Elucidation of the biosynthetic pathway for Okenone in Thiodictyon sp. CAD16 leads to the discovery of two novel carotene ketolases

Abstract

Okenone is a unique ketocarotenoid found in many purple sulfur bacteria; it is important because of its unique light absorption and photoprotection properties. Okenane, a compound formed by diagenetic reduction of okenone, is an important biomarker in geochemical analyses of sedimentary rocks. Despite its ecological and biogeochemical importance, the biochemical pathway for okenone synthesis has not yet been fully described. The genome sequence of an okenone-producing organism, Thiodictyon sp. strain CAD16, revealed four genes whose predicted proteins had strong sequence similarity to enzymes known to produce ψ-end group modifications of carotenoids in proteobacteria. These four genes encoded homologs of a 1,2-carotenoid hydratase (CrtC), an O-methyltransferase (CrtF), and two paralogs of carotenoid 3,4-desaturases (CrtD). Expression studies in lycopene- or neurosporene-producing strains of Escherichia coli confirmed the functions of crtC and crtF, but the crtD paralogs encoded enzymes with previously undescribed functions. One enzyme, CruS, was only distantly related to CrtD desaturases, was bifunctional, and performed a 3,4-desaturation and introduced a C-2 keto group into neurosporene derivatives in the presence of dioxygen. The enzyme encoded by the other crtD paralog also represents a new enzyme in carotenogenesis and was named cruO. CruO encodes the C-4/4' ketolase uniquely required for okenone biosynthesis. The identification of CruO and the demonstration of its biochemical activity complete the elucidation of the biosynthetic pathway for okenone and other related ketocarotenoids.

FIGURE 1.

Organization of carotenoid genes of PSB. A, Thiodictyon sp. CAD16; B, T. marina DSM 5653^T; and C, M. purpuratum DSM 1591^T.

FIGURE 2.

HPLC elution profile of pigments produced in in vitro assay with γ-carotene and crude extract of E. coli BL21(DE3) harboring plasmid pCDFDuet::crtC. The in-line absorption spectra of peaks 1 and 2 are shown in the numbered boxes below the elution profile. Peak 1, 1′-hydroxy-γ-carotene; peak 2, γ-carotene.

FIGURE 3.

HPLC elution profile of pigments produced in E. coli BL21(DE3) harboring plasmids pAC::LYC and pCDFDuet::crtC::crtF. The in-line absorption spectra of peaks 1 to 6 were identical; the spectrum of the compound numbered 1 is shown in the numbered box below the elution profile. Peak 1, 1,1′-dihydroxylycopene; peak 2, 1-methoxy-1′-hydroxylycopene; peak 3, 1-hydroxylycopene; peak 4, 1,1′-dimethoxylycopene; peak 5, 1-methoxylycopene; peak 6, lycopene. The insertion of a hydroxyl group into lycopene or 1-hydroxylycopene causes a compound to elute ∼4.4 min earlier. The methylation of a hydroxyl group increases the retention time by ∼2 min.

FIGURE 4.

HPLC elution profile of pigments produced in E. coli BL21(DE3) harboring plasmids pAC::LYC, pCDFDuet::crtC, and pCOLADuet::crtD1(cruO). The in-line absorption spectra of the compounds numbered 1 to 4 are shown in the numbered boxes below the elution profile. Peak 1, carotenoid with reduced fine structure and a bathochromic shift of ∼30 nm compared with lycopene; peaks 2, 3, and 4, carotenoids with reduced fine structure and bathochromic shifts of ∼15 nm compared with lycopene. For additional details, see the text.

FIGURE 5.

Test for keto groups in carotenoids by reduction with sodium borohydride. A, carotenoid corresponding to the peak of the elution profile shown in Fig. 4. Panel 1, spectrum of carotenoid dissolved in isopropanol; panel 2, spectrum 2 min after the addition of sodium borohydride; panel 3, spectrum 30 min after the addition of sodium borohydride. Complete reduction of the carotenoid occurred after 30 min, resulting in a hypsochromic shift of 29 nm and an increase in the fine structure. The resulting carotenoid had a spectrum identical to that of lycopene. B, carotenoid corresponding to peak 2 of the elution profile shown in Fig. 4. Panel 1, spectrum of carotenoid dissolved in isopropanol; panel 2, spectrum 2 min after the addition of sodium borohydride; panel 3, spectrum 30 min after the addition of sodium borohydride. Complete reduction of the carotenoid occurred after 30 min, resulting in a hypsochromic shift of 19 nm and an increase of the fine structure. The resulting carotenoid had a spectrum identical to that of lycopene.

FIGURE 6.

HPLC elution profile of pigments produced in in vitro assay with hydroxylated lycopene derivatives and crude extract of E. coli BL21(DE3) harboring plasmid pCOLADuet::crtD1(cruO). The in-line absorption spectra of the compounds numbered 1 to 5 in the elution profile are shown in the numbered boxes. Peak 1, carotenoid with reduced fine structure and a bathochromic shift of ∼15 nm compared with lycopene; peak 2, 1,1′-dihydroxylycopene; peak 3, carotenoid with reduced fine structure and a bathochromic shift of ∼15 nm compared with lycopene; peak 4, 1-hydroxylycopene; peak 5, lycopene. For additional details, see the text.

FIGURE 7.

HPLC elution profile of pigments produced in E. coli BL21(DE3) harboring plasmids pAC::NEUR, pCDFDuet::crtC, and pCOLADuet::crtD2(cruS). The in-line absorption spectra of the compounds in peaks 1 and 2 are shown in the numbered boxes below the elution profile. Peaks 1 and 3, carotenoids with reduced fine structure and a bathochromic shift of ∼40 nm compared with neurosporene; peak 2, 1,1′-dihydroxyneurosporene; peak 4, 1-hydroxyneurosporene and/or 1′-hydroxyneurosporene; peak 5, neurosporene.

FIGURE 8.

Neighbor-joining phylogenetic tree of CrtD desaturases and their homologs. Bootstrap numbers from 100 resamplings are indicated at the nodes.

FIGURE 9.

Proposed pathways for biosynthesis of okenone and methoxyspheroidenone. A, pathway leading to okenone. This pathway is not oxygen-dependent, and the enzymatic reactions were confirmed in E. coli. The reaction order shown is the most likely reaction order that has been deduced from information available in the literature and from the experiments described herein, but the timing of the ring desaturation/methyl transfer reaction is still uncertain. B, putative pathway for oxygen-dependent synthesis of methoxyspheroidenone. The dashed arrows indicate that the enzymatic functions have been confirmed in E. coli, but none of the carotenoids of this pathway were detected in Thiodictyon sp. CAD16 and T. marina cells grown under dark, microoxic conditions.