Biosynthesis of the Terpene Phenalinolactone in Streptomyces sp. Tü6071: Analysis of the Gene Cluster and Generation of Derivatives

Abstract

Phenalinolactones are terpene glycosides with antibacterial activity. A striking structural feature is a highly oxidized gamma-butyrolactone of elusive biosynthetic origin. To investigate the genetic basis of the phenalinolactones biosynthesis, we cloned and sequenced the corresponding gene cluster from the producer strain Streptomyces sp. Tü6071. Spanning a 42 kbp region, 35 candidate genes could be assigned to putatively encode biosynthetic, regulatory, and resistance-conferring functions. Targeted gene inactivations were carried out to specifically manipulate the phenalinolactones pathway. The inactivation of a sugar methyltransferase gene and a cytochrome P450 monoxygenase gene led to the production of modified phenalinolactone derivatives. The inactivation of a Fe(II)/alpha-ketoglutarate-dependent dioxygenase gene disrupted the biosynthetic pathway within gamma-butyrolactone formation. The structure elucidation of the accumulating intermediate indicated that pyruvate is the biosynthetic precursor of the gamma butyrolactone moiety.

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.…”

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

“…Very recently, a biosynthetic gene cluster of phenalinolactone, a diterpene compound, was cloned from Streptomyces sp. Tü6071 by searching for an NDP-glucose-4,6-dehydratase gene that participates in the formation of L-amicetose attached to an aglycon of phenalinolactone [34]. In the cluster, the plaT2 gene has significant similarity to isoprenoid cyclases and is annotated to be a diterpene cyclase responsible for the biosynthesis of phenalinolactone.…”

“…Among them, bra3, bra4, and bra5 products had high amino acid identities to plaT3, plaT2, and plaT1 products, respectively, all of which were recently cloned from Streptomyces sp. Tü6071 and confirmed to participate in the early step of biosynthesis of phenalinolactone, a diterpene compound [34]. Since the early step of the proposed biosynthetic pathway of phenalinolactone was thought to be identical of those of BCA (Fig.…”

Tracking Down Biotransformation to the Genetic Level: Identification of a Highly Flexible Glycosyltransferase from Saccharothrix espanaensis

“…Saccharothrix espanaensis DSM 44229, Streptomyces albus J1074, and their mutants were grown on mannitol soy flour agar plates or tryptone soy agar plates (15). Liquid growth for strain maintenance was performed in tryptone soy broth (TSB), and feeding experiments were conducted in NL111 medium (16), both in double-baffled flasks at 28°C and 180 rpm on a rotary incubator. Apramycin, spectinomycin, and thiostrepton were added to the medium to final concentrations of 100 g/ml, 100 g/ml, and 50 g/ml, respectively.…”