Abstract
Erythromycins produced by Saccharopolyspora erythraea have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.
Highlights
The high G+C Gram-positive bacterial actinomycetes are well known as one of the most abundant sources of bioactive secondary metabolites (Barka et al, 2016)
Results showed that pIB139 had no effect on the yield of erythromycin by testing the fermentation extracts of A226/pIB139 and SACE_0303/pIB139 (Figure 1D) erythromycin A (Er-A) yield of SACE_0303/pIB-0303 was nearly recovered to the parental level, and overexpression of SACE_0303 in A226 increased the Er-A yield by ∼31% (Figure 1D)
Five TFRs from Sac. erythraea were successively shown to be involved in the repression or activation of erythromycin biosynthesis, in which SACE_7301 and SACE_3446 exerted a direct interaction to the promoters of the ery cluster (Wu et al, 2014a, 2016), and SACE_3986 and SACE_5754 indirectly controlled the transcription of the ery cluster (Wu et al, 2014b, 2019)
Summary
The high G+C Gram-positive bacterial actinomycetes are well known as one of the most abundant sources of bioactive secondary metabolites (Barka et al, 2016). At least 20 families of transcription factors (TFs) have been found in the antibiotic-producing actinomycetes (RomeroRodriguez et al, 2015) Engineering of those regulators and their targets that control the biosynthesis of pharmaceutical antibiotics is an effective manner to boost the productivity of these fermentation products (Martín and Liras, 2010). There is lack of insight into the transcriptional regulation of the ery cluster owing to the absence of a CSR gene (Mironov et al, 2004). This implicates the unique mechanism for regulating erythromycin biosynthesis, and increases the difficulty in regulatory engineering of Sac. erythraea for erythromycin biosynthetic titer improvement
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