Force Of Amphotericin Biosynthetic Genes Gene Sgcr3 Function And Its Relationship With Sgcr1 And Sgcr2 Regulation

Lidamycin(C-1027),produced by Streptomyces globisporus C-1027,is a novel enediyne antitumor antibiotic and has entered phaseâ…¡clinical trial in China recently.C-1027 biosynthetic gene cluster has been cloned,sequenced and characterized.Bioinformatic analysis and biochemical studies revealed a distinct iterative typeâ… enediyne polyketide synthase(PKSE) and provided a convergent biosynthetic strategy for C-1027 from four biosynthetic building blocks.However,almost nothing was known about the transcriptional regulation of enediyne antibiotic production prior to the present work.The biosynthetic gene cluster for 9-membered enediyne C-1027 contains at least three putative regulatory genes,i.e.,sgcR1,sgcR2 and sgcR3.The predicted gene products of sgcR1,sgcR2 and sgcR3 share sequence similarities to StrR,regulators of AraC/XylS family and TylR,respectively,sgcR1 and sgcR2 are two adjacent genes transcribed in the same direction with a gap of only 25 bp,suggesting that they might be transcriptionally coupled within an operon,sgcR2 and sgcR3 contain a TTA leucine codon respectively. Furthermore,the biosynthetic gene clusters for three 9-membered enediynes(C-1027, neocarzinostatin and maduropeptin) show high similarity in the organization of genes around these regulatory genes.Despite varying degrees of chromophore structural uniqueness,all homologues of three genes are located adjacent to the genes of PKSEs (sgcE,ncsE and mdpE) and the tailoring enzymes(E1 to E11),which are responsible for the biosynthesis of enediyne core.This may represent a further indication for a regulatory role of the three genes in C-1027 biosynthesis.Disruption of sgcR3 abolished C-1027 production,suggesting that it is a positive regulator of C-1027 production.Complementation of the sgcR3 disrupted strain R3KO with intact sgcR3 gene restored C-1027 production and overexpression of sgcR3 in wild type strain resulted in a substantial increase in C-1027 production,confirming their positive regulatory role in C-1027 biosynthesis.SgcR3 was expressed and purified in E. coli BL21(DE3).Purified N-terminal His₆-tagged SgcR3 showed specific DNA-binding activity for upstream sequence of sgcR1 and sgcR2 of C-1027 biosynthetic gene cluster.sgcR1,sgcR2 and sgcR1R2 with its own upstream sequence or the strong constitutive promoter ermE^*p were cloned into the vector pKC1139 and the recombinant plasmids were introduced into S.globisporus C-1027.Overexpression of sgcR1,sgcR2 and sgcR1R2 in wild type strain increased C-1027 production respectively,suggesting their positive role in C-1027 biosynthesis.The cross-complementation studies showed sgcR1R2 could functionally complement sgcR3 in trans,suggesting that sgcR1R2 occupies a lower rung than sgcR3 in the hierarchy of these genes.Consistent with this, the results from gene expression analysis via real time reverse transcriptase polymerase chain reaction(RT-PCR) showed that transcripts of sgcR1 and sgcR2 were significantly lower in R3KO mutant than in wild type strain,while sgcR3 transcripts were statistically at similar level in R1R2KO mutant as in wild type strain.A positive effect of sgcR1R2 and sgcR3 gene products on the transcription of biosynthetic structural genes such as sgcA1,sgcC4,sgcD6 and sgcE8 was also observed.To further investigate the functions of the sgcR1 and sgcR2,SgcR1 and SgcR2 were expressed and purified in E.coli BL21(DE3).In this work,the results from disruption,complementation and overexpression analyses suggested a positive regulatory role of sgcR3 in C-1027 biosynthesis.Furthermore, purified SgcR3 showed specific DNA-binding activity for the upstream region of sgcR1R2.Consisting with this,cross-complementation experiments suggested that sgcR3 occupies a higher rung than sgcR1 and sgcR2 in the hierarchy of C-1027 regulatory genes. Additional evidence was observed through the study of the gene expression in mutants by using quantitative real time RT-PCR.The data presented in this work set the stage for subsequent studies to delineate the complexity of the regulation of C-1027 biosynthesis, as well as for designing strategies for the construction of strains with enhanced C-1027 production.