The Regulatory Pathway Of Ste1(DasR) In Streptomyces Sp.139

Polysaccharides are important natural compounds,which can be divided into animal polysaccharides,plant polysaccharides and microbial polysaccharides by their sources.Up to now,although more and more attention has been paid to the research of polysaccharides,there is still a big gap compared with proteins and nucleic acids in its research level.Compared with plant and animal polysaccharides,the application of microbial polysaccharides in the field of medicine lags behind.A new exopolysaccharide Ebosin had been isolated from Streptomyces sp.139 in our laboratory,which has significant anti-rheumatoid arthritis and anti-psoriasis activity.To the best our knowledge,Ebosin is the first pharmacologically active exopolysaccharides discovered in Streptomyces.The biosynthesis gene cluster of the polysaccharide consists of 27 biosynthesis genes(ste1-ste27).Sequence alignment analysis shows that stel may be a negative regulatory gene of exopolysaccharide Ebosin biosynthesis.After blocking ste1 in the Ebosin biosynthesis gene cluster,it was found that the yield of Ebosin increased significantly.However,it is still unclear how Stel regulate exopolysaccharide biosynthesis.Sequence alignment analysis showed that Ste1 was highly homologous to the global regulatory protein DasR in Streptomyces coelicolor.The global regulatory protein DasR is the intersection of balancing metabolism of carbon source and nitrogen source in Streptomyces.By binding with intermediate metabolites GlcNAc-6P and GlcN-6P,it plays an important role in Streptomyces mediating nutrition sensing,morphological differentiation.transformation between primary and secondary metabolism,etc.DasR and its homologous proteins can regulate the biosynthesis of all known antibiotics in Streptomyces coelicolor,and it has also been reported that they can regulate antibiotic biosynthesis in actinomycetes such as Saccharopolyspora erythraea.However,studies on DasR proteins regulating polysaccharide synthesis have not been reported outside of our laboratory.Accordingly,on the basis of the previous research about ste1 mutant,this study focused on the correlation between the primary metabolism of aminoglycans and secondary metabolism regulated by Ste1,especially the regulation of exopolysaccharide Ebosin biosynthesis.The study consists of the following three parts:In the first part,the utilization rates of different sugars(Glc,GlcNAc,(GlcNAc)2)were determined by HPLC in wild-type strain of Streptomyces sp.139 and ste1 mutant strain.The results indicated that the rate of GlcNAc utilization by mutant strain Streptomyces sp.139 D1 decreased significantly compared with wild-type strain,but there were no significant change in the utilization rate of Glc and(GlcNAc)2.The results were consistent with the regulation of GlcNAc by DasR in Streptomyces coelicolor.Subsequently,transcriptome sequencing was performed on wild-type strain and ste1 mutant strain and wild-type strain of Streptomyces sp.139.The sequencing results included 6848 genes,of which 15 genes were significantly up-regulated and 85 genes were significantly down-regulated.According to the functional classification of significantly changed genes,it was found that their functions were mainly related to metabolism,especially related to amino sugar metabolism.Among the significantly up-regulated genes are chitinase gene S139GL004512(F3L20_RS21775)and PTS N-acetylglucosamine transporter subunit EIIA coding gene S139GL002188(F3L20_RS1 0900),corresponding to amino-saccharide degradation and transport,while significantly downregulated genes are S1 39GL000716(F3L20_RS03630),which encode polysaccharide deacetylase.Interestingly,these genes contain consensus binding sites of DasR,which points out the direction for further research.According to the results of the first part,the second part is to obtain two recombinant proteins,Stel and DasR.the simple work of obtaining recombinant proteins is listed as a separate part,not only because the soluble expression of these two proteins is low,but also very difficult to purify.We constructed different expression vectors using pET28a(His tagged),pGEX4T1(GST tagged)and pCold(cold-shock vector for high-level expression of soluble proteins,His tagged),and tried N-terminal tag,C-terminal tag and double-terminal tag in pET system.Finally,enough Stel and DasR proteins were purified from E.coli using pCold plasmids,which were used in the third part of EMSA.Then,in the third part,combined with transcriptome sequencing which revealed the genes with significantly changing on expression level in ste1 mutant strain,16 candidate genes were identified by searching dre(DasR responsive element)in whole Streptomyces sp.139 genome.It was found that Stel can specifically bind to amino sugar metabolism related chitinase gene(F3L20_RS21775)and PTS N-acetylglucosamine transporter subunit EIIA coding gene(F3L20_RS 10900),as well as polysaccharide deacetylase gene(F3L20_RS03630)related to polysaccharide modification and degradation,indicating that Stel may be related to amino sugar metabolism and exopolysaccharide accumulation.In addition,it is also found that the sequence that Ste1 can bind is not exactly the same as that of DasR,and the differences in specific regulation patterns need to be further studied.Due to the difficulties of epidemic situation and protein purification,a complete closed loop was not formed in our study,but an experimental method for screening target gene segments that could bind to Stel and DasR was established,and 6 genes regulated by Stel were identified,which provided a reference and basis for bioengineering Streptomyces sp.139 to obtain high-yield Ebosin strains and research on the regulatory networks related to amino sugar metabolism and polysaccharide biosynthesis in Streptomyces.