Dataset Information

The two-component system CepRS regulates the cephamycin C biosynthesis in Streptomyces clavuligerus F613-1.

ABSTRACT: During industrial fermentation, Streptomyces clavuligerus F613-1 simultaneously produces primary product clavulanic acid (CA) and cephamycin C. The cephamycin C biosynthetic gene cluster and pathway have been basically elucidated and the CcaR positive regulator was found to control the cephamycin genes expression. However, additional mechanisms of regulation cannot be excluded. The BB341_RS13780/13785 gene pair in S. clavuligerus F613-1 (annotated as SCLAV_2960/2959 in S. clavuligerus ATCC27064) encodes a bacterial two-component system (TCS) and were designated as CepRS (for cephamycin regulator/sensor). CepRS significantly affects cephamycin C production but only slightly affects CA production. To further understand the regulation of cephamycin C biosynthesis, the cepRS genes were deleted from S. clavuligerus F613-1. The deletion mutant resulted in decreased cephamycin C production but had no phenotypic effects. Real-time quantitative polymerase chain reaction analysis revealed that CepRS regulates the expression of most genes involved in cephamycin C biosynthesis, with electrophoretic mobility shift assays showing that CepR interacts with the cefD-cmcI intergenic region. These results demonstrate that the CepR response regulator serves as a transcriptional activator of cephamycin C biosynthesis, which may provide an approach for metabolic engineering methods for CA production by S. clavuligerus F613-1 in future.

SUBMITTER: Fu J

PROVIDER: S-EPMC6661058 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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The two-component system CepRS regulates the cephamycin C biosynthesis in Streptomyces clavuligerus F613-1.

Fu Jiafang J Qin Ronghuo R Zong Gongli G Zhong Chuanqing C Zhang Peipei P Kang Ni N Qi Xiaoyu X Cao Guangxiang G

AMB Express 20190727 1

During industrial fermentation, Streptomyces clavuligerus F613-1 simultaneously produces primary product clavulanic acid (CA) and cephamycin C. The cephamycin C biosynthetic gene cluster and pathway have been basically elucidated and the CcaR positive regulator was found to control the cephamycin genes expression. However, additional mechanisms of regulation cannot be excluded. The BB341_RS13780/13785 gene pair in S. clavuligerus F613-1 (annotated as SCLAV_2960/2959 in S. clavuligerus ATCC27064) ...[more]

PMID: 31352530

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Project description:Clavulanic acid (CA) is the preferred clinical drug for the treatment of infections by β-lactam antibiotic-resistant bacteria. CA is produced by Streptomyces clavuligerus, and although there have been many reports on the effects of carbon and nitrogen sources on CA production, the mechanisms involved remain unclear. In this study, we found that CA accumulation in S. clavuligerus F613-1 was increased significantly in MH medium, which is rich in organic nitrogen, compared with that in ML medium, which contains half the amount of organic nitrogen present in MH medium. Transcriptome analysis revealed that genes involved in CA biosynthesis, such as ceas1, ceas2, bls1, bls2, cas2, pah2, gcaS, and cad, and arginine biosynthesis, such as argB, argC, argD, argG, argH, argJ, and argR, were upregulated under rich organic nitrogen. Metabolome data revealed notable differences between cultures of F613-1 grown in MH and ML media with regard to levels of key intracellular metabolites, most of which are involved in arginine metabolic pathways, including arginine, glutamine, and glutamic acid. Additionally, supplementation of ML medium with arginine, glutamine, or glutamic acid resulted in increased CA production by S. clavuligerus F613-1. Our results indicate that rich organic nitrogen mainly affects CA biosynthesis by increasing the levels of amino acids associated with the arginine metabolic pathway and activating the expression of the CA biosynthetic gene cluster. These findings provide important insights for improving medium optimization and engineering of S. clavuligerus F613-1 for high-yield production of CA. IMPORTANCE The bacterium Streptomyces clavuligerus is used for the industrial production of the broad-spectrum β-lactamase inhibitor clavulanic acid (CA). However, much remains unknown about the factors which affect CA yields. We investigated the effects of different levels of organic nitrogen on CA production. Our analyses indicate that higher organic nitrogen levels were associated with increased CA yields and increased levels of arginine biosynthesis. Further analyses supported the relationship between arginine metabolism and CA production and demonstrated that increasing the levels of arginine or associated amino acids could boost CA yields. These findings suggest approaches for improving the production of this clinically important antibiotic.

Project description:Streptomyces bingchenggensis is an industrial producer of milbemycins, which are important anthelmintic and insecticidal agents. Two-component systems (TCSs), which are typically situated in the same operon and are composed of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Here, an atypical TCS, AtcR/AtcK, in which the encoding genes (sbi_06838/sbi_06839) are organized in a head-to-head pair, was demonstrated to be indispensable for the biosynthesis of multiple secondary metabolites in S. bingchenggensis. With the null TCS mutants, the production of milbemycin and yellow compound was abolished but nanchangmycin was overproduced. Transcriptional analysis and electrophoretic mobility shift assays showed that AtcR regulated the biosynthesis of these three secondary metabolites by a MilR3-mediated cascade. First, AtcR was activated by phosphorylation from signal-triggered AtcK. Second, the activated AtcR promoted the transcription of milR3. Third, MilR3 specifically activated the transcription of downstream genes from milbemycin and yellow compound biosynthetic gene clusters (BGCs) and nanR4 from the nanchangmycin BGC. Finally, because NanR4 is a specific repressor in the nanchangmycin BGC, activation of MilR3 downstream genes led to the production of yellow compound and milbemycin but inhibited nanchangmycin production. By rewiring the regulatory cascade, two strains were obtained, the yield of nanchangmycin was improved by 45-fold to 6.08 g/L and the production of milbemycin was increased twofold to 1.34 g/L. This work has broadened our knowledge on atypical TCSs and provided practical strategies to engineer strains for the production of secondary metabolites in Streptomyces.IMPORTANCEStreptomyces bingchenggensis is an important industrial strain that produces milbemycins. Two-component systems (TCSs), which consist of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Coupled encoding genes of TCSs are typically situated in the same operon. Here, TCSs with encoding genes situated in separate head-to-head neighbor operons were labeled atypical TCSs. It was found that the atypical TCS AtcR/AtcK played an indispensable role in the biosynthesis of milbemycin, yellow compound, and nanchangmycin in S. bingchenggensis. This atypical TCS regulated the biosynthesis of specialized metabolites in a cascade mediated via a cluster-situated regulator, MilR3. Through rewiring the regulatory pathways, strains were successfully engineered to overproduce milbemycin and nanchangmycin. To the best of our knowledge, this is the first report on atypical TCS, in which the encoding genes of RR and HK were situated in separate head-to-head neighbor operons, involved in secondary metabolism. In addition, data mining showed that atypical TCSs were widely distributed in actinobacteria.

Dataset Information

The two-component system CepRS regulates the cephamycin C biosynthesis in Streptomyces clavuligerus F613-1.

Publications

The two-component system CepRS regulates the cephamycin C biosynthesis in Streptomyces clavuligerus F613-1.

Similar Datasets

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