The Research Of Modifier Genes On Gentamicin Key Groups

Gentamicin and its intermediate metabolites have important clinical value.A series of groups are modified in gentamicin biosynthesis,such as methylation,amination,aminomethylation,however,the modifier genes refer to the gentamicin biosynthesis have not been elucidated.In this study,we explored the modifier genes of gentamicin key groups at the molecular level.The genT,genN,genY,genBl and genMl were knocked out use the technology of molecular genetics.The modifier genes on gentamicin key groups were confirmed by the variation analysis of gentamicins biosynthesis when the relevant gene was disrupt.Firstly,explored the function of genT.Using the plasmid pJTU412 as a vector,the recombinant plasmid pFT104 was constructed and introduced into the micromonospora purpurea G1008 by conjugation.Then the single crossover mutant GDT105 was acquired.The disruption mutant GT106 was screened out by replica plating and PCR analysis.The metabolites were analyzed by using MS.Compared with the original strain G1008,GT106 still synthetic gentamicin C1,C2,C2a,C2b and Cla.It suggested that genT was not involved in the C-6'-aminomethylation.Secondly,the research of functional genes genN.Using the plasmid pKC1139 as vector.the recombinant plasmid pFTN203 was constructed for blocking-up genN and introduced into the GT106 by conjugation.The mutant strain GTN205 was obtained by PCR analysis.Based on the role of genK in C-6'-methylation,the mutant strain GTNK308 was obtained by knocking out genK in the GTN205.The metabolites were analyzed by using MS.The mutant strain GTN205 no longer produced gentamicin C1 and C2b,but accumulated gentamicin C1a and C2.what's more,GTNK308 no longer produced gentamicin C2b,but accumulated gentamicin C1a,A2 and A.These results showed that the inactivation of genN led to blocking the synthesis of gentamicin C1 and C2b.This result indicated that genN plays an critical role in the modification of aminomethylation at C-6' of purpurosamine.The genN may codes N-methyl transferase.Thirdly,the research of functional genes genY.The recombinant plasmid pFY303 was constructed by the same method as the genN research.The plasmid pFY303 introduced into the G1008 and GK1101 by conjugation.The mutant strain GY305 and GKY307 were obtained by PCR analysis.MS analysis indicated that GY305 and GKY307 produced sisomicin,gentamicin C1a and C2b instead of gentamicinC1 and C2.This result indicated that genY involved in the modification of methylation at C-6' of purpurosamine.Fourthly,study on the function of genBl.Taking the same method and parent strain as the genY research,The recombinant plasmid pFB1403 was constructed,the plasmid pFY303 introduced into the G1008 and GK1101 by conjugation.The mutant strain GB1405 and GKB1407 were obtained by PCR analysis.MS analysis the metabolites of GB1405 and GKB1407.Compared with the original strain G1008,the metabolites of GB1405 and GKB1407 without changed.Combination of genBl^ genB2,genB3 and genB4 gene in vitro expression found that GenB1,GenB2,GenB3 and GenB4 each plays an role in the C-6'-amination.This chapter through the inactivation of genBl has not completed the synthesis of block gentamicin C,This result indicated that the other aminotransferase genes genB2,genB3 and genB4 coded aminotransferase catalytic C-6'-amination,and make the gentamicin C flows were not blocked.Fifthly,the research of functional genes genMl.The recombinant plasmid pFM1503 was constructed by the same method.The genMl was knocked out in G1008,and the mutant strain GM1505 was obtained by PCR analysis.MS analysis indicated that GM1505 produced 2-DOS.These results showed that the genMl coded D-glucose transferase.The genMl play an critical role in the first step of glycosylation.