Enzyme Fusion Removes Competition for Geranylgeranyl Diphosphate in Carotenogenesis

Abstract

Geranylgeranyl diphosphate (GGPP), a prenyl diphosphate synthesized by GGPP synthase (GGPS), represents a metabolic hub for the synthesis of key isoprenoids, such as chlorophylls, tocopherols, phylloquinone, gibberellins, and carotenoids. Protein-protein interactions and the amphipathic nature of GGPP suggest metabolite channeling and/or competition for GGPP among enzymes that function in independent branches of the isoprenoid pathway. To investigate substrate conversion efficiency between the plastid-localized GGPS isoform GGPS11 and phytoene synthase (PSY), the first enzyme of the carotenoid pathway, we used recombinant enzymes and determined their in vitro properties. Efficient phytoene biosynthesis via PSY strictly depended on simultaneous GGPP supply via GGPS11. In contrast, PSY could not access freely diffusible GGPP or time-displaced GGPP supply via GGPS11, presumably due to liposomal sequestration. To optimize phytoene biosynthesis, we applied a synthetic biology approach and constructed a chimeric GGPS11-PSY metabolon (PYGG). PYGG converted GGPP to phytoene almost quantitatively in vitro and did not show the GGPP leakage typical of the individual enzymes. PYGG expression in Arabidopsis resulted in orange-colored cotyledons, which are not observed if PSY or GGPS11 are overexpressed individually. This suggests insufficient GGPP substrate availability for chlorophyll biosynthesis achieved through GGPP flux redirection to carotenogenesis. Similarly, carotenoid levels in PYGG-expressing callus exceeded that in PSY- or GGPS11-overexpression lines. The PYGG chimeric protein may assist in provitamin A biofortification of edible plant parts. Moreover, other GGPS fusions may be used to redirect metabolic flux into the synthesis of other isoprenoids of nutritional and industrial interest.

Figure 1.

Carotenoid biosynthesis and branching pathways. The methylerythritol phosphate pathway (MEP) produces IPP and its isomer DMAPP which is condensed into geranylgeranyl diphosphate (GGPP) in plastids by GGPP synthase (GGPS). GGPS11 is the most abundant among the six plastid-localized GGPS isoforms. GGPP is metabolized into plastoquinones via solanesyl-diphosphate synthase (SPS), into gibberellins via ent-copalyl diphosphate synthase (CPS), and into phythyl-diphosphate via geranylgeranyl reductase (GGR), which represents a precursor for chlorophyll and tocopherols. Phytoene synthase (PSY) is the first enzyme of the carotenoid pathway producing colorless phytoene, which is subsequently converted into red-colored lycopene and orange-colored β-carotene. Lycopene synthesis requiring four enzymes in plants is carried out by the enzyme CrtI in bacteria. In this work, we employed a translational fusion between GGPS11 and PSY in order to direct isoprenoid pathway flux into carotenoid biosynthesis.

Figure 2.

Analysis of interaction between Arabidopsis GGPS isoforms with PSY. Yeast strains expressing Nub (N) or N-terminal Nub fusions with Arabidopsis GGPS isoforms 2, 6, 8, 9, 10, and 11 were combined with yeast strains expressing Cub only (C) or C-terminal Cub fusion with Arabidopsis PSY, respectively. Corresponding Nub/Cub fusions with the Arabidopsis kation channel protein KAT1 were used as negative controls whereas dimerization of Nub-KAT1/KAT1-Cub served as a positive control. A, Interaction assay. Growth on selective medium, supplemented with 150 µM Met after 2 d incubation. B, β-Gal activity determined by ONPG assays of yeast strains coexpressing combinations with PSY-Cub. C, Nub-GGPS protein levels in PSY-Cub combinations. Nub-GGPS proteins carried an N-terminal 3-HA tag and were detected using a 3-HA antibody. D, Phytoene amounts in PSY-Cub combinations, quantified by HPLC. E, Absorption spectrum of phytoene determined in yeast strains coexpressing PSY-Cub and GGPS11. Results in B and D are mean +/− SEM of three biological replicates. * Significant difference to that in Nub control (P > 0.05).

Figure 3.

Enzyme properties of Arabidopsis GGPS11 and PSY. A, Dependence of GGPS11 activity on substrate IPP concentrations. DMAPP was supplied with constant 20 µm whereas IPP concentrations varied between 1 and 30 µm. Incubation time was 2 min. Data (R² = 1.0) were fitted with the Michaelis-Menten equation using the GraphPad Prism software (for equations, see Methods). B, Time-course experiment of GGPP formation by GGPS11 under standard assay conditions. C, Time-course experiment of GGPP and phytoene formation under standard assay conditions with equimolar amounts of GGPS11 and PSY. Round open circles represent the sum of GGPP and phytoene. Product concentrations are expressed in IPP equivalents in order to facilitate direct comparison. Note that phytoene synthesis stops almost completely after 30 min although GGPP is still available. Standard assay conditions were 20 µm DMAPP and 20 µm IPP with 138 nm enzyme concentrations. Partial GGPP adhesion on plastic surfaces during sample incubation and transfer explains apparent incongruence of substrate and product amounts. Data are mean ± SEM of three replicate experiments.

Figure 4.

Enzymatic activity of GGPS11, PSY, and translational fusion proteins in E. coli. GGPS11 (GGPS), PSY, and fusion proteins GGPS11-PSY (GGPY) and PSY-GGPS11 (PYGG) in various combinations were coexpressed with CrtI in E. coli cells. A, Lycopene amounts; acetone extracts are shown below. B, Immunoblot with 60 µg bacterial lysates using anti-PSY antibodies. GGPS11 alone was included as a negative control. Data are means ± SEM of three replicate experiments.

Figure 5.

Standard enzyme assay with PSY-GGPS fusion protein. A, Time-course experiment of phytoene and GGPP formation and IPP consumption under standard enzyme assay conditions with PSY-GGPS11 fusion protein (PYGG) and 20 µm DMAPP/20 µm IPP. Round open circles represent the sum of GGPP and phytoene. Product concentrations are expressed in IPP equivalents in order to facilitate direct comparison. Note that phytoene synthesis continues with almost unchanged velocity for about 2 h. B, Substrate dependancy of recombinant PYGG. IPP was provided with 5, 10, 20, 40, 60, 80, 100, and 150 µm, incubation time was 10 min. Data (R² = 0.98) were fitted with the Michaelis-Menten equation using the GraphPad Prism software (for equations, see Methods). Results in A and B are means of three replicate experiments. C, Coomassie-stained SDS-PAGE of recombinant purified proteins of His₆-GGPS11 (35.7 kD), His₆-PSY (44.2 kD), and the fusion protein His₆-PYGG (79.6 kD).

Figure 6.

Complementation of mutated chimeric PSY-GGPS proteins. A and B, Recombinant PYGG was incubated with 20 µm DMAPP/20 µm IPP for 1 h, then amounts of GGPP and phytoene were determined and expressed in nmol IPP equivalents. A, Assay of PYGG fusion protein and versions with impaired PSY (pyGG1 and pyGG2) complemented with wild-type PSY (+PSY). B, Assay of PYGG fusion protein and versions with impaired GGPS11 (PYgg1 and PYgg2) complemented with wild-type GGPS11 (+GGPS). C to E, Standard enzyme assay with equimolar concentrations of GGPS11 and PSY (C), PYGG (D) and GGPS11 and PSY in a molar ratio of 1:10 (E). All assays were performed with 138 nm PSY and PYGG and 20 µm DMAPP/20 µm IPP. Data are means of three replicate experiments.

Figure 7.

Carotenoid amounts in PYGG-overexpressing calli. Arabidopsis GGPS11, PSY, and the fusion protein PYGG were constitutively overexpressed in Arabidopsis. Callus was developed from seeds for 5 d in the light, followed by two weeks etiolation on callus-inducing medium. A, Detection of GGPS11, PSY, and PYGG in calli. Callus protein extracts from the wild type and one line overexpressing GGPS11 (GGPS), PSY, and PYGG were probed with anti-PSY antibodies (aPSY) and anti-GGPS11 (aGGPS) antibodies, respectively. Arrows indicate band positions corresponding to PYGG, PSY, and GGPS11. B to D, Phenotype of wild-type and PYGG-expressing seedlings grown for 7 d under long-day conditions (B, left and right), dark-grown seedlings (C), and detached primary leaves of dark-grown seedlings (D). E and F, Representative images (E) and carotenoid contents (F) of calli. Results are means ± SEM of three biological replicates. Asterisk indicates significant difference compared to that in the wild type (Student’s t test, P < 0.05).