从植物共生菌宏基因组文库筛选新的生物催化酶基因

Screening of new genes with biocatalytic potential from a plant microbiota metagenomic library

摘要:

【目的】通过建立宏基因组文库的高通量保存与基于探针洗脱的多次膜杂交筛选方法, 从植物共生菌宏基因组文库筛选具有生物催化潜力的新酶基因。【方法】首先根据滴度将初始文库噬菌体包装颗粒感染到EPI300?-T1R E. coli, 过夜培养后对应保存于96孔板; 提取粘粒进行文库的杂交筛选。【结果】描述的洗脱条件可完全去除尼龙膜上与靶DNA结合的探针, 并且尼龙膜上的靶DNA至少可用于7次探针杂交, 从而明显提高宏基因组文库的筛选效率。【结论】以Enoate reductase (ER)和短链脱氢酶(SDR)的同源基因片段为探针, 运用该方法经两轮筛选获得候选单克隆并进行了部分粘粒的测序, 发现了新的ER和SDR同源基因, 并克隆到相应的全长基因序列用于后续的表达与酶化学研究。

Abstract:

[Objective] We aim to find the new genes with biocatalytic potential from the plant microbiota metagenomic library by the means of high-throughput screening combined with multiple hybridizations based on probe stripping. [Methods] First, the phage particles were used to infect EPI300?-T1R E. coli cells according to the titer of the phage particles as a primary library. After incubation the mixture was then divided into aliquots of 96 and cultured in the medium overnight, followed by storage in 2-mL 96-well plates. The resulting fosmids were hybridized to screen the library for the new enzyme genes. [Results] We found a thoroughly removal of the probe by striping the nylon membrane as described here, and the target DNA on the nylon membrane can be used repeatedly for at least 7 times. All these resulted in a highly efficient means for storage and screening of the metagenomic library. [Conclusion] By using the enoate reductase and short-chain dehydrogenase (SDR) as probes, candidate fosmid clones were obtained after two cycles of screening. Based on fosmid sequence analyses, new homologues of enoate reductase and SDR were found and cloned for subsequent heterologous expression and enzymology.