The Research Of Genes Related In Gentamicin Pseudotrisaccharide Biosynthesis

The biosynthesis metabolic flux of gentamicin pseudotrisaccharide had been speculated in detail.Many genes in gentamicin biosynthesis gene cluster involved in the modification of gentamicin intermediate.They were important resource to exploit gentamicin A,B and X compounds.This study was based on the established Micromonospora purpurea conjugative transfer system and chose genA,genF,genB4 as the target genes.It revealed the relation between amino oligosaccharide biosynthesis and genes by knocking out these genes and analysising the change of metabolites.Firstly,research of the function of genA.Using the temperature-sensitive plasmid pKC1139 as vector,the recombinant plasmid pAB103 was constructed and introduced into the micromonospora purpurea G1008 by conjugation.Then the single crossover mutant GA1 was obtained.Apramycin resistance,PCR amplification and sequencing were used to confirm Micromonospora purpurea GA1048.MS was used to analysis the secondary metabolites.Gentamicin A2,instead of gentamicin C complex,was accumulated in Micromonospora purpurea GA1048,comparing with Micromonospora purpurea G1008.The inactivation of genA led to the changes of gentamicin biosynthesis flow,and genA might be responsible for the methylation at C-3" of garosamine.Secondly,research of the function of genF.Using the plasmid pJTU412 as vector,the recombinant plasmid pFB104 was constructed and introduced into the E.coli ET12567 by transformation.Taking the same method and parent strain as the genA research,the.genF in-frame deletion engineering strain GF208 was obtained.HPLC and MS were used to analysis the metabolites,and the results showed that the metabolic flow of gentamicin biosynthesis had no change and still accumulated getamicinC compounds.This indicated that the gene genF was not responsible for the methylation and didn't participate in the biosynthesis modification of gentamicin.Thirdly,research of the function of genB4.Taking the same method and parent strain as the genA research,the plasmid pGB403 was constructed for the genB4 disruption.The engineering strain GB4408 was obtained.Its metabolites was analysised by TLC,HPLC and MS.The engineering strain GB4408 mainly produced intermediate G418,sisomicin and verdamycin instead of gentamicinC compounds.The metabolic flux from sisomicin to gentamicinCla and from verdamycin to getamicinC2 were blocked.This result indicated that genB4 might be responsible for the dehydrogenation at C-4'-C5' of purpurosamine.Fourth,The construction of engineering strain mainly producing sisomicin.The E.coli ET12567(pUZ8002/pGB403)was as donor bacteria and then conjugated with the micromonospora purpurea GbK.Taking the same method,both the gene genK and the gene genB4 in-frame deletion engineering strain GbKB4 was obtained.TLC,HPLC and MS were used to analysis the metabolites.The result indicated that GbKB4 mainly accumulated intermediate sisomicin and getamicinX2 instead of G418 and verdamycin,comparing with the engineering strain GB4408.The strain mainly produced sisomicin was successful constructed.