An atypical orphan carbohydrate-NRPS genomic island encodes a novel lytic transglycosylase

Abstract

Microbial genome sequencing platforms have produced a deluge of orphan biosynthetic pathways suspected of biosynthesizing new small molecules with pharmacological relevance. Genome synteny analysis provides an assessment of genomic island content, which is enriched in natural product gene clusters. Here we identified an atypical orphan carbohydrate-nonribosomal peptide synthetase genomic island in Photorhabdus luminescens using genome synteny analysis. Heterologous expression of the pathway led to the characterization of five oligosaccharide metabolites with lysozyme inhibitory activities. The oligosaccharides harbor a 1,6-anhydro-β-D-N-acetyl-glucosamine moiety, a rare structural feature for natural products. Gene deletion analysis and biochemical reconstruction of oligosaccharide production led to the discovery that a hypothetical protein in the pathway is a lytic transglycosylase responsible for bicyclic sugar formation. The example presented here supports the notion that targeting select genomic islands with reduced reliance on known protein homologies could enhance the discovery of new metabolic chemistry and biology.

Figure 1. Biosynthetic gene cluster and characterized oligosaccharides 1–5

(A) Atypical carbohydrate-NRPS genomic island. Wildtype genetic organization and reconstructed genetic organization used in heterologous expression studies are shown. Peripheral genes marked in grey are predicted to not be a part of the pathway. See also Figure S1. (B) Structures of oligosaccharides 1–5.

Figure 2. LC/ESI-MS Extracted Ion Count (EIC) analysis of oligosaccharide 1–5 production in the deletion mutants

(A) Oligosaccharide 1; (B) 2; (C) 3; (D) 4; and (E) 5. The reconstructed pathway (pE-reconstructed = pE2408–2413) was compared to gene deletions (plu2403–plu2413) and an empty control vector (pET28a) by LC/MS. Positive ion signals ([M+H]⁺) are shown for 1–4. Negative ion signal ([M−H]⁻) is shown for phosphorylated 5. See also Figure S2.

Figure 3. Reactions catalyzed by Plu2411 and Plu2409

(A) Conversion of α-UDP-GlcNAc to 1–3 by Plu2411 and Plu2409 in the presence of Mn²⁺ or Mg²⁺; LC/ESI-MS(+) Total Ion Count (TIC) chromatogram of the reaction in the presence of Mn²⁺ (B) and Mg²⁺ (C).

Figure 4. Reaction catalyzed by hypothetical Plu2409 with chitin substrate

(A) Proposed transformations catalyzed by GT Plu2411 and hypothetical Plu2409; (B) LC/ESI-MS(+) TIC chromatogram for Plu2409 catalyzed reaction with chitin as a substrate; see also Figure S4. (C) Proposed biosynthesis of representative 1 catalyzed by Plu2409.