The Research Of Glycosylation Modification Genes Of Fortimicin

Fortimicin is a new t-ype of pseudodisaccharide aminoglycoside antibiotic with aminocyclitol fortamine as glycosidic substituent.The relationship between the biosynthetic pathway of fortimicin and every gene has not been elucidated,and the domestic researches about fortimicin focuses on strain screening breeding and protoplast preparation.It is still blank for the studies on the gene functions of the biosynthetic pathway.This study focuses on disrupting some target genes using gene knock-out technique,studying the influence of gene disruption on secondary metabolites biosynthesis to predict the function and modification site of glycosylation modification genes.Establishment of conjugational transfer system for Micromonospora olivoasterospora.Apramycin resistance gene aac(3)IV was used as screening mark by sensitivity test.The recombinant plasmid pUQ502 was constructed with the plasmid pJTU412 containing aac(3)IV as the vector,and introduced into E.coli ET12567 with pUZ8002 as a donor.Spores were cultivated 48 hours at 32? after heat treated at 50? for 15 min;Spores and E.coli ET12567 were mixed at a ratio of 108:109 and painted in medium,then covered with apramycin at 25?g/ml concentration and nalidixic acid at 25?g/ml concentration after 48 hours.Firstly,study of the function of gene forQ.The recombinant plasmid pUQ502 constructed was introduced into the Micromonospora olivoasterospora FOM868 by conjugation,and the single crossover mutant QD1 was obtained.The double crossover mutant FQ61 was screened out by replica plating after QD1 was continuously cultured in the absence of apramycin.Ferment and extract the metabolites,TLC and MS was used to analysis the metabolites.Compared to the parent strain FOM868,the engineering strain FQ61 no longer produced fortimicin A and B,while accumulated the inermediatte metabolite FTM-Ao.The result showed that gene forQ was involved in the C-6' dehydrogenation of purpurosame in the fortimicin biosynthesis pathway.Secondly,study of the function of gene forO.The recombinant plasmid pOB32 was constructed using the thermal sensitive plasmid pKC1139 as vector and introduced into the micromonospora olivoasterospora FOM868 by conjugation.Then the double crossover mutant F05 was screened out.The results of fermentation and product analysis showed that the mutant strain F05 no longer synthesised fortimicin A and B but accumulated the inermediatte metabolites.Combine with bioinformatics technology,the result indicated that forO was the important gene for the C-3 oxygen methylation in the fortimicin biosynthesis pathway.Thirdly,study of the function of gene forK.Using the same method of studying the function of gene forO,the double crossover mutant FK6 was obtained.Ferment and extract the metabolites.The results of MS detection analysis showed that the fortimicin biosynthetic pathway of strain FK6 was blocked,which just accumulated the inermediatte metabolites.C-6'methylation of purpurosamine of fortimicin was blocked.This indicated that forK might be responsible for the C-6' methylation of purpurosamine of fortimicin.Fourthly,study of galactosylated modification gene sisl replacing the genP.Using the thermal sensitive plasmid pKC1139 as Vector,the recombinant plasmid pIP303 for sisI-genP gene replacement was constructed and introduced into Micromonospora purpurea G1008 by conjugation.The genetic recombination engineering strain GIP95 was obtained after screening.Use TLC and MS to analysis the components and structure changes of the metabolites.The results showed that the metabolic flow of the strain GIP95 was not blocked,which can still synthesize gentamicin C components.Gene sisI presumed to be 3',4'-double dehydroxylase gene from Micromonospora inyoensis was responsible for 3',4'-double dehydroxylation of the magenta sugar amine in the biosynthesis of gentamicin,completing the heterologous expression in Micromonospora purpurea G1008.