Enzyme Fusion Removes Competition for Geranylgeranyl Diphosphate in Carotenogenesis1[OPEN]

Maurizio Camagna,2 Alexander Grundmann, Cornelia Bär,3 Julian Koschmieder, Peter Beyer, and Ralf Welsch4,5 Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany ORCID IDs: 0000‑0001‑6340‑8079 (M.C.); 0000‑0003‑3901‑3063 (J.K.); 0000‑0003‑0041‑6543 (P.B.); 0000‑0002‑2865‑2743 (R.W.) Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021

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.

Isoprenoids comprise a large group of plant metab- 2014). In contrast to plants, most animals, including olites with divergent functions, ranging from photo- humans, are incapable of synthesizing carotenoids and synthesis to plant growth regulation and interaction rely either on a regular dietary intake of β-carotene and with the environment. Carotenoids are a class of iso- other provitamin A carotenoids or the consumption of prenoids that contribute to light harvesting, provide retinoids from animal sources for maintaining visual protection against excess light via nonphotochemical function, providing antioxidative protection, ensuring quenching, and color flowers and fruits to attract pol- developmental processes, and preventing disease (von linators and to facilitate seed dispersal. Carotenoids Lintig, 2012; Johnson, 2014). are also cleaved to form phytohormones like abscisic Plant carotenoid biosynthesis takes place in plastids acid and strigolactones (Cazzonelli, 2011; Giuliano, and is characterized by a dramatic change in the solu- bility of the metabolites involved (Moise et al., 2014). Isopentenyl diphosphate (IPP), emerging from the 1This work was supported by the HarvestPlus research consor- plastid-localized methylerythritol phosphate (MEP) tium (grant 2014H6320.FRE) to R.W. pathway (Fig. 1), represents the water-soluble building 2 Current address: Graduate School of Bioagricultural Sciences, block common to all isoprenoids. One molecule of its Nagoya University, Nagoya 464-8601, Japan isomer, namely dimethyl allyl diphosphate (DMAPP), 3Current address: Agroscope, Schwarzenburgstrasse 161, 3003 Bern, Switzerland is required as the starter molecule, and three molecules 4Author for contact: [email protected] of IPP are condensed by the enzyme GGPP synthase 5Senior author. (GGPS, Kloer et al., 2006) to form the strongly am- The author responsible for distribution of materials integral to phipathic C20 geranylgeranyl diphosphate (GGPP). the findings presented in this article in accordance with the policy Subsequently, phytoene synthase (PSY) catalyzes the described in the Instructions for Authors (www.plantphysiol.org) is: symmetric condensation of two molecules of GGPP, Ralf Welsch ([email protected]). which eliminates the diphosphates and generates the R.W. and P.B. conceived the research plan and supervised the experi- highly lipophilic hydrocarbon phytoene, the first com- ments; M.C. performed most of the experiments and data analysis, A.G. mitted step in the carotenoid pathway. This colorless contributed to project conception and performed vector construction, compound is desaturated and isomerized via four en- C.B. contributed to enzyme assay establishment and performed experiments; R.W. generated and analyzed the transgenic lines; R.W. wrote zymes into the red-colored lycopene, the main pigment the article with support from J.K. and P.B.; R.W. agrees to serve as the of tomatoes. Lycopene is cyclized by two different cy- author responsible for contact and ensures communication. clases into α- and β-carotene, the major carotene compo- [OPEN]Articles can be viewed without a subscription. nents of carrot roots for example (Giuliano et al., 2002; www.plantphysiol.org/cgi/doi/10.1104/pp.18.01026 Maass et al., 2009; Arango et al., 2014). The predominant

chlorophylls, the phytyl residue can be remobilized for tocopherol synthesis (Tanaka et al., 1999; Vom Dorp et al., 2015; Almeida et al., 2016). Furthermore, GGPP provides the lipophilic chain for plastoquinone after be- ing converted into solanesyl diphosphate by solanesyl diphosphate synthase (SPS, Hirooka et al., 2005; Hedden and Thomas, 2012). Cyclization of GGPP by ent-copalyl diphosphate synthase followed by dephos- phorylation and further ring modification catalyzed by ent-kaurene synthase liberates this precursor from the plastid to undergo numerous cytosolic oxygenation reactions toward biologically active gibberellins (Prisic Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 and Peters, 2007). GGPSs are encoded by a gene family in Arabidopsis (Beck et al., 2013). Whereas some GGPS isoforms are localized in mitochondria, ER and cytoplasm, at least seven of them reside in plastids (Coman et al., 2014; Ruiz-Sola et al., 2016b). Recently, several plastid- Figure 1. Carotenoid biosynthesis and branching pathways. The meth- localized isoforms (GGPS6, -7, -9, and -10) were found ylerythritol phosphate pathway (MEP) produces IPP and its isomer to predominantly synthesize the C25 prenyl diphosphate DMAPP which is condensed into geranylgeranyl diphosphate (GGPP) geranyl farnesyl diphosphate (GFDP) but only minor in plastids by GGPP synthase (GGPS). GGPS11 is the most abundant amounts of GGPP in vitro which raised question on among the six plastid-localized GGPS isoforms. GGPP is metabolized into plastoquinones via solanesyl-diphosphate synthase (SPS), into gib- the exact biochemical function of individual members berellins via ent-copalyl diphosphate synthase (CPS), and into phyth- of this family in planta (Nagel et al., 2015). The isoform yl-diphosphate via geranylgeranyl reductase (GGR), which represents GGPS11 is the most abundant one in almost all plant a precursor for chlorophyll and tocopherols. Phytoene synthase (PSY) tissues, whereas the remaining five isoforms are pref- is the first enzyme of the carotenoid pathway producing colorless phy- erentially expressed in roots (GGPS6, GGPS7) or weakly toene, which is subsequently converted into red-colored lycopene expressed in flowers or seeds (Beck et al., 2013). Inter- and orange-colored β-carotene. Lycopene synthesis requiring four en- action studies by coimmunoprecipitation, bimolecular zymes in plants is carried out by the enzyme CrtI in bacteria. In this fluorescence complementation (BiFC), and split-ubiquitin work, we employed a translational fusion between GGPS11 and PSY assays revealed that GGPS11 interacted with enzymes in order to direct isoprenoid pathway flux into carotenoid biosynthesis. of the major off-branching pathways for chlorophyll, plastoquinone, and carotenoid biosynthesis (Ruiz-Sola et al., 2016b). Interestingly, a cytoplasmic alternative leaf carotenoids lutein and violaxanthin represent splicing product of GGPS11 encoding a truncated but hydroxylated and epoxidated derivatives of α- and functional enzyme is essential for embryo develop- β-carotene, respectively. ment (Ruiz-Sola et al., 2016a). Therefore, GGPS11 is In most cases, PSY controls the entire pathway flux considered an essential enzyme for the synthesis of as the rate-limiting enzyme and thus, increased PSY these GGPP-derived compounds and downstream en- protein levels achieved either through overexpression zymes are thought to compete for the GGPP substrate. or by providing enhanced PSY stability increase the For instance, constitutive overexpression of PSY in to- total carotenoid content in plant tissues (Fraser et al., mato plants is reported to frequently result in dwarf- 2002; Maass et al., 2009; Farré et al., 2010; Welsch et al., ism caused by reduced gibberellins levels (Fray et al., 2010, 2018; Zhou et al., 2015; Álvarez et al., 2016; 1995). Chayut et al., 2016). In biotechnological approaches, Because of its high amphipathicity, GGPP is un- PSY overexpression is sometimes combined with the likely to diffuse in monodisperse form. This suggests coexpression of the bacterial desaturase CrtI, which a channeled distribution of GGPP into the different functionally replaces the four plant enzymes required branching pathways allowing its directed conversion for carotene desaturation toward lycopene. When the by subsequent enzymes avoiding GGPP “leakage”. downstream pathway enzymes are sufficiently active, Metabolite channels can thus be envisioned as (maybe β-carotene and xanthophylls can be formed (Beyer transient) multienzyme complexes that are hardly di- et al., 2002; Paine et al., 2005; Schaub et al., 2005; Zhu rectly experimentally accessible. Substrate channeling et al., 2008). has been implicated in the synthesis of various plant However, in addition to carotenoid biosynthesis, the natural products, e.g. cyanogenic glycosides, phenyl- intermediate GGPP acts as the precursor for several propanoids, alkaloids, and flavonoids (Burbulis and essential isoprenoid biosynthetic pathways. In chloro- Winkel-Shirley, 1999; Jørgensen et al., 2005; Bassard plasts, a large fraction is reduced by GGPP reductase et al., 2012; Laursen et al., 2016). to phytyl diphosphate for phylloquinone and chloro- Some studies are in support of enzyme complexes. phyll biosynthesis, the latter via chlorophyll synthase For instance, gel permeation chromatography stud- (CHLS) catalysis. Subsequently, upon degradation of ies suggested that PSY forms high molecular mass

1014 Plant Physiol. Vol. 179, 2019 A Chimeric Carotenogenic Enzyme complexes containing GGPS and IPP/DMAPP isomerase (Maudinas et al., 1977; Camara, 1993; Fraser et al., 2000). Similarly, high-molecular mass membrane complexes containing carotene desaturases and cyclases (Bonk et al., 1997; Lopez et al., 2008) as well as protein-protein interactions observed for carotene hy- droxylases (Quinlan et al., 2012) have been interpreted in terms of a structural basis for metabolite channeling. However, although the interaction between GGPS11 and PSY has recently been shown by yeast two-hybrid protein interaction studies and BiFC, direct biochemical evidence for substrate channeling is missing Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 (Ruiz-Sola et al., 2016b). In the present work, we approached this channeling conundrum using recombinant GGPS11 and PSY. We identified a strict requirement forde-novo synthesized GGPP to effectively carry out phytoene synthesis which is in favor of protein-protein interaction. Since this interaction could not be shown by classical biochemical methods, we pressure-tested the idea by bringing GGPS11 and PSY together artificially using a synthetic biology approach, i.e. by translationally fusing the two enzymes. The characterization of this “mini-metabolon” in vitro and in planta revealed improved substrate directionality into carotenogenesis. This also suggests that enzyme fusions at central pathway hubs like GGPS are capable of directing metabolite biosynthesis in a targeted manner.

RESULTS

Interactions between GGPS Isoenzymes and PSY The role of GGPS11 serving several isoprenoid pathways including carotenogenesis was scrutinized by investigating whether other plastid-localized GGPS Figure 2. Analysis of interaction between Arabidopsis GGPS isoforms isoforms show differences in their interaction with with PSY. Yeast strains expressing Nub (N) or N-terminal Nub fusions with Arabidopsis PSY (PSY). We included GGPS2, GGPS6, Arabidopsis GGPS isoforms 2, 6, 8, 9, 10, and 11 were combined GGPS8, GGPS9, GGPS10, and GGPS11 for interaction with yeast strains expressing Cub only (C) or C-terminal Cub fusion with assays using the split ubiquitin system (Fig. 2). GGPS7 Arabidopsis PSY, respectively. Corresponding Nub/Cub fusions with was omitted as it shares 95% identity to GGPS6 and the Arabidopsis kation channel protein KAT1 were used as negative both enzymes diverged recently through tandem du- controls whereas dimerization of Nub-KAT1/KAT1-Cub served as a positive control. A, Interaction assay. Growth on selective medium, plication, suggesting largely redundant functions (Co- supplemented with 150 µM Met after 2 d incubation. B, β-Gal activity man et al., 2014). The split ubiquitin assays showed determined by ONPG assays of yeast strains coexpressing combina- that, in addition to GGPS11, the isoenzymes GGPS2, tions with PSY-Cub. C, Nub-GGPS protein levels in PSY-Cub combina- GGPS6, GGPS8, and GGPS9 interacted with PSY and tions. Nub-GGPS proteins carried an N-terminal 3-HA tag and were the resulting yeast cells accumulated phytoene. The detected using a 3-HA antibody. D, Phytoene amounts in PSY-Cub only exception was GGPS10, which neither showed combinations, quantified by HPLC. E, Absorption spectrum of phy- interaction nor formed phytoene, although its iden- toene determined in yeast strains coexpressing PSY-Cub and GGPS11. tity as a GGPS has been reported (Beck et al., 2013). Results in B and D are mean +/− SEM of three biological replicates. Corroborating previous data, the interaction strength * Significant difference to that in Nub control (P > 0.05). and phytoene levels were highest for the combination of PSY and GGPS11 (Beck et al., 2013; Ruiz-Sola et al., 2016b). However, phytoene levels were similarly high Individual Kinetic Investigation of Recombinant in GGPS8/PSY coexpressing yeast cells, although their Arabidopsis GGPS11 and PSY interaction strength was about 70% lower compared to The physical interaction between GGPS and PSY that of the GGPS11/PSY combination. might be required for efficient phytoene synthesis.

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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.

We therefore investigated the properties of GGPS11 and N-terminal His6-tagged fusion protein. The protein PSY individually to compare them with those in cou- was purified after chaotropic refolding, as reported pled assays containing both enzymes. GGPS11, trun- elsewhere (Welsch et al., 2010). The substrate GGPP cated by its transit peptide sequence, was expressed was synthesized upfront by carrying out a GGPS11 as an N-terminally His6-tagged protein in E. coli and assay under standard conditions (20 µM DMAPP/20 purified. Enzyme assays were performed in a biphasic µM IPP), providing 4 µM [14C]GGPP as PSY substrate. system including phosphatidyl cholin/Tween 80 mi- However, upon subsequent addition of recombinant celles with a constant 20 µM DMAPP and varying [14C] PSY (1.22 µg, corresponding to 138 nM), [14C]phytoene IPP concentrations (1–30 µM) using isotope-dilution was synthesized very slowly. Only 3% of the [14C] with unlabeled IPP. The [14C]GGPP formed was quan- GGPP substrate was converted after a 2 h incubation tified by scintillation counting after extractive separa- (see Supplemental Table S1). In line with this inac- tion from unincorporated IPP (Fig. 3). For Arabidopsis cessibility of presynthesized GGPP, the conversion of GGPS11, fitting GGPP formation from IPP and DMAPP externally added [3H]GGPP in the range of 0.5–4 µM with the Michaelis-Menten equation allowed the deter- was lesser still with only 0.1% conversion into [3H] mination of an apparent KM = 8.47 ± 0.73 µM IPP and a phytoene after an incubation time of 2 h. Thus, the −1 −1 Vmax = 1.9 ± 0.06 nmol GGPP s mg , corresponding to kinetic parameters for PSY could not be determined −1 kcat = 0.069 ± 0.002 s . These values are comparable to with presynthesized GGPP. those determined for the GGPS11 homolog from Sina- pis alba (Kloer et al., 2006), with Vmax = 1.8 nmol GGPP −1 −1 Phytoene Synthesis in a Coupled System with s mg , but with lower KM = 4.4 µM IPP (kcat = 0.065 s−1). Under standard assay conditions, 1 µg (138 nM) Recombinant GGPS11 and PSY of Arabidopsis GGPS11 was capable of converting 4 Alternatively, the simultaneous presence of PSY and nmol of IPP at saturating DMAPP amounts (4 nmol) GGPS11 was tested as a means of improving phytoene into GGPP within 30 min of incubation time (Fig. 3). formation. These coupled assays contained the same In order to determine kinetic parameters of PSY, protein and substrate concentrations as in the individ- the PSY cDNA was truncated, removing the sequence ual assays described above. In stark contrast to the sit- encoding the transit peptide, and expressed as an uation with presynthesized GGPP, the coupled system

1016 Plant Physiol. Vol. 179, 2019 A Chimeric Carotenogenic Enzyme carried about 60% of the IPP substrate into phytoene Using the in vivo activity assay in E. coli (Welsch within 2 h (Fig. 3). After a short lag phase, the forma- et al., 2000), we evaluated whether an N-terminal or tion of phytoene proceeded almost linearly within the C-terminal translational fusion of GGPS11 and PSY initial 30 min, it then slowed down and fully ceased generates a functional enzyme. The additional co- after 1 h. In addition to phytoene, 30% of the IPP sub- expression of the bacterial carotene desaturase CrtI strate was converted into the intermediate GGPP with- converting the colorless phytoene into red-colored ly- in 20 min. Then, GGPP amounts slowly decreased to copene provided a photometrically accessible readout 25% of substrate IPP as the conversion into phytoene for the combined GGPS-PSY activity. No enzymatic exceeded GGPP formation. In this time-course experi- activity was observed when GGPS was fused to the N ment, the sum of both products (GGPP and phytoene) terminus of PSY (GGPS11-PSY, GGPY; Fig. 4). However, matched quantitatively with GGPP formation in an in- activity was recovered by the additional coexpression dividual GGPS11 assay (Fig. 3). Incubations with half of individual GGPS11 enzyme, indicating that GGPS11 Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 of the initial IPP concentrations (10 µM IPP with 20 µM activity in GGPY was impeded. In contrast, GGPS11 DMAPP) preceded with similar product proportions fused to the C terminus of PSY was enzymatically ac- of 60% phytoene and 30% GGPP (Supplemental Fig. tive (Fig. 4). We therefore focused on this fusion vari- S1). In contrast to assays with 20 µM IPP, the increase of ant, which was denoted PYGG (for PSY-GGPS11). GGPP ceased earlier, already after about 10 min and remained constant thereafter. Readdition of PSY after the synthesis of phytoene ceased was incapable of reinitiat- Kinetic Investigation of the PYGG Fusion ing phytoene synthesis from accumulated GGPP. This Whereas recombinant GGPS11 could be recovered as excludes progressive PSY inactivation by denaturation soluble enzyme from E. coli with the expression system which is frequently observed as an artifact with highly used (Welsch et al., 2000; Kloer et al., 2006), PYGG be- purified proteins (e.g. Koschmieder et al., 2017). haved like PSY in that it was found largely insoluble in Thus, the phytoene synthesis rate of recombinant inclusion bodies (Supplemental Fig. S3). Accordingly,

PSY increased dramatically when GGPP was supplied His6-PYGG was purified with the same chaotropic re- by an actively synthesizing GGPS11, while preformed folding protocol developed for His6-PSY (see “Material GGPP was hardly accessible to PSY. However, even and Methods”). This yielded the monodisperse PYGG in coupled assays, GGPP accumulated in addition to fusion protein with the expected molecular mass of phytoene. Thus, GGPP remained unmetabolized sug- 79.6 kD, composed of 35.7 kD for GGPS11 and 44.2 kD gesting a similar PSY inaccessibility as that seen for for PSY (Fig. 5). preformed or externally added GGPP. Enzyme assays were run with same protein concen- The amphipathic character of GGPP led us to as- trations as previously used under standard concen- sume that GGPP sequestration into the micelles of the trations (20 µM DMAPP/20 µM IPP; 138 nM PYGG). biphasic system used might cause its inaccessibility. As the PYGG fusion protein provided an equimolar We investigated the partition of GGPP into the lipid ratio of the GGPS11 and PSY moieties, the results were phase using phosphatidyl choline liposomes (Bozzuto directly compared with the coupled enzyme assays. and Molinari, 2015). In line with the above assumption, Phytoene synthesis proceeded almost linearly for 2 h, [14C]GGPP was almost quantitatively recovered in li- but with an approximate 4-fold lower velocity than posomal fractions obtained by ultracentrifugation after that in coupled assays, and thereafter continued with synthesis from DMAPP/[14C]IPP by GGPS11 (Supple- further reduced reaction velocity (Fig. 5). However, it is mental Fig. S2). Thus, rapid sequestration of GGPP into interesting to note that in contrast to the coupled assay, membranes and the inability of this substrate to reach [14C]IPP was almost quantitatively converted into [14C] the active site of PSY once membrane-bound might ex- phytoene after 4 h. Only trace amounts of [14C]GGPP plain why GGPP, once liberated and diffusing, is not “leakage” was observed and this did not increase with efficiently used for phytoene synthesis. continued reaction time (Fig. 5). This supports efficient GGPP channeling from GGPS11 to PSY in the PYGG protein fusion (Bera et al., 2000). Kinetic constants for Altered IPP Conversion Efficiency through Direct Protein PYGG were determined by fitting phytoene forma- Fusion tion from IPP and DMAPP with the Michaelis-Menten

The necessity for immediate GGPP conversion by equation and revealed an apparent KM = 11.2 ± 1.8 µM −1 −1 PSY, in the simplest explanation, calls for close prox- IPP and a Vmax = 0.019 ± 0.001 nmol phytoene s mg −1 imity of GGPS11 and PSY to enable substrate chan- (kcat = 0.00069 ± 0.00004 s ; Fig. 5). neling. Consequently, the availability of GGPS11 in a correct stoichiometric ratio with PSY might limit the carotenoid biosynthetic flux. A feasible approach to Determination of Single Enzyme Activities in the Fusion overcome these potential restrictions is to directly fuse Protein GGPS11 with PSY, thereby eliminating potential vari- Despite the strongly increased proportion of phy- ables required for the recruitment of GGPS11 by PSY toene formed upon enzyme fusion, the concomitantly and also potentially eliminating competition from other reduced phytoene synthesis rate may indicate that the GGPP-utilizing pathways. individual kinetics of the GGPS11 and/or PSY moieties

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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).

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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.

might also be affected. To decipher these possibilities, These mutant fusion proteins were named PYgg1 and we reciprocally inactivated one or the other of the two PYgg2, respectively. enzymes in PYGG by introducing loss-of-function Prior to introducing these mutations into PYGG, mutations. We then complemented the mutant PYGG we confirmed their inactivating effect through heter- versions by adding the respective wild-type enzyme. ologous expression of individually mutated PSY and If this restored phytoene formation even beyond the GGPS11 enzymes in our E. coli in vivo assay system levels formed with wild-type PYGG, the enzyme moi- coexpressing CrtI. In agreement with their intended ety impaired in the fusion protein would be identified. function, not even trace amounts of lycopene were To test the activity of the GGPS11 fusion moiety, observed with any of the mutants whereas positive PSY loss-of-function mutants were generated by in- control combinations with wild-type enzymes accu- troducing single-amino acid changes known to in- mulated up to 32 ng lycopene OD600−1 (Supplemental activate squalene synthase, which shares structural Fig. S4). and catalytic similarities with PSY (Gu et al., 1998). The fusion protein mutants were purified and incu- The two independent PSY mutant versions were in bated under standard assay conditions, either individ-

the Asp-rich motif, namely PSY D313E and PSY D317E. ually or supplemented with wild-type recombinant The mutant fusion proteins were named pyGG1 and PSY or GGPS11. As expected, both pyGG mutants were pyGG2, respectively. To test whether PSY activity was incapable of synthesizing phytoene and accumulated impaired in PYGG, GGPS11 mutants were designed GGPP exclusively (Fig. 6). Addition of equimolar based on amino acid substitutions known to strongly amounts of recombinant wild-type PSY completely reduce farnesyl diphosphate synthase activity (Mar- rescued phytoene synthesis, but the levels were identi- rero et al., 1992; Joly and Edwards, 1993). In one mu- cal to those obtained with the wild-type PYGG fusion tant, two Asp residues in the second Asp-rich motif protein. This indicates that enzymatic properties of (SARM) were substituted by Lys, yielding GGPS11 the PSY enzyme moiety remained largely unaffected

D297E-D298E. In the other mutant, two Arg residues through the fusion with GGPS11. closely adjacent to the first Asp-rich motif (FARM) As expected, both PYgg mutants were enzymatically were replaced by Lys, yielding GGPS11 R166K-R167K. inactive and did not produce even trace amounts of

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GGPP (Fig. 6). Upon addition of recombinant wild- individual enzyme overexpression, the competition for type GGPS11, slightly higher phytoene amounts com- GGPP from other branches of the isoprenoid pathway pared to the wild-type PYGG control were produced. may be altered. PYGG was expressed under control However, the levels of free GGPP increased dramati- of the constitutive CaMV-35S promoter in Arabidop- cally to about 4-fold the amounts of phytoene. This in- sis. For control lines, we also overexpressed GGPS11 dicated that the activity of GGPS11 in wild-type PYGG individually under control of the nos promoter and was strongly reduced upon the N-terminal fusion with included lines constitutively overexpressing PSY as PSY. published (Maass et al., 2009). In contrast to the recombinant proteins used so far in vitro, GGPS11 and PSY carried their native N-terminal transit peptides and Emulating PYGG Kinetics with Adjusted Stoichiometry PYGG was translationally fused with the PSY transit of Individual Enzymes peptide. Efficient transit peptide processing was con- Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 In conclusion, the reduced activity of the GGPS11 firmed by immunoblot analysis using callus protein moiety in PYGG accounts for the reduced velocity of extracts (see Fig. 7) from one selected GGPS11, PYGG, phytoene synthesis. However, this circumstance might and PSY-overexpressing line each. PYGG was detected also contribute to the improved efficiency, i.e., the with its correct molecular mass, calculated as 63.5 kD strongly reduced GGPP leakage upon phytoene for- after plastid import. mation by the fusion protein. Accordingly, the coupled From a total of 12 PYGG-expressing lines, viable system with individual enzymes would be expected homozygous lines could be generated only from five to behave similarly when the GGPP synthesis rate is lines which did not show phenotypic deviation from reduced, e.g. through an altered GGPS11:PSY ratio. the wild type. In contrast, all other lines exhibited Based on the previously determined Michaelis-Menten about 25% of seedlings with orange cotyledons and/ parameters, we performed in silico assays of GGPS11 or primary leaves that did not continue to grow on soil and PSY, which relied on the discrete solving of the and thus did not set seeds (Fig. 7). Upon continued Michaelis-Menten equations. The use of these computer growth on MS medium, leaves turned only slightly simulations allowed us to examine the consequences of green with large white/transparent areas and showed varying GGPS11 concentrations at constant PSY con- extreme dwarfism. The remaining seedlings from centrations on phytoene formation in the coupled sys- these lines grew without phenotypic deviations from tem. Using this approach, a GGPS11:PSY molar ratio the wild type (Supplemental Fig. S6). Homozygous of approximately 1:10 was predicted to match the phy- GGPS11-overexpressing lines were generated and toene formation velocity determined for PYGG, thus showed no differences to Wt seedlings, as already ob- much lower than the 1:1 ratio used in the previous served in Nagel et al. (2015). Increased GGPS11 protein coupled assays (Supplemental Fig. S5). Confirmatory, levels were confirmed by immunoblot analysis (Fig. 7). coupled enzyme assays with individual enzymes pro- In order to determine carotenoid pathway flux in vided in a 1:10 ratio resulted in reaction kinetics identi- nongreen cells, we employed callus tissue generated cal to those observed with PYGG (Fig. 6). In both cases, from germinating seedlings. This system was es- IPP conversion efficiency into phytoene was maximal tablished recently for routine analysis of carotenoid with only trace amounts of accumulated GGPP. pathway fluxes (Schaub et al., 2018). Calli from lines overexpressing GGPS11 showed no increase in carotenoid content whereas those overexpressing PSY in- Expression of the Chimeric Protein in Arabidopsis creased total carotenoid content to 1800 µg mg DW−1 The data from mutagenesis, the simulation results, in the strongest lines. Interestingly, carotenoids in and their verification by coupled assays indicate that PYGG-expressing callus of lines from which homozy- substrate channeling may not be the underlying cause gous plants could not be obtained showed a further of the PYGG characteristics. Rather, considerably re- increase in carotenoid levels by almost 50% to up to duced GGPP leakage might be the result of altered 3000 µg mg DW−1 (Fig. 7). kinetics of the fused two-enzyme cascade. However, adjusting the conditions of the coupled assay to meet the characteristics of PYGG does not rule out channeling. Logistically, it is not straightforward to test for DISCUSSION true channeling with GGPS and PSY. Substrate competition experiments (with GGPP and IPP) that are clas- PSY Interacts with Most Plastid-Localized GGPS sic in the investigation of metabolite channels (Møller and Conn, 1980) are not applicable here because of the GGPSs constitute a small gene family with twelve inaccessibility of GGPP upon membrane or micellar members in Arabidopsis, of which at least seven are sequestration (see above). plastid-localized (Beck et al., 2013; Coman et al., 2014). Assuming that PYGG is, in fact, capable of improv- The reason for this redundancy remains unclear. Only ing the metabolite flux into carotenogenesis through GGPS11 knock-out lines are lethal whereas mutant substrate channeling, the fusion was expressed in planta. lines of almost all other plastid-targeted GGPS isoen- This was guided by the expectation that, compared to zymes do not show aberrant phenotypes (Ruiz-Sola

1020 Plant Physiol. Vol. 179, 2019 A Chimeric Carotenogenic Enzyme

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 Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 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 im- ages (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).

et al., 2016b). Interestingly, GGPS6, -7, -9, and -10 were Moreover, again with GGPS10 as an exception, all unable to rescue GGPS11 knock-out lines following GGPS isoforms included were capable of providing their overexpression (Nagel et al., 2015). Moreover, substrate GGPP for phytoene synthesis via PSY fol- GGPS11 has the highest expression level in most Ara- lowing coexpression in yeast cells. bidopsis tissues, especially in photosynthetic tissues, The incapability of GGPS10 to functionally inter- whereas expression of other GGPS isoforms dominate act with PSY following coexpression in yeast conflicts in roots, seeds, and flower organs (Beck et al., 2013). with the observed phytoene synthesis following its Thus, GGPS11 is considered as the essential enzyme coexpression with the bacterial phytoene synthase for the major GGPP-consuming plastid-localized path- CrtB in bacteria (Beck et al., 2013). However, CrtB is ways, including carotenogenesis, and it is assumed active even with a GGPS isoform that does not colocal- that there is functional specialization for the remaining ize with PSY in plastids e.g. with ER-localized GGPS3 GGPS isoforms. (Beck et al., 2013). It is therefore somewhat surprising that PSY inter- This suggests that plant and bacterial phytoene acted not only with GGPS11 (Ruiz-Sola et al., 2016b), synthases might differ in their modes of accepting but with all other plastid-localized GGPS isoforms substrate GGPP. Whereas under the conditions used, (GGPS2, -6, -8, and -9), except GGPS10 (Fig. 2). PSY activity strictly depends on actively synthesized

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GGPP supply in vitro, as demonstrated here, and PSY GGPS11 and PSY may well ensure the close proximity is incapable of metabolizing free or liposome-bound between GGPS and PSY, thereby allowing efficient GGPP, recombinant CrtB was shown to efficiently con- GGPP metabolization. vert substrate GGPP into phytoene, independent of an The advantage of efficient substrate conversion in active GGPS (Iwata-Reuyl et al., 2003). Assuming that microdomains is not restricted to amphipathic com- this property was present in phytoene synthases of pounds and is also observed, for instance, for soluble early photosynthetic prokaryotes, its loss in plant phy- metabolites like cAMP (between adenyl cyclases and toene synthases might be correlated with the diver- phosphodiesterases) and ATP (between creatine ki- sification of GGPP-derived metabolites and the need nases and myosin ATPase; Selivanov et al., 2007; Bail- to more tightly coordinate carotenogenesis with other lie, 2009). Considering the amphipathic properties of pathways. This might be facilitated by the establish- GGPP it appears possible that these observations are ment of functional dependencies in the form of pro- not restricted to the GGPS11/PSY metabolon, but may Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 tein-protein interactions. as well apply to other branched pathways in planta. The capability of GGPS2, -6, -8, -9, and -11 to synthe- Although recombinant oat CHLS accepts free GGPP size GGPP, which is concluded from this study and the for chlorophyllide esterification and Arabidopsis work of Beck et al. (2013), conflicts with results from SPS2 accepts free GGPP for prenylquinone synthesis, in vitro approaches with recombinant enzymes (Nagel CHLS enzyme activities are weak and data on com- et al., 2015). This revealed the C25 prenyl diphosphate parative assays done in the presence of active GGPS geranylfarnesyl diphosphate (GFDP) as the predomi- are not available (Schmid et al., 2001; Hirooka et al., nant product for GGPS6 and -9, whereas GGPP was 2003, 2005). Thus, regulated composition and stabili- found only in trace amounts. Only GGPS2 and -11 ty of GGPS-containing metabolons might be essential were found to exclusively synthesize GGPP whereas to distribute GGPP into different metabolite avenues. GGPS8 did not show any enzymatic activity in these Interestingly, Zhou et al. (2017) recently identified a investigations. However, the fact that phytoene was GGPS-recruiting protein in rice (OsGRP) that efficiently formed for those GGPSs previously characterized in competes with stromal rice GGPS1 dimerization and vitro as (mainly) GFDP-synthesizing enzymes doubt- recruits it into large thylakoid membrane protein lessly shows that GGPP is indeed produced. Moreover, complexes including GGPP reductase, CHLS, as well GGPP is synthesized in amounts which are high com- as other proteins involved in chlorophyll biosynthe- pared with those synthesized by GGPS11 (Fig. 2; Beck sis. They found that OsGRP increases GGPP synthe- et al., 2013). This discrepancy to the in vitro situation sis activity and specificity, evidently functioning as a might suggest that some GGPS isoforms are capable complex organizer to support efficient substrate chan- of altering their product pattern. This might be caused neling toward chlorophyll biosynthesis. by different conditions of the system (in vitro versus in vivo when expressed in yeast or bacterial cells), but more likely depends on GGPP turnover by the activity Protein Complexes in the Carotenoid Pathway of subsequent enzymes. Additional biochemical in- Several observations support substrate channeling vestigations with combinations of GGPS isoforms and from IPP to phytoene in the carotenoid pathway. For downstream enzymes are required to answer these instance, native plastid extracts from tomato fruits, questions. daffodils flowers, and Capsicum fruits were shown to synthesize phytoene directly from IPP (Porter and Spurgeon, 1981; Lützow and Beyer, 1988; Fraser et al., GGPS11-PSY Interaction Facilitates Efficient Substrate 2000). Interestingly, intermediates did not accumulate Metabolization nor were they accepted efficiently as substrates when Recombinant PSY was almost inactive when pre- added to the assay. This indicates that the entry of dif- formed GGPP was used as substrate, either when add- fusible metabolites is largely prevented which is in ed from solution or provided in a presynthesized form agreement with our findings. by GGPS11. In contrast, the simultaneous presence of In contrast, the accessibility of lipophilic substrates active GGPP-synthesizing GGPS11 allowed phytoene in vitro to carotenogenic enzymes downstream to formation at high rates (Fig. 3). This observation sug- phytoene synthesis appears completely different. Both gests the existence of microdomains between GGPS11 recombinant phytoene desaturase (PDS), β-carotene and PSY allowing GGPP to reach high local concen- cyclase as well as carotenoid isomerase were enzymat- trations and to be metabolized into phytoene before ically very active when lipophilic substrates were sup- it escapes from these microdomains (Lee et al., 2012). plied in liposomes (Isaacson et al., 2004; Yu and Beyer, Moreover, GGPP incorporates into liposomal mem- 2012; Gemmecker et al., 2015; Brausemann et al., 2017; branes, which renders it inaccessible for phytoene syn- Koschmieder et al., 2017). It was shown that PDS and thesis. Thus, the spacial proximity of GGPS11 and PSY ζ-carotene desaturase interacts monotopically with is a precondition for efficient phytoene synthesis. This membranes to access their substrates (Koschmieder is ensured by protein-protein interaction, as shown et al., 2017). Mathematical modeling implied that sub- by the split-ubiquitin system as well as through BiFC strate channeling occurred between subunits of PDS (Fig. 2; Ruiz-Sola et al., 2016b). Enzyme fusion between homotetramers, whereas no kinetic information is

1022 Plant Physiol. Vol. 179, 2019 A Chimeric Carotenogenic Enzyme available to date to show whether a channeling relation well as an approximate doubling of carotenoids in also exists between the different membrane-bound en- Arabidopsis callus, as compared with that in the stron- zymes. These different properties compared with the gest PSY-overexpressing lines. clear dependency of PSY on an active GGPS11 may The novelty in the PYGG-expression experiment re- reflect different substrate solubilities: the highly am- sides in the fact that, for the first time after years of phipathic character of GGPP may necessitate a “spe- overexpressing individual or combined individual cial treatment”, whereas this is not the case with the carotenoid genes in Arabidopsis, green leaves (cotyle- uniformity of the lipophilic downstream metabolites. dons and primary leaves) show an orange phenotype It may as well reflect the pronounced pathway branch- (Fig. 7). This confirms, first, that the fusion protein is ing point at the GGPP stage of the pathway. stable in plants and not subjected to nonspecific or targeted protein degradation and, second, the fusion is capable of increasing the IPP conversion efficien- Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 The Advantage of the Translational Fusion between cy specifically toward the synthesis of carotenoids. GGPS and PSY Moreover, we find a most evident impairment with The translational fusion between GGPS11 and PSY chlorophyll formation. In light of the channeling dis- did not show accelerated phytoene formation. How- cussion raised, the simplest explanation may be that ever, it exhibited improved substrate conversion ef- the CHLS-branching point is in fact GGPP depleted ficiency by strongly reducing GGPP leakage, which and that, consequently, the fusion provides a directed generates a proportion of this intermediate that rep- and channeled metabolic flux toward carotenogenesis. resents a dead end. One reason is the reduced GGPS11 activity (Fig. 5). This leads to a more balanced rate of GGPP formation for PSY to cope with, which is the Other Natural and Synthetic Chimeric Enzymes rate-limiting step in this two enzyme cascade. It ap- Bifunctional enzymes are widespread in various pears confirmatory, at first glance, that GGPP accumu- plants and sometimes exist even in parallel to their lation can be abolished by reducing GGPS11 amounts. individual counterparts in related species (Hagel and Thus, one might argue whether reduced GGPS activ- Facchini, 2017). For instance, the epimerization of re- ity or improved channeling—or both—contribute to ticuline in the benzylisoquinoline alkaloid pathway the improved efficiency observed. The only feasible occurs via two individual enzymes in field poppy way of testing a contribution of channeling is to test whereas a single protein combining both enzymes sep- whether phytoene synthesis by PYGG is independent arated by a short spacer region catalyzes the identical from competitive interactions with other GGPP-con- reaction in opium poppy. Moreover, natural enzyme suming reactions in vivo occurring at the GGPS11 hub fusions are also known from the carotenoid pathway, (Ruiz-Sola et al., 2016b). e.g. a bifunctional enzyme in the fungus Mucor circinel- In fact, carotenogenesis responded differently to loides carries phytoene synthase and lycopene cyclase PYGG expression compared to that following the activity, which are encoded by separate genes in other overexpression of individual GGPS11 and PSY. Higher organisms (Velayos et al., 2000). Bifunctional lycopene levels of GGPS11 did not increase carotenoids and cyclases are known from the cyanobacterium Prochlo- chlorophylls in leaves or carotenoids in the nongreen rococcus marinus and the prasinophyte algae Ostreococ- Arabidopsis callus system (Fig. 7). This may not be cus lucimarinus generating β,β-, β,ε-, or ε,ε-carotene in surprising assuming that specific GGPS11-containing various proportions (Stickforth et al., 2003; Blatt et al., metabolons control GGPP distribution, which would 2015). One further example with remarkable similarity in turn be affected by stoichiometrically determined to the GGPS11-PSY fusion is known from the precur- relative enzyme abundances of branching pathways sor biosynthesis for fungal fusicoccins, which involve or regulated complex formation. However, overex- GGPP as substrate. The corresponding chimeric en- pression of PSY is known to increase carotenoid lev- zyme includes a GGPS as well as a terpene cyclase do- els, but only in nongreen tissues (Fraser et al., 2007; main (Toyomasu et al., 2007). Maass et al., 2009; Welsch et al., 2010; Bai et al., 2011). Moreover, several synthetic fusions of enzymes In green tissues, PSY overexpression has little effect involved in isoprenoid biosynthesis revealed simi- on carotenoid accumulation just like the overexpres- larly improved efficiency as observed for PYGG. For sion of GGPS. This is partially due to compensation instance, a fusion between farnesyl diphosphate syn- by enhanced cleavage of xanthophylls (Lätari et al., thase (FPS) and aristolochene synthase revealed more 2015) but can as well reflect the GGPS/PSY protein efficient substrate to product conversion compared stoichiometry. It remains to be investigated whether with single enzyme reactions in vitro (Brodelius et al., simultaneous overexpression of both GGPS11 and PSY 2002). Expression of a chimeric enzyme composed of is capable of increasing the abundance of active com- amorpha-4,11-diene synthase (ADS), the rate-limiting plexes and carotenoid amounts also in green tissues. enzyme of artemisinin biosynthesis, and FPS revealed These restrictions regarding complex formation, which high product levels, which were explained by higher might be different for green and nongreen systems, local concentration of FPP at ADS than in the single are overridden by PYGG-expression which results in enzyme situation that might enable ADS to operate increased carotenoid amounts in nongreen tissues as at substrate saturation (Han et al., 2016). For further

Plant Physiol. Vol. 179, 2019 1023 Camagna et al.

investigation, we propose the exploitation of PYGG expression was induced with 1 mm isopropylthiogalactoside (IPTG) for 4 h. in other nongreen plant tissues, such as tomato fruit, Cells were centrifuged (10 000 g/10 min), resuspended in 10 mL buffer A (20 mm Tris/HCl, pH 8.0, 100 mm NaCl, 10 mm MgCl2, 10% [v/v] glycerol), disrupted to show whether this synthetic bifunctionality might with a French Press (Amicon), and centrifuged (10 000 g, 10 min). Subsequently, boost the pathway beyond the overexpression of PSY 600 µL TALON Co2+ resin (Clontech), equilibrated in buffer A, was added to alone. This potential is already indicated by the strong- the supernatant. After 30 min incubation on ice, the suspension was loaded ly increased carotenoid formation in the callus system onto an empty 5-mL TALON column (Clontech), then retained TALON resin was washed with 20 mL buffer A and His6-GGPS11 was eluted with 5 mL (Fig. 7). In light of the positive findings with other buffer A supplemented with 100 mm imidazol. Protein concentration was de- engineered enzyme fusions, e.g. combining xylanase termined with Bradford assay (Bio-Rad) and the protein was stored at -20°C. and laccase activity (Ribeiro et al., 2011) or combining 4-coumarate CoA-ligase and stilbene synthase (Zhang et al., 2006), we find this a potentially valuable synthet- Enzyme Assays Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 ic avenue to be pursued, which would also provide In vitro enzyme assays were performed in a final volume of 200 µL. Activi- sufficient amounts of biological material required for ty of recombinant PSY depends on detergents (Iwata-Reuyl et al., 2003), which in-depth biochemical analyses. did not affect GGPS activity; thus the same buffer was used for all recombinant enzymes/combinations. All assay components except substrates were premixed in enzyme reaction buffer (100 mm Tris/HCl, pH 7.6; 0.08% [v/v]

Tween 80; 20% [v/v] glycerol, 2 mm MnCl2; 10 mm MgCl2; 1 mm TCEP; 600 mm NaCl; 62.5 ng µL−1 phosphatidylcholin liposomes). For individual PSY assays 3 −1 MATERIALS AND METHODS [ H]GGPP (50 Ci mmol ; in ethanol:NH4OH:H2O [6:3:1]; American Radiola- beled Chemicals) and for coupled and PYGG assays the substrate mixture (20 µM DMAPP, 16.25 µM IPP [isoprenoids.com] and 3.75 µM [14C]IPP [50 mCi mmol−1; in ethanol:NH OH:H O [6:3:1]; American Radiolabeled Chemicals]) Yeast Split Ubiquitin Assay 4 2 was added to start the reaction, respectively. For standard assays, enzymes The split-ubiquitin system was used as described (Obrdlik et al., 2004; were used at 138 nm each. Reactions were stopped by adding 50 µL 160 mm Welsch et al., 2018). ORFs were truncated by the length of transit peptides EDTA. For extraction, 200 µL BuOH was added, mixed, and centrifuged (21 14 14 predicted by ChloroP (Emanuelsson et al., 1999; see Supplemental Table S2). 000 g, 5 min). The BuOH hyperphase containing [ C]GGPP and [ C]phytoene 14 GGPS11 cDNAs were cloned into pNXgate in THY.AP4 and mated with PSY- ([ C]IPP remained in the hypophase) was transferred into a new tube and the Cub, present in THY.AP5 (Cub) and the resulting diploid cells were cultured extraction was repeated with 100 µL BuOH. Combined BuOH extracts were in synthetic complete medium lacking Leu and Trp. Interaction growth tests mixed with 300 µL of 1 m MgCl2 in MeOH, inverted, centrifuged, 300 µL hep- 14 were performed on synthetic minimal agar, supplemented with 150 µM Met tane was added, centrifuged, and the heptane hyperphase (containing [ C] 14 to reduce background activation of reporter genes. For β-galactosidase assays phytoene; [ C]GGPP remained in the hypophase) was transferred into a new and phytoene extraction, yeast strains were grown overnight in synthetic com- tube. The extraction was repeated with 200 µL of heptane. All fractions were plete medium supplemented with adenine and His at 28°C in order to eliminate measured by scintillation counting (Tri-Carb 2900 TR, Perkin-Elmer) in 6-mL growth differences caused by different interaction strengths. β-Galactosidase scintillation cocktail (Rotiszint eco plus, Roth). 14 activity was determined with ortho-nitrophenyl-β-galactoside (ONPG) in bi- For GGPP sequestration assays, 20 µM DMAPP/[ C]IPP were converted 14 ological triplicates as described (Chayut et al., 2016) and expressed relative to into [ C]GGPP using 10 µg GGPS11 overnight in enzyme reaction buffer de- −1 cell density measured at 600 nm. void of detergents and liposomes. 62.5 nmol µL liposomes were added For phytoene extraction, yeast cells from 200 mL of culture were pelleted for 5 and 30 min, samples were centrifuged at 100 000 g for 1 h, pellets and 14 and lyophilized. Four mL of ethanol containing 2 Sudan IV (Sigma-Aldrich) supernatant were differentially extracted, and amounts of [ C]GGPP were de- as internal standard was added. For saponification, 120 µL KOH (1 mg mL−1) termined by scintillation counting. was added and samples were incubated at 85°C for 5 min. After cooling on For GGPP distribution measurements, enzyme reaction buffer was pre- 3 ice, 6 mL 1% NaCl (w/v) and 2 mL of petroleum ether:diethyl ether (2:1, v/v) pared like above without Tween 80. Four pmol [ H]GGPP and 4 nmol GGPP was added. Samples were mixed and centrifuged. The petroleum ether:di- (isoprenoids.com) were added, incubated for 5 and 30 min and centrifuged for ethyl ether step was repeated, and the combined epiphases were dried and 1 h at 100 000 g. The supernatant was transferred into a new tube and the li- dissolved in 30 µL of chloroform. Ten microliters of each sample was injected posome pellet was resuspended in 200 µL H2O. Supernatant and resuspended into the HPLC system equipped with a C30 column (YMC Europe) using a liposomes were measured by scintillation counting. gradient system (Hoa et al., 2003). Phytoene peaks were normalized relative to the internal standard and quantified according to Schaub et al. (2005). Protein extracts from yeast cells were prepared according to Wang et al. Protein Co-Expression in E. coli (2004). Cells were lysed in 50 µL of 1.85 M NaOH per 3 A units and incubat- 600 pRSF-PSY and pACYC-GGPS11 vectors contained corresponding cDNAs ed on ice for 10 min. An equal volume of 50% (w/v) trichloroacetic acid was devoid of sequences encoding transit peptides in the plasmids pRSFDuet added, and proteins were recovered by centrifugation for 5 min. The pellet and pACYCDuet, respectively. pRSF-GGPS-PSY and pRSF-PSY-GGPS were was suspended in 50 µL of SDS sample buffer containing 8 m urea and com- made by overlap extension PCR with the GGPS11 cDNA (amplified from bined with 20 µL of 1 m Tris and incubated for 1.5 h at 37°C. Proteins were sep- pACYC-GGPS11) and the PSY cDNA (amplified frompRSF-PSY ) and com- arated by SDS-PAGE, then blotted onto PVDF membrane. Immunodetection bined by Gibson assembly in NcoI-digested pRSFDuet (Gibson et al., 2009). A was performed using anti-HA antibodies (Sigma). 20 amino acid linker containing alternating Gly and Ser residues (Chen et al., 2013) was introduced between the two proteins. Protein Purification and Analysis Mutations were introduced in GGPS11 and PSY by overlap extension PCR (primers see Supplemental Table S2) from plasmids pET-GGPS11 (R166K-R167K;

For recombinant His6-PSY expression, the PSY cDNA devoid of its se- D297E-D298E) and pCOLD1-PSY (D313E; D317E), respectively. pCOLD1-PYGG vec- quence encoding the transit peptide was subcloned into the vector pCOLD1, tors containing mutated GGPS11 versions were made from corresponding pET revealing pCOLD1-PSY (Welsch et al., 2010). For pCOLD1-PYGG, the NcoI/ vectors exchanging a XhoI/Bsu36I fragment with pCOLD1-PYGG. pCOLD1- PstI fragment from pRSF-PYGG (see below) was subcloned into pCOLD1, and PYGG vectors containing mutated PSY versions were made from corresponding

digested similarly. Purification of His6-PSY and His6-PYGG was performed as pCOLD1 vectors by subcloning amplified PCR fragments containing intro- described in Welsch et al. (2010). Aliquots of recombinant proteins were frozen duced EcoRI/SmaI sites. in liquid nitrogen and stored at -80°C until use. For lycopene assays, pRSFDuet containing separate PSY and GGPS11 ORFs

The recombinant His6-GGPS11 was created using the cDNA of GGPP11 was combined with pCDF-CrtI and used as positive control. pRSF-GGPS-PSY from Arabidopsis (base 238 to 1189, accession no AK227130) was inserted into and pRSF-PSY-GGPS were combined with pCDF-CrtI. pCOLD1-PSY and mu- vector pETDuetTM-1 (Novagen) creating pET-GGPS11 that was transformed tants thereof were combined with pACYC-GGPS and pCDF-CrtI while pET- into E. coli BL21 (Novagen). Cells were grown at 37°C until an OD600 of 0.5 and GGPS11 and mutants thereof were combined with pRSF-PSY and pCDF-CrtI.

1024 Plant Physiol. Vol. 179, 2019 A Chimeric Carotenogenic Enzyme

For lycopene extraction, E. coli pellets were resuspended in 300 µL acetone, ACKNOWLEDGMENTS centrifuged (4 000 g, 5 min), and the supernatant transferred into a new tube. The pellet was re-extracted two times with 300 µL acetone each. Combined We wish to thank Prof. Paul Fraser (University of London, UK) for critical acetone supernatants were evaporated in a speed vac and resuspended in pet- reading of the manuscript. We thank Carmen Schubert (University of Freiburg)

rol ether. Lycopene was determined photometrically using ε474nm = 185 230 L for her skillful technical assistance. We acknowledge the ABRC (Arabidopsis mol−1 cm−1. Biological Resource Center) and S.P. Dinesh-Kumar and M. Snyder for providing Arabidopsis GGPS cDNA clones. Received August 17, 2018; accepted October 1, 2018; published October 11, Generation of Transgenic Arabidopsis Lines and Growth 2018. Conditions For PYGG-expressing Arabidopsis lines, the GGPS11 cDNA was amplified by PCR thereby providing an in-frame 5 extension encoding a 20-amino acid ′ LITERATURE CITED Gly/Ser linker (Chen et al., 2013). The ORF was fused with the PSY ORF by Downloaded from https://academic.oup.com/plphys/article/179/3/1013/6116483 by guest on 28 September 2021 overlap extension PCR and subcloned into pCAMBIA1390-35Spro (Álvarez Almeida J, Azevedo M da S, Spicher L, Glauser G, vom Dorp K, Guyer L, et al., 2016), revealing pCAMBIA1390-35S::PYGG. For GGPS11-overexpressing del Valle Carranza A, Asis R, de Souza AP, Buckeridge M, Demarco D, lines, the GGPS11 ORF was subcloned into pCAMBIA1390-nospro. Arabidopsis Bres C, (2016) Down-regulation of tomato PHYTOL KINASE strongly (eco-type Wassilewskija) was transformed by vacuum infiltration (Bechtold impairs tocopherol biosynthesis and affects prenyllipid metabolism in an and Pelletier, 1998). Homozygous T2 progenies were identified by the segrega- organ-specific manner. J Exp Bot 67: 919–934 tion pattern of the corresponding T3 progenies on Murashige and Skoog plates Álvarez D, Voß B, Maass D, Wüst F, Schaub P, Beyer P, Welsch R (2016) Ca- containing antibiotics. Plant and seed-derived callus growth and carotenoid rotenogenesis Is Regulated by 5'UTR-Mediated Translation of Phytoene analysis were performed as described (Maass et al., 2009). Synthase Splice Variants. Plant Physiol 172: 2314–2326 Arango J, Jourdan M, Geoffriau E, Beyer P, Welsch R (2014) Carotene Hy- droxylase Activity Determines the Levels of Both α-Carotene and Total Ca- Bioinformatics rotenoids in Orange Carrots. Plant Cell 26: 2223–2233 The kinetic constants were evaluated by nonlinear least squares fitting of Bai C, Twyman RM, Farré G, Sanahuja G, Christou P, Capell T, Zhu C (2011) the data to the Michaelis-Menten equation using the software GraphPad Prism A golden era—pro-vitamin A enhancement in diverse crops. In Vitro Cell. Dev.Biol.-Plant 47: 1–1710.1007/s11627-011-9363-6 (v7.01): V = (Vmax*[S])/(KM+[S]). In silico reactions of Michaelis-Menten protein kinetics were realized in Baillie GS (2009) Compartmentalized signalling: spatial regulation of cAMP Python, where the Michalis-Menten equations were solved via numerical in- by the action of compartmentalized phosphodiesterases. 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Proc Natl Acad Sci USA 96: Supplemental Figure S5: In silico-emulation of PYGG reaction kinetics. 12929–12934 Camara B (1993) Carotenoids Part B: Metabolism, Genetics, and Biosynthesis. Supplemental Figure S6: Carotenoid and chlorophyll levels in seedlings. Methods Enzymol Supplemental Table S1: Conversion of GGPP into phytoene by recombi- Cazzonelli C (2011) Goldacre Review: Carotenoids in nature: insights from nant PSY. plants and beyond. Funct Plant Biol 38: 833–847

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