Occurrence of Two 5-Aminolevulinate Biosynthetic Pathways in Streptomyces nodosus subsp. asukaensis Is Linked with the Production of Asukamycin

Abstract

We report the results of cloning genes for two key biosynthetic enzymes of different 5-aminolevulinic acid (ALA) biosynthetic routes from Streptomyces. The genes encode the glutamyl-tRNA Glu reductase (GluTR) of the C 5 pathway and the ALA synthase (ALAS) of the Shemin pathway. While Streptomyces coelicolor A3(2) synthesizes ALA via the C 5 route, both pathways are operational in Streptomyces nodosus subsp. asukaensis, a producer of asukamycin. In this strain, the C 5 route produces ALA for tetrapyrrole biosynthesis; the ALA formed by the Shemin pathway serves as a precursor of the 2-amino-3-hydroxycyclopent-2-enone moiety (C 5 N unit), an antibiotic component. The growth of S. nodosus and S. coelicolor strains deficient in the GluTR genes (gtr) is strictly dependent on ALA or heme supplementation, whereas the defect in the ALAS-encoding gene (hemAasuA) abolishes the asukamycin production in S. nodosus. The recombinant hemA-asuA gene was expressed in Escherichia coli and in Streptomyces, and the encoded enzyme activity was demonstrated both in vivo and in vitro. The hemA-asuA gene is situated within a putative cluster of asukamycin biosynthetic genes. This is the first report about the cloning of genes for two different ALA biosynthetic routes from a single bacterium.

Biochemical and Genetic Insights into Asukamycin Biosynthesis

“…ChcA, encoding the 1-cyclohexenylcarbonyl CoA reductase of ansatrienin biosynthesis in S. collinus (12), identified two cosmids, 2B9 and 10D6, which revealed a 1.8-kb overlapping region of the cloned DNA inserts. AsuD2, encoding a 2-oxoamine synthase, hybridized only with cosmid 10D6 (15). Cosmids 2B9 and 10D6 were mapped and sequenced to disclose a total combined 63,922 bp of DNA inserts (GenBank TM accession number GQ926890).…”

“…The BamHI-KpnI-cleaved fragment was then used to replace the corresponding region in the pALS3 plasmid. The further steps were identical with the pALS4 construction (15).…”

“…AsuD2, previously identified as HemA-AsuA, belongs to a group of pyridoxal phosphate-dependent 2-oxoamine synthases, including 5-aminolevulinate synthase (ALAS), FUM8, L-serine palmitoyltransferase, and 8-amino-7-oxononanoate synthase, which catalyze the decarboxylative condensation between an amino acid and an acyl-CoA (15, 39 -42 instead of a strictly invariant Thr 83 of the ALAS subgroup. AsuD2 functions differently from the reported ALAS forming 5-ALA in many organisms, as no asukamycins were detected in the asuD2 mutant even in the presence of 5-ALA, but the asukamycin production was restored by reintroducing the plasmid pALS4 carrying the asuD2 gene (15). The pALS4-S83T with a S83T replacement in asuD2 was constructed but failed to restore asukamycin production in the asuD2 truncated mutant (supplemental Fig.…”

“…A gene set involved in the CHCCoA biosynthesis of ansatrienin has been well characterized in Streptomyces collinus (12), but the genes involved in 3,4-AHBA and polyketide chain assembly of asukamycin have not been identified. The C 5 N ring moiety, found in several natural products including reductiomycin, moenomycin A, and ECO-02301, was suggested to be derived from a 5-aminolevulinate (5-ALA) intermediate (13)(14)(15)(16). The only reported gene product of asuD2 (hemA-asuA), a member of the 2-oxoamine synthases, was found to be involved in asukamycin biosynthesis and to catalyze the condensation of succinyl-CoA and glycine (15).…”

“…The C 5 N ring moiety, found in several natural products including reductiomycin, moenomycin A, and ECO-02301, was suggested to be derived from a 5-aminolevulinate (5-ALA) intermediate (13)(14)(15)(16). The only reported gene product of asuD2 (hemA-asuA), a member of the 2-oxoamine synthases, was found to be involved in asukamycin biosynthesis and to catalyze the condensation of succinyl-CoA and glycine (15). Protoasukamycin C1, the polyketide assembly product, is further oxygenated to give the final product asukamycin (1,10,17).…”