d-Sedoheptulose-7-phosphate is a common precursor for the heptoses of septacidin and hygromycin B

Abstract

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l-glycero-β-d-manno-heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes sepD and sepH, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d-glycero-β-d-altro-heptose together with GmhA and HldE, two enzymes from the Escherichia coli LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.

Keywords: Streptomyces; biosynthesis; heptose; lipopolysaccharide; natural product.

Fig. 1.

Representative bacterial natural products with seven-carbon-chain–containing carbohydrate structures. (A) Group I compounds with heptofuranoses or heptothiofuranose; (B) group II compounds with highly reduced heptopyranoses; (C) group III compounds with l-heptopyranoses; (D) group IV compounds with d-heptopyranoses. The seven-carbon-chain–containing carbohydrate moiety of each compound is shown in red.

Fig. 2.

The biosynthetic gene cluster and a proposed biosynthetic pathway of septacidin. (A) Genetic organization of the sep gene cluster from S. fimbriatus CGMCC 4.1598. (B) A proposed pathway for septacidin biosynthesis based on in vitro and in vivo experiments. The enzymes involved in gram-negative bacteria LPS heptose biosynthesis are bracketed and indicated in green.

Fig. 3.

Metabolic profiles of the sep gene cluster heterologous expression strain S. albus 1598 and the sep gene in-frame deleted mutants upon HPLC and LC-MS analysis. (A) HPLC traces of the mycelia methanolic extracts of a negative control S. albus 153, S. albus 1598, and the sep gene mutants. (B) LC-MS–extracted ion count chromatograms of m/z 327.1 [M+H]⁺ in the supernatants of S. albus 1598, S. albus ∆sepD, and ∆sepH fermentations. (C) LC-MS extracted ion count chromatograms of m/z 384.2 [M+H]⁺ in the supernatants of S. albus 1598, S. albus ∆sepD, and ∆sepH fermentations. EIC, extracted ion count.

Fig. 4.

Representative assays of the septacidin and hygromycin B heptose biosynthesis enzymes. (A) HPLC analysis of enzymatic assays of SepB, SepC, SepL, and HygP. (B) A proposed ADP-d-glycero-β-d-altro-heptose biosynthetic pathway catalyzed by HygP, HldE, and GmhB. The enzymes from gram-negative bacteria LPS heptose biosynthesis are bracketed and indicated in green.

Fig. 5.

The biosynthetic gene cluster and a proposed biosynthetic pathway of hygromycin B. (A) Genetic organization of the hyg gene cluster from S. hygroscopicus DSM 40578. (B) A proposed pathway for hygromycin B biosynthesis.