Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites: hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis

Abstract

Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11 carrying pKU462∷ptl-clusterΔptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an extra-copy of the sav7469 gene to SUKA11 Δsav7469 carrying pKU462∷ptl-clusterΔptlH restored the production of pentalenic acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites.

Figure 1

AseI-physical map of S. avermitilis and distribution of biosynthetic genes encoding terpenoid compounds (sav76; gene for avermitilol biosynthesis, crt; genes for isoneriatene biosynthesis, hop; genes for squalene and hopanoid biosynthesis, sav2163; gene for germacradienol and geosmin biosynthesis, sav3032; gene for epi-isozizaene biosynthesis) (A), and gene cluster for neopentalenoketolactone biosynthesis (sav2989; gene encoding MarR-family transcriptional regulator, gap1; gene for pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase, ptlH; gene for 1-deoxypentalenic acid 11-β hydroxylase, ptlG; gene for transmembrane efflux protein, ptlF; 1-deoxy-11β-hydroxypentalenic acid dehydrogenase; ptlE; gene for Baeyer-Villiger monooxygenase, ptlD; gene for dioxygenase, ptlC; gene for hypothetical protein, ptlB; gene for farnesyl diphosphate synthase, ptlA; gene for pentalenene synthase, ptlI; gene for pentalenene C13 hydroxylase CYP183A1, sav3000; gene for AraC-family transcriptional regulator, sav3001; gene for lyase, sav3002; gene for hypothetical protein) (B). Two genes, sav7469 and sav7470 encode cytochrome P450 (CYP105D7) and ferredoxin (FdxH), respectively.

Figure 2

Proposed neopentalenoketolactone biosynthetic pathway in S. avermitilis. α-KG indicates α-ketoglutarate.

Figure 3

GC-MS analysis of EtOAc extracts treated with TMS-diazomethane of S. avermitilis SUKA5 (A) and SUKA7 (SUKA5 Δsav7469::aadA). The two strains were cultured at 28 °C with shaking at 200 rpm for 96 h.

Figure 4

GC-MS analysis of EtOAc extracts treated with TMS-diazomethane of SUKA11 carrying pKU462::ptl-clusterΔptlH (A), SUKA13 (SUKA11 Δsav7469::aadA) carrying pKU462::ptl-clusterΔptlH (B), and SUKA13 carrying pKU462::ptl-clusterΔptlH and pKU493::rpsJp-sav7469-sav7470-sav3129-sav5675 (C).

Figure 5

UV spectra of oxidized, reduced and CO-bound forms of C-terminal His₄-tagged CYP105D7 (A) and its CO-difference spectrum (B). UV difference binding spectra of CYP105D7 and 1-deoxypentalenic acid (5) binding (C), saturation curve for CYP105D7 and 1-deoxypentalenic acid (5) (D).

Figure 6

GC-MS analysis of EtOAc extracts treated with TMS-diazomethane from incubation of 1-deoxypentalenic acid (5) with (B) or without (A) CYP105D7 protein in the presence of a NADPH-generating system, electron-transport partners (spinach ferredoxin and ferredoxin reductase) and Michaelis-Menten plot of initial velocity of formation of pentalenic acid (1) (C).