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Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea.

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.

SUBMITTER: Liu Y

PROVIDER: S-EPMC8476842 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Propanol stimulates erythromycin biosynthesis by increasing the supply of propionyl coenzyme A (propionyl-CoA), a starter unit of erythromycin production in Saccharopolyspora erythraea Propionyl-CoA is assimilated via propionyl-CoA carboxylase to methylmalonyl-CoA, an extender unit of erythromycin. We found that the addition of n-propanol or propionate caused a 4- to 16-fold increase in the transcriptional levels of the SACE_3398-3400 locus encoding propionyl-CoA carboxylase, a key enzyme in propionate metabolism. The regulator PccD was proved to be directly involved in the transcription regulation of the SACE_3398-3400 locus by EMSA and DNase I footprint analysis. The transcriptional levels of SACE_3398-3400 were upregulated 15- to 37-fold in the pccD gene deletion strain (?pccD) and downregulated 3-fold in the pccD overexpression strain (WT/pIB-pccD), indicating that PccD was a negative transcriptional regulator of SACE_3398-3400. The ?pccD strain has a higher growth rate than that of the wild-type strain (WT) on Evans medium with propionate as the sole carbon source, whereas the growth of the WT/pIB-pccD strain was repressed. As a possible metabolite of propionate metabolism, methylmalonic acid was identified as an effector molecule of PccD and repressed its regulatory activity. A higher level of erythromycin in the ?pccD strain was observed compared with that in the wild-type strain. Our study reveals a regulatory mechanism in propionate metabolism and suggests new possibilities for designing metabolic engineering to increase erythromycin yield.IMPORTANCE Our work has identified the novel regulator PccD that controls the expression of the gene for propionyl-CoA carboxylase, a key enzyme in propionyl-CoA assimilation in S. erythraea PccD represses the generation of methylmalonyl-CoA through carboxylation of propionyl-CoA and reveals an effect on biosynthesis of erythromycin. This finding provides novel insight into propionyl-CoA assimilation, and extends our understanding of the regulatory mechanisms underlying the biosynthesis of erythromycin.

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Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea.

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