Identification Of Phenazine Metabolites In Streptomyces Lomondensis S015 And Construction Of High-yield Strains

Streptomyces lomondensis S015,a strain isolated from rizosphere soil of Shanghai suburb,could inhibit the growth of many bacteria and fungi.Previous studies identified a phenazine antibiotic lomofungin from the culture broth of S.lomondensis S015.In this study,a new phenazine metabolite was purified from strain S015 and four high-yield genetic engineered strains were also constructed.Firstly,the new product was purified from the culture broth of S.lomondensis S015 through 2-butanone extraction and preparative HPLC.Total amount of 21.4 mg of product A and 18.9 mg of lomofungin were obtained from 20 L cultivations of strain S015.The molecular weight of product A was 300.22 according to UPLC-MS analysis.The structure of product A was identified as 4,7,9-trihydoxyl-6-aldehyde-phenazine-1-carboxylic acid(THAPCA)according to ¹H-NMR and ¹³C NMR analysis.Secondly,phoP single gene knocked out mutant S015?phoP and complementary mutant S015?phoP::phoP were contstructed to study the effects of phoP on phenazine metabolites biosynthesis in S.lomondensis S015.No phenazine products were detected in the strain without gene phoP,and phenazine products were detected in the phoP gene complementation strain.The results suggested that phoP could improve the biosynthesis of phenazine compounds in strain S015.Finally,we overexpressed genes phoP,adpA,metK and vHbS in S.lomondensis S015 respectively.The production of both lomofungin and THAPCA in the four genetic engineered strains,strains S015 P,S015 A,S015 K and S015 S,were analyzed and compared with the wild type S015as well as the vector control srain S015 N0.The results showed that both the regulatory genes(phoP and adpA)and the heterologous genes(metK and vHbS)could improve phenazine metabolites biosynthesis in S.lomondensis S015.The maximum production of lomofungin in strain S015 P,S015 A,S015 K and S015 S were 136.4±11.3 mg/L,190.2±20.5 mg/L,187.8±15.1mg/L and 155.1±8.7 mg/L respectively,which were about 2.2,3.1,3.0 and2.5 times of that in vector control srain S015 N0.The maximum production of THAPCA in strain S015 P,S015 A,S015 K and S015 S were 84.6±7.7mg/L,63.3±6.8 mg/L,61.5±1.6 mg/L and 60.3±5.3 mg/L respectively,which were about 2.8,2.1,2.0 and 2.0 times of that in vector control srain S015 N0.The results in this study showed that genes phoP,adpA,metK and vHbS could be used to improve phenazine metabolites biosynthesis in other Streptomyces strains.