SACE_3986, a TetR family transcriptional regulator, negatively controls erythromycin biosynthesis in Saccharopolyspora erythraea

Abstract

Erythromycin, a medically important antibiotic, is produced by Saccharopolyspora erythraea. Unusually, the erythromycin biosynthetic gene cluster lacks a regulatory gene, and the regulation of its biosynthesis remains largely unknown. In this study, through gene deletion, complementation and overexpression experiments, we identified a novel TetR family transcriptional regulator SACE_3986 negatively regulating erythromycin biosynthesis in S. erythraea A226. When SACE_3986 was further inactivated in an industrial strain WB, erythromycin A yield of the mutant was increased by 54.2 % in average compared with that of its parent strain, displaying the universality of SACE_3986 as a repressor for erythromycin production in S. erythraea. qRT-PCR analysis indicated that SACE_3986 repressed the transcription of its adjacent gene SACE_3985 (which encodes a short-chain dehydrogenase/reductase), erythromycin biosynthetic gene eryAI and the resistance gene ermE. As determined by EMSA analysis, purified SACE_3986 protein specifically bound to the intergenic region between SACE_3985 and SACE_3986, whereas it did not bind to the promoter regions of eryAI and ermE. Furthermore, overexpression of SACE_3985 in A226 led to enhanced erythromycin A yield by at least 32.6 %. These findings indicate that SACE_3986 is a negative regulator of erythromycin biosynthesis, and the adjacent gene SACE_3985 is one of its target genes. The present study provides a basis to increase erythromycin production by engineering of SACE_3986 and SACE_3985 in S. erythraea.

TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis

“…It is worthwhile to note that genetic engineering of TFR elements has been shown to increase the yields of valuable antibiotics in industrial settings (13)(14)(15). Nonetheless, distinct from research related to TFRs for the biosynthesis of avermectin and erythromycin (13,(15)(16)(17)(18)(19), insights into the regulatory role of TFRs in lincomycin biosynthesis have not been reported. In the genome of S. lincolnensis LCGL, a derivative of S. lincolnensis LC-G with artificial synthetic 4ϫattB ⌽C31 (20), we identified 123 putative TFRs based on BLAST analysis with Pfam PF00440 (TetR_N) and the genome annotation of S. lincolnensis LC-G.…”

Regulation of Bottromycin Biosynthesis Involves an Internal Transcriptional Start Site and a Cluster-Situated Modulator

“…There is surprisingly little known about the regulation of specialized metabolites that do not contain master regulators in their gene clusters. A rare example is erythromycin biosynthesis in Saccharopolyspora erythraea, which is regulated by BldD, a key regulator of actinomycete development, as well as two other regulatory genes which are not located within its biosynthetic cluster (Chng et al, 2008;Kirm et al, 2013;Wu et al, 2014). We sought to identify the master regulators of bottromycin biosynthesis using a DNA affinity capture experiment with the intergenic region between btmB and btmC, which contains the major promoters that control the pathway.…”

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

“…Since extensive investigations have proved that TFRs are involved in the antibiotic biosynthesis in actinomycetes through gene inactivation and bioassay experiments [33], we have identified several TFRs pertinent to erythromycin production in S. erythraea, including the repressor SACE_3986 [32], and the activator SACE_7301 currently studied. By homologous recombination of linearized fragments ( Figure 1A), SACE_7301 was replaced by the thiostrepton resistance gene (tsr) in S. erythraea A226, and the desired mutant, named as ΔSACE_7301, was isolated and confirmed by PCR analysis ( Figure 1B).…”

“…Then, 3 ml cultures were inoculated into 250 ml baffled flasks containing 30 ml R5 liquid fermentation medium [38], and shaken for 6 days at 30°C (220 rpm). For flask fermentation of the erythromycin high-yielding strain WB and its derivatives, a liquid industrial medium was used as previously described [32]. After 24 h fermentation, 0.3 ml of n-propanol was added into the cultures and further incubated for 5 days at 30°C (220 rpm).…”

“…With the availability of the complete genome sequence, 101 putative TFRs in S. erythraea were inferred [29]. Based on the homologous recombination with linearized DNA fragments to inactivate specific genes, a number of the S. erythraea TFRs were examined, resulting in the identification of two TFRs (SACE_7040 and SACE_0012) having an association with morphological differentiation [30,31], and one TFR (SACE_3986) negatively controlling the erythromycin biosynthesis [32]. Nevertheless, functions of most TFRs in S. erythraea remain to be elucidated.…”