The Putative TetR-family Regulator SACE₇301 Controls Erythromycin Production In Saccharopolyspora Erythraea

Saccharopolyspora erythraea,a Gram-positive actinomycetes,makes erythromycin which is a macrolide antibiotic with broad-spectrum antibacterial effect.Since 1952,erythromycin and its derivatives has been developed to the third generation and now ketolide antibiotics,the third generation of erythromycin,is considered to be the next generation of the treatment of drug-resistant bacteria antibiotics due to its acid stability and inhibition of a number of drug-resistant bacteria.Erythromycin has been one of a leading anti-infective drugs in the clinical.The research to the synthesis of erythromycin focused on the erythromycin biosynthetic pathway and transformation of erythromycin in the past.In recent years,Rodriguez and Lum studies show that the main difference between the erythromycin overproducing strain and the wild strain is regulation stage of the synthesis of erythromycin,causing more and more people interested in the regulation of gene of Sac.erythraea.However,the gene cluster for erythromycin biosynthesis lacks any regulatory genes in Sac.erythraea,hampering efforts to improve erythromycin productivity.So far there are no regulatory genes have been reported except the global regulator BldD?SACE₂077?which is essential for erythromycin biosynthesis and morphological differentiation.With the increase in market demand,erythromycin overproducing strain has become increasingly important.To examine its biological function,the SACE₇301 gene was deleted in Sac.erythraea A226 by linearized fragment homologous recombination.Firstly,with Sac.erythraea A226 genomic DNA?gDNA?as a template,two 1.5-kb DNA fragments flanking the SACE₇301 gene were amplified by PCR method.The two PCR products were digested with EcoRI/Kpnl and Xbal/Hind?,respectively,and inserted into the corresponding sites of pUCTSR,generating pUCTSR-A7301.By linearized fragment homologous recombination,and the SACE₇301 gene was replaced with the thiostrepton resistance cassette,generating ?SACE₇301.When grown on R3M agar medium,ASACE₇301 and A226 had comparable aerial mycelium formation rates.Bioassay analysis of erythromycin production showed that the SACE₇301 mutant displayed lower inhibitory activity against B.subtilis relative to its parental strain A226,but higher inhibitory activity than the ?bldD mutant,indicating that SACE₇301 was a positive regulator for erythromycin production in Sac.erythraea,and had no effect on spore formation.Moreover,the wild-type A226 and the ?SACE₇301 mutant were cultivated at 30? for 6 days in the R5 liquid medium,and the extracts of those cultures were analyzed by HPLC.As expected,the erythromycin A yield of ?SACE₇301 mutant was decreased by 34.5%relative to that in A226.In order to exclude the possibility that the decreased erythromycin production was due to a random mutation in another locus,the ?SACE₇301 mutant was complemented with cloned SACE₇301,with an empty pZMW vector control.The apramycin resistant clones,?SACE₇301/pZMW-7301 and ?SACE₇301/pZMW,were confirmed by PCR analysis.The erythromycin A yield in ?SACE₇301/pZMW-7301 was recovered to 84.17%level of original strain A226,and improved to 152.09%compared to?SACE₇301/pZMW,confirming that the reduced production of erythromycin A in?SACE₇301 resulted from the absence of the SACE₇301 gene.To substantiate the role of SACE₇301 as a positive regulator of erythromycin A production,SACE₇301 over-expression in Sac.erythraea A226 was investigated.We introduced pZMW-7301 or pZMW into the Sac.erythraea A226 to obtain correct transformants A226/pZMW-7301 and A226/pZMW,respectively.HPLC analysis of fermentation products showed that A226/pZMW-7301 had enhanced erythromycin A production by 23.1%compared with A226/pZMW.These findings,like those from the SACE₇301 deletion and complementation experiments,indicate that SACE₇301 plays a positive regulatory role in erythromycin production.To examine whether S ACE₇301 might directly regulate erythromycin biosynthesis through transcriptional regulation,a His6-SACE₇301 protein was expressed in E.coli BL21?DE3?.EMSA was performed by combining the promoter region of eryAI with purified SACE₇301.A nonspecific poly dI/dC DNA probe was added into the binding reaction as a control.Intriguingly,the eryAI promoter probe exhibited strong shifts by the addition of purified SACE₇301 at a range of protein concentrations,the same as the positive control of BldD,suggesting that recombinant SACE₇301 specifically binds the promoter regions of eryAI.A TetR family regulator?SACE₇301?was identified to positively regulate the erythromycin production.In contrast,SACE₇031 does not appear to be involved in the morphological differentiation in Sac.erythraea indicated by no appreciable differences in spore formation.In addition,since the EMSA of purified SACE₇301 protein to eryAI promoter DNA demonstrates direct associations,we suggest that the influence of SACE₇301 on erythromycin A production is likely to be manifested by direct associations.Our findings here not only form a new understanding of the unusual regulatory mechanism of erythromycin biosynthesis in Sac.Erythraea,but provide a theoretical basis for the use of genetic engineering to increase erythromycin production.