An atypical two-component system, AtcR/AtcK, simultaneously regulates the biosynthesis of multiple secondary metabolites in Streptomyces bingchenggensis

Abstract

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.

Keywords: Streptomyces bingchenggensis; milbemycins; nanchangmycin; rewiring regulatory network; strain engineering; two-component system.

Fig 1

AtcR and AtcK affect the production of specialized metabolites in the S. bingchenggensis TMB strain. (A) Genetic organization of atcR/atcK and its flanking genes. (B) Deletion of atcR or atcK blocks the yellow compound production. TMB-C, strain TMB contains the pSET152 plasmid; D38, the atcR deletion strain contains the pSET152 plasmid; D39, the atcK deletion strain contains the pSET152 plasmid; C38, the atcR deletion strain contains the pSET152-derived plasmid with PkasO*-driven atcR; C39, the atcK deletion strain contains the pSET152-derived plasmid with PkasO*-driven atcK. (C) HPLC chromatogram of milbemycin A3/A4 production in extracts from TMB-C, D38, D39, C38, and C39. (D) HPLC chromatogram of nanchangmycin production in extracts from TMB-C, D38, D39, C38, and C39. (E) The production of milbemycin A3/A4, yellow compound, and nanchangmycin in TMB-C, D38, D39, C38, and C39. Error bars show standard deviations.

Fig 2

qRT-PCR analysis of the transcription of representative genes from mil, nan, and kel clusters in TMB-C, D38, and D42. (A) The relative transcription levels of the milA2, milR, and milF genes at different time points. (B) The relative transcription levels of the nanA8, nanR2, and nanR4 genes at different time points. (C) The relative transcription levels of the sbi_06844, milR3, and atcR genes at different time points. The relative transcription levels of these genes are illustrated with saturation of color in the heatmap. The relative transcriptional abundances of each gene were calibrated with their transcription level at 12 h in TMB-C.

Fig 3

The regulatory cascade of AtcR/AtcK in S. bingchenggensis. (A) Verification of specific binding of AtcR to the upstream region of milR3 with EMSA. (B) Verification of specific binding of MilR3 to the upstream region of nanR4 with EMSA. (C) The repression of nanchangmycin production by nanR4. Strain TMB/RnanR4 was the strain in which transcription of nanR4 was repressed by ddCpf1-based CRISPRi interference. (D) The proposed regulatory cascade of AtcR/AtcK for the production of milbemycin, yellow compound, and nanchangmycin. Solid arrows represent activation. Dashed lines with bars represent repression.

Fig 4

Engineering milbemycin A3/A4 and nanchangmycin overproduction strains by rewiring the regulatory network in S. bingchenggensis. (A) The schematic strategy for improving the titer of milbemycin A3/A4 by strengthening the regulatory pathway of AtcR/AtcK. (B) Milbemycin A3/A4 production in engineered strains. (C) The schematic strategy for improving the titer of nanchangmycin by disrupting the regulatory cascade of AtcR/AtcK. (D) Nanchangmycin production by engineered strains. Error bars show standard deviations from three replicates.

Fig 5

The distribution of gene clusters similar to the atypical TCS contains clusters from atcR to sbi_06846. Cluster BLAST was analyzed on the antiSMASH server. (A) The genetic organization of gene sets from different Streptomyces strains carrying Type-II PKS similar to that of the kel cluster. (B) Genetic organization of gene sets from different Streptomyces strains exhibits similar regulatory islands and different kinds of putative BGCs. Red arrows indicate the genes encoding response regulators; green arrows indicate the genes encoding histidine kinase; blue arrows indicate genes encoding TOMM precursor leader peptide-binding protein; gray arrows indicate the genes encoding hypothetical protein; aqua green arrows indicate genes encoding SARP family regulators; orange arrows indicate genes encoding mini PKS; brown arrows indicate genes encoding FAD-dependent monooxygenases; purple arrows indicate genes encoding helix-turn-helix domain-containing proteins; yellow arrows indicate the genes that encode SDR family NAD(P)-dependent oxidoreductases; dark purple arrows indicate genes encoding glycosyltransferases; and dark orange arrows indicate genes encoding thioesterases.