新型预苯酸脱氢酶基因的克隆和生物信息学分析

Cloning and Bioinformatics Analysis of a Novel Prephenate Dehydrogenase Gene from a Soil Metagenome

采用基于序列特异性直接测序策略,从所构建的碱性污染土壤宏基因组文库中分离得到一个编码预苯酸脱氢酶的新基因pdhE-1,并对新基因进行生物信息学分析和保守区域特征研究。结果表明,pdhE-1编码一个由287个氨基酸编码组成的多肽。在DNA水平上,该基因与来自新鞘氨醇杆菌属(Novosphingobiumsp.)的预苯酸脱氢酶相似性较高,为82%;在氨基酸水平上,与来自运动发酵单胞菌(Zymomonas mobilis)的预苯酸脱氢酶序列相似性为65%,一致性为42%。新型预苯酸脱氢酶基因pdhE-1的克隆和生物信息学分析研究为进一步完成酶基因的功能鉴定奠定了基础。

The metagenomic library technology provides an effective platform for the mining of unknown genes. A novel prephenate dehydrogenase gene designated as pdhE-1 was cloned by sequence-based screening of a plasmid metagenomic library from uncultured alkaline-polluted microorganisms. The gene had an open reading frame of 864 base pairs and encodes a 287 amino acid polypeptide with a predicted molecular mass of about 31 kDa. The deduced amino acid se- quence comparison and phyl0genetic analysis indicated that pdhE-1 and other putative prephen- ate dehydrogenase,were closely related. The possible ORF shares 82~ identical in the level of DNA with the prephenate dehydrogenase of Novosphingobium sp. PPIY and 420//oo identical and 650/40 similarity at amino acid level with the prephenate dehydrogenase of Zymomonas mobilis according to the BLAST program. The bioinformatic analysis above showed that the novel en- zymes can be isolated from soil metagenome. The findings indicated the advantage of met- agenomic library in cloning novel prephenate dehydrogenase genes through sequence-based screening using the E. coli host.