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

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.