Regulatory Mechanism Of Signaling Molecule H₂O₂ On Highly Efficient Natamycin Synthesis In Streptomyces Gilvosporeus

Streptomyces species is a natural storehouse for bioactive substances like antibiotics.Natamycin,produced by certain kinds of Streptomyces,also known as pimaricin and is a natural,broad-spectrum and efficient polyene macrolide antifungal antibiotic.It is widely used in the treatment of clinical fungal infection and the prevention of food fungal contamination.In recent years,the antitumor activity of natamycin has opened its new applicatio prospect.Cholesterol oxidase gene is an essential gene for the biosynthesis of natamycin,mechanism of action has been a puzzle in this field.In addition,ROS oxidative stress,the development and differentiation of mycelia and the synthesis of secondary metabolites are closely related in Streptomyces,but the interaction mechanism among them is still unknown.Thus,it will be a theoretical significance to analyze the signaling pathway of cholesterol oxidase gene acting on natamycin biosynthesis,to build a bridge between the morphological development of Streptomyces and the synthesis of secondary metabolites,to enrich the regulatory mechanism of natamycin biosynthesis,and to guide the construction of high yield strains of natamycin.In this thesis,we used Streptomyces gilvosporeus F607 as the research object and explained the metabolism of sgn E,a cholesterol oxidase gene,on activating synthesis of natamycin through H₂O_2,which is one of the enzyme metabolite and acts as a signal molecule.Then,coordinated regulation of Oxy R and Sgn R on the expression of sgn M was investigated.Thirdly,regulatory pathway of Sox R and RS12540/12545（Mac R/S）affecting the morphological development and secondary metabolite synthesis was clarified.Based on this ROS activation mechanism,we constructed an efflux gene overexpression strain S.gilvosporeus F-EX with high efficiency of natamycin synthesis.Then main results of this thesis:（1）The regulation of cholesterol oxidase Sgn E on the biosynthesis of natamycin was accomplished by its enzymatic reaction product H₂O₂.Natamycin production was reached to6.15 g·L^-1（increased by 36.7%）when H₂O₂was added at 8 h and reduced to 1.85 g·L^-1 when20μM DMTU was added.Effect of ROS level on the biosynthesis of natamycin clearly affects indicates that H₂O₂plays an important role in regulating the biosynthesis of natamycin and the efficiency of natamycin synthesis.In addition to blocking the biosynthesis of natamycin,the deletion of sgn E gene significantly reduced the intracellular H₂O₂concentration to<5μmol·g^-1.The blocking effect,caused by sgn E gene knockout,on natamycin synthesis can be recovered by adding sgn E gene or H₂O₂.Sgn E has the catalytic activity of cholesterol oxidase,in which,stigmasterol and ergosterol are effective catalytic substrates.（2）The effects of H₂O₂,redox protein Oxy R and Sox R on natamycin biosynthesis were elucidated.It was verified that H₂O₂addition could promote the biosynthesis of natamycin.Compared to the control TC-24,56 genes were up-regulated in the redox pathway of H₂O₂treatment group TH-24.The expression levels of PKS encoding genes（sgn S4,sgn S3,sgn S2,sgn S0,sgn S1）in the polyketone synthesis pathway increased by more than 1.2 times compared with the control strain.sox R and oxy R gene knockout and complement strains DS9,DO12 and CS2,CO7 were constructed.It was found that both sox R and oxy R gene knockout had inhibitory effects on natamycin biosynthesis.The yield of DS9 was 44.6%of F607 at 120h,and the yield of DO12 was only 0.53 g·L^-1 at 120 h（about 10%of F607）.Two redox proteins positively regulate natamycin biosynthesis,and both Oxy R and Sox R significantly affect the expression of pathway specific regulatory genes sgn M and sgn R.（3）The target of Oxy R on regulating natamycin biosynthesis and the co-regulatory of Sgn R and Oxy R on pathway specific regulation gene sgn M were analyzed.The redox residues Cys212 and Cys221 of Oxy R_Sg in S.gilvosporeus were analyzed.Cys212 was the preferred oxidation residue and played an important role in the oxidation process.The DNA binding domain and allosteric site of Oxy R tetramer were analyzed by molecular simulation,and the binding sequence of target DNA was predicted.Oxy R-His recombinant protein was expressed and purified.Combined with DNA base mutation and EMSA,target gene of Oxy R regulating natamycin biosynthesis was studied.It was proved that"GGT/ACC"was the key sequence of Oxy R binding target gene,and the mutation of"CGGC"sequence had little effect on the binding ability.Through spatial structure analysis and EMSA validation,the mechanism of Oxy R regulating the target gene of natamycin biosynthesis was analyzed.By adding H₂O₂ and DTT to simulate the reduction and peroxidation of Oxy R,it was found that Oxy R realized the change of binding ability to sgn M promoter region through the isomerism during the transition of oxidation state,and thus regulated the expression of specific regulatory genes in this pathway.We analyzed the interaction between Sgn R and Oxy R.It was found that random coil domain of Sgn R inserts into the Oxy R tetramer to form a larger DNA-binding domain,which binds the DNA fragment that covering BS_{Sgn R} and BS_{Oxy R}.Meanwhile,self-combination of Sgn R through TPR and TPR_L at four polymerization sites AS1-AS4,completes oligomerization and"self-recruitment".This mode of action,combined with Oxy R"mutual recruitment",constitutes a complete SARP regulatory mode that regulatory protein regulates the-35 region of its target genen,such as sgn M.（4）The mechanism of Sox R-Mac RS cascade regulation on natamycin biosynthesis and spore development was revealed.Through domain and function prediction analysis,it was found that Sox R in S.gilvosporeus had"CCGCCAA-5nt-TTGCGG"sequence in mac RS gene promoter region.After Mac RS knockout,the wet weight of bacteria decreased slightly,but the biosynthesis of natamycin in S.gilvosporeus was inhibited.Compesation of this gene restored natamycin biosynthesis to 3.24 g·L^-1.Meanwhile,the deletion of Mac RS had a great effect on spore formation.The results of scanning electron microscopy and transmission electron microscopy showed that the absence of Mac RS affected the normal differentiation of mycelia and spore formation,which was caused by the inhibition of the septum required for mycelia differentiation and spore production.Further validation confirmed that the expression of spore and septum genes ssg A,ssg R,adp A,ads A and fts L were significantly decreased in Mac RS deletion strains,and most natamycin biosynthesis genes,including pathway specific regulation genes sgn M,sgn R and PKS genes such as sgn S2 and sgn S0,were silenced.By analyzing the structure and function of Mac R,protein expression and purification,and EMSA experiment,Mac R can directly bind the promoter region that between pathway specific regulatory genes sgn M and sgn R,and the promoter region of septum formation gene fts L.（5）The efflux mechanism and the promotion effect of efficient efflux on natamycin synthesis were investigated.ade IJK gene from Acinetobacter johnsonii M19 and sgn A/B gene from S.gilvosporeus were used to construct overexpression strains S.gilvosporeus F-EX and S.gilvosporeus F-EA with natamycin production of 7.38 g·L^-1 and 6.4 g·L^-1.S.gilvosporeus F-EX showed significant improvement in extracellular/intracellular ratio compared to original strain F607.sgn A/B gene overexpression is beneficial to S.gilvosporeus in maintaining intracellular ROS levels and promoting the continuous efficient synthesis of natamycin.ade IJK gene overexpression strain S.gilvosporeus F-EA slightly increased the yield of natamycin,but the effect was weaker than S.gilvosporeus F-EX.Furthermore,the mechanism of Sgn A/B transport natamycin was analyzed.Sgn A/B is an inwards opening hetero-disubunit ABC transporter.The transport channel formed by Sgn A and Sgn B has two natamycin binding sites DBC1 and DBC2,located in NBD and TMD domains respectively.However,the weak affinity of the transmembrane region is conducive to the extracellular transport of natamycin by the outgoing transporter,and the inhibition effect of intracellular accumulation of natamycin against ROS accumulation is relieved.Transcriptomic analysis showed that the effect of sgn A/B overexpression was more significant in the phase of natamycin log-synthesis.sgn A/B overexpression not only improved the extracellular transport efficiency of natamycin,but also indirectly promoted the synthesis of natamycin by promoting the primary metabolism of S.gilvosporeus and increasing the supply of precursors required for efficient synthesis of natamycin.