Cloning and Heterologous Expression of Cyanobacterial Genes Encoding Phosphinothricin N- acetyltransferase in E. coli

Phosphinothricin N-acetyltransferase (Pat) is a bacterial enzyme that is introduced into plants to confer resistance to herbicides containing phosphinothricin (PPT). The expression of the Pat protein in transgenic plants allows them to tolerate the herbicide glufosinate. Although the enzyme has been identified in cyanobacteria through sequence analysis, its activities have not yet been thoroughly investigated. In this study, we cloned the putative Pat enzyme-encoding genes alr4468 and sll1647 from Anabaena sp. PCC7120 and Synechocystis sp. PCC6803, respectively, into the His-Tagged pET28a+ expression vector. The recombinant vectors containing these genes were introduced into protease-free E. coli BL21 cells. We successfully expressed the Alr4468 and Sll1647 polypeptides in the recombinant E. coli BL21 cultures that were induced with 1 mM IPTG. The highest in vitro Pat activities were measured using enzyme assays from IPTG-induced cell crude extracts, showing increases of 3.47 times for rAlr4468 and 2.53 times for rSll1647 compared to controls. As a result, the alr4468 and sll1647 genes from cyanobacteria, which exhibit similar physiological properties to those of high-structured plants, may serve as potential sources for developing herbicide-resistant transgenic plants in future research.