Study On Key Genes Of Non-ribosomal Peptide Biosynthesis Gene Cluster Of Phaeodactylum Tricornutum

Iron is necessary for the life activities of organisms as a trace element.It usually exists in the form of chelate or ferric hydroxide in the ocean,which the iron content proportion is far below than in the earth's crust.In order to adapt to the low-iron environment in the ocean,marine diatoms have evolved a variety of adaptation strategies.Diatoms can synthesize and secrete siderophores in a low-iron environment to enhance iron absorption,and most siderophores are usually synthesized by non-ribosomal peptide synthetases?NRPSs?,a multimodule type of enzyme that responsible for the core skeleton of siderophores.The typical NRPSs consist of adenylation domain?A?,thiolation domain?T?,condensation domain?C?and thioesterase domain ?TE?.Phaeodactylum tricornutum as a representative diatom of pinnate diatoms,can be found in both the surface sea water and the deep sea.However,its mechanism of response to low iron stress was remains unclear.In the early stage of this research team,four potential NRPSs gene clusters were found in the genome of P.tricornutum through gene mining technology.Of all the four potential NRPSs gene clusters,the NRPS in the NRPS1 gene cluster had three domains of A-T-TE,and genes related to secondary metabolism such as Nit1 and NAT were found in both its up stream and down stream.What's more,the Nit2 gene was found on chromosome 20 of P.tricornutum which had 40%of sequence identity with the Nit1 gene.The NRPS1 and other key genes were cloned and heterologously expressed in E.coli.After purification,recombinant proteins of 140 k Da,40 k Da,40 k Da and 84 k Da were obtained.At the same time,in order to mad NRPS have catalysis,the sfp protein and coenzyme A are needed to convert inactive apo-type NRPS into active holo-type NRPS,so gene cloning and heterologous expression of sfp were also carried out to obtain a 32 k Da recombination protein.Then,with 20 amino acids,?-ketoglutarate,oxaloacetic acid and D-aspartic acid?-amide were tested to determine substrate specificity of recombinant His₆-NRPS1 adenosine domain.The results showed that L-aspartate was the most suitable substrate.Subsequently,using L-asparagine as a substrate,the enzymatic product of His₆-NRPS1 was explored,and the method of HPLC-MS was used to detect that there may be interference of the substance synthesized by E.coli.itself.The yeast will be used to express NRPS1 protein to explore enzymatic products that follow.With 20 kinds of amino acids were tested to determine substrate specificity of recombinant Nit1 and Nit2 by PLP and spectral scanning methods,and the results showed that they all use cysteine as the substrate.After pre-column derivatization,it was found by HPLC-Fluorescence that the substrate of NAT was L-asparagine and corresponding products were generated.Then the product was detected by HPLC-MS method,and it was also found that there may be the effect of E.coli's own synthetic products.The method of expressing recombinant protein in yeast will be followed to explore its enzymatic products.Through the study of the P.tricornutum NRPS1 and its surrounding key genes,it is helpful to explore the function of each gene in the synthesis of secondary metabolites,which provides a basis for exploring the potential siderophores synthesis pathways in diatoms.