Effects Of Iron Stress And Strong Promoter A4 Insertion On The Secondary Metabolism Of Streptomyces Luteus TRM45540

Streptomyces luteus TRM45540 is isolated from the saline soil of Lop Nur in Xinjiang and is capable of producing a cyclic peptide antibiotic actinomycin D.In this paper,the response of secondary metabolism of TRM45540 was investigated by iron stress and insertion of strong promoter A4.It was found that the production of actinomycin D from S.luteus TRM45540 was increased under the stress of high concentration of ferrous sulfate.When ISP4 was used as the fermentation medium and the amount of ferrous sulfate added was 1000 mg/L,the yield of actinomycin D was increased by 52.06% compared with the normal amount of ferrous sulfate(1 mg/L).When the fermentation medium was oat-soybean medium,the yield of actinomycin D of TRM45540 was increased by 56.47%,up to 1013.36 mg/L.Based on the analyses of the differences of the three transcriptome samples treated with 1 mg/L,100 mg/L and 1000 mg/L of ferrous sulfate,the metabolic response rules of TRM45540 under ferrous sulfate stress was presumed and summarized.Under high-iron stress,the porphyrin-related ion transport system was firstly activated to transport ferrous ions from the outer-part of the cell membrane to the inner-part of the cell membrane,consuming a large amount of ATP at the same time,resulting in the lack of phosphate ions.Thereby the SenX3-RegX3 pathway of the two-component system was activated,which regulated the biosynthesis of actinomycin D and triggered the expression of polyene-like silencing gene clusters.Furthermore,a tetraene compound was isolated and purified by macroporous resin adsorption chromatography,silica gel chromatography and high performance liquid chromatography.The purified compound was identified as JBIR-13 by 1D & 2D NMR method.The lack of phosphate ions as well as the high concentration of ferrous sulfate was proved to take the same effect on the secondary metabolism of S.luteus TRM45540.In addition,glutathione wasabundantly expressed and taken part in detoxification metabolism of ferrous ions.The production of actinomycin D was increased by random integration of the global strong promoter A4 on the TRM45540 genome.The strong promoter A4 was integrated into plasmid pSET152,transformed into E.coli,and the promoter A4 fragment was randomly inserted into the TRM45540 genome by conjugative transfer of E.coli to TRM45540.The yield production of actinomycin D in the conjugate TRM45540::pYS001 was increased by 26.85%.