Dissecting and engineering of the TetR family regulator SACE_7301 for enhanced erythromycin production in Saccharopolyspora erythraea

Abstract

Background: Saccharopolyspora erythraea was extensively utilized for the industrial-scale production of erythromycin A (Er-A), a macrolide antibiotic commonly used in human medicine. Yet, S. erythraea lacks regulatory genes in the erythromycin biosynthetic gene (ery) cluster, hampering efforts to enhance Er-A production via the engineering of regulatory genes.

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

“…We previously identified and dissected the two TFRs, SACE_3986 and SACE_7301, involved in the erythromycin production, and SACE_3986 inactivation or SACE_7301 overexpression in the industrial S. erythraea WB resulted in the yield improvement. 24 , 25 Herein, we examined their interaction relationship to SACE_3446 via qRT-PCR and EMSA analysis. Compared with wild-type A226, Δ SACE_3446 did not exhibit differential expression of SACE_7301 and SACE_3986 ( Fig.…”

“…5a ). A previous study demonstrated that overexpression of SACE_7301 in WB dramatically elevated its erythromycin A yield, 25 thereby we expressed SACE_7301 with 3 extra copies under the control of P ermE * in the WBΔ 3446 Δ 3986 , to yield WBΔ 3446 – 3986 /3 × 7301 . Interestingly, erythromycin A yield in WBΔ 3446 – 3986 /3 × 7301 was dramatically decreased relative to that in WBΔ 3446 – 3986 , but a little higher than that in WB ( Fig.…”

“…Flask Fermentation of A226, WB and their derivatives were done as previously described. 25 For bioassay-based erythromycin analysis, 5 µL above fermentation supernatants were added to LB agar plates, which were sprayed with an overnight culture of B. subtilis PUB110. The plates were incubated at 37 °C for 12 h, and the erythromycin production was estimated by detecting and measuring the growth-inhibition zones.…”

“…Samples (50 mL) were taken every 24 h and erythromycin A was extracted from above cultures in terms of previous reported method. 25 HPLC analysis was carried out on Agilent 1260 HPLC system equipped with an Agilent Extend-C18 column (5 µm; 250 × 4.6 mm), which was equilibrated with 40% solution A (acetonitrile, chromatographic grade) and 60% solution B (potassium dihydrogen phosphate, 0.032 mol/L, pH 6.8). An isocratic program was carried out at a flow rate of 1.0 mL/min at 29 °C using UV detector at 210 nm.…”

“…To date, except for two characterized TFRs (SACE_7040 and SACE_0012) that negatively controlled S. erythraea morphogenesis, 22 , 23 we recently identified two additional TFRs (SACE_3986 and SACE_7301) negatively or positively regulating the synthesis of erythromycin. 24 , 25 Nonetheless, little is still known about remaining TFRs encoded by the S. erythraea genome. Therefore, we herein selected six TFRs with three groups of genome contexts for gene inactivation to define potential regulators of erythromycin biosynthesis.…”