The Optimization On The Structure Of Nucleoside Antibiotics Capuramycin And Albomycin

Objectives 1 To obtain Capuramycin homologues through replacing and transformating the caprolactam ring,and view to produce a lead compound with higher value of druggability.Clarify the catalytic mechanism of the enzyme responsible for the coupling of lactam ring and glycoside skeleton, and provide useful information to build the "nonnatural capuramycin homologues library" with combinatorial biosynthesis approach. 2 To get the information on Albomycin kind compound, and establish biosynthesis model with that special structural feature. Identify the function of the major biosynthetic gene through inactivating the target gene and purificating its homologues from mutant strains.In addition,study the enzymes which are responsible for the siderophore biosynthesis and coupling,and elucidate its catalytic mechanism.Provide the basis to build up a "ferrimycin" homologues library by means of genetic engineering, combinatorial biosynthesis as well as semisynthetic and other strategies.Methods Inactivation and complementation the target gene,fermentation on the wild and the corresponded mutants,purification on A-503083 F and siderophore,protoplast transformation,heterologous expression, protein purification,enzyme catalysis in vitro,HPLC detection on analysis of enzyme activity.Results 1 The mutant strain producing Capuramycin homologues A-503083 F established with PCR-targeting successfully. The high quality A-503083 F was purified and prepared by using SP-207, HW-40 F and HPLC based on optimized fermentation conditions, characterized on feeding lactam ring deversities based on the chemical structure of capuramycin.The HPLC results explored that the catalytic reaction happened as plan after heterologous gene expression and the followed enzymatic catalysis of desired product in vitro. 2 The pure siderophore was successfully separated and purificated as a standard comparison for combinatorial biosynthesis of siderophore based on the information of Albomycin biosynthesis.The technology of knockout and biocombination was used to achieve the heterologous expression of the siderophore biosynthetic gene cluster. And it laid a foundation for the construction of the siderophore biosynthesis system by adding substrates and adjusting PH to optimize the engineering strain’s fermentation conditions.Conclusions 1 Taking the method of adding substrates and adjusting PH during the fermentation can optimize strain’s fermentation conditions. 2 Using the host S. lividans TK-64 can heterologous expresse the Cap W and siderophore. 3 It is feasible that Capuramycin homologues obtained in this research verify the construction "non-natural capuramycin homologues library" in vitro. 4 The information of siderophore biosynthesis that has been preliminary obtained can provide much useful information for the construction of " ferrimycin" homologues library.