Inactivation of SACE_3446, a TetR family transcriptional regulator, stimulates erythromycin production in .

Erythromycin A is a widely used antibiotic produced by ; however, its biosynthetic cluster lacks a regulatory gene, limiting the yield enhancement via regulation engineering of . Herein, six TetR family transcriptional regulators (TFRs) belonging to three genomic context types were individually inactivated in A226, and one of them, SACE_3446, was proved to play a negative role in regulating erythromycin biosynthesis. EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene , the resistant gene and the adjacent gene (encoding a long-chain fatty-acid CoA ligase), and repressed their transcription. Furthermore, we explored the interaction relationships of SACE_3446 and previously identified TFRs (SACE_3986 and SACE_7301) associated with erythromycin production. Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis, we individually and concomitantly inactivated them in an industrial WB. Compared with WB, the WBΔ and WBΔΔ mutants respectively displayed 36% and 65% yield enhancement of erythromycin A, following significantly elevated transcription of and . When cultured in a 5 L fermentor, erythromycin A of WBΔ and WBΔΔ successively reached 4095 mg/L and 4670 mg/L with 23% and 41% production improvement relative to WB. The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.