Construction And Industrialization Of Gentamicin C1a Metabolic Branch

Take the metabolic branch of gentamicin Cla as the main line,the three engineering strains of GenB102,GenA102 and TI102 were successfully constructed by blocking the expression of three genes of gmrB,gmrA and sisl.In the meantime,the fermentation conditions of bacteria GbK10 producing gentamicin Cla were optimized by means of statistical theory and then the industrial capacity of bacteria GbK10 was tested and verified.Firstly,the construction of engineering strain GenB102 and the study on the function of gmrB.The plasmid pKC1139 was used as the shuttle vector to construct the homologous recombinant plasmid pKBE2 to inactivate the gmrB gene.Then the recombinant palsmid was introduced into Micromonospora purpurea G1008 by conjugation.A single exchange strain GenB 101 was obtained by resistance screening and the gmrB deletion mutant strain GenB102 was screened out after GenB101 was continuously cultured in the absence of apramycin.TLC and MS were used to analyzing the metabiolites.The results showed that GenB 102 was able to synthesize gentamicin C components and the metabolites components were roughly the same as that of the parent strain G1008.It suggested that gmrB was not involved in the modification of N-methylation at C-6' of purpurosamine and might be an auxiliary resistance gene.Secondly,the construction of engineering strain GenA102 and the research of the function of gmrA.The gmrA inactivated engineering bacteria GenA102 was obtained by the same method as the gmrB.the metabolites of strain GenA102 was analyzied by means of TLC and MS.The result showed that the main ion peaks of GenA102 metabolites were 511.3,525.3 and 539.3,which were different from those of the parent strain G1008 of 450.3,464.3 and 478.3.It indicated that the gmrA might be involved in the process of gentamicin biosynthesis,but was not the C-6' N-methylase gene.The investigation of the growth characteristics of GenA102 showed that GenA102 couldn't grow when the concentration of gentamicin reached 50 u/mL.The gmrA was cloned and expressed in vitro and it showed that the Escherichia coli DA2 integrated with gmrA could sustain the concentration of gentamicin more than 1000 u/mL,which proved that gmrA was a key resistance gene of Micromonospora purpurea.Third,the construction of engineering strain that mainly produced JI-20A.The recombinant plasmid pKTI2 was constructed by using pKC1139 as vector for knocking out sisl and introduced into Micromonospora inyoensis TS388 by conjugation.Then the sisi gene deletion mutant strain TI102 was obtained and its metabolites were analyzed by TLC and MS.The results showed that TI102 mainly accumulated intermediate metabolite JI-20A instead of sisomicin,which indicated that sisI might participate in the double dehydroxylation modification of purpurosamine at position C-3' and C-4'.In addition,an engineering strain that mainly produced JI-20A was obtained.Fourth,study on functional identity of genP and sisl.The recombinant engineering strain TPI102 including genP instead of sisl was constructed on TS388.The metabolites of TPI102 were analyzed by TLC and MS.The results showed that the engineering bacteria couldn't accumulate JI-20A but accumulate sisomicin,which was consistent with the parent strain.It suggested that the genP could heterologous express in Micromonospora inyoensis.In addition,this result not only clarified that the sisl and genP were the gene responsible for the 3',4'-didehydroxylation of purpurosamine,but also revealed that the gene combinations between Micromonospora is feasibility.Fifth,the optimization of fermentation condition and the industrialization experiment of GbK10.The four factors,which were significant for the fermentation unit of GbK10,were selected by Plackett-Burman design method.The interaction effect of these four factors was studied by response surface method and the best combination of these factors was calculated.The optimum conditions were as follows:starch 59.8 g/L,soybean meal 28 g/L,initial pH 7.17,speed 264 rpm.Under the condition,the fermentation unit of GbK10 in shake bottle increased from 520 u/mL to 767.9 u/mL.Then GbK10 was expanded to culture in 200 L fermenter and its fermentation unit reached 972.3 u/mL.The fermentation unit of GbK10 in 35 t fermenter could ultimately reach 1100.8 u/mL,which had met the industrial demands.