The Mining Of Hidden Natural Products In Streptomyces Sp. CB00271 By Disruption Of Gene Clusters

Streptomyces species have been a rich source of novel natural product discovery and drug development.Streptomyces sp.CB00271,a high-yield β-rubromycin producer,was obtained by our group through genome mining in the early stage,and later a copper chelate complex chalkophomycin(CHM)with a diazenium structure was discovered from it.Whole-genome sequencing and anti SMASH analysis showed that CB00271 possesses 36 biosynthetic gene clusters(BGCs)of secondary metabolites,which have a promising potential for the development of natural product resources.However,under the existing fermentation conditions,CB00271 mainly produced a series of RUB compounds,and it was difficult to detect the existence of other compounds in different fermentation media.Therefore,it is urgent to activate the silent BGCs in CB00271 and to explore and develop the potential natural product resources.In this study,Streptomyces sp.CB00271 was our target strain.We aims to inactivated the target BGC core gene by in-frame-deletion,thereby eliminating the interference of major secondary metabolites and then activating other silent BGCs in the genome to exploit natural product resources.First,the mutant 271-ΔKO(Δrub)was successfully obtained by disrupting the BGC rub,which is responsible for the synthesis of RUBs.Metabolite analysis of 271-ΔKO in different fermentation media revealed that a series of secondary metabolites was activated.Through structural characterization,we identified that these compounds can be divided into three classes,including type II polyketides: gaudimycin D,non-ribosomal polypeptides: skyllamycins and 2,3-dihydroxybenzoate(2,3-DHB)and 2,3-dihydroxybenzoic acid(2,3-DHBA).At the same time,we ensured their biosynthetic locus,the BGC gau encoding gaudimycin D located in cluster 13,the BGC sky encoding skyllamycins located in cluster 20,and the BGC str containing core genes dhb A,dhb B and dhb C of 2,3-DHB located in cluster 34 of CB00271 genome.Subsequently,we disrupted the core gene of gau and sky BGC respectively,and obtained the double-BGCs-knockout mutant 271-DΔKO(Δrub:Δgau)and the triple-BGCs-knockout mutant 271-TΔKO(Δrub:Δgau:Δsky).The results of fermentation metabolites showed that the yield of2,3-DHBA in different BGCs-knockout mutants was significantly improved.By optimizing the medium carbon source of 271-TΔKO,the yield of 2,3-DHBA was increased to 308.9±12.8 mg/L;the highest titer ever been reported.Besides,the yield of CHM in 271-DΔKO strain was inproved by more than 1.7 times compared with wild-type CB00271 strain.In addition,the mechanism of high production of 2,3-DHBA was explained by metabolic pathway analysis.Polyketides such as RUBs and gaudimycin D,as well as the cinnamoyl lipid side chains of skyllamycins are synthesized using acetyl-Co A as a precursor,while amino acids like phenylalanine(L-Phe),tryptophan(L-Trp)and tyrosine(L-Tyr)in skyllamycins are derived from the chorismate precursor in the shikimate pathway.Therefore,the inactivation of competitive BGCs can enrich the carbon source flow to synthesis of 2,3-DHB,thereby greatly increasing the production of 2,3-DHBA.This study demonstrates that disruption of BGCs of major secondary metabolites is an effective strategy to activate silent BGCs and increase the yield of target products.At the same time,we have successfully constructed "engineered strains" with relatively clean genetic background and abundant carbon source supply: 271-DΔKO and 271-TΔKO,which are not only beneficial to the subsequent development of high-yielding strains of CHM and 2,3-DHBA.what’s more,it provided a basis for us to construct the Streptomyces chassis microorganism,a prospected heterologous expression host of the natural products from shikimate pathway.