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基因改组选育高产杆菌肽地衣芽孢杆菌及改组菌株差异分析
引用本文:贾 锐,张 充,吕凤霞,别小妹,赵海珍,陆兆新.基因改组选育高产杆菌肽地衣芽孢杆菌及改组菌株差异分析[J].食品科学,2016,37(23):135.
作者姓名:贾 锐  张 充  吕凤霞  别小妹  赵海珍  陆兆新
作者单位:南京农业大学食品科技学院,江苏 南京 210095
摘    要:为了提高地衣芽孢杆菌肽产量,采用全基因组改组技术进行菌株选育。以地衣芽孢杆菌TJ12为原始菌株,通过紫外诱变、亚硝基胍诱变、常压室温等离子体诱变构建了TJ12菌的突变体库。在优化其原生质体制备和再生条件的基础上,以其中4 株诱变菌株(U11、U23、H1、L71)作为亲本,采用聚乙二醇介导的方法进行3 轮多亲本的全基因组改组,同时,结合双亲灭活的筛选方法,最终选出1 株杆菌肽产量提高并能稳定遗传的优良菌株F3,对其摇瓶发酵36 h,杆菌肽A产量达760 mg/L,为野生菌株TJ12的1.7 倍。与野生菌株TJ12相比,改组菌株提前进入生长稳定期,两者发酵过程pH值几乎无差异;最终改组菌生长量虽低于野生菌,但菌株单位细胞还原糖产量及杆菌肽产量都高于野生菌株;其合成基因和调控基因表达量相对野生菌株都上调,其中合成基因上调幅度较大。推测改组菌株自身有了更强大的杆菌肽耐受机制,且合成基因相关部位可能发生了改变。

关 键 词:杆菌肽  地衣芽孢杆菌  基因组改组  诱变  原生质体  

Breeding and Comparative Analysis of High-Yield Bacitracin-Producing Bacillus licheniformis by Genome Shuffling
JIA Rui,ZHANG Chong,Lü Fengxia,BIE Xiaomei,ZHAO Haizhen,LU Zhaoxin.Breeding and Comparative Analysis of High-Yield Bacitracin-Producing Bacillus licheniformis by Genome Shuffling[J].Food Science,2016,37(23):135.
Authors:JIA Rui  ZHANG Chong  Lü Fengxia  BIE Xiaomei  ZHAO Haizhen  LU Zhaoxin
Affiliation:College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
Abstract:Genome shuffling technique was used for breeding of Bacillus licheniformis TJ12 for enhanced bacitracin production. Firstly, UV light, nitroso-guanidin (NTG) or atmospheric and room temperature plasma (ARTP) was used to obtain a candidate mutant library. Based on the optimized conditions for protoplast preparation and regeneration, multiparental whole genome shuffling was carried out with 4 mutant strains (U11, U23, H1, and L71) by polyethylene glycol (PEG) mediated protoplasts fusion. As a result, the shuffled strain F3 was identified by biparental inactivation method to show excellently improved antibacterial activity and good genetic stability. The maximum production of bacitracin A of 760 mg/L was obtained when the strain F3 was cultured in shake flasks for 36 h, which was increased 1.7 folds compared with the initial strain TJ12. The strain F3 moved earlier into the stable growth period, but its final biomass was lower than that of the wild strain. There was almost no difference in pH during the fermentation process. The production of reducing sugar and bacitracin by the strain F3 was higher compared with the wild strain. Additionally, the expression of regulatory genes was upregulated in the strain F3, and the expression of synthetic gene was upregulated more significantly. With a higher production of bacitracin in each cell of the strain F3, we speculated that the strain had a more powerful mechanism of self-tolerance to bacitracin and the relevant parts of the synthetic genes might be changed when compared with the initial strain.
Keywords:bacitracin  Bacillus licheniformis  genome shuffling  mutagenesis  protoplast  
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