Transcriptome-guided target identification of the TetR-like regulator SACE-5754 and engineered overproduction of erythromycin in Saccharopolyspora erythraea

Abstract

Background: Erythromycin 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. Results: Here, 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 ΔSACE-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 WBΔSACE-5754/pIB139-0388 and WBΔSACE-5754/pIB139-6149 was successively increased by 42 and 30% compared to WB. Co-overexpression of SACE-0388 and SACE-6149 in WBΔSACE-5754 resulted in enhanced Er-A production by 64% relative to WB. In a 5-L fermenter, WBΔSACE-5754/pIB139-0388-6149 produced 4998 mg/L Er-A, a 48% increase over WB. Conclusion: We 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.