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Background: Erythromycin A (Er-A) produced by the actinomycete Saccharopolyspora erythraea 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 S. erythraea. Results: Here, we identified a TetR family transcriptional regulator (TFR), SACE_5754, negatively controlling erythromycin production. SACE_5754 indirectly repressed the transcription of ery cluster and cannot regulate itself and its adjacent gene SACE_5753. RNA-seq coupled with EMSAs and qRT-PCR was performed to identify the targets of SACE_5754, and confirmed that transcription of SACE_0388 (encoding a pyruvate, water diknase), SACE_3599 (encoding an antibiotic resistance macrolide glycosyltransferase) and SACE_6149 (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, SACE_0388 and SACE_6149 exhibited the positive effect on erythromycin production. Overexpression of either SACE_0388 or SACE_6149 in ∆SACE_5754 further increased the Er-A production. By engineering the industrial strain S. erythraea WB with deletion of SACE_5754 combined with overexpression of either SACE_0388 or SACE_6149, Er-A production in WB∆SACE_5754/pIB139–0388 and WB∆SACE_5754/pIB139–6149 was successively increased by 42 and 30% compared to WB. Co-overexpression of SACE_0388 and SACE_6149 in WB∆SACE_5754 resulted in enhanced Er-A production by 64% relative to WB. In a 5-L fermenter, WB∆SACE_5754/pIB139–0388-6149 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 SACE_5754 and its target genes, SACE_0388 and SACE_6149, resulted in enhanced erythromycin production in both wild-type and industrial S. erythraea 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. [ABSTRACT FROM AUTHOR]

In this work, we found that the Lrp/AsnC family protein SACE_5717 negatively regulated erythromycin biosynthesis in S. erythraea. Disruption of SACE_5717 led to a 27% improvement in the yield of erythromycin in S. erythraea A226. SACE_5717 directly repressed its own gene expression, as well as that of the adjacent gene SACE_5716 by binding to the target sequence 5′-GAACGTTCGCCGTCACGCC-3′. The predicted LysE superfamily protein SACE_5716 directly influenced the export of lysine, histidine, threonine and glycine in S. erythraea. Arginine, tyrosine and tryptophan were characterized as the effectors of SACE_5717 by weakening the binding affinity of SACE_5717. In the industrial S. erythraea WB strain, deletion of SACE_5717 (WBΔSACE_5717) increased erythromycin yield by 20%, and by 36% when SACE_5716 was overexpressed in WBΔSACE_5717 (WBΔSACE_5717/5716). In large-scale 5-L fermentation experiment, erythromycin yield in the engineered strain WBΔSACE_5717/5716 reached 4686 mg/L, a 41% enhancement over 3323 mg/L of the parent WB strain. [ABSTRACT FROM AUTHOR]

Streptomyces lincolnensis is generally utilized for the production of lincomycin A (Lin-A), a clinically useful antibiotic to treat Gram-positive bacterial infections. Three methylation steps, catalyzed by three different S-adenosylmethionine (SAM)-dependent methyltransferases, are required in the biosynthesis of Lin-A, and thus highlight the significance of methyl group supply in lincomycin production. In this study, we demonstrate that externally supplemented SAM cannot be taken in by cells and therefore does not enhance Lin-A production. Furthermore, bioinformatics and in vitro enzymatic assays revealed there exist two SAM synthetase homologs, MetK1 (SLCG_1651) and MetK2 (SLCG_3830) in S. lincolnensis that could convert l-methionine into SAM in the presence of ATP. Even though we attempted to inactivate metK1 and metK2, only metK2 was deleted in S. lincolnensis LCGL, named as ΔmetK2. Following a reduction of the intracellular SAM concentration, ΔmetK2 mutant exhibited a significant decrease of Lin-A in comparison to its parental strain. Individual overexpression of metK1 or metK2 in S. lincolnensis LCGL either elevated the amount of intracellular SAM, concomitant with 15% and 22% increase in Lin-A production, respectively. qRT-PCR assays showed that overexpression of either metK1 or metK2 increased the transcription of lincomycin biosynthetic genes lmbA and lmbR, and regulatory gene lmbU, indicating SAM may also function as a transcriptional activator. When metK1 and metK2 were co-expressed, Lin-A production was increased by 27% in LCGL, while by 17% in a high-yield strain LA219X. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]