Cloning, Expression And Functional Idendification Of Sare0357, A Gene Of Adenylation Domain From Marine Actinomycete S.arenicola CNS-205

Nonribosomal peptide synthetases (NRPSs) are critical enzymes for the biosynthesis of nonribosomal peptide (NRP) compounds, a large class of biologically active natural products. Amino acid adenylation domains (A domains) are essential components within NRPSs and recognize specifically amino acid building blocks to be incorporated during the biosynthesis of NRPSs. Salinispora arenicola CNS-205was a firstly-isolated obligate marine actinomycete. Several bioactive metabolites, including NRP compounds, have been isolated from this strain. Existence of a high number of secondary metabolites biosynthetic clusters on its genome revealed its strong potential to accumulate more natural products.Here, a gene sare0357annotated as "amino acid adenylation domain" located on the genome of Salinispora arenicola CNS-205, was cloned and characterized. The recombinant target protein Sare0357was expressed efficiently but as inclusion bodies in a prokaryotic system. After denaturation and renaturation, enzymatic properties of the purified Sare0357were investigated using a newly-developed nonradioactive malachite green colorimetric assay in vitro.It was found that Sare0357could recognize specifically and activate tryptophan (Try) and phenylalanine (Phe). The basic kinetic parameters of it for these two substrates were detected as Km=0.04033±0.00367and0.02765±0.00151(mM), Vmax=2.13505±0.02943and1.55806±0.0097(μM/min) and kcat=14.2336±0.1962and10.3871±0.0646(min-1) for Try and Phe respectively.Biochemical characterization of Sare0357from the marine actinomycete Salinispora arenicola CNS-205elucidated its enzymatic activity and substrate specificity, supporting its involvement in Try/Phe-activation and-adenylation. Combining its location within a cryptic NRPS gene cluster, the present data will offer clear evidences to identify a new gene cluster for NRP metabolite biosynthesis in this strain. Moreover, its chemical features will allow to introduce it into the combinatorial biosynthesis of other NRP compounds in the future.