Research On The Modification Of Toyocamycin Producing Strain Streptomyces Diastatochromogenes 1628 By Using Ribosome Engineering Technology

Toyocamycin?TM?was one of the important members of the family of nucleoside antibiotics.It had been recognized as a promising fungicide utilized in controlling the occurrence of plant disease in the agricultural industry field because of its high efficient,low toxicity and low residue.In the previous study,a strain S.diastatochromogenes 1628 was isolated,which could produce TM.However,its poor TM yield was still not to meet large-scale industrial production.A large number of recent studies indicated that ribosome engineering technology could improve the synthesis ability of microbial secondary metabolites efficiently.In this context,in order to increase the TM production of wild-type strain 1628,this work attempt to modifying S.diastatochromogenes 1628 by using ribosome engineering technology,Meanwhile,characterization of the representative mutants was also studied.It will provide theoretical basis for the further study of the molecular mechanism of high efficient biosynthesis of TM.The main contents were performed as follows:Firstly,rifampicin?Rif?,gentamicin?Gen?and streptomycin?Str?was utilized for screening antibiotic resistant mutants of S.diastatochromogenes 1628.TM production and genetic stability of different kinds of mutants were determined.The experimental results showed that 2 in 40 Strr mutants had no significant improve in TM production?<1 fold?.3 in 100 Genr mutants only increase 1-2 fold TM production comparing to that of control,also genetic stability of Genr mutants was poor.Strikingly,9 in 120 Rifr mutants improved TM output dramatically.Moreover,genetic stability was very good.The highest TM yield of Rifr mutant 1628-T15 reached 689.3mg/L,which was 4.5 times higher than that of the wild-type strain.Among all the Rifr mutants,Rifr mutant 1628-T62 showed the strongest decline in TM production.Secondly,both mutants 1628-T15 and 1628-T62 were used for further studies.The mutations in rpoB gene which encodes polymerase ? subunit were sequenced and analyzed.The mutants 1628-T15 and 1628-T62 showed one of the following rpoB mutations: A1310 G and C1328 G,respectively.The resulting change of corresponding amino acid in mutant 1628-T15 and mutant 1628-T62 were His437 Arg and Ala443 Gly.By using scanning electron microscope,it showed that no significant difference was found between mutant 1628-T15 and S.diastatochromogenes 1628,while the ability of growth and sporulation of mutant 1628-T62 was impaired comparing to that of strain1628.The transcriptional level of toyG and adpA_sd which were related to the synthesis of TM and morphology differentiation in S.diastatochromogenes 1628,respectively,was investigated in the parental strain as well as in both mutants.The result showed that in mutants 1628-T15,while there was no difference between mutant 1628-T15 and S.diastatochromogenes1628 in terms of transcriptional level of adpA_sd.In contrast,expression of adpA_sd and toyG gene was found to be severely suppressed in mutant 1628-T62.The above results were in agreement with changes related to TM production and morphological differentiation in mutants.In addition,it was striking that both mutant 1628-T15 and 1628-T62 exhibited similar level of protein synthesis compared to that of parental strain 1628 by using GUS as reporter gene.Finally,adpA_sd gene was cloned and placed under control of the PermE* in plasmid pIB139 to generate pIB139-adpA_sd.The resulted plasmid pIB139-adpA_sd was introduced and integrated into chromosome DNA of mutant 1628-T62 by conjugation to create recombinant strain 1628-T62 A.The experimental results indicated that ability of sporulation of recombinant strain 1628-T62 A was enhanced comparing to that of control strain 1628-T62.Moreover,TM production of 1628-T62 A strain was improved 3-times higher than that of strain 1628-T62.These data suggested that adpA_sd gene was required for morphological differentiation in S.diastatochromogenes 1628.The over-expression of adpA_sd gene in S.diastatochromogenes 1628 could improve its TM yield in some extent.