Study On Biosynthesis Of Carbamycin And

Bitespiramycin, a mixture with 4"-O-isovalerylspiramycin Ⅰ, Ⅱ, Ⅲ (ISP) as the major compoents, is produced by recombinant Streptomyces spiramyceticus F21 harboring a 4"-O-isovaleryltransferase gene (ist) from Streptomyces thermotolerans. Enhancing expression level of ist gene might increase bitespiramycin production and the proportion of ISP in fermentation product, which can simplify industrial process and reduce cost. Characterization of pathway-specific regulatory genes of ist gene provided a way to increase the level of ist expression in this work.Although acyB2 is presumed to be a positive regulatory gene of ist in the biosynthetic gene cluster of carbomycin from S. thermotolerans, it was not confirmed by gene inactivation and complement experiment. In this work, the disruption of acyB2 eliminated carbomycin production, and its complementation restored carbomycin production. Overexpression of acyB2 increased drastically carbomycin production, which proved that the AcyB2 is the positive regulator of cabomycin biosynthesis. Linked with acyB2, a new putative regulatory gene, cbmR, had been cloned and identified from this cluster. The inactivation of cbmR gene inhibited the carbomycin production while intact original cbmR gene restored carbomycin production. However, overexpression of cbmR by ermE*p blocked the carbomycin production. Gene expression analysis was performed by real-time quantitative PCR for the genes of the cluster in the wild-type strain and in the acyB2 and cbmR deletion mutants. The results indicated that AcyB2 and CbmR were activators controlling most of the carbomycin biosynthetic genes, including ist gene. The level of these target genes expression were increased in acyB2 overexpression mutant, but inhibited in cbmR overexpression mutant. The acyB2 and cbmR were successfully expressed in E. coli and the purifed products will used for studying their regulatory mechanism.There are homologous regulatory genes with acyB2 and cbmR. including srm40 and srm22 from spiramycin biosynthetic gene cluster, orf27 and or/28 from medimycin biosynthetic gene cluster. As 16-member macrolides, carbomycin, spiramycin and medimycin have similar structure and biosynthetic genes. Therefore these regulators maybe have similar regulatory function. Recombinant plasmids of pWHM3 containing these regulatory genes and ist gene were transformed into S. lividans TK24 for biotransformation of spiramycin. The results showed the yield of ISP biostransformed by the S. lividans TK.24 transformants containing the two regulatory genes cbmR-acyB2 or srm22-srm40 was lower than that of single acyB2 or srm40, but the orf28-orf27 was much better than a single orf27. Among them, acyB2 and orf28-orf27 had the best effect on improving the expression of ist gene, and even more than the strong promoter ermE*p.4 mM L-amino acids in the fermentation medium could significantly improve the yield of ISP, and the effect of L-Leu was the best.