Cloning of the Gene Cluster Responsible for the Biosynthesis of Brasilicardin A, a Unique Diterpenoid

Abstract

Brasilicardin A (BCA), produced by Nocardia brasiliensis IFM 0406 (currently referred to as N. terpenica), has a unique structure consisting of a diterpene skeleton with L-rhamnose, N-acetylglucosamine, amino acid, and 3-hydroxybenzoate moieties, and exhibits potent biological activities. To understand the biosynthetic machinery of this unique compound, we have cloned the corresponding gene cluster. Firstly, we cloned a gene by PCR that encodes geranylgeranyl diphosphate synthase (GGPPS), which produces a direct precursor of diterpene compounds. We obtained four candidate genes and one of the genes was confirmed to encode a GGPPS. By sequence analysis of regions flanking the GGPPS gene, we identified eleven genes (bra1-11 ), all oriented in the same direction. We did not, however, detect any genes related to Lrhamnose and N-acetylglucosamine biosyntheses in the flanking regions. A gene disruption experiment did indeed show that this gene cluster was responsible for BCA biosynthesis.

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

“…Brasilicardin contains a 3HBA moiety. Bra8 was provisionally assigned an oxidative function by these authors (43). However, our recognition that RapK and FkbO are chorismatases suggests an alternative role for Bra8 in generating 3HBA directly from chorismate (Fig.…”

“…5). This analysis identified as their closest relatives a group of three mutually very similar proteins: an unidentified predicted protein from a Streptomyces species; another predicted protein (Hyg5) from the rapamycin-producing strain (42), and, most tellingly, a protein (Bra8) from Nocardia brasiliensis IFM 0406 encoded within the biosynthetic gene cluster for the glycosylated diterpene natural product brasiliocardin (43). Brasilicardin contains a 3HBA moiety.…”

Structure of the ent-Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase

“…Sequence alignment of all functionally characterized type II DTSs of bacterial origin that use GGPP as a substrate reveals that this histidine is conserved and may be an indicator, along with the DxDD motif, of bacterial type II DTS function (Figure S5). Both Bra4 and PlaT2, bacterial DTSs that cyclize epoxyGGPP, 42,43 have glutamines, instead of histidines, in this position.…”

Functional Conservation of the Capacity for ent-Kaurene Biosynthesis and an Associated Operon in Certain Rhizobia

“…In the organization of the rhizobial diterpenoid biosynthesis operons, it is notable that the genes predicted to be involved in oxidation are in the 5= region, with all those predicted to be involved in the formation of the cyclized olefin ent-kaurene falling in the 3= region. This includes the putative GGPP synthase (GGPS), as bacteria do not necessarily produce GGPP, leading to the presence of a GGPS in all of the identified bacterial diterpenoid biosynthetic gene clusters (21,(32)(33)(34)(35)(36)(37). The observed organization of the rhizobial diterpenoid biosynthetic operon suggests that the 3= and 5= regions might form nominally independent subclusters, although no such subclusters appear in the currently available sequence information.…”