The Expression Of NRPS/PKS In Three Different Hosts

Inonotus obliquus parasites on the birch tree trunk,its secondary metabolites are polyphenols and melanoids.Where melanin is a secondary metabolite that protects fungal cells from UV,oxidative and ionizing radiation.Previous studies have shown that the expression of the heterozygous nonribosomal peptide synthase-polyketide synthase(NRPS/PKS)is proportional to watersoluble melanin production.Therefore,the production of NRPS/PKS is proportional to the water-soluble melanin of Inonotus obliquus.In this study,we cloned the gene of NRPS/PKS,heterologous expressed in different hosts and obtained the corresponding protein,the protein as a catalyst for catalytic reaction,in vitro validation NRPS/PKS product coding for the Inonotus obliquus water-soluble melanin.And provide the experimental basis for the study of water-soluble melanin synthesis.Previous studies have obtain the recombinant strain of NRPS/PKS integrated into the WA site of Aspergillus nidulans.The product of the recombinant strain was fermented and extracted,and the free radical scavenging test was carried out with the product of wild betel leaf fungus as control.The results showed that the scavenging free radicals of the recombinant strain were similar to the control,but did not capture the corresponding protein.In order to further validate NRPS/PKS through in vitro experiments to produce water-soluble melanin,label the NRPS/PKS protein,and NRPS/PKS was expressed by fusion protein expression and segmentation expression respectively.The protein was used for in vitro enzyme promote reaction.First,the fusion expression vector was constructed by Saccharomyces cerevisiae.The fusion expression vector was transformed into Aspergillus nidulans by protoplast method and fused with the highly expressed 3'end of the glucoamylase gene of Aspergillus nidulans.The results showed that although the NRPS/PKS protein was not detected by Westernblot,the color of the fermentation broth changed significantly,and speculation the expression of NRPS/PKS protein was low.Secondly,according to the different domains of NRPS/PKS gene,the gene was divided into NRPS/PKS_NRPS and NRPS/PKS_PKS.The results showed that NRPS/PKS_NRPS fragment could express protein in E.coli BL21,while NRPS/PKS_PKS could express protein in Saccharomyces cerevisiae(BJ5464-NpgA).The expressed proteins were subjected to catalytic reactions in vitro,and the reaction product was analyzed by HPLC.It was found that the heterologous protein expression of two structural domain were active.The chemical structure of the product remains to be further research.