SACE₃446,a TetR Family Transcriptional Regulator Controls The Biosynthesis Of Erythromycin In Saccharopolyspora Erythraea

Erythromycin,a clinically important 14-membered broad spectrum macrolide antibiotic,is produced by actinomycete Saccharopolyspora erythraea.The sales of erythromycin and its further developed semi-synthetic derivatives,including clarithromycin,azithromycin,dirithromycin,roxithromycin,and telithromycin,have reached billions of dollars per year.The prominence of erythromycin in pharmaceutical field makes overproducing strains necessary for industrial setting.Traditionally,strain improvement was mainly accomplished by multiple rounds of random mutagenesis and screening.Not only is random mutagenesis time consuming,but these overproduction mechanisms are inaccessible for the development of new strains.In recent years,the transfer of either exogenous or endogenous genes has been used to increase erythromycin production,but only a global regulator BldD controlling the synthesis of erythromycin was reported.Besides,Rodriguez reported that the production of high-yield strains is controlled by regulatory genes other than structural genes.The genome of Sac.erythraea was completely sequenced and annotated in 2007.There are 101 predicted TetR-like transcriptional regulator genes in the Sac.erythraea genome.The TetR family is a common class of transcriptional regulators in bacteria,which contain a conserved helix-turn-helix(HTH)DNA-binding motif and generally act as transcription repressors.Based on the key role of the TetR family transcriptional regulators in Streptomyces(e.g.rrdA,SCO1135,AtrA-g,SAV3818),we attempt to select TetR regulators which are related to the biosynthesis of erythromycin in Sac.erythraea.By homologous recombination of linear fragments,gene disruption mutant of SACE 3446 was constructed.Firstly,two 1.5 kb DNA fragments(the upstream and downstream fragments of SACE 3446 gene)of the adjacent regions were amplified from the genome of Sac.erythraea A226.Then the amplified products were digested and sequentially inserted into the corresponding sites of pUCTSR,yielding pUCTSR?3446.Secondly,the fragments(tsr-?SACE₃446),which carrying a thiostrepton resistance gene and the upstream and the downstream fragments of SACE₃446,were introduced into protoplasts of Sac.erythraea A226 through PEG-mediated transformation.By isolating on R3M agar medium containing 30?g/ml of Thio,SACE₃446 gene disruption mutant was obtained.When?SACE₃446 and the control(A226,bldD)were inoculated into the slope of R3M at 30?,the results showed that ?SACE₃446 mutant formed aerial mycelium as the same as A226.Besides,as ?SACE₃446 and the control(A226,?bldD)were simultaneously inoculated in TSB medium at 30? for 168h to preliminarily analysis the change of erythromycin titer by the size of Inhibition zone,the result is that the titer of erythromycin in ?SACE₃446 was higher than that in A226.The results implied that SACE₃446 gene might regulate the biosynthesis of erythromycin but not participate in the morphological differentiation of Sac.erythraea.HPLC analysis revealed that the titer of erythromycin A in SACE₃446 disruption mutant increased by around 53%compared with the original strain A226.In order to test whether the changes of erythromycin production in strain ?SACE₃446 was solely due to the deletion of SACE₃446 gene,a signal copy of SACE₃446 gene was subcloned to the E.coli-Sac.erythraea integrative shuttle expression plasmid pZMW and then introduced into the ?SACE₃446 strain by PEG-mediated protoplast transformation to generate the complementation strain ?SACE₃446/pZMW3446.Then the level of erythromycin A in TSB medium was measured,the strain?SACE₃446/pZMW3446 produced equal erythromycin to the original strain A226.To further verify the biological function of SACE₃446,the plasmid pZMW3446 was then introduced into Sac.erythraea A226 to obtain A226/pZMW3446.The HPLC analysis showed that there was a 54%decrease of erythromycin A compared with the original strain A226.Together,these events indicate that SACE₃446 exactly encodes a regulator negatively regulating the biosynthesis of erythromycin.In order to understand whether SACE₃446 is associated with other regulators controlling the synthesis of erythromycin,we chose a global regulator BldD which concurrently regulates both the synthesis of erythromycin and the morphological differentiation.A double disruption mutant ?bldD/ASACE₃446 was successfully constructed based on ?bldD.?bldD/ASACE₃446 produced higher level of erythromycin than ?bldD strain,and this change could be restored by complementation of a single copy of SACE₃446 gene.These results suggest that target gene of SACE₃446 may locate in downstream of bldD.To study whether SACE₃446 is commonly regulates the biosynthesis of erythromycin,the industrial strain ZMD was selected as an object.When knocking out SACE₃446 gene in industrial strain ZMD,it produces higher erythromycin than the original strain ZMD.Together,these results imply that SACE₃446 gene is a universal regulator which can negatively regulate the biosynthesis of erythromycin in Sac.erythraea A226 and industrial strain ZMD.SACE₃446 gene negatively controlling erythromycin biosynthesis in Sac.erythraea was screened by directional inactivation of genetic engineering.Besides,we speculated the relationship between two regulators by constructing a double deletion mutant.Not only do we herein report a horizon for erythromycin industrial setting,but our research provides a theoretical basis for speculating the relationships between regulators by genetic engineering.