Genetic Engineering Strain Reconstruction Of FK506 And Strain Screening & Process Improvement

FK506,produced by Streptomyces tsukubaensis,is a 23-membered polyketide macrocyclic natural product,which has been applied in clinic for reducing the rate of allograft rejection.At present,the strains of FK506 production are mostly improved by traditional mutagenesis breeding methods.Now,with the rapid development of microbial metabolic engineering and genome-sequencing technologies,genetic engineering becomes a novel breeding method.In this study,by using the methods of conjugal transfer,another copy of the positive regulatory fkb R gene,production modification gene fkb DM,and structural gene tcs C in FK506 biosynthesis gene clusters were introducted into Streptomyces tsukubaensi,and the fermentation technology of the best strain was optimised and scaled up.Using the genome of Streptomyces tsukubaensis No.9993 as a template,the positive regulatory fkb R gene,ketonization enzyme Fkb D and O-methyltransferase Fkb M gene,reduced carboxylase Tcs C gene were obtained by PCR,which were ligated with pSET152 digested by enzyme and produced three recombinant plasmids named as p SET152-fkbR,pSET152-fkb DM and p SET152-tcs C.The three kinds of recombinant engineering strains were obtained by conjugational transfering.Fermentation studies verified that the production levels of FK506 by the engineering strains were higher than that of the starting strain in which the strain with more fkb DM genes showed the highest production titer for FK506,more 74% than that of the starting strain.It suggests that overexpressing fkb R,fkb DM,tcsC is effective for improving the yield of FK506.From five engineering strains constructed previously in our laboratory,the strain with overexpressed rapL/P which catalyzed the synthesis of piperidine ring in mcrocyclic skeleton was selected and using as the investigated strain and its fermentation process was optimized and scaled up.The composition and content of its seed and fermentation media were optimized by single factor experiment,and the best ratio of medium composition was obtained by response surface experiments.Under the condition of optimized fermentation,the FK506 production level in flasks increased 127% and is about 890 ?g/ml.Then,the fermentation technology was scaled up in 10 L fermentor and FK506 production level reached 1000 ?g/ml,suggesting that this strain and optimized technology is valuable for industrial production.