Characterization of the Alnumycin Gene Cluster Reveals Unusual Gene Products for Pyran Ring Formation and Dioxan Biosynthesis

Abstract

Alnumycin is closely related to the benzoisochromanequinone (BIQ) polyketides such as actinorhodin. Exceptional structural features include differences in aglycone tailoring that result in the unique alnumycin chromophore and the existence of an unusual 4-hydroxymethyl-5-hydroxy-1,3-dioxan moiety. Cloning and sequencing of the alnumycin gene cluster from Streptomyces sp. CM020 revealed expected biosynthesis genes for polyketide assembly, but several genes encoding subsequent tailoring enzymes were highly atypical. Heterologous expression studies confirmed that all of the genes required for alnumycin biosynthesis resided within the sequenced clone. Inactivation of genes aln4 and aln5 showed that the mechanism of pyran ring formation differs from actinorhodin and granaticin pathways. Further inactivation studies identified two genes, alnA and alnB, involved in the synthesis and attachment of the dioxan moiety, and resulted in the production of the polyketide prealnumycin.

Biosynthetic pathway toward carbohydrate-like moieties of alnumycins contains unusual steps for C-C bond formation and cleavage

“…3C). This finding was surprising because based on previous molecular genetic evidence, the aldo-keto reductase Aln4 (354 amino acids, 39.2 kDa) was implicated to be involved in cyclization of the third ring of the polyketide aglycone (14). Nonetheless, the aldoreduction of position 3′ of 4 by Aln4 was in agreement with the proposed function of the homologous enzyme AFB 1 -AR (26% identity) involved in the degradation of aflatoxin (25), although the reaction appeared to be more complicated due to the additional rearrangement of the dioxolane unit of 4 to the dioxane moiety seen in 1.…”

“…1) as its main metabolite, but crucially the biosynthesis of 1 was completely abolished in the strain. At the time, Aln4 was annotated to be involved in formation of the alnumycin chromophore due to the incorrect cyclization of the third ring in 3 (14). The unidentified metabolite was duly isolated and purified from a S. albus/pAlnuoriΔaln3 cultivation, and structure elucidation of the compound (SI Appendix) confirmed the presence of a unique dioxolane (cis-bicyclo[3.3.0]-2′,4′,6′-trioxaoctan-3′β-ol) moiety in place of the dioxane unit.…”

“…To date the biosynthetic pathway utilized for synthesis of the dioxane unit has been unknown, although we have previously cloned and successfully expressed the alnumycin gene cluster heterologously in Streptomyces albus (Fig. 2 A and B) to enable efficient manipulation of the pathway (14). Initial knockout studies also identified the involvement of two genes, alnA and alnB, in the biosynthesis and transfer of the dioxane moiety as both single-gene mutants produced prealnumycin (2, Fig.…”

“…Initial knockout studies also identified the involvement of two genes, alnA and alnB, in the biosynthesis and transfer of the dioxane moiety as both single-gene mutants produced prealnumycin (2, Fig. 1) as their main metabolite (14).…”

Characterization of the Two-Component Monooxygenase System AlnT/AlnH Reveals Early Timing of Quinone Formation in Alnumycin Biosynthesis

“…In order to understand how the structural diversity of BIQ metabolites is reflected in the composition of the biosynthetic enzymes, the alnumycin gene cluster has previously been cloned and expressed heterologously in Streptomyces albus (35). Molecular genetic studies have clarified that the biosynthesis is initiated from a butyryl starter unit (46), while cyclization of the third ring is catalyzed by enzymes belonging to entirely different enzyme families than those found from the actinorhodin and granaticin pathways (35).…”