Study On The Structural And Functional Genes Of Chlortetracycline

Streptomyces aureofaciens was a kind of important medicinal microorganisms.Chlortetracycline(CTC),tetracycline(TC)and demethychlortetracycline(DMCTC)were its main secondary metabolites.Although the biosynthesis metabolic of CTC had been speculated in detail,most of the structural genes involved in the biosynthesis has not been clarified.In this research,we explored the functional genes modifying key structures of CTC at the molecular level.The ctcK,ctcN,ctcJ,ctcL and ctcO were knocked out by means of molecular genetics,and the effect of structural and functional genes in CTC biosynthesis were revealed by the variation analysis of the metabolites when the relevant gene was disrupted.First,research of the function of ctcK.Using the plasmid pJTU412 as vector and apramycin resistance gene aac(3)IV as screening mark,the recombinant plasmid pJTK2 was constructed and introduced into the Streptomyces aureofaciens J13 by conjugation.Then the single crossover mutant SK11 was acquired.The ctcK in-frame deletion mutant strain SK12 was screened out by replica plating after SKI 1 was continuously cultured in the absence of apramycin.HPLC and MS were used to analysis the metabolites.The results showed that the mutant strain SK12 mainly accumulated DMCTC instead of CTC and TC compared with the original strain J13.It was suggested that ctcK participate in the methylation modification of CTC at the C-6 position.In addition,the reverse mutant SK14 was obtained by complementation of the ctcK gene,and the result of HPLC analysis indicated that its metabolites chiefly reaccumulated the production of CTC,which showed no significant difference compared with the metabolites of J13.This clarified that ctcK plays a critical role in the methylation modification of CTC at the C-6 position.Second,study on the function of ctcN.Taking the same method and parent strain as the functional research of ctcK,the ctcN was knocked out and the mutant strain SN12 was received.The metabolites of mutant strain SN12 were analyzed by MS.The result showed that SN12 didn't synthesize 6-dehydroxy-chlortetracycline or anhydrotetracycline,which indicated that ctcN may not be responsible for the hydroxylation of CTC at the C-6 position.In addition,compared with the metabolites of parent strain J13,the relative content of CTC was significantly reduced and 6-methylpretrtramid was mostly accumulated in the metabolites of SN12,indicating that the metabolic flux of CTC biosynthesis were blocked partially and the intermediate metabolite of 6-methylpretrtramid was accumulated correspondingly as the result of ctcN deleted.Therefore,we speculated that ctcN might involve with the hydroxylation modification of CTC at the C-4 or C-12a position.Third,study the function of ctcJ.The ctcJ deletion mutant strain SJ12 was constructed on J13 by using the same methods.The MS analysis of metabolites showed that SJ12 mainly accumulated CTC and the metabolites components were roughly the same as that of the parent strain J3.So it can be infered that ctcJ may not be the essential gene which is closely related to the biosynthesis of CTC,and it might be a regulatory gene or the gene encoding auxiliary protein.Fourth,study the function genes ctcL.The ctcL was knocked out on J13 and mutant strain SL12 was acquired by using the same ways.The metabolites of SL12 were analyzed by MS and the result revealed that the ion peak of 4-keto-anhydrotetracycline instead of CTC was detected in the metabolites of SL12.It was suggested that ctcL may be the gene encoding C-4 aminotransferase,involving in the transamination from 4-keto-anhydrotetracycline to 4-amino-anhydrotetracycline.Fifth,explore the function of ctcO.Similarly,the ctcO deletion mutant strain SO12 was constructed on J13 by disrupting the ctcO.MS were used to analyze the metabolites,and the result showed that S012 accumulated other intermediate metabolites instead of CTC,4-amino-anhydrotetracycline or 4-dedimethylamino-chlortetracycline.Therefore,we supposed that ctcO may not be the gene encoding C-4 aminomethyltransferase,but may be the indispensable gene relating to the biosynthesis of CTC.However,the illumination of specific function of ctco needs further exploration.