Preliminary Biosynthetic Study And Yield Improvement Of A Novel Chalkophore Chalkophomycin

Objectives: Chalkophomycin(CHM)is a novel copper binding natural product isolated from Streptomyces sp.CB00271.It contains a rare diazeniumdiolate functional group coordinated with a cupric ion.This compound and its ligand have certain potential to be developed as antitumor drugs or to treat diseases due to copper overload,such as Wilson’s diseases.Biosynthetic studies of Streptomyces natural products may help to discover CHM derivatives and improve their yields.Therefore,a preliminary study on the biosynthesis and yield improvement of CHM was carried out.The main studies in this thesis are as follows:The preliminary study on the biosynthesis of chalkophomycin.Methods and results: Our group has previously identified the biosynthetic gene cluster(BGC)chm responsible for CHM biosynthesis in CB00271 through bioinformatic analysis and gene replacement of its core polyketide synthase(PKS)/non-ribosomal peptide synthase(NRPS)gene(unpublished data).S.sp.CB00271 also produces a series of rubromycin aromatic polyketides.Our laboratory has obtained a mutant strain CB00271-DΔks with the core polyketide synthase gene knocked out.Its metabolic profile is clearer.In this study,we further predicted the functions of other genes in chm by Blast P.Along with the structural characteristics of CHM,we selected five genes for gene deletion experiments using homologous recombination-based double crossover knockout method,i.e.,orf13,orf19,orf20,orf23,and orf24,encoding an acetyl-Co A dehydrogenase,a N-N bond synthase,a N-OH synthase,an Omethyltransferase,and a flavin reductase,respectively.Gene replacement of orf20 and orf23 led to the abolishment of CHM production,while introduction of p SET152-based containing orf20 and orf23 can partly complement CHM production.This suggests that both ORF20 and ORF23 are involved in the biosynthesis of CHM.Gene replacement of orf13 no longer produced CHM.Gene replacement of orf19 decreased CHM production and increased production of CHM analogs as possible intermediates.Gene replacement of orf24 had no effect on CHM production,and the mutants also have no production of new metabolites.After overexpression of orf7 and orf8 encoding putative pathway-situated regulators,the production of CHM was greatly reduced,indicating that they are negative regulators.Both overexpression of orf6 encoding a transporter and gene replacement of orf25 encoding a chitin synthase exhibited no effect on the yield of CHM.Discovery of chalkophomycin in Streptomyces sp.CB03629 and its yield improvement.Methods and results: We first discovered that Streptomyces sp.CB03629 could produce higher amount of CHM,and its yield was about 22 mg/L.This yield was about twice that of CB00271 or CB00271-DΔks.Therefore,we further optimized the production medium of CB03629 by a single factor optimization and the Box-Behnken design.The yield of CHM was increased to 219 ± 21.27 mg/L,which was about11-fold of the original yield and 22-fold of the yield in both CB00271 strains.Subsequently,16 S r RNA of CB03629 and its whole-genome sequence CB03629 was obtained.A highly similar chm BGC in CB03629 could also be identified by bioinformatics analysis.Conclusion: six gene replacement mutant strains,three overexpressing strains,and two complementing strains were constructed in CB00271-DΔks.Fermentation analysis confirmed the essential role of orf13,orf20,and orf23 for the biosynthesis of CHM;ORF7 and ORF8 were discovered to be potential negative regulators for CHM biosynthesis.In addition,the yield of CHM was increased to 219 ± 21.27 mg/L in CB03629 through medium optimization.The above studies not only revealed certain biosynthetic mechanisms for CHM,but also should facilitate future functional and mechanistic study of CHM,due to the higher titer of CHM in CB03629.