Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules

Abstract

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O- and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.

Keywords: deoxysugar; drug discovery; microbial genomics; polyketide; secondary metabolite.

Fig. 1.

Structure and biosynthesis of GNPs. (A) Selected GNP structures exemplify the diverse biosynthetic origin of the aglycone (black). (B) The simplified genetic organization of the avermectin biosynthetic pathway shows that it can be differentiated into aglycone biosynthetic genes (gray) and sugar biosynthetic genes (red). In a deoxysugar pathway, there are common biosynthetic genes (NT, nucleotidylyltransferase; 4,6-DH, 4,6-dehydratase; GT, glycosyltransferase; blue arrows) and specific biosynthetic genes (here: 2,3-DH, 2,3-dehydratase; 3-KR, 3-ketoreductase; 5-E, 5-epimerase; 4-KR, 4-ketoreductase; O3-MT, O3-methyltransferase; brown arrows). Sugar groups are indicated in red. Abbreviations: Glc1P, D-glucose-1-phosphate; PKS, polyketide synthase; TDP-Glc, 1-TDP-D-glucose.

Fig. 2.

The glycogenomic workflow for characterization of GNPs from genome-sequenced microbes. (A) Tandem mass-spectrometric analysis of microbial metabolic samples can reveal biosynthetic building blocks such as amino acids and sugar monomers of natural products via tandem MS fragment ions. (B) Identification of putative GNPs in a LC-MSⁿ analysis as peaks in EICs of known sugar fragment masses. (C) Verification of putative GNPs by characterization of candidate sugar monomers by sugar neutral losses and corresponding sugar fragment ions in tandem MS spectra. (D) Connection of putative GNP chemotype with corresponding GNP genotype in target microbial genome by genome mining of GNP pathway with glycosylation genes matching observed sugar fragments. (E) Characterization of GNP chemotype by analysis of aglycone biosynthetic genes of candidate GNP pathway and further structure elucidation.

Fig. 3.

Glycogenomic characterization of anthracycline polyketide cinerubin B from Streptomyces sp. SPB74. (A) LC-MSⁿ analysis of an metabolic extract yielded a putative GNP fraction via a product ion corresponding to an aminodeoxysugar (EIC, 158.12 m/z; red). (B) The MSⁿ analysis of the candidate GNP yielded sugar mass shifts for three different groups of candidate MSⁿ sugars, including aminodeoxysugars (red B-ion). (C) Genome mining of Streptomyces sp. SPB74 characterized a candidate pathway for target GNP with the biosynthetic genes corresponding to, e.g., the candidate MSⁿ aminodeoxysugars (red) and biosynthetic genes of a type II PKS aglycone (gray). (D) Chemotype prediction of a glycosylated anthracycline polyketide from tandem MS and genetic data. The target GNP was further characterized as cinerubin B with the aminodeoxysugar L-rhodosamine (red) by NMR. Abbreviations: BPC, base peak chromatogram; EIC, extracted ion chromatogram. For 2,3DH, 3,4DH, 3KR, 4KR, E, AmT, N,N-MT, and OxRed, see Dataset S2.

Fig. 4.

Glycogenomic characterization of arenimycin B genotype and chemotype from Salinispora arenicola CNB-527. (A) LC-MSⁿ analysis of a metabolic extract yielded a putative GNP fraction via product ions corresponding to a dimethylaminotrideoxysugar (EIC, 142.1 m/z; red). (B) The MSⁿ analysis of a candidate GNP (809 m/z, z = 1) yielded sugar mass shifts for a candidate MSⁿ forosamine sugar (red) and methyldeoxysugars (blue). (C) Genome mining of S. arenicola CNB-527 characterized a candidate GNP pathway with the biosynthetic genes corresponding to the MSⁿ candidate forosamine sugar (red) and O-methyldeoxysugars (blue) and biosynthetic genes of a type II PKS aglycone (gray). (D) Chemotype prediction of a glycosylated aromatic polyketide from MSⁿ and genetic data, which was further characterized by NMR as a GNP, arenimycin B. Coproduced arenimycin A is shown for comparison.