Isolation of novel Pseudomonas syringae promoters and functional characterization in polyhydroxyalkanoate-producing pseudomonads
Isolation of novel Pseudomonas syringae promoters and functional characterization in polyhydroxyalkanoate-producing pseudomonads
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DOI：
10.1016/j.nbt.2009.12.003
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发表时间：
2010-02-28
影响因子：
5.4
通讯作者：
Swingle, Bryan M.
中科院分区：
文献类型：
--
作者：
Solaiman, Daniel K. Y.;Swingle, Bryan M.
构建了一个假单胞菌致病变种基因组DNA片段文库。番茄DC 3000构建在含lacZ α的质粒pBS 29中。该文库用于初步的基于α互补的筛选，以鉴定在大肠杆菌中具有启动子活性的克隆。对10个阳性克隆进行了测序，并对它们在DC 3000株染色体DNA中的位置进行了定位。进一步测定了五个阳性克隆（P2、P3、P4、P6和P8）在三种产生多羟基烷酸盐的假单胞菌（树脂噬假单胞菌、波纹假单胞菌和绿针假单胞菌）中的启动子活性。为此，绿色荧光蛋白基因（gfp）克隆下游推定（DC 3000）启动子在穿梭质粒。我们发现，只有假单胞菌转化窝藏的gfp-含有质粒驱动的推定启动子P2显示荧光，表明该启动子是在三个测试的假单胞菌的功能。P2序列的计算机分析结果进一步支持P2作为真正启动子的分配，通过在该序列中定位推定的-10和-35启动子区域以及转录因子结合位点rpoD 17。利用启动子P2成功地驱动天蓝色链霉菌α-半乳糖苷酶基因在绿针假单胞菌中表达，为利用大豆糖蜜低聚糖生产聚羟基脂肪酸酯生物聚合物或鼠李糖脂生物表面活性剂奠定了基础。
A library of genomic DNA fragments of Pseudomonas syringae pv. tomato DC3000 was constructed in a lacZ alpha-containing plasmid, pBS29. The library was used in a preliminary alpha-complementation-based screen to identify clones with promoter activity in Escherichia coli. Ten positive clones were sequenced and their locations in the chromosomal DNA of DC3000 strain were mapped. Five positive clones (P2, P3, P4, P6 and P8) were further assayed for promoter activity in three polyhydroxyalkanoate-producing pseudomonads: Pseudomonas resinovorans, P. corrugata and P. chlororaphis. To this end, a green-fluorescent-protein gene (gfp) was cloned downstream from the putative (DC3000) promoter in a shuttle plasmid. We found that only Pseudomonas transformants harboring the gfp-containing plasmid driven by putative promoter P2 showed fluorescence, indicating that this promoter is functioning in the three tested pseudomonads. Results of an in silico analysis of the P2 sequence further support the assignment of P2 as a bona fide promoter by the localization of putative -10 and -35 promoter regions and a transcription-factor-binding site, rpoD17, in this sequence. We successfully applied promoter P2 to drive the expression in P. chlororaphis of a recombinant alpha-galactosidase gene of Streptomyces coelicolor, which should be useful for the utilization of oligosaccharides of soy molasses for the production of polyhydroxyalkanoate biopolymer or rhamnolipid biosurfactant.