芽孢杆菌fmb70进行紫外诱变、亚硝基胍诱变、常压室温等离子体诱变,获得3株短杆菌素产量提高的诱变菌株。随后以诱变菌株为亲本进行两轮基因组改组,获得融合子F₂-24,其短杆菌素产量为(340.5±16.35) μg/mL,是野生菌株fmb70短杆菌素产量的1.92倍。融合子传代5代后,该菌株短杆菌素产量无明显差异,说明菌株稳定性良好。最后对该菌株产短杆菌素的培养基和发酵条件进行优化,优化后的培养基为:4%蔗糖、2%牛肉膏、0.5%氯化镁,发酵温度30℃、培养24 h、培养基初始pH6.0。优化后的短杆菌素产量可达(442.45±9.58)μg/mL,是初始培养条件的2.50倍。

关键词： 短杆菌素; 侧孢短芽孢杆菌; 基因组改组; 培养基优化

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

收稿日期: 2021-03-15 出版日期: 2021-08-31

ZTFLH:

Q933

基金资助: * 国家自然科学基金资助项目(31771948)

通讯作者: 陆兆新 E-mail: fmb@njau.edu.cn

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引用本文:

王晓洁,孟凡强,周立邦,吕凤霞,别小妹,赵海珍,陆兆新. 利用基因组改组技术提高短杆菌素产量及其培养条件优化^*[J]. 中国生物工程杂志, 2021, 41(8): 42-51.

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.

链接本文:

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

表1 HPLC检测短杆菌素程序

表2 培养基主要成分正交试验因素水平

表3 传统理化的条件及正突变率

图1 不同条件对原生质体形成与再生的影响

图2 紫外线照射和热处理对原生质体存活率的影响

图3 不同条件对原生质体融合率的影响

图4 第一轮基因组改组融合子的筛选

表4 第二轮基因组改组融合子产量稳定性分析

图5 不同碳源对短杆菌素产量的影响

图6 不同氮源对短杆菌素产量的影响

图7 不同无机盐离子对短杆菌素产量的影响

表5 培养基主要成分正交试验结果

图8 不同发酵条件对短杆菌素产量的影响

图9 野生菌株与融合子F2-24的发酵验证

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