Construction Of Micromonospora Engineering Strain And Optimization Of The Secondary Metabolites

Micromonospora purpurea is gentamicin producing strains.In this study,By treating Micromonospora purpura as the research object,blocking genK,genDl and genX and genP four genes by gene knockout method,which lie in biosynthesis gene cluster of gentamicin.And the micromonospora engineering strains(GbK987,GD1989,GX322,KP310 and GP408)were constructed.The specific contents include the following four aspects:First,construction of engineering bacteria with high yield of gentamicin C 1a.Firstly,screening Microromonospora purpurea Gb987 as parent strain.Followed by the recombinant plasmid pFD308,which was used for bloking genK,was introduced into the micromonospora purpurea Gb987 by conjugation.The disruption mutant GbK110(Gb987?genK)was screened out by replica plating and PCR analysis.According to the results of TLC,MS and HPLC analysis,we substantiated that the engineering strain GbK110 accumulates gentamicin C1 a.Compared with the original strain,gentamicin C1a increased from 62.83%to 85.24%.Optimization studies were carried out on genetic stability and fermentation cultural characters,it was turned out that GbK110 has good genetic stability and the shake flask fermentation titer up to 668 ?/mL,increasing 12.84%compared to the parent strain.Second,construction of engineering bacteria of gentamicin A.Using the temperature sensitive plasmid pKC1139 as vector,the recombinant plasmid pGD14 was constructed and introduced into the micromonospora purpurea GK1101 by conjugation.The disruption mutant GD1989(GK1101?genD1)was screened out by replica plating and PCR verification.Analysis of the secondary metabolites of TLC and MS,showed that GD1989 no longer synthesis of gentamicin C family products,only accumulated gentamicin A.The results showed that genDl gene is involved in gentamicin A C-4" methylation,knocking out genDl interrupt gentamicin A to gentamicin X2.Third,Construction of engineering strain GX322,KP310 and GP408.Using the same methods of construction engineering strain GD1989 and constracting engineering strain GX322(G1008?genX).According to the results of TLC,MS and HPLC analysis,secondary metabolites of C1,C2 and C2a component ratio changes,through it remain synthesis gentamicin Cs.It was speculatied that the genX gene may be closely related to the synthesis of gentamicin,and,involved in gentamicin C1 branch synthesis metabolism.Fourth,Construction of engineering strain KP310.Using the same methods of construction engineering strain GD1989 and constracting engineering strain KP310(GbK110?AgenP).According to the results of TLC and MS analysis,KP310 no more synthesis gentamicin C family products,KP310 accumulated JI-20A.It was demonstrated that genP involved in 3',4' dehydroxylation process.Firth,Construction of engineering strain GP408.Using the same methods of construction engineering strain GD1989 and constracting engineering strain GP408(G1008?genP).According to the results of TLC and MS analysis,GP408 no more synthesis gentamicin C family products,either.Different from the metabolites of KP310,GP408 accumulated JI-20B.