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

BACKGROUND

Erythromycin A (Er-A) produced by the actinomycete 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 .

RESULTS

Here, we identified a TetR family transcriptional regulator (TFR), SACE_5754, negatively controlling erythromycin production. SACE_5754 indirectly repressed the transcription of cluster and cannot regulate itself and its adjacent gene . RNA-seq coupled with EMSAs and qRT-PCR was performed to identify the targets of SACE_5754, and confirmed that transcription of (encoding a pyruvate, water diknase), (encoding an antibiotic resistance macrolide glycosyltransferase) and (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, and exhibited the positive effect on erythromycin production. Overexpression of either or in ∆ further increased the Er-A production. By engineering the industrial strain WB with deletion of combined with overexpression of either or , Er-A production in WBpIB139-0388 and WBpIB139-6149 was successively increased by 42 and 30% compared to WB. Co-overexpression of and in WB resulted in enhanced Er-A production by 64% relative to WB. In a 5-L fermenter, WBpIB139-0388-6149 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 and its target genes, and , resulted in enhanced erythromycin production in both wild-type and industrial 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.