Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking

Abstract

Background Herbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC–MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development. Results The genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum. Conclusions A herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the “cryptic” herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

New Polyketides from the Antarctic Fungus Pseudogymnoascus sp. HSX2#-11

“…Molecular networking is an outstanding methodology for time-effective analysis of secondary metabolites in biological samples based on LC-MS/MS fragmentation profiles, and has been proved to be a valuable approach for chemical de-replication and the discovery of new specialized metabolites [ 10 , 11 , 12 , 13 ]. Meanwhile, research on the Antarctica fungal metabolic profiles using molecular networking has not been reported.…”

UPLC-Q-TOF-MS/MS Analysis of Seco-Sativene Sesquiterpenoids to Detect New and Bioactive Analogues From Plant Pathogen Bipolaris sorokiniana

“…Most of researchers used this technology to identify (new/novel) metabolites or dereplicate in different crude extracts ( Jin et al, 2018 ; Wang et al, 2020 ). Recently, a molecular networking technique based on (U)HPLC-MS/MS combined with different databases was used in the dereplication and targeting of new natural products from diverse biological resources ( Watrous et al, 2012 ; Yang et al, 2013 ; Aksenov et al, 2017 ; Hou et al, 2019 ; Ramos et al, 2019 ; Rivera-Chaìvez et al, 2019 ; Shi et al, 2019 ; Wu et al, 2019 ; Chao et al, 2020 ; Zang et al, 2020 ; Lei et al, 2021 ; Lin et al, 2021 ). The molecular networking technique used the known or new compound as the “seed” to realize the visualization of analogues.…”

Complete Genome Sequence of Streptomyces albus Strain G153

“…D-GENIES ( 9 ) comparison with S. albus J1074 revealed only 0.04% mismatched regions, and the Mauve version 2.4.0 ( 10 ) alignment revealed an 11,997-bp long insertion sequence (3,292,629 to 3,304,629 bp) in the G153 genome, in which a total of six CDSs were coded, including those annotated as LuxR family transcriptional regulators. LuxR family proteins are often involved in the quorum sensing mechanisms ( 11 ) and activate biosynthetic gene clusters in Streptomyces strains ( 12 ).…”