Metathramycin, a new bioactive aureolic acid discovered by heterologous expression of a metagenome derived biosynthetic pathway

Abstract

Bacterial natural products have been a rich source of bioactive compounds for drug development, and advances in DNA sequencing, informatics and molecular biology have opened new avenues for their discovery. Here, we describe the isolation of an aureolic acid biosynthetic gene cluster from a metagenome library derived from a New Zealand soil sample. Heterologous expression of this pathway in Streptomyces albus resulted in the production and isolation of two new aureolic acid compounds, one of which (metathramycin, 6) possesses potent bioactivity against a human colon carcinoma cell line (HCT-116, IC₅₀ = 14.6 nM). As metathramycin was a minor constituent of the fermentation extract, its discovery relied on a combination of approaches including bioactivity guided fractionation, MS/MS characterisation and pathway engineering. This study not only demonstrates the presence of previously uncharacterised aureolic acids in the environment, but also the value of an integrated natural product discovery approach which may be generally applicable to low abundance bioactive metabolites.

Fig. 1. Biosynthesis of aureolic acids mithramycin and chromomycin A₃, and the structure of chromocyclomycin.

Fig. 2. Discovery and heterologous expression of an aureolic acid biosynthetic gene cluster from a soil metagenome library. (A) Comparison of our metagenome derived aureolic acid cluster (MMY cluster) to the known clusters for mithramycin and chromomycin A₃. Genes are coloured based on broad function classifications. Amino acid identities for alignments of homologous genes are provided in Table 1. (B) HPLC traces for extracts from S. albus harbouring the MMY biosynthetic gene cluster (S. albus::MMY, red) and an empty vector control (S. albus, blue). Metabolite peaks with the characteristic aureolic acid UV absorbance spectrum (inset, green) are indicated with an asterisk (*). Compound 5 is the major metabolite peak, with a retention time of 13 min. Select minor metabolite peaks were identified by EIC of the LCMS trace; compound 6 is a minor metabolite with retention time 9.4 min, 4-demethylpremithramycinone 7.3 min and premithramycinone (3) peak at 9.8 min. The retention time on the reversed-phase C₁₈ column is shown on the X-axis, and absorbance at 420 nm on the Y-axis.

Fig. 3. Key NMR correlations used in elucidation of the structure of premetathramcyin: Key HMBC and COSY correlations in the NMR spectra of 5 consistent with the aglycone core of the proposed structure of chromocyclomycin as well as Key HMBC and COSY/TOCSY correlations in the NMR spectra of premetathramcyin providing the glycosidic linkages are shown.

Fig. 4. MS/MS fragmentation of mithramycin and metathramycin: MS/MS spectra obtained with a collision energy of 60.0 eV are depicted. (A) Tandem MS showing a possible fragmentation pattern from a sample of mithramycin. Two of the observed daughter ions also occur in panel B. (B) Tandem MS fragmentation pattern of metathramycin. The non-conserved fragment ions m/z differ from those of mithramycin by 116 (i.e. 935–819 and 530–414), equivalent to the mass difference between mithramycin and metathramycin. (C) Putative structures for observed fragment ions.

Fig. 5. Pathway engineering provides evidence that metathramycin is the product of direct biosynthetic conversion from premetathramycin: (A) reaction catalysed by OIV, W enzymes in the biosynthesis of mithramycin. (B) Overexpression of the OIV, W homologues under the control of a strong constitutive promoter results a 1.9 fold increase in production of metathramycin as compared to a strain containing these genes under the control of a native promoter, data for three biological replicates are shown. (C) MTS assay against the human tumour cell line HCT-116 (n = 4) shows metathramycin possesses potent cytotoxicity comparable to that of mithramycin.