In vitro characterization of 3-chloro-4- hydroxybenzoic acid building block formation in ambigol biosynthesis

摘要

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the encoded enzymes, i.e. Ab2 and Ab3, were identified by in vitro and in vivo assays as cytochrome P450 enzymes responsible for biaryl and biaryl ether formation. The key substrate for these P450 enzymes is 2,4-dichlorophenol, which in turn is derived from the precursor 3-chloro-4-hydroxybenzoic acid. Here, the biosynthetic steps leading towards 3-chloro-4-hydroxybenzoic acid were investigated by in vitro assays. Ab7, an isoenzyme of a 3-deoxy-7-phosphoheptulonate (DAHP) synthase, is involved in choris-mate biosynthesis by the shikimate pathway. Chorismate in turn is further converted by a dedicated chor-ismate lyase (Ab5) yielding 4-hydroxybenzoic acid (4-HBA). The stand alone adenylation domain Ab6 is necessary to activate 4-HBA, which is subsequently tethered to the acyl carrier protein (ACP) Ab8. The Ab8 bound substrate is chlorinated by AblO in meta position yielding 3-CI-4-HBA, which is then trans-fered by the condensation (C) domain to the peptidyl carrier protein and released by the thioesterase (TE) domain of Ab9. The released product is then expected to be the dedicated substrate of the halogenase Abl producing the monomeric ambigol building block 2,4-dichlorophenol.