Study On The Regulation Of Biosynthesis Of Piracetin

Bitespiramycin(BT)has completed the phase III of clinical trial,and 4"-isovaleryl group was verified to increase the drug distribution in tissue and antibacterial activity in vivo,as well extend the half-life of antibiotics.Clinical trials showed that BT had a very significant effect on upper respiratory tract infection caused by bacteria and the feature of low oral dose with few adverse reactions.A comprehensive study of the bitespiramycin biosynthetic genes can provide help for rationally increasing the production of bitespiramycin.By means of analysis of the genomic data of BT producing strain,we characterized the biosynthesis gene cluster of BT.There were four endogenous regulatory genes and an exogenous regulatory gene,acyB2,in the gene cluster.orf23 and orf42 were homologous to srm22 and srm40 from S.ambofaciens respectivly.srm22 and srm40 were identified to play positive regulator in the biosynthesis of spiramycin and AcyB2 also had 69%identities with Srm40 by Blast program.Cloning and expression of acyB2?srm40 and srm22 in E.coli,we only obtained soluble purified Srm22 protein.Srm22 could bind the promotors of acyB2 and srm40 instead of binding its own promoter and other structural genes' promotors by EMSA test.It suggested that Srm22 regulated BT biosynthesis by acting on acyB2 and srm40 genes rather than structural genes of BT gene cluster.We infered global regulatory genes,lrp that encodes a leucine-responsive regulatory protein and bldD related to the synthesis of spores,might participate in the regulation of BT biosynthesis by the means of bioinformatics.The Lrp and BldD proteins were successfully expressed in E.coli and purified by His-tag affinity chromatograph culture.EMSA tests showed that Lrp could form obviously retardation bands with promotors of acyB2?srm40 and srm22,which suggested Lrp involved in regulation of the transcrtiption of the three regulatory genes.On the contrary,BldD could not bind anyone of acyB2 and srm22 promoters specifically.The results demonstrated BldD didn't directly regulate on transcription of these regulatory genes.Addition of the L-Leu exogenously can significantly raise the proportion of BT in fermentation products.Furthermore,L-Leu is closely connected to the synthesis of isovalerylation chain in BT.Lrp participated in regulating multiple metabolic pathways including L-Leu biosynthesis pathway.We intended to relieve the feedback of L-Leu concentration and increase isovaleryl-CoA substrate by deleting the L-Leu binding domain of Lrp.Detecting SP and BT productions by HPLC,the gene truncated lrp in S.spiramyceticus 1941(?lrp-SP)resulted in a 88.6%increasing in spiramycin ?,and the proportion of total SP reaching to 81.5%compared to 76.3%in wild type strain.The truncation of lrp in S.Spiramyceticus 1941-BT(?lrp-BT)enhanced ISP ? production by 212.1%,and the proportion of total ISP reached to 18.9%compared to 17.7%in S.spiramyceticus 1941-BT strain.These results indicated that gene truncation of lrp could increase the production and proportion of SP and BT,especially SP ? and ISP ?.Amino acids assay showed that BCAAs concentration existed in ?lrp-SP(Val:32.0 gg/g;Leu:37.8 ?g/g;Ile:13.5 ?g/g)is lower than that of S.spiramyceticus 1941(Val:65.1?g/g;Leu:62.6 ?g/g;Ile:25.1 ?g/g),which suggested gene truncation of lrp could promote intracellular BCAA to catabolic pathway.Transcriptome data indicated that the expression of genes related to SP?BT and Ala biosynthetic pathways and acyl-CoA metabolic pathway was all increased.There are three glycosyl groups,important to antibacterial activity,in BT chemical structure.We inferred orf21(NDP-hexosamine-N-dimethyl transferase gene)may take part in forosamine biosynthesis of BT.Gene disruption of orf21 in.S.spiramyceticus 1941 had devoid of antimicrobial activity,and could not detect the intact SP components by TLC and HPLC assays.However,?orf21 might produce spiramycin precursors which chemical structure is under study.