The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, L-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn 2؉ ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C 3 and C 4 primary metabolites.The shikimate pathway (Fig. 1B), involving seven enzymatic steps that produce chorismate via shikimate from phosphoenolpyruvate (PEP) 2 and erythrose-4-phosphate, is well established as the common pathway for the biosynthesis of aromatic amino acids in bacteria, fungi, algae, and higher plants. Not only aromatic amino acids but also most biogenic benzene derivatives, such as p-aminobenzoic acid, m-aminobenzoic acid, 2-amino-3-hydroxybenzoic acid, 2-amino-6-hydroxybenzoic acid, and many vitamins, are derived from chorismate (1, 2). The shikimate biosynthesis pathway is also employed by Archaea, although the genes encoding the first two enzymes involved in 3-dehydroquinate (DHQ) synthesis are missing in the genomic sequences of many Archaea (3). In one of Archaea, Methanocaldococcus jannaschii, DHQ is synthesized from aspartate 4-semialdehyde (ASA) and 6-deoxy-5-ketofructose-1-phosphate by two alternative enzymes and supplied to the shikimate pathway (4). Recent studies (5, 6) showed that 3-amino-5-hydroxybenzoic acid, a precursor for ansamycin antibiotics, is also synthesized through the aminoshikimate pathway, a variant of the shikimate pathway. Thus, the benzene ring as one of the primary chemical structures in nature is extensively formed through the shikimate pathway, although some benzene derivatives are formed from aliphatic acyl-CoA by polyketide synthases (7, 8).We recently isolated grixazone (Fig. 1A), a mixture of yellow pigments grixazone A and grixazone B, containing a phenoxazinone chromophore, as secondary metabolites of Streptomyces griseus (9, 10). In the present study on the grixazone biosynthesis, we ...
