The Role Of Globin In Pyrroloformamides Biosynthesis And Heterologous Synthesis Of Aminobenzoic Acid

Research purposes:Dithiolopyrrolone（DTP）natural products are a class of compounds with excellent antibacterial and antitumor activities,including holomycin,thiolutin,thiomarinol,and pyrroloformamides.The antibacterial mechanism of holomycin is that the reduced holomycin can chelate the zinc ion in the active site of key bacterial metalloproteins.Both holomycin A and pyrroloformamide C（PYF C）with the unique 1,3-dithiacyclohexene structure were previously discovered.The destruction of their disulfide bonds leads to the disappearance of their antibacterial activity,which may be a self-protection mechanism of DTP-producing bacteria.However,the biosynthetic mechanism of 1,3-dithicyclohexene is still unclear.As an ancient and widely distributed protein family,globins have recently been found to catalyze the insertion of carbene into various chemical bonds,resulting in many diverse small molecules.The globin genes are highly conserved in the discovered DTP natural product biosynthetic gene clusters（BGCs）,but their functions are unclear.The first part of this thesis focuses on the globin Pyf K in the pyf gene cluster for the biosynthesis of PYF in Streptomyces sp.CB02980 and Streptomyces sp.CB02009,and the role of Pyf K in PYF biosynthesis is explored.Antioxidants inhibit biological oxidation processes and are widely used in the food and pharmaceutical industries.2,4-dihydroxy-3-aminobenzoic acid is a key precursor of natural product platensimycin,which is biosynthesized by the proteins encoded by the ptm B₁B₂B₃ gene cassette in the platensimycin BGC.In the second part of this thesis,the heterologous synthesis of ptm B₁B₂B₃ in model Streptomyces strains was studied,and the antioxidant activity of the obtained heterologous expression products was evaluated.Research Methods and Results:（1）The role of globin in the biosynthesis of pyrroloformamides.Bioinformatic analysis first suggested that the homologous proteins of Pyf K in bacteria were widespread and are clustered into four categories.We next overexpressed and purified Pyf K from CB02980 in Escherichia coli and discovered that Pyf K can use ethyl diazoacetate as a carbene donor to catalyze the insertion of its carbene into the disulfide bond of PYF A to form its 1,3-dithicyclohexene derivative.However,Pyf K could not catalyze the insertion of this carbene into other tested compounds containing disulfide bonds,indicating its high substrate selectivity.After overexpressing pyf K in CB02980 and CB02009,the total yield of PYFs decreased,while the relative yield of PYF C increased.In addition,the sporulation ability of the CB02980::erm Ep*-pyf K mutant strain also decreased.After knockout of pyf K in CB02980 and CB02009,PYFs were no longer produced.After overexpression of pyf K in Streptomyces albus J1074,this strain showed even higher susceptibility to PYF A.Taken together,these data suggest that Pyf K can regulate the ratio of PFY A/C in CB02980 and CB02009 through its unique catalytic function,with the potential role of a resistance gene.In addition,the negative correlation between the expression level of pyf K and the production of PYFs also indicates that it may regulate the production of these natural products,while the regulatory mechanism needs to be further studied.（2）Heterologous expression studies of the ptm B₁B₂B₃ gene cassette.We first constructed plasmids for heterologous expression of ptm B₁B₂ and ptm B₁B₂B₃ in model Streptomyces strains using p SET152.We discovered that two compounds were produced when the overexpression plasmid were introduced into Streptomyces albus J1074.Through fermentation,isolation,and structural identification,we identified these two compounds as N-acetylated derivatives of 3-amino-2,4-hydroxybenzoic acid and 3-amino-4-hydroxybenzoic acid,respectively.We further found that they have some antioxidant activity.Conclusions:We preliminarily studied the function of the conserved globin in PYF BGC in two Streptomyces strains,and showed that pyf K may be a resistance gene for PYF biosynthesis and also participate in its regulation.In addition,we obtained aminobenzoic acid derivatives with certain antioxidant activity after heterologous expression of ptm B₁B₂B₃.Our study may not only facilitate the discovery of new functions for orphan globins from natural product BGCs,but also provide a convenient route for the preparation of hydroxybenzoic acid derivatives by microbial fermentation.