Functional analysis of bifidobacterial promoters in Bifidobacterium longum and Escherichia coli using the α-galactosidase gene as a reporter
Functional analysis of bifidobacterial promoters in Bifidobacterium longum and Escherichia coli using the α-galactosidase gene as a reporter
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DOI：
10.1016/j.jbiosc.2014.05.002
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发表时间：
2014-11-01
影响因子：
2.8
通讯作者：
Fukiya, Satoru
中科院分区：
文献类型：
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作者：
Sakanaka, Mikiyasu;Tamai, Saki;Fukiya, Satoru
双歧杆菌中的异源基因表达需要弱启动子、强启动子和诱导型启动子，具体取决于不同表达研究的目标。大肠杆菌中的弱启动子对于稳定的异源基因克隆也是理想的。在这里，我们使用长双歧杆菌α-半乳糖苷酶基因开发了一个报告系统，并研究了长双歧杆菌中七个双歧杆菌启动子的活性和诱导性以及它们在大肠杆菌中的活性。这些研究揭示了不同的启动子活性。三个启动子在长双歧杆菌中高度活跃，但在大肠杆菌中仅有轻微活性。其中，两个磷酸酮醇酶基因 (xfp) 启动子在葡萄糖上生长的长双歧杆菌细胞中表现出很强的活性。相比之下，果糖转运蛋白操纵子（fruERFG）的启动子活性受到除葡萄糖之外的碳水化合物（包括果糖、木糖和核糖）的强烈诱导。这些启动子将允许在双歧杆菌中强或高度诱导表达，并在大肠杆菌中实现稳定的基因克隆。与这些启动子的功能相反，蔗糖利用操纵子cscBA的启动子在大肠杆菌中表现出非常高的活性，但在长双歧杆菌中表现出低活性。其他三个启动子在长双歧杆菌和大肠杆菌中均具有功能。特别是，两个蔗糖磷酸化酶基因（scrP）启动子在长双歧杆菌中显示出蔗糖和棉子糖的诱导活性，表明它们适用于调节表达研究。本研究揭示的多种启动子功能将有助于双歧杆菌研究中异源基因的调控表达。 (C) 2014 年，日本生物技术协会。版权所有。
Heterologous gene expression in bifidobacteria requires weak, strong, and inducible promoters depending on the objectives of different expression studies. Weak promoters in Escherichia colt can also be desirable for stable heterologous gene cloning. Here, we developed a reporter system using the Bifidobacterium longum alpha-galactosidase gene and investigated the activity and inducibility of seven bifidobacterial promoters in B. longum and their activities in E. colt. These studies revealed diverse promoter activities. Three promoters were highly active in B. longum, but only slightly active in E. coli. Among these, two phosphoketolase gene (xfp) promoters exhibited strong activity in B. longum cells grown on glucose. In contrast, the promoter activity of the fructose transporter operon (fruERFG) was strongly induced by carbohydrates other than glucose, including fructose, xylose, and ribose. These promoters will allow strong or highly inducible expression in bifidobacteria and stable gene cloning in E. con. In contrast to the functions of these promoters, the promoter of sucrose-utilization operon cscBA showed very high activity in E. coli but low activity in B. longum. Other three promoters were functional in both B. longum and E. con. In particular, two sucrose phosphorylase gene (scrP) promoters showed inducible activity by sucrose and raffinose in B. longum, indicating their applicability for regulated expression studies. The diverse promoter functions revealed in this study will contribute to enabling the regulated expression of heterologous genes in bifidobacteria research. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.