The Heterologous Expression Of Two Kinds Of Antibiotic Biosynthesis Genes In S. Lividans TK24

Bitespiramycin is a new antibiotic biosynthesized by a recombinant spiramycin producing strain, Streptomyces spiramyceticus WSJ-1 (for short, WSJ-1) that harbored a 4"-O-acyltransferase gene (ist) for the addition of an acyl group, mainly an isovaleryl group, at the 4"-OH of spiramycin. BT had passed phaseⅢclinical trial. It exhibited an excellent therapeutic effect.Bitespiramycin is a mixture of spiramycin derivatives, with 4"-isovalerylspiramycin as its main component. It has some minor components, such as 4"-butyl,4"-propionyl spiramycin, etc. As the proportion of the main components of bitespiramycin (4"-isovalerylspiramycin I, II andⅢ; for short, BT I, II and III respectively) produced by WSJ-1 is not high, it brings some difficulties in the its production process such as the fermentation, extraction, purification and quality control. It is, therefore, desired to increase the proportion of the main component of bitespiramycin produced by its fermentation.HPLC analysis of the fermentation products of WSJ-1 showed that there existed still a significant level of spiramycin. They were not 4"-isovalerylated. An increase of 4"-acyltransferase activity and/or supplying of isovaleryl group should promote the biotransformation of spiramycin to 4"-isovalerylspiramycin.To improve the bioconversion level of 4"-isovalerylspiramycin from spiramycin, and provide directions for constructing new generation of bitespiramycin producing strains with 4"-isovalerylspiramycin as major component, A group of two recombinant pasmids, pKC-e-ist-ist and pKC-ist-ist, carrying tandem-repeated 4"-isovaleryltransferase gene with PermE* at its 5’-upstream and tandem-repeated 4"-isovaleryltransferase gene, repectively, were constructed and introduced into Streptomyces lividans TK24 by protoplast transformation. The bioconversion abilities from spiramycin to 4"-isovalerylspiramycin by the above transformants were compared. Streptomyces lividans TK24 [pKC-e-ist-ist] showed nearly four times higher of bioconversion ability than that of Streptomyces lividans TK24 [pKC-ist-ist] upon addition of spiramycin at a final concentation of 50μg/ml; other group of two recombinant pasmids, pSET-e-ist-ist and pSET-e-ist, carrying tandem-repeated 4"-isovaleryltransferase gene with PermE* at its 5’-upstream and 4"-isovaleryltransferase gene with PermE* at its 5’-upstream, repectively, were constructed and introduced into Streptomyces lividans TK24 by protoplast transformation. The bioconversion abilities from spiramycin to 4"-isovalerylspiramycin by the above transformants were compared. Streptomyces lividans TK24 [pSET-e-ist-ist] showed nearly one times higher of bioconversion ability than that of Streptomyces lividans TK24 [pSET-e-ist].Therefore, PermE*, the strong promotor of erythromycin resistance gene,and tandem-repeated could promote the expression of 4"-isovaleryltransferase gene and impove the bioconversion of spiramycin to 4"-isovalerylspiramycin significantly in Streptomyces lividans TK24.Geldanamycin (GDM) is a benzoquinone antibiotic. The biosynthetic gene cluster of GDM has been cloned and sequenced. A general knowledge about the biosynthesis of GDM had been obtained, but some of its biosynthetic details, especially the post-PKS modification process and the enzymes associated with this process remains to be understood in depth.Gene disruption experiment had proved that the post-PKS modification gene, gdmN, was responsible for the C-7 carbamoylation in GDM biosynthesis. In this paper, a strong erythromycin resistance gene promoter PermE* was palced at the 5’upstream of gdmN gene, and then ligated into an expression vector of Streptomyces. This recombinant plasmid was introduced into Streptomyces lividans TK24 by transformation, for its heterologous expression and then microbial transformation of CT-1-7 (4,5-dihydro-7-descarbamol-7-hydroxylgeldanamycin) which was presumed to be the native subtrate of the carbamoyltransferse encodes by gdmN gene. The expected bioconversion compound,4,5-dihydrogeldanamycin, was detected by silica gel TLC. Therefore, we confirmed the gene function of gdmN in GDM biosynthesis by its heterologous expression in Streptomyces lividans TK24, which was a type strain for heterologous expression of foreign gene(s) in Streptomyces. The heterologous expression system for gdmN gene, which was established by us, should also be useful for studying the substrate tolerance of the carbamoyltransferase in the future.