Molecular Detection of Cellulase -Encoding Gene in Thermophilic Streptomyces and Its Cloning in Escherichia coli Origami strain

Backgrounds: Streptomyces is an aerobic filamentous Gram-positive bacterium frequently found in various environments worldwide. Cellulases are a group of glycosyl hydrolase enzymes that are frequently produced by bacteria. Thus, the aim of this study was to detect cellulase-encoding gene (celA) in soil-living Streptomyces strains and evaluate its cloning in Escherichia coli Origami strain. Materials & Methods: Soil samples were collected from a depth of ۵-۱۰ cm in Tehran, Iran. After identification of Streptomyces isolates by morphological and biochemical tests, genomic DNA was extracted. Polymerase chain reaction (PCR) test was employed to identify Streptomyces strains harboring the cellulase gene. The celA gene was positively transmitted to the host bacterium E. coli via a vector and cloned through the TA technique. Real-time PCR was used to measure the overexpression of this gene. ClustalX and Mega۵ software were used to draw the phylogenetic tree. Findings: Out of ۱۲ Streptomyces isolates, ۲۵% were found to carry the celA gene. After cloning the celA gene, the cloned strains were chosen by colony selection (blue/white). The real-time PCR test showed the expression of the celA gene in the transformed strains. Phylogenetic analysis results using the neighbor-joining assay showed that Streptomyces spp. with ۸۱% bootstrap were located in the same clade, indicating their close relatedness. Conclusion: Soil is one of the high-potential sources of the production of secondary metabolites, which could be used as a valuable source of various biological products such as cellulase.

Keywords:

Streptomyces , Cellulase , Cloning , TA cloning