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解淀粉芽孢杆菌基因组改组提高抗菌脂肽产量及其相对基因表达的 FQ RT-PCR 分析。

Genome shuffling of Bacillus amyloliquefaciens for improving antimicrobial lipopeptide production and an analysis of relative gene expression using FQ RT-PCR.

机构信息

College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095 Jiangsu, People's Republic of China.

出版信息

J Ind Microbiol Biotechnol. 2012 Jun;39(6):889-96. doi: 10.1007/s10295-012-1098-9. Epub 2012 Feb 17.

Abstract

Genome shuffling is an efficient approach for the rapid improvement of the yield of secondary metabolites. This study was undertaken to enhance the yield of surfactin produced by Bacillus amyloliquefaciens ES-2-4 using genome shuffling and to examine changes in SrfA expression of the improved phenotype at the transcriptional level. Six strains with subtle improvements in lipopeptide yield were obtained from populations generated by ultraviolet irradiation, nitrosoguanidine, and ion beam mutagenesis. These strains were then subjected to recursive protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant F2-38 strain that exhibited 3.5- and 10.3-fold increases in surfactin production in shake flask and fermenter respectively, was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR. Delta CT (threshold cycle) relative quantitation analysis revealed that surfactin synthetase gene (srfA) expression at the transcriptional level in the F2-38 strain was 15.7-fold greater than in the ES-2-4 wild-type. The shuffled strain has a potential application in food and pharmaceutical industries. At the same time, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.

摘要

基因组改组是提高次生代谢产物产量的有效方法。本研究旨在通过基因组改组提高解淀粉芽孢杆菌 ES-2-4 产生表面活性剂的产量,并在转录水平上研究改进表型中 SrfA 表达的变化。从紫外线照射、亚硝基胍和离子束诱变产生的群体中获得了 6 株在脂肽产量上有细微提高的菌株。然后,这些菌株被进行递归原生质体融合。通过融合紫外线照射和热处理获得的致死原生质体,创建了一个可能产生阳性菌落的菌株文库。经过两轮基因组改组,获得了一个高产重组 F2-38 菌株,该菌株在摇瓶和发酵罐中的表面活性剂产量分别提高了 3.5 倍和 10.3 倍。使用 FQ(荧光定量)RT-PCR 对初始菌株和改组菌株的合成酶基因表达进行了比较分析。Delta CT(阈值循环)相对定量分析表明,F2-38 菌株表面活性剂合成酶基因(srfA)的转录水平表达是 ES-2-4 野生型的 15.7 倍。改组菌株在食品和制药工业中有潜在的应用。同时,改进表型的分析将为反向代谢工程提供更有价值的数据。

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