Study On Maturation Mechanism Of Blasticidin S And Improvement Of Its Yields

As a serious rice disease,rice blast can infect leaves,stems,stalks and roots of rice at a fast spreading velocity,and it’s impossible to completely eradicate it once an area is infected.Blasticidin S(BS)was the first non-mercurial fungicide that has good inhibitory effects on mould and yeast,and thus applied on a large scale in replace of organomercury pesticides in Japan to prevent rice blast.Currently BS is widely used as a selection agent in transgenic studies.Mark Zabriskie’s team has cloned and sequenced the biosynthetic gene cluster and proposed a biosynthetic pathway,but few studies on its relative functional genes since the genetic manipulation in the native producer is very difficult.Intermediate metabolites of demethylblasticidin S(DBS),leucyldemethylblasticidin S(LDBS)and leucylblasticidin S(LBS)were detected in the BS fermentation broth in certain conditions.Leucylation of DBS and/or BS was proved to be a novel self-resistance as LBS and LDBS displayed greatly reduced cytotoxicity.This study aims to find the enzyme(s)responsible for hydrolysis of LBS to final product,and elucidate the regulation of loading and removal of the leucine during the biosynthetic pathway.After tremendous failures in cloning of gene coding for LBS hydrolysis near BS gene cluster,we accidentally observed that CFE(cell free of extract)of both fungus and bacteria can hydrolyze LBS to BS.It was proved not to be spontaneous but a process governed by the conserved LBS hydrolase across various organisms.Through tracking of LBS hydrolytic activity in fractional precipitation of proteins,we identified 5 candidates of LBS hydrolases based on LC-MS/MS assay,and finally determined the Pep N as the sole LBS hydrolase in E.coli.In vivo and in vitro results showed that Pep N hydrolyzes LBS in a p H-sensitive way with most appropriate of p H 7~8 but is inactive when the p H is below 5 or above 10.This observation in part explained the variation in both of BS yield and ratio of LBS to BS in different fermentations of S.lividans WJ2.Pep N has three homologs here named as Pep N1,Pep N2 and Pep N3 both in the BS heterologous producer S.lividans WJ2 and the native producer S.griseochromogenes.In vitro results showed that all of them have LBS hydrolytic activities and Pep N1 is 102 to 104 times greater than Pep N2 and Pep N3.Likewise,the transcription data of Streptomyces lividans also indicated that pep N1: pep N2: pep N3=31:11:1,which confirmed Pep N1 was the highest one of them.Thus,Pep N1 was proved to be the principal hydrolase for LBS hydrolyzing in S.lividans WJ2 and native producer S.griseochromogenes.Overexpression of pep N1 in WJ2 enhanced BS yield by 100% and lowered the ratio of LBS to BS from 2:1 to 2:3.To further investigate the roles of Pep N1,Pep N2 and Pep N3 in maturation of blasticdin S,seven WJ2 mutants with pep N1,pep N2 and pep N3 disrupted individually and assembly have been produced.The LBS hydrolytic efficiency of their CFE and cell again supported the conclusion of Pep N1 was the principal hydrolase for LBS hydrolyzing,while Pep N3 located in the cytoplasm tended to hydrolyze LDBS.Surprisingly,the pep N1-N3 triple-mutant S.lividans YGY14 produces blasticidin S and leucylblasticidin S at a titer and ratio like that by WJ2 in the fermentation medium,demonstrating there exist cryptic hydrolase(s)in addition to Pep N1-N3.We later found that the weak hydrolytic activities can be enhanced by 1 to 3 times with the addition of Mg2+,K+,Fe2+ that are present in the fermentation medium.Using similar strategy for purification of Pep N in E.coli,24 cryptic hydrolases candidates had been isolated and determination of their role in BS maturation is underway.LBS was isolated from S.griseochromogenes in p H<4 condition,whereas variable LBS is coupled with BS production in WJ2.Besides,the hydrolyzing efficiency of LBS by the native producer is greater than that by WJ2.However,comparison of in vitro and in vivo activities of the principal hydrolase of Pep N1 showed that Pep N1 in WJ2 is like the native producer.Decay assay showed that Pep N1 from both strain are still stable after 30 days incubation at 37℃.Monitoring of p H value of fermentation broth showed that the native strain always in the optimal p H of 7~8 condition.Pep N1 in the native strain was highly expressed in the late fermentation process.Thus,the efficiency of LBS maturation and the yield of BS in the native strain is higher.The low productivity of BS makes WJ2 a potential host to study the BS enhancement,each of BS biosynthetic genes was overexpressed under the control of promoter of erm E* in the WJ2 to find out the limitation step for BS biosynthesis.In parallels,the genes responsible for precursor and/or intermidate supply,such as cytosine,UDPglucuronic acid,Leu-t RNALeu and LDBS were also overexpressed.The yield of BS has been improved from 2.02 g/L to 2.84 g/L in shake flask fermentation by overpressing Bld A and leucyl-t RNA synthase,which provided rational guidance for the industrial production of BS.