Cloning, Expression And Enzyme Assay Of Amino Acid Adenylation Domains From Marine Actinomycete

Nonribosomal peptides (NRPs) represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during NRP biosynthesis. Salinispora arenicola CNS-205is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters.In order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, sareO357and sare0718annotated "putative amino acid adenylation domain", were amplified from the genomic DNA by PCR. Firstly, the general features and possible functions of the two genes were predicted by bioinformatics analysis. Subsequently, a series of prokaryotic expression plasmids were constructed, including pET23a-sare0357, pET2Sa-sare0357and pET42a-sare0357, as well as pE723a-sare0718, pET28a-sare0718, pET42a-sare0718and pGEX-2T-sare0718. E.coli BL21(DE3) competent cells were then transformed with the above recombinant plasmids, respectively. After induction with IPTG, His-tagged recombinant proteins of Sare0357and Sare0718were expressed in E. coli BL21(DE3) mainly as Inclusion body whereas the GST-tagged Sare0718fusion protein was largely in the supernatant. All the recombinant proteins were purified with Affinity chromatography and verified by Western blotting. At the end, enzyme assays were performed to explore the adenylation activity of Sare0718in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that, among the20proteinogenic amino acids, Sare0718could in vitro specially recognize and activate L-alanine under the consumption of ATP. The basic kinetic parameters of Sare0718for L-alanine are Km=0.1164±0.0159(mM), Vmax=3.1484±0.1278(μM/min), kcat=12.5936±0.5112(min-1).In summary, this dissertation reported the molecular cloning and protein expression of sare0357and sare0718. By revealing the biochemical role of sare0718gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic NRPS-related gene cluster covering sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on.