Activation and mechanism of a cryptic oviedomycin gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes

Abstract

Genome sequencing analysis has revealed at least 35 clusters of likely biosynthetic genes for secondary metabolites in Streptomyces ansochromogenes. Disruption of adpA encoding a global regulator (AdpA) resulted in the failure of nikkomycin production, whereas other antibacterial activities against Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis were observed with the fermentation broth of ΔadpA but not with that of the wild-type strain. Transcriptional analysis showed that a cryptic gene cluster (pks7), which shows high identity with an oviedomycin biosynthetic gene cluster (ovm), was activated in ΔadpA. The corresponding product of pks7 was characterized as oviedomycin by MS and NMR spectroscopy. To understand the molecular mechanism of ovm activation, the roles of six regulatory genes situated in the ovm cluster were investigated. Among them, proteins encoded by co-transcribed genes ovmZ and ovmW are positive regulators of ovm AdpA directly represses the transcription of ovmZ and ovmW Co-overexpression of ovmZ and ovmW can relieve the repression of AdpA on ovm transcription and effectively activate oviedomycin biosynthesis. The promoter of ovmOI-ovmH is identified as the direct target of OvmZ and OvmW. This is the first report that AdpA can simultaneously activate nikkomycin biosynthesis but repress oviedomycin biosynthesis in one strain. Our findings provide an effective strategy that is able to activate cryptic secondary metabolite gene clusters by genetic manipulation of global regulatory genes.

Keywords: AdpA; Streptomyces ansochromogenes; cryptic gene clusters; gene regulation; global regulators; metabolism; natural product biosynthesis; oviedomycin; polyketide; transcriptional coactivator.

Figure 1.

Bioassays of fermentation broth of wild-type strain and its derivatives. WT, S. ansochromogenes; ΔadpA, adpA disruption mutant; ΔadpAc, strain complemented by integrating a copy of adpA into the chromosome of ΔadpA. The strains were cultured in liquid MS for 4 days, and 200-μl supernatants from culture broths were assayed for bioactivity against C. albicans, S. aureus, B. cereus, and B. subtilis.

Figure 2.

Transcriptional analysis of PKS and NRPS biosynthetic gene clusters in S. ansochromogenes and ΔadpA by RT-PCR. A, cDNA templates were synthesized from RNA of S. ansochromogenes cultivated in three different liquid media (MS, SP, and GP). The strain name is indicated at the top of the gel, and growth time is below the strain name. The media used are indicated at the bottom of the gels. S. ansochromogenes genomic DNA (+) and double-distilled H₂O (−) were used as positive and negative controls for each primer pair. Each gene cluster is given at the left of the gels. B, genetic organization of pks7 cluster in S. ansochromogenes. The gene cluster contains 26 ORFs, and the gene ovmW was marked in this study.

Figure 3.

Identification and bioassay of oviedomycin. A, HPLC analysis of oviedomycin production in WT strain and ΔadpA. B, HPLC analysis of purified oviedomycin and the bioassay against S. aureus. The solute methanol was used as the negative control. mAU, milli-absorbance unit. C, MS/MS analysis of oviedomycin. D, structure of oviedomycin. E, cytotoxic assay of oviedomycin. The cell lines used in the cytotoxin assay were MCF-7, A549, and HepG2. Error bars, S.D.

Figure 4.

Heterologous expression of oviedomycin biosynthesis gene cluster. A, schematic representation of the plasmid used for heterologous expression of oviedomycin. B, heterologous expression of oviedomycin in M1146.

Figure 5.

Transcriptional analysis of oviedomycin biosynthetic genes by qRT-PCR. Total RNAs were isolated from mycelia of S. ansochromogenes and its derivatives after fermentation for 24 h. The constitutive hrdB transcript was used as an internal control. Error bars, S.D. A, transcriptional analysis of structural genes in WT strain and ΔadpA. B, transcriptional analysis of regulatory genes in WT strain and ΔadpA. C, transcriptional analysis of structural genes in WT strain, ΔadpA, XJJ104, and ΔadpA/ΔovmZ.

Figure 6.

HPLC analysis of oviedomycin produced by S. ansochromogenes and its derivatives. A, the injection volume for ΔadpA is 10 μl, and the others are 100 μl. B, injection volume is 100 μl. C, injection volume is 50 μl.

Figure 7.

EMSA and DNase I footprinting assays. A, EMSA of His₆-tagged AdpA protein with the probe P_ovmZ containing the promoter of ovmZ. Each lane contains 50 ng of DNA probes and 2 μg of poly(dI-dC). S, unlabeled specific probe (10-fold) was added; N, nonspecific probe (10-fold) was added. B, binding sequence (sites I, II, and III) of AdpA on the upstream region of ovmZ. The binding sites are shaded in red, and the translation start codons are indicated in italic type. C, DNase I footprinting assays of the His₆-tagged AdpA protein binding to the probe P_ovmZ. Each line contains 200 ng of DNA probe incubated with increasing concentrations of AdpA protein (0, 2.5, 5, 10, and 50 nm). The binding sites are marked in the dashed rectangle, and the sequences are given below the dashed rectangle.

Figure 8.

In vivo determination of the interaction between OvmZW and P_ovmOI. A, the plasmid pKC1139::P_hrdBZW was used to express OvmZ and OvmW. The plasmid pIJ10500::P_ovmOI::gusA contains the promoter of ovmOI and the reporter gene gusA. They were introduced into M1146 to detect the activity of the ovmOI promoter. B, chromogenic assay on AS-1 plate containing the substrate X-Gluc. XJJ105 contains the plasmid pIJ10500::P_ovmOI::gusA; XJJ106 contains the plasmid pKC1139::P_hrdBZW; XJJ107 contains the plasmid pKC1139::P_hrdBZ; XJJ108 contains the plasmid pKC1139::P_hrdBW; XJJ109 contains the plasmids pKC1139::P_hrdBZW and pIJ10500::P_ovmOI::gusA; XJJ110 contains the plasmids pKC1139::P_hrdBZ and pIJ10500::P_ovmOI::gusA; and XJJ111 contains the plasmids pKC1139::P_hrdBW and pIJ10500::P_ovmOI::gusA.

Figure 9.

Model for the regulation of nikkomycin and oviedomycin biosynthesis by AdpA in S. ansochromogenes.