Proteomics Comparison Of Three Strains Producing Toyocamycin And Preliminary Study On Its Regulatory Proteins

Streptomyces diastatochromogenes 1628 can produce the nucleoside antibiotic toyocamycin(TM),which was found to be highly efficient against a broad range of plant pathogenic fungi.In earlier study,the rifampin-resistant mutants1628-T15 and 1628-T62 which showed the highest(4.5 times higher)and lowest 78%lower)TM production comparing to wild-type strain 1628,respectively,was isolated by using ribosome engineering technology.In this study,in order to explore the key factors involved in metabolism and regulatory of TM overproduction,the proteomics approach was employed to analyze and identify the distinct differences of protein expression among the two mutants(1628-T15 and 1628-T62)and WT strain.Three differential regulation proteins were cloned and their relevance to secondary metabolism was studied.The main contents were performed as follows:Firstly,based on isobaric tags for relative and absolute quantification(iTRAQ)technology,distinct differences of protein expression among the two mutants1628-T15(H)and 1628-T62(L),and wild-type strain1628(N)were identified and analyzed.There was no significant difference in whole protein expression level among the three groups of tested strains(H,L,N).This result suggested that the high-yield of TM may be due to the downregulation of competed pathway rather than the upregulation of whole protein level.In order to study on the regulatory mechanism of TM biosynthesis on the molecular level,three regulation proteins X0P338(GntR),X0MUI0(Crp),and A0A0C2B1U9(IclR)which exhibited the differential expression level were studied.Their corresponding genes gntr(918 bp),crp(1443 bp),and iclr(771 bp)were cloned.Next,amplified gene gntr was subjected to BLAST alignment,which has highly homology with amino acids of FadR protein in some Streptomycetes,this suggested that the protein X0P338 from 1628 belongs to the FadR subfamily of GntR transcriptional regulation family.Using plant-pathogenic fungus Fusarium oxysporum f.sp.cucumerinum as an indicator,the antifungal activity of gntr overexpression strain 1628-G was significantly higher than that of the wild-type strain 1628.The TM production of 1628-G was 1.77-fold higher than that of the wild-type strain 1628.Inaddition,the production of tetraene macrolide antibiotics was also increased:tetramycin A,tetramycin P,and tetraenol B was increased by approximately 46%,25%,and 45%,respectively.Real-time fluorescence quantitative PCR results showed that the expression level of gntr gene in 1628-G was higher than that of WT strain1628 during the whole fermentation process,and the relative expression level of toy genes(toyD,toyF,toyM)which involved in TM biosynthesis was higher than corresponding values of wild-type strain 1628 in the early and middle fermentation period,whereas their expression levels were lower comparing with WT strain 1628 in the later fermentation.These data suggested that X0P338(GntR)may be a positive global regulator not only for promoting TM biosynthesis but also for accelerating other secondary metabolites production.Finally,the amino acid sequences of X0MUI0(Crp)showed 99% homology with the transcription regulators of the Crp/Fnr family in other Streptomycetes.The antifungal activity of overexpression strain 1628-C was slightly higher than that of WT strain 1628.Also,the yield of TM was also slightly increased comparing with WT strain(265.93 mg/L vs 235.34 mg/L),indicating that as far as X0MUI0(Crp)is concerned the correlation of positive regulation and TM biosynthesis was not strong.The amino acid sequence of A0A0C2B1U9(IclR)has high homology with the IclR family transcription regulators identified in Streptomycetes.The antifunal ability of the overexpression strain 1628-I was slightly worse than that of the WT strain,and the yield of TM was reduced by about 50.3%.These experimental results indicated that IclR is a negative regulator of TM biosynthesis.