Effect Of Aminopeptidase N1 Expression In Streptomyces Lividans WJ2 On The Biosynthesis Of Blasticidin S

Blasticidin S?BS?is a peptide nucleoside antibiotic produced by Streptomyces griseochromogenes,it was the first antibiotic used on large scale in replace of mercurial to fight against rice blast because of its good fungicidal activity as well as the harmlessness to fishes.The final step of the BS biosynthesis pathway is the hydrolysis of leucylblasticidin S?LBS?to BS.The intact cells of BS native producer Streptomyces griseochromogenes and the heterologous producer Streptomyces lividans WJ2 can both catalyze this step.Earlier work showed that both strains encode three PepN homologues,of which PepN1 from each strain is mainly responsible for the hydrolysis of LBS,as well as leucyldemethylblasticidin S?LDBS?at a comparable catalytic efficiency.However,comparing with the native producer which only synthesizes BS,S.lividans WJ2 can also generate LBS and LDBS.This result indicates that the regulation of PepN1 in these two producers differs.To this end,PepN1 levels in both of cells cultured from 2^nd to 6^th day were compared by Western blot using PepN1 antibody.The results showed that the content of PepN1 in the native producer remained constant from the 2^nd to 6^th day,whereas PepN1 started to diminish from the 4^th day and totally disappeared on the 6^th day in the heterologous producer.The disappearance of PepN1 in S.lividans WJ2 may be due to being secreted into the culture medium or degraded by proteases.The proteins in the culture medium of the two strains were collected by ammonium sulfate precipitation and resuspended.The presence of PepN1 in culture medium was detected by Western blot and the LDBS hydrolytic activity was assayed in vitro.The results showed that PepN1was contained in the culture medium of BS native producer,but not in the S.lividans WJ2,demonstrating that BS native producer can secrete PepN1out of the cells.Bioinformatic analysis of all secretion systems in the genomes of S.griseochromogenes ATCC14511 and S.lividans TK24revealed 5 secretion proteins with low similarity in the two strains,and a secretion system protein only exist in S.griseochromogenes.The 6secretion system proteins in S.griseochromogenes were heterologously introduced into S.lividans WJ2.In 5 resultant mutants,PepN1 was detected in the culture medium,indicating that these secretion system proteins are involved in the secretion of PepN1.Analysis of the fermentation products of these mutants,the secretion of PepN1 into the culture medium facilitates the hydrolysis of LBS to BS.We then explored whether PepN1 is able to self-proteolytic degradation,western blot revealed that only a small amount of PepN1 was degradated spontaneously,and Mg²⁺had a stabilizing effect on PepN1.Proteases in cell free extracts can also cleave PepN1 weakly in a non-specific way.In general,PepN1sgr?derived from S.griseochromogenes?is more stable than PepN1sli?derived from S.lividans WJ2?.However,above two degradations of PepN1sli cannot account for the complete disappearance of PepN1sli in cells.The localization of PepN1 in cells was also studied using the fluorescent protein labeling method.In this study,the genes of encoding the red fluorescent protein mRuby3 and PepN1 were fused,and a strong promoter KasOp was added to enhance fusion gene expression.The fusion gene was introduced into S.griseochromogenes and S.lividans WJ2respectively.The distribution of PepN1 was investigated by observing the position of fluorescence at the cells.The results showed that the fluorescence was mainly located in the cells,indicating that PepN1 was located inside of the cell,and the distribution of PepN1 in the two strains is the same.Compared with S.griseochromogenes,the secretion system of S.lividans WJ2 can not secrete PepN1 to the outside of the cell.For the purpose of self-protection,the efficiency of hydrolysis of LBS into BS by S.lividans WJ2 cells is reduced,so the yield of BS is lowered and the fermentation product contains other intermediates such as LBS.