Discovery of the Tyrobetaine Natural Products and Their Biosynthetic Gene Cluster via Metabologenomics

Abstract

Natural products (NPs) are a rich source of medicines, but traditional discovery methods are often unsuccessful due to high rates of rediscovery. Genetic approaches for NP discovery are promising, but progress has been slow due to the difficulty of identifying unique biosynthetic gene clusters (BGCs) and poor gene expression. We previously developed the metabologenomics method, which combines genomic and metabolomic data to discover new NPs and their BGCs. Here, we utilize metabologenomics in combination with molecular networking to discover a novel class of NPs, the tyrobetaines: nonribosomal peptides with an unusual trimethylammonium tyrosine residue. The BGC for this unusual class of compounds was identified using metabologenomics and computational structure prediction data. Heterologous expression confirmed the BGC and suggests an unusual mechanism for trimethylammonium formation. Overall, the discovery of the tyrobetaines shows the great potential of metabologenomics combined with molecular networking and computational structure prediction for identifying interesting biosynthetic reactions and novel NPs.

Figure 1

Structure elucidation of tyrobetaine and chlorotyrobetaine. (a) The structure of tyrobetaine and chlorotyrobetaine with key NMR correlations indicated. (b) The MS² spectrum for tyrobetaine with key masses indicated. *Indicates the mass after removal of the trimethylammonium. L^OH = hydroxyleucine. Red masses indicate observed masses. Δppm values are indicated in parentheses after the key masses. Key mass losses are in purple.

Figure 2

Tyrobetaine BGC and heterologous expression. (a) The BGC for the tyrobetaines from Streptomyces sp. WC-3703. Arrows indicate genes. The color of the arrow corresponds to the type of gene (indicated below the arrows). See Table S5 for BLAST analysis. (b) AntiSMASH NRPS domain predictions for TybD, TybE, and TybF. A = adenylation domain with subscript indicating the predicted amino acid (Y = tyrosine, X = no consensus, A = alanine). MT = N-methyltransferase. T = thiolation domain. C = condensation domain. See Table S6 for NRPS adenylation domain predictions. (c) Extracted ion chromatogram for tyrobetaine (587.30–587.31) in wild type S. lividans 66, S. lividans 66 pRAM6 (heterologous expression of NRPS_GCF.432), purified tyrobetaine, and S. lividans 66 pRAM6 spiked with purified tyrobetaine.

Figure 3

Feeding experiments with stable isotope labeled amino acids. Mass spectrum from spent media from Streptomyces sp. NRRL WC-3703 grown on (a) medium alone or medium with (b) D₄-L-tyrosine, (c) D₃,¹³C-methionine, (d) D₁₀-L-leucine, or (e) D₄-L-alanine. (f) Structure of tyrobetaine with likely location of stable isotopes. Yellow bar indicates the m/z for tyrobetaine. Colored bars indicate isotopically labeled tyrobetaine. See Figure S6 for chlorotyrobetaine.

Figure 4

Phylogenetic analysis of TybD N-methyltransferase. (a) Phylogenetic tree for the N-methyltransferase of TybD compared to that of known methyltransferases from the UniProtKB/Swiss-Prot database. NMT = N-methyltransferase. Functions of proteins are indicated by color and defined by text of the same color. Black numbers are bootstrap support percentages. (b) A portion of the phylogenetic tree for the N-methyltransferase of TybD compared to that of nonredundant protein sequences from NCBI. The products of known NRPS N-methyltransferases are indicated in purple text. Black numbers are FastTree support values. See Figure S7B for the full figure.