Construction Of The High Yield Erythromycin Producing Strain And The Heterologous Expression Of Three Putative Secondary Metabolite Biosynthetic Gene Clusters In Streptomyces Sp.SH-62

In this study,two different research works are described,which include construction of the high yield erythromycin producing strain and heterologous expression of three putative secondary metabolite biosynthetic gene cdusters in Streptomyces sp.SH-62.1.Erythromycins are a kind of macrolide antibiotics produced by Saccharopolyspora erythraea with broad spectrum active against pathogenic Gram-positive bacteria.The fact that second and third generation erythromycin antibiotics are synthesized from erythromycin A results in the great market demand of this compound.In this study,we used the BAC 13G3 which contains a complete erythromycin biosynthetic gene cluster to construct two compatible plasmids pYPQ01 and pYPQ02 via ? RED recombinase system.These two plasmids were successively introduced into S.erythraea by conjugation and we obtained two recombinant strains.One is S.ery::pYPQ01 containing two copies of the erythromycin biosynthetic cluster,the other is S.ery::pYPQ01+pYPQ02 which has three copies of the erythromycin biosynthetic cluster.HPLC and LC-MS analyses demonstrated that two recombinant strains produced respectively 3.1 and 6.4 times more erythromycin A than the wild type strain.It is the first time to improve the erythromycin A production by increasing the copy number of its biosynthetic cluster and increase of the product yield is much larger than that of reported in literatures.2.Streptomyces sp.SH-62 isolated from Fangxian Country,Hubei Province,by Professor Qi Zhou,exhibits strong antibacterial and antifungal activities.Bioinformatics analyses found that there is a putative hygromycin A biosynthetic gene cluster(hgm),a putative validamycin biosynthetic gene cluster(vlm)and a putative lantibiotics biosynthetic gene cluster(Ian)in the genome of this strain.The bacterial artificial chromosome(BAC)genomic library of SH-62 was screened by PCR with seven pairs of specific primers.Eight positive BACs were selected out and introduced into Streptomyces albus and Streptomyces lividans for heterologous expression,respectively.Fermentation metabolites of these heterologous expression strains were detected by bioassay,HPLC and LC-MS.We found that heterologous expression of the hgm cluster produced an unknown antifungal compound instead of hygromycin A.LC-MS analysis found its molecularformula may be C28H2gN6O4 or C27H32N2O8.Heterologous expression of the vlm cluster also produced an antifungal compound instead of validamycin and LC-MS analysis showed its molecularformula may be C11H9NO2.One recombinant strain with activity against Gram positive and negative bacteria and fungi was obtained by heterologous expression of the lan cluster in S.albus.Meanwhile,two recombinant strains were achieved by introducing the lan cluster into S.lividans ZX1 for heterologous expression.Both of them exhibited activity against Gram positive and negative bacteria and one of them also showed weak antifungal activity.