Midecamycin 3-0-Acyltransferase Gene Expression In S.mycarofaciens Var.464

The content of main composition A₁ of the home-made Midecamycin （MDM） is lower thanthat of Japanese, including considerable LMA₆ （the difference between MDMA₁ and LMA isthat the former C-3 is propionylation the latter C-3 is acetylation）. There are.two reasons,the first isthat the amount of 3-0-Acyltransferase is insufficient or lack of activity, the second is the3-0-Aeyltransferase lack of specificity. In order to increase the production of the MDMA₁, westarted with increasing the dose of 3-0-Acyltransferase Gene （mdmB）. mdmB gene linked withPmerR and PermE^* promoter and to terminator was successfully transformed intoS.mycarofaciens var.464, for the first time. A plurinatality strain was gained.In order to increase the dose of mdm B gene, construted on the plasmid vector pKC1139,recombinant plasmids pSPU271 carrying PermE^* promoter, to terminator and mdmB gene,pSPU277 carrying PmerR promoter, to terminator, mdmB gene were constructed. Protoplasttransformation and conjugal transfer were inspected to transform the plasmid pSPU271 andpSPU277 to S.mycarofaciens var.464, transformant was not gained.A new efficient transforming system conjugal transfer was established using plasmidpSPU240 construted on the plasmid vector pSET152. Construted on the plasmid vectorpSET152, recombinant plasmids pSPU281 carrying PermE^* promoter and toterminator andmdmB gene, pSPU287 carrying PmerR promoter and to terminator, mdmB gene wereconstructed, pSPU287and pSPU281 were transformed into S.mycarofaciens var.464 usingconjugal transfer system. PCR authentication was adopted in the experiment, in which thestrap of mdmB genes and exogenous promoter were found.After the experiments of screening 200 conjugons of pSPU287and pSPU281,aplufinatality strain 287-53 has been obtained Single crossing over homologousrecombination has been proved in the recombinant plurinatality strain 287-53’s integration siteanalysis. In the experiment of transfer of culture secondary screenings, after being addedpreeurosor 01, its F4, F5 generations’ biotic titers in broth are higher than the original strain’sby 30.61% and 93.74％. The result of HPLC test （external reference method） demonstratedthat MDMA₁ content in broth was increased respectively by 59.34%and 42.44%.TheMDMA₁ content, however, of recombinant plurinatality strain 287-53 and the original strainon MDMA₁ are at the same level. Compared with without being feeded precurosor 01,F4,F5 generations’ MDMA1 content of 287-53 in broth were increased respectively by 346％and239％,the original strain respectively by 192％and 152％.Compared with the originalstrain,the more rapidly increase on MDMA₁ content in broth after being feeded precurosor 01,recombinant plurinatality strain 287-53 showed higher catalytic capability on acylation theMDMC₃ oxygen,which demonstrated that the amount of 3-0-Acyltransferase expressed inrecombinant pludnatality strain 287-53 probably increased.Apart from 287-53. Other recombinant plurinatality strains 287-18、287-23、287-36、287-62 were also proved holding the same integration way Single crossing over homologousrecombination. The second copy of mdmB gene linked with PmerR promoter was integratedto the S.mycarofaciens var.464 chromosome. It could be presumed that MDM biosyntheticgene cluster in the single crossing over homologous recombination recombinant plurinatalitystrains was not destroyed.After the experiments of screening 200 Recombinants, five of the seven recombinantplurinatality strains are conjugons of pSPU287, the unique stable recombinant plurinatalitystrain was obtained after transfer of culture secondary screenings. Consequently, theconclusion could be drawed that PmerR promoter was stronger than PermE^* promoter inS.mycarofaciens var.464.The study of fermentation of recombinant plurinatality strain 287-53 demonstrated thatthere was no difference in thalline growth between recombinant plurinatality strain 287-53and the original strain. Fermentation cycle, however, was extended and fermentation levelwas inhanced.