New Antitumor Antibiotic Lidamycin Biosynthetic Genes Gene Sgcr And Sgcm The Functional Study

Lidamycin, a novel enediyne antitumor antibiotic produced by S. globisporusC-1027, is composed of a chromophore and an apoprotein. Lidamycinbiosynthetic gene cluster has been cloned, sequenced and characterized. Most ofgenes encoding biosynthetic enzymes for the building blocks of the chromophorehave been annotated, according to which the principle biosynthetic pathways oflidamycin have been determined. However, in the lidamycin biosynthetic cluster,there are still some genes whose functions remain unknown, and based on thebioinformatics analysis, some of these genes might encode potential regulatoryproteins in lidamycin biosynthetic pathway in S. globisporus C-1027.In this thesis, two unknown genes, sgcR and sgcM, were chosen for the functionalanalysis. In search of database, sgcR, with a length of 1,148 bp, encodes a proteindemonstrating some homology to a transcriptional regulator protein whichbelongs to XRE (Xenobiotic Response Element, XRE) family; and sgcM, with alength of 1,040 bp, encodes a protein which has a highly homologous counterpartin neocarzinostatin biosynthetic gene cluster of Streptomyces carzinostaticus.Blast results also reveal that SgcM shows some similarity with a putativeDNA-binding protein with a helix-turn-helix DNA-binding motif.To investigate the roles these two genes might involve in the biosynthetic pathwayof lidamycin, gene inactivation experiment was conducted. Two apramycinresistant vector plasmids, pKC1139 and pOJ260 were used for constructingdisruption plasmids that consisted of a fragment whose sequence is identical to theleft-side flanking region of the target disruption area (left arm), athiostrepton-resistance gene (tsr), and another fragment identical to the right sideflanking region of the disruption area (right arm). Two arms amplified from thegenome DNA of S. globisporus C-1027 as well as tsr gene were cloned into themultiple cloning sites of pKC1139 and pOJ260. The recombinant plasmid wastransferred into S. globisporus C-1027 through conjugation between E. coliET12567/pUZ8002 and S. globisporus C-1027. Homologous recombination mightoccur between wild-type gene in the lidamycin biosynthetic gene cluster and thedisrupted gene on the constructed plasmid. The transconjugatants that had undergone gene replacement via double cross-over were resistant to thiostreptonand sensitive to apramycin. Disruption of sgcR and sgcM were confirmed by PCRand Southern blot analysis of restriction enzyme digested total genomic DNA.Both sgcR disruption mutant and sgcM disruption mutant were obtained andnamed S. globisporus RKO and S. globisporus MKO, respectively, in either ofwhich sgcR or sgcM was "knocked out" and replaced by tsr.Fermentation broth of S. globisporus RKO was collected for bioassay against B.subtilis and an increasing antibacterial activity of S. globisporus RKO wasobserved, suggesting an overproduction of lidamycin in sgcR deleted mutant. Forthe complementation of sgcR, two vectors, pKC1139 and pSET152 were used toconstruct complementation plasmids. The fragment of sgcR with a strongconstitutive promoter ermE~*p was cloned into the vector pKC1139 and pSET152,respectively, and complementary plasmids were introduced into the sgcR deletedmutant via conjugation. The complementary strain was obtained and confirmed.Fermentation and bioassay revealed that the production of lidamycin incomplementary strain was inhibited compared with sgcR deleted mutant. Thecombined results above suggested SgcR might be an important negativeregulatory protein involved in biosynthetic pathway of lidamycin.Fermentation broth of S. globisporus MKO was collected for bioassay against B.subtilis, and no significant change in antibacterial activity was observed in sgcMdeleted mutant. To construct complementation plasmids of sgcM, the fragment ofsgcM with a strong constitutive promoter ermE~*p was cloned into the vectorpKC1139 and pSET152, respectively, and complementation plasmids wereintroduced into S. globisporus MKO via conjugation, sgcM complementarystrains were obtained and confirmed. In sgcM complemented strain, we found anobvious decrease in its antibacterial activity, indicating the production oflidamycin had been affected by the over-expression of SgcM. It is quite possiblethat SgcM might exist as a negative regulatory protein involved in biosyntheticpathway of lidamycin. For some unknown reasons, deletion of SgcM did notcause much effect on the production of lidamycin under the detected circumstance,whereas ove-expression of SgcM will significantly inhibit the production oflidamycin.