Overexpression Of SACE₇301 Improve Erythromycin Production In Saccharopolyspora Erythraea

Antibiotics derived from the secondary metabolism of the Streptomyces and other actinobacteria include antibacterial, anticancer and antifungal or other activities of agents. The biosynthesis of secondary metabolites is subjected to complex regulation. Elucidation of the regulatory mechanisms could provide clues to improve the production levels of secondary metabolites. Erythromycin produced by Saccharopolyspora erythraeais one of the most clinically important macrolide antibiotics with broad-spectrum macrolide antibacterial activity. This work focused on the positive TetR-family regulator SACE₇301 controls erythromycin production in Sac.erythraea.Bioinformatics predicts that SACE₇301 is a TetR family transcriptional regulator.Its gene size is 660bpand protein molecular weight is 24.97kDa.We deleted SACE₇301 in strain A226 preliminary,named △SACE₇301.Then bacteriostatic assays of bacillus subtilis revealed that compared to A226,inhibition zone size was significantly smaller of △SACE₇301 mutant.The result indicated SACE₇301 is a positive regulator oferythromycin biosynthesis.We also constructed a pET22b-7301 vector,and obtained pure water-soluble proteins of SACE₇301.In order to explain the mechanism clearly and improve the erythromycin production,we carried out the following works.In the first place,we did a systematic fermentation by R5Medium, then the extracts of those cultures were analyzed by HPLC.Compared with the parental strain A226, △SACE₇301 had a 38%reduction in the Er-A yield, while A226/pZMW-7301 was 14% increased relative to A226.To investigate the effect of SACE₇301 overexpression on the transcriptionof ery genes, eryAl, ermE were selected for transcriptional analysis among A226/pZMW-7301 and A226/pZMW by qRT-PCR. The results showed that the transcription of eryAI、ermE in A226/pZMW-7301 were increasedobviously. Although SACE₇301 controls erythromycin production positively, the △SACE₇301 and A226 had comparable growth rates and cell densities. TetR family members regulate the transcription of target genes by specifically binding to their promoters, we found SACE₇301 can bind to the promoter regions of eryAI and ermE byEMS A analysis. SACE₇301 playsed a positive role in the erythromycin biosynthesis by potentially increasing the transcription of its several structural and resistance genes.Finally,To further improve the production of erythromycin, an overexpression tactic was designed and utilized for expressing SACE₇301 in wild-type strain A226 and industrial overproducer WB.SACE₇301 under the control of PermE* was cloned into pSET152, constructed vectors with 1,2, or 3 copies of SACE₇301 respectively.Thentheseplasmids were introduced into A226,obtained A226/7301, A226/2×7301 and A226/3×7301 strains.By fermentation and HPLC analysis, the three strains all presented Er-A product improvement,with the 3 copy number ofSACE₇301,reaching the highest 1.44 folds. qRT-PCRwas usedto comparethe transcriptional levels of eryAI and ermE betweenA226 and itSACE₇301-overxpressed strains.With the increase of SACE₇301 copy numberthe transcripts of eryAI and ermE in those mutants were gradually increased compared to the original level.In order toverify the structural stability of the expression cassette in theengineered strain, we cultured A226/3x7301 in the R5 liquid medium for 6 days, and found that the size of the gene expression cassette containing the 3 copies SACE₇301 from plasmid pSET152-3×7301, did not change by PCR analysis.To examinetheapplicabilityofoverexpression of SACE₇301 for enhancing erythromycin production in industrially-relevant strain WB.the desired mutants WB/7301, WB/2×7301 and WB/3×7301 were obtained.In flask experiments with an industrial medium for 6 days, the Er-A yields of three strains were respectively increased, with the 3 copy number of SACE₇301 increased 42% in comparison to that of WBreaching the highestincreasement.