| Ficellomycin is an antibiotic produced by Streptomyces ficellus NRRL8067 that contains aziridine functional groups,which plays an important role in the synthesis of pharmaceutical intermediates and natural active products.At present,the research on aziridine at home and abroad mostly focuses on the optimization of chemical synthesis processes,and the research on its biosynthesis methods is still in its infancy.In this study,the key genes of putative aziridine functional group biosynthetic pathway in ficellomycin were studied,including key genes sulfate adenylyltransferase,adenylsulfate kinase and a putative sulfotransferase.Focus on the role of a putative sulfotransferase(fic28)in the process of aziridine biosynthesis and the feasibility of synthesis of aziridine ring compounds in vitro.Gene blocking and remediation(some gene blocking strains have been obtained in the early stage)experiment,bacteriostatic experiment of Penicillium oxalate from fermentation broth of original strain and blocking strain,color reaction of aziridine and feeding experiment of 3’-adenosine phosphate-5’-phosphate sulfate(PAPS)during fermentation showed that the sulfate adenylyltransferase,adenylsulfate kinase and sulfotransferase genes were indeed involved in the biosynthesis of aziridine in ficellomycin and were the necessary genes for its biosynthesis.The sulfotransferase(fic28),a key gene in the biosynthesis of aziridine,was constructed and heterogenously expressed.Enzymatic reaction analysis showed that the sulfotransferase could specifically transfer a sulfonyl from PAPS to the hydroxyl group of(R)-(-)-2-pyrrolidine methanol.The sulfonyl group leaves in alkaline condition,and then transforms.into a aziridine by intramolecular nucleophilic attack of adjacent secondary amines.Aziridine cyclic compounds can be detected with TLC by the chromogenic reaction of 4-(4-nitrobenzyl alcohol)pyridine perchlorate(NBP-C104)and alkylating agent 1,2-dimethoxyethane.The enzyme catalyzed substrate(R)-(-)-2-pyrrolidine methanol generates aziridine cyclofinal product(R)-1-azabicyclo[3.1.0]hexane and the final color is blue.In this study,the biosynthesis mechanism of aziridine in ficellomycin was verified,and the mechanism of sulfonation/molecular cyclization mediated by sulfotransferase was revealed.It provides new insights for the study of new enzyme chemistry,lays a theoretical foundation for the green synthesis and manufacture of aziridine derivatives and provides important clues for the study of functional diversity of sulfotransferases. |
