Molecular Breeding Manipulation Of Streptomyces Thermotolerans To Increase The Bioconversion Ability Of Tylosin

Acetyl-isovaleryl tylosin(AIV),a macrolide antibiotic,has an important value of application in the industry.AIV is produced by bioconversion of tylosin,which acylated hydroxyl groups at the 3- and 4″-positions,by Streptomyces thermotolerans.In this study, we would introduce acyB1-B2 gene ermE gene and disruption nsdA gene respectively into Streptomyces thermotolerans to increasing the bioconversion ability of tylosin to AIV.The fermentation conditions,including inoculation amount,medium capacity, inoculation time and tylosin bioconversion time,that effect the bioconversion abilities of tylosin were studied in shake flask.A frozen suspension was inoculated into seed medium, and then added to the seed medium again.After 24h of culture,5mL seed broth was inoculated into a 500-mL flask containing 35mL of fermentation medium.5mL solution containing tylosin and L-leucine was fed after 48h.AIV was separated and measured by HPLC after 48h of culture.In this optimal flask condition the bioconversion ability of tylosin to AIV of the starting strain reached 70%.Three Genes encoding 3-O- acyltransferase(acyA),4″-O-acyltransferase(acyB1) and its regulatory factor(acyB2) were responsible for converting tylosin to AIV.In this study,recombinant plasmid pBIB425 containing structural gene of acyB1-B2,was derived from pIJ8600.The recombinant strain BIB425 was obtained by introducing pBIB425 into the industrial strain BIB0830 of S.thermotolerans through intergeneric conjugal transfer. HPLC assays indicated that compared with the start strain BIB0830,in the level of flask, the bioconversion abilities of tylosin to AIV of recombiant BIB425 increased 14%.Only increasing the copy of acyB1 gene can not enhance the acyltransferase activity;the activation of acyB2 gene is importance for converting tylosin to AIV.ErmE similar to CarB encodes a ribosome dimethytransferase.In this study,the recombinant plasmid pBIB304 containing up-mutant promoter of ermE(ermEp~*) and structural gene of ermE,was derived from pIJ8600.The recombinant strain BIB304 was obtained by introducing pBIB304 into the industrial strain BIB0830 of S.thermotolerans through intergeneric conjugal transfer.The resistance experiment showed the resistance to tylosin of BIB304 dramatically increased from 100μg/mL to 900μg/mL.But the bioconversion ability of tylosin to AIV of BIB304 was not affected.The negative regulator of Streptomyces differentiation(nsdA) was also investigated in S.thermotolerans.The nsdA~-mutant BIB308 was obtained by introducing pBIB308 containing disruption cassette of nsdA gene into the industrial strain BIB0830 through intergeneric conjugal transfer.HPLC assays showed that,in the level of flask,the bioconversion ability of tylosin to AIV of recombinant BIB308 increased 14%.