Transcriptome-guided target identification of the TetR-like regulator SACE_5754 and engineered overproduction of erythromycin in .

BackgroundErythromycin A (Er-A) produced by the actinomycete Saccharopolyspora erythraea is an important antibiotic extensively used in human medicine. Dissecting of transcriptional regulators and their target genes associated with erythromycin biosynthesis is crucial to obtain erythromycin overproducer strains through engineering of relevant regulatory elements in S. erythraea.ResultsHere, we identified a TetR family transcriptional regulator (TFR), SACE_5754, negatively controlling erythromycin production. SACE_5754 indirectly repressed the transcription of ery cluster and cannot regulate itself and its adjacent gene SACE_5753. RNA-seq coupled with EMSAs and qRT-PCR was performed to identify the targets of SACE_5754, and confirmed that transcription of SACE_0388 (encoding a pyruvate, water diknase), SACE_3599 (encoding an antibiotic resistance macrolide glycosyltransferase) and SACE_6149 (encoding a FAD-binding monooxygenase) were directly repressed by SACE_5754. A consensus palindromic sequence TYMAGG-n2/n4/n11-KKTKRA (Y: C/T, M: A/C, K: T/G, R: A/G) was proved to be essential for SACE_5754 binding using DNase I footprinting and EMSAs. During the three target genes of SACE_5754, SACE_0388 and SACE_6149 exhibited the positive effect on erythromycin production. Overexpression of either SACE_0388 or SACE_6149 in SACE_5754 further increased the Er-A production. By engineering the industrial strain S. erythraea WB with deletion of SACE_5754 combined with overexpression of either SACE_0388 or SACE_6149, Er-A production in WBSACE_5754/pIB139-0388 and WBSACE_5754/pIB139-6149 was successively increased by 42 and 30% compared to WB. Co-overexpression of SACE_0388 and SACE_6149 in WBSACE_5754 resulted in enhanced Er-A production by 64% relative to WB. In a 5-L fermenter, WBSACE_5754/pIB139-0388-6149 produced 4998mg/L Er-A, a 48% increase over WB.ConclusionWe have identified a TFR, SACE_5754, as a negative regulator of erythromycin biosynthesis, and engineering of SACE_5754 and its target genes, SACE_0388 and SACE_6149, resulted in enhanced erythromycin production in both wild-type and industrial S. erythraea strains. The strategy demonstrated here may be valuable to facilitate the manipulation of transcriptional regulators and their targets for production improvement of antibiotics in industrial actinomycetes.