Identification of an Endogenous Strong Promoter in Burkholderia sp. JP2-270

文献类型： 外文期刊

第一作者： Ke, Jing

作者： Ke, Jing;Shen, Jiamin;Wang, Haoran;Zhang, Xinxin;Wang, Yucong;Chen, Guoqing;Feng, Guozhong

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关键词： Burkholderia sp. JP2-270; pyrrolnitrin; promoter; overexpression; RNA-Seq

期刊名称：MICROORGANISMS （ 影响因子：4.2； 五年影响因子：4.6 ）

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年卷期： 2024 年 12 卷 9 期

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收录情况： SCI

摘要： Burkholderia is the second largest source of natural product bacteria after Actinomyces and can produce many secondary metabolites including pyrrolnitrin (PRN). Natural products of microbial origin are usually found in trace amounts, so in metabolic engineering, promoter engineering is often used to regulate gene expression to increase yield. In this study, an endogenous strong promoter was identified based on RNA-seq to overexpress biosynthetic genes to increase the production of PRN. By analyzing the transcriptomic data of the antagonistic bacterium Burkholderia sp. JP2-270 in three different development periods, we screened 50 endogenous promoters with high transcriptional activity, nine of which were verified by an obvious fluorescent signal via fluorescence observation. Then, combined with RT-qPCR analysis, P-hp, the promoter of a hypothetical protein, was found to be significantly expressed in all three periods. In order to increase the suitability of endogenous promoters, the promoter P-hp was shortened at different lengths, and the results show that a sequence length of 173 bp was necessary for its activity. Moreover, this promoter was used to overexpress the PRN biosynthesis genes (prnA, prnB, prnC and prnD) in JP2-270, resulting in a successful increase in gene expression levels by 40-80 times. Only the overexpression of the prnB gene successfully increased PRN production to 1.46 times that of the wild type. Overall, the endogenous strong promoters screened in this study can improve gene expression and increase the production of secondary metabolites in JP2-270 and other strains.

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