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ARTICLE IN PRESS
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doi:10.1016/j.ym
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Metabolic Engineering 7 (2005) 384–400
www.elsevier.com/locate/ymben
Metabolically engineered oilseed crops with enhanced seed tocopherol
Balasulojini Karunanandaaa,1, Qungang Qia,1, Ming Haoa, Susan R. Baszisa,Pamela K. Jensena, Yun-Hua H. Wonga,2, Jian Jianga,3, Mylavarapu Venkatrameshb,4,
Kenneth J. Gruysa,5, Farhad Moshiria, Dusty Post-Beittenmillera,James D. Weissa, Henry E. Valentina,�,5
aMonsanto Company, 800 N. Lindbergh Boulevard, St. Louis, MO 63167, USAbRenessen LLC, 800 N Lindbergh Boulevard, St. Louis, MO 63167, USA
Received 30 January 2005; received in revised form 8 May 2005; accepted 18 May 2005
Available online 25 August 2005
Abstract
Tocochromanols (tocopherols and tocotrienols) are important lipid soluble antioxidants and are an essential part of the
mammalian diet. Oilseeds are particularly rich in tocochromanols with an average concentration 10-fold higher than other plant
tissues. Here we describe a systematic approach to identify rate-limiting reactions in the tocochromanol biosynthetic pathway, and
the application of this knowledge to engineer tocochromanol biosynthesis in oilseed crops. Seed-specific expression of genes
encoding limiting tocochromanol pathway enzymes in soybean increased total tocochromanols up to 15-fold from 320 ng/mg in WT
seed to 4800 ng/mg in seed from the best performing event. Although WT soybean seed contain only traces of tocotrienols, these
transgenic soybean accumulated up to 94% of their tocochromanols as tocotrienols. Upon crossing transgenic high tocochromanol
soybean with transgenic high a-tocopherol soybean, the vitamin E activity in the best performing F2-seed was calculated to be 11-
fold higher than the average WT soybean seed vitamin E activity.
r 2005 Elsevier Inc. All rights reserved.
Keywords: Vitamin E; Tocochromanol; Tocopherol; Tocotrienol; Oilseed crop; Nutrition
1. Introduction
Tocochromanols are lipophilic antioxidants that aresynthesized in the plastids of plants, and by some greenphotosynthetic bacteria. They are composed of a groupof structurally related compounds with eight majorforms that can be distinguished by the number and
e front matter r 2005 Elsevier Inc. All rights reserved.
ben.2005.05.005
ing author.
ess: [email protected] (H.E. Valentin).
rs contributed equally to the manuscript.
ress: Chemir Analytical Services, 2672 Metro Blvd.
hts, MO 63043, USA
ress: BiogenIDEC, Pharmacokinetics and Drug Meta-
bridge Center, Cambridge, MA 02142, USA
ress: Exelixis Plant Sciences, 16160 SW Upper Boones
rtland, OR 97224, USA
ress: Monsanto Company, Calgene Campus, 1920
vis, CA 95616, USA
position of methyl groups on the aromatic head group(a-, b-, g-, d-tocopherol, and a-, b-, g-, d-tocotrienol) andthe presence of a saturated (tocopherols) or unsaturated(tocotrienols) isoprenoid side chain. Their biopotencyfor animals and humans is expressed as vitamin Eactivity (Nomenclature rules for vitamin E (972.31),1990; IUPAC-IUP Joint Commission on BiochemicalNomenclature, 1982) with a-tocopherol having thehighest vitamin E activity (Traber and Sies, 1996;Sheppard et al., 1993; Bramley et al., 2000). The vitaminE activity of 1mg chemically synthesized all racemic a-tocopherol is defined as 1 IU. Natural isomeric pureRRR-a-tocopherol is 1.5-fold more active than syntheticvitamin E (Chow, 2001). The National Institute ofHealth (NIH) currently suggests a recommended dailyallowance (RDA) of 22–28 IU for humans (http://www.cc.nih.gov/ccc/supplements/vite.html#rda).
ARTICLE IN PRESS
Fig. 1. Schematic drawing of the tocochromanol biosynthetic pathway. Abbreviations: DMAPP, dimithylallyldiphosphate; GGH, geranylger-
anyldiphosphate hydratase; HPPD, p-hydroxyphenylpyruvate dioxygenase; IPP, isopentenyldiphosphate; MEP, 2-C-methyl-D-erythritol 4-
phosphate; TYRA, bifunctional chorismate mutase-prephenate dehydrogenase; VTE1, tocopherol cyclase; VTE2, homogentisate phytyltransferase;
VTE3, 2-methyl-6-phytylbenzoquinol methyltransferase; VTE4, g-tocopherol methyltransferase.
B. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400 385
Higher vitamin E doses of 100–1000 IU have beenassociated with cancer reduction, improved immuneresponse, and cardiovascular benefits (Buring andHennekens, 1997; Tangney, 1997; Bramley et al.,2000). Such high doses are currently realized onlythrough supplemental tocopherol intake. Tocopherolsare also used in the pharmaceutical and cosmeticsindustry (Edwards, 2001), and as animal feed additivesto improve the quality and shelf life of meat (Sanderset al., 1997).
The combined annual consumption of tocopherols forhuman and animal applications was estimated at about40,000 t in 2002, with only 10% coming from naturalsources. Natural vitamin E originates almost exclusivelyfrom soybean oil, and is used predominantly for humanapplications due to its premium price and limitedavailability.
The first committed reaction in tocochromanolbiosynthesis is the prenylation of homogentisic acid(HGA) catalyzed by the homogentisate phytyltransfer-ase (VTE2) (Fig. 1) (Schledz et al., 2001; Collakova andDellaPenna, 2001; Savidge et al., 2002), or the homo-gentisate geranylgeranyl transferase (HGGT) (Cahoonet al., 2003). It is generally accepted that prenylation ofHGA with phytyldiphosphate (PDP) results in theformation of a�, b�, g�, and d–tocopherol, whileHGA-prenylation with geranylgeranyldiphosphate(GGDP) results in the formation of the correspondingtocotrienols (Fig. 1). While most of the biochemicalreactions leading to tocochromanol formation have
been known for more than two decades (Soll andSchultz, 1980; Soll et al., 1980, 1983, 1985), the genesinvolved in tocochromanol biosynthesis have beendiscovered only very recently (Rohmer, 2003; Shintaniand DellaPenna, 1998; Collakova and DellaPenna,2001; Savidge et al., 2002; Porfirova et al., 2002; Chenget al., 2003; Van Eenennaam et al., 2003), and analysisof tocochromanol pathway regulation and identificationof rate limiting reactions of the pathway has just beeninitiated (Savidge et al., 2002; Collakova and DellaPen-na, 2001, 2003a,b).
This manuscript provides experimental evidenceindicating limiting tocochromanol intermediates andrate-limiting biochemical reactions for tocochromanolbiosynthesis and it presents strategies and results onhow to apply this information to engineer commercialoilseed to produce oils with substantially enhancedtocochromanol and vitamin E content.
2. Materials and methods
2.1. Plant growth conditions, and transformation
procedures
Arabidopsis thaliana var. Columbia was grown under16 h photoperiod at 130–180 mE PPFD, 20 1C, and 70%relative humidity in Conviron MTPC432 chambers(Conviron, Winnipeg, Canada). The plants were grownin Metro Mix 200 soil (Hummert International, Earth
ARTICLE IN PRESSB. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400386
City, MO) in 2.500 pots at a density of 32 plants/flat,three flats to a group of 96. The three flats of a groupwere grown side by side at all times. For Agrobacterium
mediated germline transformation, Arabidopsis plantswere grown in a 20 1C chamber under 130–180 mE PPFDwith a 16 h photoperiod. Transgenic plants were selectedby plating on agar plates with kanamycin selection.Transformed hemizygous plants were grown to maturityand the R2 segregating seed were harvested fortocochromanol analysis.
Agrobacterium mediated germline transformation ofsoybean (Glycine max) cultivar A3244 was carried out aspreviously described (Martinell et al., 2002) using freshlygerminated soybean meristems that were induced toform shoots directly. Transgenic plants were grown in agreenhouse with a 10 h photoperiod, a daytime tem-perature of 29 1C and a nighttime temperature of 24 1C.Plants were grown to maturity and seeds were harvestedfor analysis.
Canola, cultivar Ebony, was transformed as describedby Radke et al. (1992), using glyphosate resistance asselectable marker.
2.2. Bacterial strains, growth conditions and cell sample
preparation
Wild type and recombinant cells of Synechocystis sp.PCC 6803 (ATCC 27184) were cultivated photoauto-trophically in BG-11 medium (Sigma, St Louis, MO)buffered with 10mM TES-NaOH (pH 8.0) at 30 1Cunder a light intensity of 1500 LUX and shaken at225 rpm on a rotary shaker unless otherwise indicated.For growth on solid medium, 1.5% (wt/vol) agar(Difco) was supplemented to the medium. Recombinantplasmids were transformed into Synechocystis viaconjugative method as described by Poster (1988) andstabilized by supplementation of the growth mediumwith 25 mg/ml of kanamycin, 10 mg/ml of gentamycin, or30 mg/ml of spectinomycin, depending on the plasmidused in the experiments. The transformed cells werespread on a 0.45 mM cellulose membrane filter (What-mans) and placed on non-selective solid medium,incubated for 24 h as described above, and transferredto selective medium plates containing the appropriateantibiotics. Positive colonies were inoculated and grownin 2ml of BG-11 liquid medium supplemented with theappropriate antibiotic for 2 days and then transferred to50ml liquid culture. These cell cultures served as pre-cultures for the final 300ml liquid cultures. Cell densitywas monitored spectrophotometrically (Spectra MAX,Molecular Devices, CA) at a wavelength of 730 nm.When the A730 value of the cell culture reached 0.6–0.8,cells were sub cultured in 300ml of fresh BG-11 mediumsupplemented with the appropriate antibiotics, and A730
was adjusted to 0.2. Cell samples were harvested atdifferent time points for measurements of tocopherols,
tocotrienols, intermediates, gene expression and enzymeactivities. Gene expression of the bifunctional Erwinia
herbicola prephenate dehydrogenase (Eh-TYRA), ma-ture Arabidopsis thaliana homogentisate dioxygenase(At-HPPD), and mature Arabidopsis thaliana GGDPdehydrogenase (At-GGH) was confirmed by immunoblot analysis (Harlow and Lane, 1988).
2.3. Synechocystis expression vectors and gene
expression
Gene expression in Synechocystis was driven by theLac promoter (Dickson et al., 1975), which expressedconstitutively in this organism. The coding regions oftocochromanol related genes from plant sources wereexpressed as mature genes. For the expression ofmultiple genes, trans-genes were arranged as operonstructures downstream of the Lac promoter. Geneexpression for Eh-TYRA, At-HPPD, and At-GGH wasconfirmed by immuno blot analysis using peptidedirected antibodies specific for each gene. The growthcharacteristics of all transgenic Synechocystis culturesused for these experiments remained unchanged com-pared to Synechocystis WT-cultures.
The base vector for expression of pathway genes waspSL1211 (Ng et al., 2000). Trans-genes tested in thepresent study were Eh-TYRA, At-HPPD, a mature At-
GGH, and Synechocystis GGH (Syn-GGH), a matureArabidopsis thaliana VTE2 (At-VTE2), and Synechocys-
tis sp. PCC6803 VTE2 (Syn-VTE2). The pSL1211-derived expression vectors harboring single or multiplegene(s) were constructed as operon structures usingstandard molecular cloning techniques and designatedas pMON36531, pMON36550, pMON36538,pMON36567, and pMON36549 (Table 1). The structur-al genes of Eh-TYRA, mature At-GGH, Syn-VTE2, andmature At-VTE2 were designed to be preceded by thenucleotide sequence AGGAAACAGCC ATG, withAGGA serving as Shine-Dalgarno sequence. At-HPPD
was preceded by AGGAGGACAGCC ATG, withAGGAGG as Shine-Dalgarno sequence. The trans-geneorganization of tocopherol pathway genes in theseexpression vectors was as depicted in Table 1. Restric-tion digestions and DNA sequencing were performed toverify correct orientations and sequences of geneticelements and gene(s) in these plasmids.
2.4. Tocochromanol analysis
Tocochromanol analysis was performed as describedby Van Eenennaam et al. (2003). The tocochromanolcomposition is provided on a wt/wt base. Statistical dataanalysis was performed using JMP statistical discoverysoftware (www.jmp.com).
ARTICLE IN PRESS
Table 1
Synechocystis expression vectors used in this study
Plasmid Relevant genetic elements Reference
pSL1211 Kanr Ng et al. (2000)
pMON36550 Specr, PLac-At-HPPD This study
pMON36531 Specr, PLac-Eh-TYRA This study
pMON36538 Specr, PLac-Eh-TYRA-At-HPPD This study
pMON36567 Specr, PLac-Eh-TYRA-At-HPPD-At-GGHm This study
pMON36549 Kanr, PLac-Eh-TYRA-At-HPPD-Syn-GGH-Syn-VTE2 This study
Synechocystis multi-gene vectors were assembled as operon structures.
Abbreviations: At-GGHm, mature A. thaliana geranylgeranyldiphosphate hydratase (accession # NM_106107); At-HPPD, A. thaliana p-
hydroxyphenylpyruvate dioxygenase (accession # AF060481); Kanr, kanamycin resistance gene; PLac, E. coli Lac promoter (Dickson et al., 1975);
Specr, spectinomycin resistance gene; Syn-GGH, Synechocystis sp. PCC 6803 GGH (accession # NP_441659); Syn-VTE2, Synechocystis sp. PCC 6803
homogentisate phytyltransferase (accession # NP_441094).
B. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400 387
2.5. Enzyme assays
To confirm gene-functionality of At-VTE2, Syn-
VTE2, At-HPPD, Eh-TYRA, At-GGH, and Syn-GGH
prior to transformation of these genes into a plantsystem, enzyme assays were performed on E. coli crudeextracts harboring expression constructs for each ofthese enzymes, and assays were performed according tothe procedures described in Savidge et al. (2002), Secor(1994), Cotton and Gibson (1970), and Keller et al.(1998); respectively. Using this approach functionalitywas confirmed for of At-VTE2, Syn-VTE2, At-HPPD,and Eh-TYRA. Enzyme activity of E. coli expressed At-
GGH and Syn-GGH was measured using radiolabeledGGDP (Amersham Biosciences UK Ltd, Buckingham-shire, UK), or geranylgeranyl bacteriochlorophyll iso-lated from Rhodospirillum rubrum (Katz et al., 1972).However, neither Syn-GGH, nor At-GGH-functionalitycould be confirmed using the assay procedures asdescribed by Keller et al. (1998).
2.6. PCR screen of F1 soybean crosses for presence of
trans-genes
F1 plants obtained by crossing homozygous soybeanlines transformed with pMON67227 (At-VTE4 and At-
VTE3) and pMON69943 (Eh-TYRA, At-HPPD, andSyn-VTE2) were screened by genomic PCR analysis.Leaf tissue (30–50mg) harvested from the F1 plants wasground in liquid nitrogen and genomic DNA wasisolated using the Qiagens genomic DNA isolationKit (Qiagen Inc., Valencia, CA). Eh-TYRA and At-
VTE4 served as representative genes for the trans-genesets in pMON69943, and pMON67227, respectively.The presence of these two genes was confirmed by PCRamplification using approximately 30 ng genomic DNAand the Invitrogen PCR SuperMix system (InvitrogenCorporation, Carlsbad, CA). Primer pairs Eh-TYRA-primers (forward: 50- GATCTAGAACAATGGCTTC-CTCTAT-30 and reverse: 50- CTTATTATGGGCG-
GCTGTCATTGG-30) and At-VTE4-primers (forward:50- AGAACCGGGTACCGAGCTCGAGAT -30 andreverse: 50- TATTAGAGTGGCTTCTGGCAA -30)were used for the screen. The PCR program consistedof 5min incubation at 94 1C, followed by 25 cycles of94 1C for 15 s, 55 1C for 15 s, and 72 1C for 2.15min. Thefinal PCR-product was incubated for 10min at 72 1C.The PCR products were separated on 0.8% agarose gelsto identify double positive lines.
2.7. Northern blot analysis
Gene expression was usually confirmed on selectedevents by western blot analysis. However, antibodies forSyn-VTE2 and At-VTE2 were not sufficiently specific toconfirm expression of these genes in plant tissues.Expression of these genes was therefore confirmed onselected Arabidopsis and canola events by NorthernBlot analysis.
RNA isolation was performed on 100mg fullymatured but not dried, Arabidopsis silique tissue and50mg immature canola seed collected 40 days afterpollination. Samples were ground with 0.25 g polyvi-nylpolypyrrolidone in liquid nitrogen. While theground tissues were frozen, 10ml REC.8+ buffercontaining 50mM Tris-HCl (pH 9.0), 800mM NaCl,10mM EDTA, 0.5% CTAB and 0.5% b-mercaptoetha-nol was added to each sample, mixed and centrifugedfor 5min at 10,000 rpm in a Sorvall centrifuge, modelSuper T21. The supernatant was filtered through mira-cloth (Calbiochem, http://www.emdbiosciences.com/html/CBC/home.html). After extraction with 3mlchloroform, the supernatant was separated and ex-tracted twice with equal volume of phenol:chloroformmix (1:1) and then ethanol precipitated. The ethanolpellet was re-suspended in 50mM EDTA. Equalamounts of total RNA (20 mg) were fractionatedon 1.2% agarose gels containing 1M formaldehyde.Gels were blotted onto nylon membrane (Ambion,Inc., Austin, TX) according to the manufacturer’s
ARTICLE IN PRESSB. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400388
instructions and hybridized to a random primer-labeledDNA probe in Sigma PerfectHybTM buffer (Sigma,St. Louis, MO, catalog # H-7033), containing 10mg/mlof salmon sperm DNA at 65 1C. A cDNA fragment(PvuI/PvuI) of At-VTE2 was labeled and used as agene-specific probe. Probe labeling with 32P-dCTP(50 mCi/reaction) was done using Amersham Redipri-meTM labeling kit (Amersham Biosciences Corp., Piscat-away, NJ, catalog # RPN1633). Blots were washed in2X SSC, 0.1% SDS at 65 1C and exposed to X-ray film(BioMax MS film, Sigma, St. Louis, MO, catalog# Z36,307-3).
2.8. Immuno blot analysis
To extract the total protein from seed, single soybeanseed or 20–22mg of Arabidopsis or a comparableamount of canola seed were mixed with 0.5mmzirconia/silica micro beads (Biospec Products, Bartles-ville, OK) and 500 ml protein extraction buffer [50mMTris (pH 7.4), 1mM EDTA, 0.1% Triton X-100,Complete protease inhibitor (Roche Molecular Bio-chemicals, Indianapolis, IN; http://www.biochem.ro-che.com) (1 tablet/50ml buffer)] and shaken in a beadbeater for 45 s twice and subsequently cooled on ice. Theextracted sample was spun in an Eppendorf table topcentrifuge at 14,000 rpm for 10min. The supernatantwas removed. Protein measurements were performedaccording to Bradford (1976), using bovine serumalbumin as standard, and spectrophotometric measure-ments were done in a computer-directed microtiter platereader (Spectra Max Plus 384, Molecular Devices Corp.,Sunnyvale, CA). Sample buffer containing 100mM Tris-HCl, pH 6.8, 4% SDS, 0.1% bromophenol blue, 20%glycerol, and 10% b-mercaptoethanol, was mixed with80mg total protein from each sample, heated for 2minat 100 1C, and centrifuged at 12,000 rpm for 5min at4 1C (Sorvall centrifuge, model Super T21). Totalproteins from each sample were separated in 10% SDSPAGE gel and blotted onto PVDF membrane (Bio-RadLaboratories, Hercules, CA) at 300mA for 2 h using aBio-Rad Transblot SD wet electroblotting apparatus(Bio-Rad Laboratories, Hercules, CA). Eh-TYRA, At-HPPD, and At-GGH specific antibodies were obtainedfrom Sigma-Genosys (http://www.sigma-genosys.co.uk/)by injecting rabbits with synthetic peptidesCSMLASRRKEAEALG (Eh-TYRA), CMMKDEEG-KAYQSGG (At-HPPD), CLPPEIIDRRVRKMK(Syn-GGH), and CDAYLRERAEKSGAT (At-GGH).Blots were treated with rabbit Eh-TYRA-, At-HPPD-,or GGH-specific antiserum (1:1000), and immunecomplexes were detected using alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies (Sig-ma-Alrich, www.sigma-aldrich.com) and nitroblue tet-razolium plus 5-bromo-4-chloro-3-indolyl phosphate(Harlow and Lane, 1988).
2.9. Extraction of intermediates for liquid
chromatography/mass spectrometry (LC/MS) analysis
Extraction from Arabidopsis thaliana seeds was accom-plished by weighing 1274mg of seeds into a 2mlmicrotube. Approximately 1 g of 0.5mm zirconia/silicamicrobeads (Biospec Products, Bartlesville, OK) and500ml of the appropriate extraction solvent was added tothe tube and shaken in a Fast Prep (FP120; SavantInstruments Inc., Holbrook, NY) for two sessions of 45 seach. The extract was allowed to settle and the super-natant was filtered, using the appropriate membrane, intoan LC vial for analysis. For tyrosine, HPPA and HGAanalysis, the extraction solvent was MeOH:water (50:50)with 0.1% formic acid, and the filter material used wasNylon. In the case of 2M6PBQ and 2M6GGBQ, theextraction solvent was pure ethanol and the filter wasmade of polytetrafluoroethylene (PTFE).
For soybean and canola, the seeds were ground priorto extraction in order to obtain homogenous flour usinga machine built in-house. For grinding bulk canola, 1 gseed were placed in a 30ml polypropylene tube with a34in steel ball. The tubes were shaken for 30 s at an
amplitude of 3.81 cm at 1000 strokes per minute. Forgrinding bulk soybean, 5–10 seed were treated similarlywith the shaking frequency set at 1200 strokes perminute. For grinding single soybean, one seed was placedin a 14ml polypropylene tube with a 3
8 in steel ball. Thetubes were shaken for 90 s at an amplitude of 3.81 cm at1000 strokes per minute. After grinding, approximately30710mg soybean or canola meal was extracted andfiltered as described above for Arabidopsis.
2.10. Determination of tocopherol intermediates using
liquid chromatography/mass spectrometry
The liquid chromatography/mass spectrometry (LC/MS) system consisted of an HP1100 series HPLC(Agilent Technologies, Palo Alto, CA) connected to anAPI QSTAR Pulsar-i (Applied Biosystems, Foster City,CA) mass spectrometer using their TurboIonspray (i.e.electrospray) interface. The system was operated usingthe Analyst QS software (Applied Biosystems, FosterCity, CA). All methods described here utilized anAlltima C18 column (4.6� 50mm, 3 mm; Alltech As-sociates, Inc., Deerfield, IL) operated at a flow rate of1ml/min connected to the TurboIonspray source with asplit of approximately 1:5 (MS:waste). The massspectrometer was operated in negative ion mode for allmethods. The injection volumes for each method wereset to 20 ml for all standards and samples. Analytes werequantified against external standard curves generatedwith the sample sets. Standards for HGA, p-hydro-xyphenylpyruvate (HPPA), and tyrosine were purchasedfrom Sigma Chemicals (Sigma St. Louis, MO). Stan-dards for 2M6PBQ and 2M6GGBQ were synthesized as
ARTICLE IN PRESSB. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400 389
described previously (Soll and Schultz, 1980; Henry etal., 1987). The LC and MS parameters for each methodwere as follows.
Tyrosine and HPPA were analyzed by LC/MS usinggradient conditions. The HPLC mobile phases werewater (A), acetonitrile (B) and 10% formic acid (C). Thegradient method began with 0.1% C (throughout run)and 4% B isocratic for 0.2min, increasing to 95% B at0.6min, which was held until 0.8min, then returned toinitial conditions (4% B) at 1.0min and equilibrateduntil 3.0min. The mass spectrometer used ‘‘TOF MS’’mode to scan from 160 to 190 amu. The quantitationwas based on extracted ion chromatograms centeredaround the appropriate quasi-molecular ion of eachanalyte (tyrosine �180.15; HPPA �179.10). The reten-tion times were 1.22 and 1.14min for tyrosine andHPPA, respectively. This method could also monitor forHGA, but interferences from the seed matrix would notallow for reliable quantitation.
To obtain specificity for HGA, an MS/MS methodwas used. The mobile phases for this LC/MS/MSmethod were water (A), methanol (B) and 0.5% formicacid (C). The 5min method used 1% C throughout thegradient and started at 5% B. The gradient increased to98% B over 1.80min, was held at 98% B until 2.0min,and then returned to 5% B at 2.2min, where itconditioned the column for the remaining time. HGAeluted around 2.2min. The mass spectrometer used‘‘Product Ion’’ mode, where 167.1 amu was selected asthe parent ion. The mass spectrometer was set to useLOW resolution and a collision energy (CE) of �15 kV.The TOF mass range scanned from 123.05 to123.15 amu to detect the daughter ion mass. Thesesettings allowed for the specificity needed to quantitateHGA in matrix. The HGA detection limit under theseconditions was 0.25 ppm.
For LC/MS analysis of the 2M6PBQ and 2M6GGBQ,the HPLC separation was performed isocratically at96% B for 4.5min, where mobile phase (A) was waterand (B) was 0.1% acetic acid in methanol. The retentiontimes for 2M6GGBQ and 2M6PBQ were approximately1.5 and 1.9min, respectively. The mass spectrometer wasset to ‘‘TOFMS’’ mode and used two scan functions (i.e.‘‘Experiments’’): Experiment 1 scanned from 401.25 to401.45 amu to monitor for 2M6PBQ, and Experiment 2scanned from 395.20 to 395.45 amu to monitor for2M6GGBQ. Detection limits for 2M6PBQ and2M6GGBQ were 0.2 and 2 ppm, respectively.
3. Results
3.1. Feeding experiments/identification of rate limiting
tocopherol intermediates
To identify tocochromanol pathway intermediatesand biochemical reactions that limit tocochromanol
biosynthesis, cotyledon-derived soybean suspensioncultures were subjected to feeding experiments usingthe tocopherol precursors chorismic acid, tyrosine,HPPA, HGA, phytol, geranylgeraniol, and 1-deoxy-D-xylulose. All metabolites were supplied in concentra-tions ranging from 0.25 to 5mM. The strongest effectswere obtained with phytol and HGA when provided atthe 2mM level resulting each in a two-fold increase intotal tocopherol levels. Geranylgeraniol was not aseffective as phytol, and increased total tocopherols byup to 1.5-fold only. When phytol and HGA wereprovided in combination, each at 2mM, total tocochro-manol levels increased by five-fold, suggesting that theavailability of these two intermediates is critical fortocochromanol biosynthesis.
Data obtained in feeding experiments are often hardto interpret, as the rates for metabolite uptake are oftennot available. In addition some intermediates such astyrosine and chorismate exhibited inhibitory effects atthe concentration range tested above, while otherintermediates such as HPPA are unstable in aqueoussolutions. Therefore, subsequent experiments to confirmthe importance of HGA and PDP availability fortocochromanol biosynthesis were performed in trans-genic models.
3.2. Synechocystis sp. PCC 6803 as transgenic model for
tocochromanol pathway engineering
Potential key genes of the tocochromanol biosyntheticpathway, identified based on previous feeding experi-ments and literature data, were cloned from bacterialand plant sources, and expressed as single genes and incombination in the Synechocystis model. Eh-TYRA, andAt-HPPD were chosen as key genes to increase HGA,and At-GGH was chosen to increase PDP availability.Expression of these genes was combined with expressionof Synechocystis VTE2 (Syn-VTE2), which had beenidentified as rate limiting for tocochromanol biosynth-esis previously (Collakova and DellaPenna 2001,2003a,b; Savidge et al., 2002).
When expressing single genes in Synechocystis, At-
HPPD had the strongest impact on tocochromanollevels, resulting in up to seven-fold WT-tocochromanollevels (Fig. 2A). Combined expression of the key genesidentified above increased tocochromanol levels with theaddition of each new gene, and tocochromanol levels intransgenic cultures increased over the entire cultivationperiod of 24 days (Fig. 2A and B). Expression of all fourgenes (Eh-TYRA, At-HPPD, At-VTE2, and At-GGH)resulted in up to 16.5-fold WT tocopherol levels. Thesecultures contained up to 2200 ng tocopherol per mgdry cell mass (Fig. 2B). A small fraction (5–10%)of these tocopherols was accumulated in the form ofg-tocopherol, while the remaining fraction consisted
ARTICLE IN PRESS
Fig. 2. Tocopherol and tocotrienol formation in transgenic Synecho-
cystis cultures: (A) tocochromanol timecourse over a period of 24 days;
(B) tocopherol and tocotrienol content of lyophilized Synechocystis
cultures after a cultivation period of 24 days; (C) Synechocystis culture
supernatants after a cultivation period of 24 days.
B. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400390
of a-tocopherol. g-Tocopherol is not detected in WTSynechocystis cells.
Transgenic expression of At-HPPD, or At-HPPD andEh-TYRA, coincided with the occurrence of a brownculture supernatant, suggesting the presence of melaninlike pigment, a reaction product of HGA (Fig. 2C)Coon et al., 1994). Tocopherols and tocotrienols do notexhibit substantial absorption properties in the visiblelight spectrum and are not water-soluble. They aretherefore likely not directly associated with the observedcolor changes. Interestingly, these cultures contained10–20% of their tocochromanols in the form oftocotrienols, while tocotrienols in WT-cultures werebelow 2% of total tocochromanols (Fig. 2B). When At-
GGH was co-expressed with At-HPPD and Eh-TYRA,the culture supernatant was not brown, tocotrienolswere not detected, and total tocochromanol accumula-tion increased compared to the double gene construct(Fig. 2B and C).
3.3. Engineering oil seed tocochromanol content
Genes identified to be critical for tocochromanolpathway engineering in Synechocystis were expressed assingle genes and in combination in Arabidopsis, canolaand soybean. In Arabidopsis, each gene was placed intwo constructs using either the constitutive enhancedcauliflower mosaic virus promoter (Pe35S, McPherson
and Kay, 1994) or the seed-specific napin promoter(PNapin, Kridl et al., 1991). Single gene expression of At-
GGH did not result in significant changes of seedtocochromanol levels (Table 2). In contrast, seed-specific expression of At-HPPD, Eh-TYRA, At-VTE2,or Syn-VTE2 as single genes resulted in significant seedtocochromanol increases of 1.09-fold, 1.20-fold, 1.41-fold, and 1.36-fold in R2 seed populations, respectively.In our hands, expression of the same genes under thecontrol of a constitutive promoter did not significantlyincrease seed tocochromanol levels in the population ofall events (Table 2). For this reason, all subsequentexpression cassettes for the enhancement of seedtocochromanol production were designed using seed-specific promoters (Tables 3, 4, and 5).
When single genes were expressed in crops, only seed-specific expression of Eh-TYRA in canola resulted in asignificant increase of the average seed tocochromanolcontent of all events, with up to two-fold WT-tocochromanol levels for the best performing event(Table 4). However, when combinations of two or moretocochromanol genes were expressed, seed tocochroma-nol levels increased significantly in Arabidopsis, andcanola for all gene combinations (Tables 3 and 4). Thebest performing events harboring expression constructsfor Eh-TYRA, At-HPPD, and At-VTE2, or Syn-VTE2
in Arabidopsis and canola contained 5- and 3.7-foldWT-tocochromanol levels, respectively (Tables 3 and 4).In soybean seed populations tocochromanol increasedsignificantly when Eh-TYRA, At-HPPD, and Syn-VTE2
expression was combined (Table 4). The best performingconstruct harbored At-GGH in addition to Eh-TYRA,AT-HPPD, and At-VTE2. Four events obtained withthis construct (pMON77637) had on average more than10-fold WT tocochromanol levels, with up to 15-foldWT-levels for seed from the best performing event(Table 5). Interestingly, the increase in total tocochro-manols was obtained exclusively through tocotrienolaccumulation, with an average of 94% tocotrienols inseed from this event. In these seed the pool of a, b, g, andd-tocopherol was slightly reduced compared to WT-levels (Table 5). Overall d-tocotrienol was the dominat-ing tocochromanol species in these seed. Among thetocopherol pool, d-tocopherol was the dominatingtocochromanol species (Table 5). Both, Arabidopsisand soybean seed harboring At-HPPD and Eh-TYRA
expression constructs combined exhibited a dark brownseed phenotypre (Fig. 3).
Single soybean seed from transformation events withpMON66682 had total tocochromanol levels up to 4.30-fold higher than WT-seed (Table 4). Up to 82% of totaltocochromanols in these seed were accumulated astocotrienols. The transgenic expression of At-GGH wasexpected to reduce tocotrienol formation in these seed.Western analysis on pMON66682 seed extracts con-firmed transgenic expression of the At-GGH for four out
ARTICLE IN PRESS
Table 2
Impact of seed-specific and constitutive expression of single genes on tocochromanol levels in Arabidopsis R2 seed populations.
Vector designation Relevant genetic elements Reference Mean tocochromanol
level [ng/mg seed]
P-Value Average fold
tocochromanol
increase
pMON36525 PNapin-CTP2-At-HPPD-napin 30 Valentin and Mitsky (2002) 599748.9 0.0448 1.0970.10
control 552735.9
pMON26586 Pe35S-CTP2-At-HPPD-E9 30 This study 556735.4 0.3959 1.0370.07
control 541724.7
pMON36520 PNapin-CTP1-Eh-TYRA-napin 30 Valentin and Mitsky (2002) 524778.5 0.0084 1.2070.19
control 437728.6
pMON36511 Pe35S-CTP1-Eh-TYRA-E9 30 Valentin and Mitsky (2002) 406792.7 0.4112 0.9370.21
control 437728.6
pMON43852 PNapin-At-GGH-napin 30 This study 535748.6 0.4168 1.0370.09
control 519715.4
pMON26583 Pe35S- At-GGH-E9 30 This study 514742.3 0.6330 0.9870.08
control 523733.3
pCGN10822 PNapin-At-VTE2-napin 30 Savidge et al. (2002) 809767.0 0.0001 1.4170.12
control 574738.2
pCGN10800 Pe35S- At-VTE2-TML 30 Lassner et al. (2003) 493753.2 0.3469 0.9570.10
control 52078.36
pMON16602 PNapin-Syn-VTE2-napin 30 Valentin and Mitsky (2002) 679779.0 0.0002 1.3670.16
control 500739.9
pMON21698 Pe35S-Syn-VTE2-E9 30 This study 552718.4 0.5522 1.0270.03
control 544735.4
The original Arabidopsis seed tocochromanol data set that is summarized in Tables 2 and 3 is provided in Supplemental Table 1. To establish the
significance of changes in seed tocochromanol levels upon expression of various trans-genes in Arabidopsis, seed tocochromanol data from
transgenic populations were compared with seed tocochromanol data from populations of WT- seed or seed transformed with a vector control which
had been grown together with the transgenic seed. Means, standard error, and significance were calculated using Tukey–Kramer HSD. P-values
o0.05 are significant.
Abbreviations: At-GGH, A. thaliana geranylgeranyldiphosphate hydratase (accession # NM_106107); At-HPPD, A. thaliana p-hydroxyphenylpyr-
uvate dioxygenase (accession # AF060481); At-VTE2, A. thaliana homogentisate phytyltransferase (accession # AAL35412); CTP1, modified
chloroplast target peptide from the small subunit of the Arabidopsis ribulose bisphosphate carboxylase (Barry and Kishore, 1998); CTP2, chloroplast
target peptide from the Arabidopsis 5-enolpyruvylshikimate-3-phosphate synthase (Klee et al., 1987); E9 30, 408 nt of Pea rubisco 30 sequence
(Coruzzi et al., 1984); Eh-TYRA, Erwinia herbicola bifunctional chorismate mutase, prephenate dehydrogenase (accession # X60420); napin 30, napin
30 untranslated region (Kridl et al., 1991, accession # M64632); Pe35S, enhanced cauliflower mosaic virus promoter (Mc Pherson and Kay, 1994);
PNapin, Napin seed-specific promoter (Kridl et al., 1991, accession # M64632); Syn-VTE2, Synechocystis sp. PCC 6803 homogentisate
phytyltransferase (accession # NP_441094); TML 30, Agrobacterium tumefaciens (accession # AF242881, nucleotides 10303 to 9203).
B. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400 391
of seven tested events. The GGH western signal obtainedfrom transgenic soybean seed comigrated with an E. coli
expressed mature At-GGH protein (Fig. 4), suggestingthat the soybean-expressed protein was properly pro-cessed, and therefore, presumably properly targeted tothe plastid. Nevertheless, these events did not show atocotrienol reduction in favor of increased tocopherolswhen compared to transformation events from the samevector, not expressing GGH (Fig. 4). The phenotypicalchanges in seed color, shape, and seed germinationobserved previously on seed harboring the combined At-
HPPD and Eh-TYRA expression constructs were main-tained in seed from pMON77637 and pMON66682.
3.4. Accumulation of tocopherol precursors and
intermediates
LC-MS analysis of Arabidopsis seed for tocopherolprecursors and intermediates revealed that HGA incontrol seed was below the limit of detection
(o0.25 ppm), and HGA levels in seed expressing At-
HPPD under napin promoter control were approxi-mately 0.25 ppm. Expression of Eh-TYRA correspondedwith a two-fold increase in seed HPPA (Po0.02), andthree-fold increase in free tyrosine levels (Po0.0001).
Interestingly all seed harboring Eh-TYRA and At-
HPPD expression constructs combined, accumulatedtocotrienols, and all Arabidopsis and soybean seedharboring these two genes exhibited a brown to darkbrown seed phenotype, were unevenly shaped, andgerminated at lower rates than WT-seed. LC-MSanalysis of WT and brown seed revealed HGA increasesof 60 and 800-fold over the detection limit for matureArabidopsis and soybean seed, respectively (Fig. 3).
Transgenic high tocochromanol seed from Arabidop-sis, canola, and soybean exhibited a strong shift intocochromanol composition towards d-tocopherol andd-tocotrienol, suggesting a rate limitation by VTE3(Table 5, and above). In order to test this hypothesis, 2-methyl-6-phytylbenzoquinol (2M6PBQ) and 2-methyl-
ARTICLE IN PRESS
Table
3
Impact
oftrans-geneexpressiononArabidopsisseed
tocochromanollevels
Vector
designation
Relevantgenetic
elem
ents
Reference
Max.tocochromanol
increase
a[fold
wild-
typelevel]
Meantocochromanol
levelb[fold
wild-type
level]
Significance
c
pMON36525
PNapin-C
TP2-A
t-H
PP
D-napin
30
ValentinandMitsky
(2002)
1.26
1.0970.10
A
pMON36520
PNapin-C
TP1-E
h-T
YR
A-napin
30
ValentinandMitsky
(2002)
1.53
1.2070.19
AB
pCGN10822
PNapin-A
t-V
TE
2-napin
30
Savidgeet
al.(2002)
1.62
1.4170.11
BC
pMON36528
PNapin-C
TP2-A
t-H
PP
D-napin
30 -
PNapin-A
t-V
TE
2-napin
30
ValentinandMitsky
(2002)
1.75
1.4670.15
C
pMON36596
PNapin-C
TP2-A
t-H
PP
D-napin
30 -
PNapin-C
TP1-E
h-T
YR
A-napin
30
ValentinandMitsky
(2002)
2.24
1.7870.22
D
pMON69907
PNapin-C
TP1-E
h-T
YR
A-napin
30 -
PNapin-A
t-V
TE
2-napin
30
ValentinandMitsky
(2002)
2.41
1.7970.38
D
pMON69909
PNapin-C
TP2-A
t-H
PP
D-napin
30 -
PNapin-C
TP1-E
h-T
YR
A-napin
30 -
PNapin-A
t-V
TE
2-napin
30
ValentinandMitsky
(2002)
5.00
2.8870.66
E
Abbreviationsusedhereare
asdefined
under
Table
1.Abbreviations:
At-
VT
E2,
A.
tha
lia
nahomogentisate
phytyltransferase
(accession#AAL35412);CTP1,modified
chloroplast
target
peptide
from
thesm
allsubunitoftheArabidopsisribulose
bisphosphate
carboxylase
(BarryandKishore,1998);CTP2,chloroplasttarget
peptidefrom
theArabidopsis5-enolpyruvylshikim
ate-3-phosphate
synthase
(Kleeet
al.,1987);
Eh
-TY
RA,
Erw
inia
her
bic
ola
bifunctionalchorism
ate
mutase,prephenate
dehydrogenase
(accession#X60420);napin
30 ,napin
30untranslatedregion(K
ridlet
al.,1991,
accession#M64632);
PNapin,Napin
seed-specificpromoter(K
ridlet
al.,1991,accession#M64632).
aMaxim
um
seed
tocochromanolincrease
ofbestperform
ingeventanalyzedon10–20mgbulk
seed
oftheR2-generation
bAverageseed
tocochromanolincrease
ofalleventsoftheR2seed
population(approxim
ately
20–30events).Means,standard
error,andsignificance
werecalculatedusingTukey–Kramer
HSD.
cMeansfollowed
bythesamelettersare
notsignificantlydifferentfrom
each
other
(a¼
0.05).
B. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400392
ARTICLE IN PRESS
Table
4
R1seed
tocochromanollevelsin
transgenic
canola
andsoybeanseed
populations
Vectordesignation
Relevantgenotype
Reference
Tocochromanollevel
(ng/m
g)
Bestperform
ingeventa
Meanofallevents
Significance
b
Ca
no
la
WT-control
273
227722.4
A
pMON58175
PNapin-C
TP1-E
h-T
YR
A-napin
30
Thisstudy
540
370756.6
B
pMON58173
PNapin-C
TP1-S
yn
-VT
E2-napin
30
Thisstudy
299
247719.5
A
pMON58187
PNapin-C
TP2-A
t-H
PP
D-napin
30
Thisstudy
251
224723.4
A
pMON58178
PNapin-C
TP2-A
t-H
PP
D-napin
30 -
PNapin-
CTP1-E
h-T
YR
A-napin
30
ValentinandMitsky(2002)
763
5557177
D
pMON58186
PNapin-C
TP2-A
t-HPPD-napin
30 -
PNapin-
CTP1-E
h-T
YR
A-napin
30 -
PNapin-C
TP1-
Sy
n-V
TE
2-napin
30
ValentinandMitsky(2002)
829
4677197
C
So
yb
ean
WT-control
355
320733.4
A
pMON36575
P7Sa’-C
TP1-E
h-T
YR
A-E930
ValentinandMitsky(2002)
435
355763.2
A
pMON36576
P7Sa’-C
TP2-A
t-H
PP
D-E930
ValentinandMitsky(2002)
482
336756.7
A
pMON69933
PArc-5-C
TP1-S
yn
-VT
E2-A
rc30
ValentinandMitsky(2002)
357
321721.7
A
pMON36581
PArc-5-A
t-V
TE
2-A
rc30
ValentinandMitsky(2002)
456
374748.3
A
pMON69924
P7Sa’-C
TP2-A
t-H
PP
D-E930 ,
P7Sa’-C
TP1-
Eh
-TY
RA-E930
ValentinandMitsky(2002)
1090
8257336
AB
pMON69943
P7Sa’-C
TP2-A
t-H
PP
D-E930 ,
P7Sa’-C
TP1-
Eh
-TY
RA-E930 ,
PArc-5-C
TP1-S
yn
-VT
E2
-
Arc
30
ValentinandMitsky(2002)
1318
10547228
B
pMON77637
P7sa’-CTP2-A
t-H
PP
D-E930 ,
P7sa’-CTP1-
Eh
-TY
RA-E930 ,
PArc-5-
At-
VT
E2-A
rc30 ,
P7Sa–
At-
GG
H-N
os30
Thisstudy
3765
96171154
B
pMON66682
POleosin-A
t-G
GH-O
leosin30 ,
PFAE-A
t-
VT
E2-A
rc30 ,
P7Sa’-C
TP2-A
t-H
PP
D-E930 ,
P7Sa’-C
TP1-E
h-T
YR
A-E930
Thisstudy
1398
5747399
AB
Fortocochromanolanalysis,10–20mgofcanola
seed
or5-pooledsoybeanseed
per
eventwereused.Allexpressioncassettesin
multigenevectors
wereorganized
intheheadto
tailorientationin
the
order
provided
above.
Abbreviations:
Arc
30 ,
Pha
seo
lus
vulg
ari
sarcelin-5
30untranslated
region
(Wanget
al.,2002,accession
#Z50202);
At-
GG
H,
A.
tha
lia
nageranylgeranyldiphosphate
hydratase
(accession
#
NM_106107);
At-
GG
Hm,mature
A.
tha
lia
nageranylgeranyldiphosphate
hydratase;
At-
HP
PD,
A.
tha
lia
na
p-hydroxyphenylpyruvate
dioxygenase
(accession#AF060481);
At-
VT
E2,
A.
tha
lia
na
homogentisate
phytyltransferase
(accession#AAL35412);CTP1,modified
chloroplast
target
peptidefrom
thesm
allsubunitoftheArabidopsisribulose
bisphosphate
carboxylase
(Barryand
Kishore,1998);CTP2,chloroplasttarget
peptidefrom
theArabidopsis5-enolpyruvylshikim
ate-3-phosphatesynthase
(Kleeet
al.,1987);E930 ,408ntofPea
rubisco
30sequence
(Coruzziet
al.,1984);
Eh-T
YR
A,
Erw
inia
her
bic
ola
bifunctionalchorism
ate
mutase,prephenate
dehydrogenase
(accession#X60420);Kanr ,kanamycinresistance
gene;
NOS,nopalinesynthase
30UTR
(accession#
AF465641)Oleosin30 ,Oleosin30untranslatedregion(accession#AL161562,nucleotides
175567–176040);napin
30 ,napin
30untranslatedregion(K
ridlet
al.,1991,accession#M64632);P7sa’,
Gly
cine
ma
xseed
storageprotein
promoter(C
hen
etal.,1986);P7sa,
Gly
cine
ma
xseed
storageprotein
promoter(W
angandDubois,2003);PArc-5,
Ph
ase
olu
svu
lga
risarcelin-5
promoter(W
anget
al.,
2002);
PFAE,Arabidopsisfattyacidelongase
promoter(accession#AF355982);
PNapin,Napin
seed-specificpromoter(K
ridlet
al.,1991,accession#M64632);
POleosin,
A.
tha
lia
naoleosinpromoter
(accession#X62353);
Sy
n-V
TE
2,
Sy
nec
ho
cyst
issp.PCC
6803homogentisate
phytyltransferase
(accession#NP_441094).
aBestperform
ingeventforcanola,andmeantocochromanolcontentofbestperform
ingeventforsoybean;Means,standard
error,andsignificance
werecalculatedusingTukey–Kramer
HSD.
bMeansfollowed
bythesamelettersare
notsignificantlydifferentfrom
each
other
(a¼
0.05).
B. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400 393
ARTICLE IN PRESS
Table
5
Seedtocochromanolcontentandcompositionofsingle
seed
ofselected
soybeanlines
transform
edwithpMON77637
Event
Averagetocopherolcontent(ng/m
g)
a–,g–,and
Averagetocotrienolcontent
a–,g–
and
Totaltocochromanolcontent
d-tocopherol
[ng/m
g]
d-tocotrienol
(ng/m
g)
a–g–
d–(ng/m
g)
a–g–
d–(ng/m
g)
Maxim
um
aMeanb
P-value
WT-control
33.073.03
22974.5
57.371.86
319717.8
5.8372.99
0.3370.82
0.6771.63
6.874.5
362
326720.6
–
GM_A40061:@
.22.676.50
124717.2
13971.18
285722.1
27.473.97
714742.2
1,1127265
18547293
2429
21397282
6.791�10�8
GM_A40056:@
.23.478.79
114719.9
17378.87
311725.6
35.479.96
845744.3
1,759798.9
26407118
3095
29517107
2.793�10111
GM_A40044:@
.22.073.39
110726.0
162712.6
294736.9
34.672.61
1,024798.3
2,0447387
31027473
3795
33967441
6.739�10�13
GM_A40049:@
.14.874.44
69.4721.8
165727.2
249750.1
12.4717.0
947798.9
2,4997232
34587196
3901
37077208
6.209�10�14
GM_A40059:@
.18.475.59
72.0713.2
165715.7
255729.5
45.8713.9
1,000792.2
2,4647145
35107163
3980
37657144
4.036�10�14
GM_A40048:@
.20.0713.3
52.6726.2
142729.4
214760.7
56.2726.0
7527197
2,7257856
35337930
4806
37487956
4.586�10�14
Fivedark
seed
per
eventwerechosenandanalyzedseparately
fortocochromanolcontentandcomposition.b-tocopherolandb-tocotrienolwerebelow
2%
oftotaltocochromanols.
aMaxim
um
tocochromanolcontentin
bestperform
ingsingle
soybeanseed.
bMeantocochromanolcontentoffivesingleseed.Means,standard
error,andsignificance
werecalculatedusingTukey–Kramer
HSD.
P-values
are
provided
formeantocochromanolcontentof
transgenic
seed
comparedto
WT-seed,andvalues
o0.05are
significant.Plasm
idpMON77637harbors
seed-specificexpressioncassettesfor
At-
HP
PD,
Eh-T
YR
A,
At-
VT
E2,and
At-
GG
H.For
elem
entdetailseeTable
2.
Fig. 3. Seed phenotype and HGA accumulation in transgenic
Arabidopsis and soybean seed. Panels A and B show segregating
Arabidopsis and soybean seed transformed with pMON69909, and
pMON69943, respectively. These plasmids contained seed-specific
expression constructs for Eh-TYRA, At-HPPD, and Syn-VTE2
(Tables 3 and 4). Panels C and D show LC-MS chromatograms for
HGA from Arabidopsis and soybean seed extracts harboring
pMON69909, and pMON69943, respectively.
B. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400394
6-geranylgeranylbenzoquinol (2M6GGBQ) levels wereanalyzed in selected seed samples from all three plantspecies. Detection limits for 2M6PBQ and 2M6GGBQwere 0.2 and 2 ppm, respectively. In Arabidopsis andcanola seed harboring At-HPPD, and Eh-TYRA or At-
HPPD, Eh-TYRA, and Syn-VTE2 expression con-structs, 2M6PBQ levels were indeed four and six-foldincreased above the detection limit. In WT-seed2M6PBQ was below the limit of detection. In Arabi-dopsis seed 2M6GGBQ were also increased from 10.2 to15.8 ppm. In WT and transgenic soybean seed 2M6PBQand 2M6GGBQ were not detectable.
3.5. Soybean optimized for vitamin E production
Previous studies in Arabidopsis and soybeanhave demonstrated that WT-seed tocopherols can beconverted to nearly all a-tocopherol by expression of
ARTICLE IN PRESS
Fig. 4. Western blot analysis of soybean seed transformed with
pMON66682. Vector pMON66682 harbors seed-specific expression
cassettes for At-HPPD, Eh-TYRA, At-VTE2, and At-GGH. Seed
extracts were immuno detected with antisera against At-HPPD, Eh-
TYRA, and At-GGH. At-VTE2-expression was not immuno blot
confirmed due to the lack of specific antibodies. Abbreviations: MW,
molecular mass; nd, below the limit of detection.
Fig. 5. Correlation between a-tocopherol and total tocochromanol
levels in F2 segregating soybean seed obtained through crossing
homozygous soybean lines transformed with pMON69943, and
pMON67227. Plasmids pMON69943 and pMON67227 harbored the
P7Sa‘-CTP2-At-HPPD-E9 30, P7Sa‘-CTP1-Eh-TYRA-E9 30, PArc�5-
CTP1-Syn-VTE2-Arc 30, and P7sa’-At-VTE4-E9 30, P7sa’-At-VTE3-E9
30 expression cassettes, respectively.
B. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400 395
one (Shintani and DellaPenna, 1998) or two methyl-transferases (Van Eenennaam et al., 2003). To test if thisconversion can still be accomplished with seed havingsubstantially increased total seed tocochromanol levels,homozygous soybean plants transformed withpMON69943, harboring seed specific Eh-TYRA, At-
HPPD, and Syn-VTE2 expression constructs werecrossed with homozygous soybean lines transformedwith pMON67227, harboring seed-specific expressionconstructs for At-VTE3 and the Arabidopsis g-methyl-transferase (At-VTE4) (Van Eenennaam et al., 2003).
Single seed from the phenotypically segregating F2-generation were analyzed for seed tocochromanol
content and composition. The tocochromanol data fromthese seed indicated the presence of four phenotypicallydistinct seed populations: a population of WT-seed, asecond population of seed containing nearly 100%a-tocopherol, a third seed population exhibiting theelevated total tocochromanol seed phenotype, and afourth seed population combining both transgenic seedtocochromanol phenotypes (Fig. 5). Four seed out of atotal population of 300 seed did not fit this pattern andtheir tocochromanol values were not included in furthercalculations. The seed population combining the in-creased a-tocopherol phenotype with the increased totaltocochromanol phenotype exhibited a significant aver-age tocochromanol increase by 1.2-fold compared to thehigh total tocochromanol population. In addition, theaverage a-tocopherol and a-tocotrienol content in theseseed increased to 83.8%.
Using vitamin E activities for the different tocochroma-nol isoforms provided by Chow (Chow, 2001), the totalvitamin E content in F2 seed combining the high totaltocochromanol phenotype with the high a-tocopherol andhigh a-tocotrienol phenotype, was calculated to be up to11-fold higher than in WT-soybean seed. The phenotypesin seed color and shape observed previously in seedharboring both, At-HPPD and Eh-TYRA expressionconstructs (see above) were maintained. However, thegermination vigor of F1 seed from this crossing experimentwas substantially improved, and was under green houseconditions comparable to non-transgenic soybean seed.
4. Discussion
The present paper describes a systematic approach toidentify limiting factors of tocochromanol biosynthesis,and a new strategy to use this knowledge for engineering
ARTICLE IN PRESSB. Karunanandaa et al. / Metabolic Engineering 7 (2005) 384–400396
increased seed tocochromanol and vitamin E levels inoilseed crops. Feeding experiments performed withsoybean suspension cultures indicated that the avail-ability of HGA, phytol, and presumably PDP limittocopherol biosynthesis. In our hands these twometabolites appeared to have comparable effects ontocochromanol biosynthesis. Similar experiments per-formed with safflower suspension cultures also identifiedthese two metabolites as key intermediates for increasedtocochromanol biosynthesis, but found phytol toprovide a stronger effect than HGA (Furuya et al.,1987). If this difference reflects variations in the twodifferent cell suspension cultures is not known at thistime.
We chose the unicellular cyanobacterium Synechocys-
tis sp. PCC 6803 as a model to verify and extend theseexperimental data to a transgenic system with a fastturnaround time. In Synechocystis, overexpression ofAt-HPPD as a single trans gene had by far the strongestimpact on total tocochromanol levels, resulting in up toseven-fold WT-tocochromanol levels. This was in starkcontrast to HPPD-expression in oilseed. In Arabidopsisseed tocochromanol increased only by 1.09 and 1.25-fold compared to WT-levels for the population of allevents, and for the best performing event, respectively.This correlates well with data published by Tsegaye etal. (2002) who found up to 1.28-fold increased seedtocochromanol levels in Arabidopsis expressing At-
HPPD under the control of the seed-specific DC3promoter. The differences on tocochromanol accumula-tion upon HPPD expression in Synechocystis versusplants suggest substantial differences in either theregulation of tocochromanol biosynthesis or the toco-chromanol biosynthetic pathways. One known differ-ence in biochemical pathways for HGA biosynthesis inbacteria and higher plants is the presence of abifunctional prephenate dehydrogenase in bacteria,which allows HGA biosynthesis from chorismate viaprephenate and HPPA (Fig. 1). In fact, the Synecho-
cystis genome includes an open reading frame (slr2081)with a deduced amino acid sequence that is 28%identical to the deduced Bacillus subtilis TYRA aminoacid sequence (Henner et al., 1984). In plants, HGA issynthesized from chorismate, via prephenate, arogenate,tyrosine, and HPPA (Fig. 1) (Siehl, 1999).
Expression of Eh-TYRA in Synechocystis cultures andseed-specific expression of Eh-TYRA in Arabidopsis,canola, and soybean resulted in a moderate tocochro-manol increase of 1.60-, 1.20-, 1.63-, and 1.11-fold,respectively. While the average tocochromanol changesin Arabidopsis and canola were significant, the toco-chromanol increase in Eh-TYRA expressing soybeanwas not (Tables 2 and 4). The best performing eventsfrom Eh-TYRA expressing Arabidopsis and canola hadtotal seed tocochromanol levels increased to 1.53-foldand 2.37-fold WT-levels, respectively. The reasons for
the differences in the impact on total tocochromanols inArabidopsis, canola and soybean are not known at thistime. Traces of tocotrienols (o5%) were detected insome Eh-TYRA expressing seed from canola andsoybean.
Combined seed-specific expression of At-HPPD andEh-TYRA in Arabidopsis, canola, and soybean estab-lished the direct pathway for HGA biosynthesis as it ispresumed to exist in Synechocystis when expressingHPPD. Interestingly, this gene combination increasedthe average total seed tocochromanol levels to 1.78-fold,2.44-fold, and 2.58-fold versus WT-levels for Arabidop-sis, canola, and soybean seed, respectively. Thisrepresented a significant increase in total seed tocochro-manol levels in all populations when compared to WT-seed. When compared to seed populations harboringsingle gene expression constructs, the level of totaltocochromanol increase remained significant in Arabi-dopsis and canola. In addition, Arabidopsis andsoybean seed showed a brown seed phenotype thatcorrelated with a substantial increase of HGA levels(Fig. 3). In summary, these data provided strongevidence for the potency of the HPPD and TYRA genecombination, and that HGA limits tocochromanolbiosynthesis in these systems. A role of HGA as limitingprecursor is consistent with previous feeding experi-ments, and with a conclusion recently communicated byCollakova and DellaPenna, which is based on mRNAlevels of tocopherol pathway enzymes during stressconditions (Collakova and DellaPenna, 2003a,b). It isalso consistent with data published by Rippert et al.(2004), that describe substantially increased tocochro-manol levels in tobacco leaf harboring expressionconstructs for yeast TYRA, and At-HPPD.
The brown seed color in Arabidopsis and soybeanwas reminiscent of the brown coloration of culturesupernatant of Synechocystis cultures expressing At-
HPPD, or At-HPPD and Eh-TYRA. These parallels incoloration, and the impact on tocochromanol levels inSynechocystis expressing At-HPPD, and in dicotyledo-nous plants expressing Eh-TYRA and At-HPPD sup-port the previous working theory that TYRA andHPPD enzymes can create a potent shunt for HGA-biosynthesis, which can increase HGA-levels by 60 and800-fold in mature Arabidopsis and soybean seed,respectively. The high efficiency of the Eh-TYRA-At-
HPPD combination for HGA-biosynthesis presumablyresults from bypassing the tyrosine regulatory mechan-ism. Tyrosine biosynthesis is feed-back inhibited bytyrosine on the level of the chorismate mutase reaction(Siehl, 1999). Co-expression of Eh-TYRA and At-HPPD
appears to direct intermediates from chorismate toHGA without substantially altering tyrosine levels.Because tyrosine levels are affected only marginally,the increased flux towards HGA is not subjected to feed-back inhibition. In Synechocystis, the endogenous
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TYRA activity was apparently not limiting, and there-fore, the high HGA- and high tocochromanol pheno-type was achieved by expression of At-HPPD alone.Plants are not known to express a TYRA like gene andtherefore, TYRA and HPPD are both required toestablish this phenotype. Interestingly, the elevatedHGA-phenotype was accompanied by the accumulationof tocotrienols, which accounted for up to 20%, 42%,47%, and 72% of total tocochromanols in Synechocys-
tis, Arabidopsis, canola, and soybean, respectively. Thisobservation is consistent with observations from Rip-pert et al. (2004), who also reported the accumulation oftocotrienols in tobacco leaves harboring yeast TYRA
and At-HPPD expression constructs.Collakova and DellaPenna (2001) reported that At-
VTE2 does not utilize GGDP as substrate. In our handshowever, the Arabidopsis homogentisate phytyltransfer-ase utilizes both PDP and GGDP as substrates(unpublished results), but displays a marked preferencefor PDP over GGDP when assayed in vitro (S. Hunterand E. Cahoon, personal communication). With theobserved preference of At-VTE2 for PDP it is surprisinghow such large tocotrienol levels were produced inArabidopsis. An alternative might be the existence of aVTE2-paralog, as it has been detected by recentbioinformatics analysis of the Arabidopsis genome(Valentin et al., 2003). The large amounts of HGAwhich always coincided tocotrienol biosynthesis mightact as a stress signal, that either activates the VTE2
paralog, or alters At-VTE2 substrate specificity. How-ever, only altered substrate specificity would be con-sistent with the tocotrienol increase that was observedupon co-expression of At-VTE2 in addition of Eh-
TYRA and At-HPPD in Arabidopsis, and soybean(Tables 3 and 4).
Another interesting variation in tocopherol pathwayengineering between Synechocystis and oilseed wasfound in the conversion of tocotrienols to tocopherols.In Synechocystis cultures expressing At-HPPD and Eh-
TYRA, the additional expression of At-GGH appearedto be sufficient to convert tocotrienols to tocopherols.This was concomitant with the disappearance of browncoloration in the culture supernatant, and with theutilization of excess HGA for increased tocopherolformation. HGA utilization was presumably improveddue to increased availability of PDP. Increased totaltocopherols in these cultures are also consistent withPDP being the preferred substrate for Syn-VTE2 (datanot shown). A role for GGH in supplying precursors fortocopherol biosynthesis was suggested by Tanaka et al.(1999) where antisense suppression of GGH in trans-genic tobacco resulted in reduced tocopherol levels, andenzyme assay data, which suggest that E. coli expressedAt-GGH can reduce GGDP and geranylated chloro-phyll (Keller et al., 1998). In our studies however,conversion of tocotrienols to tocopherols by transgenic
expression of the At-GGH was clearly not obtained intransgenic seed. Co-expression of At-GGH with At-
HPPD, Eh-TYRA, and At-VTE2 did not reducetocotrienol levels in soybean seed transformed withpMON66682, although immuno blot analysis of ex-tracts from these soybean seed was consistent with At-
GGH being expressed, and plastid targeted, andsequence analysis of the trans-gene confirmed thesequence integrity. Comparison of the Synechocystis
results to plant seed tocochromanol data suggests thatthere may be additional unidentified factors thatfacilitate the conversion of tocotrienols to tocopherolsin presence of GGH in the bacterial system, which arepossibly lacking in Arabidopsis, canola and soybeanseed. Additional experiments are needed to explain thesedifferences.
The highest total seed tocochromanol levels wereobtained in transgenic seed systems through combinedseed-specific expression of one or two genes in additionto the TYRA and HPPD combination. In Arabidopsis,expression of At-VTE2 in addition to Eh-TYRA, and At-
HPPD increased the average seed tocochromanol levelof the R2 seed populations 2.88 fold compared to WT-seed. A comparable gene combination, using Syn-VTE2
in canola and soybean provided events with higher totaltocochromanol levels than events that harbored only Eh-
TYRA, and At-HPPD expression constructs. However,the impact on all R1 seed populations was notstatistically significant (Table 4), or was even negative,as in canola. In our data set, this may be the result oflarge individual variations in total tocochromanol levelsfrom event to event, resulting in a high standarddeviation. The reasons for high variability from eventto event are not well understood yet, but the correlationof the increase in variability from event to event withincreasing number of transgenes, and therefore, vectorssize may indicate a connection to T-DNA stability.
In soybean the highest total seed tocochromanollevels were obtained with seed transformed withpMON77637. This binary vector harbored seed specificexpression constructs for At-HPPD, Eh-TYRA, At-
VTE2, and At-GGH. Four transformation events fromthis construct produced on average 10.4–11.5-fold WT-seed tocochromanol levels (Table 3). Whether At-GGH
expression had a positive impact on total tocochroma-nol accumulation in these seed is currently unknown.However, it appears unlikely, because the tocochroma-nol increase observed in these seed appears to resultfrom tocotrienol accumulation only (Table 5). In facttocotrienols appear to increase in these seed whiletocopherol levels appear to decrease resulting in seedwith an average of 494% tocotrienols for eventGM_A40048:@. In contrast, WT soybean seed containonly traces of tocotrienols.
Compared to previous studies of tocopherol pathwayengineering the total fold tocochromanol increase
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obtained here does not appear unusual, as Cahoon et al.(2003) achieved a 15-fold total tocochromanol increasesin Arabidopsis leaf, and 4–6-fold total tocochromanolincrease in corn seed by transgenic expression of abarley homogentisate geranylgeranyl transferase. Evenhigher tocochromanol increases have been reported as aresult of stress conditions. Collakova and DellaPenna(2003b) obtained up to 18-fold tocochromanol increasein Arabidopsis leaf as a result of abiotic stress. However,the final tocochromanol levels of 900 ng/mg dryArabidopsis leaf material, and 334 ng/mg in corn seedreported by Cahoon et al. appear low compared to4800 ng/mg obtained in this study in soybean seed. Themaximum tocochromanol levels obtained by Cahoon etal. in Arabidopsis leaf appear to be of the samemagnitude as the 926 ng/mg seed tocopherol reportedby Savidge et al. (2002) in Arabidopsis seed expressingAt-VTE2. Whether these numbers suggest that expres-sion of the enzyme catalyzing the first committedreaction in tocochromanol biosynthesis (VTE2, orHGGT) reaches its maximum at 900–1000 ng/mg tissuematerial under non stress conditions remains to betested in further experiments. However, the combinedexpression of enzymes, which increase tocochromanolprecursor availability with VTE2 expression, as pre-sented here, appears to provide a potent strategy toincrease total tocochromanol levels in seed and cyano-bacterial systems.
The crossing experiment performed between soybeanlines that accumulate almost exclusively a-tocopherol asa result of transgenic At-VTE3 and At-VTE4 expression,and soybean lines with about 3-fold increased total seedtocochromanol levels as a result of At-HPPD, Eh-
TYRA, and Syn-VTE2 expression demonstrated afurther average seed tocochromanol increase by 1.2-fold in seed combining both sets of genes as compared toseed exhibiting the high tocochromanol phenotype only.This result is consistent with a flux limitation by VTE3,which had been predicted based on a shift in tocochro-manol composition towards d-tocopherol and d-toco-trienol in seed with increased total tocochromanol levels(Table 5), and it is consistent with conclusions reportedby Collakova and DellaPenna (2003b) that VTE3 maylimit tocopherol biosynthesis under high stress condi-tions. High stress conditions were correlated withincreased tocopherol biosynthesis.
A compositional tocochromanol shift towards theaccumulation of d-tocopherol and d-tocotrienol wasobserved in all plant seed when tocochromanol levelsexceeded 2-fold WT-levels (dataset for canola notshown, for Arabidopsis and soybean seed see Supple-mental Table 1, and Table 5, respectively). Thissuggested that the substrate competition betweenVTE1 and VTE3 favored the cyclase reaction, indicatingthat VTE3 was the dominating factor in flux limitationin seed systems. This appears to contrast observations
from Kumar et al. (2005) who saw increased leaftocopherol levels upon transgenic expression of VTE1
but may be the result of using different metabolicbackgrounds (leaf versus oil seed). Oil seed optimizedfor vitamin E production require that all tocopherols areconverted to the tocopherol isoforms with the highestvitamin E activity, a-tocopherol. Overexpression ofVTE1 is expected to divert flux towards d-tocopherolas observed by Kumar et al. (2005), and as documentedin this manuscript. Transgenic expression of VTE1 forengineering enhanced seed vitamin E content wouldtherefore require careful finetuning with transgenicVTE3 expression in order to keep the tocopherolcomposition at its optimum.
Interestingly, F1-seed from the crossing experimentsgerminated much more vigorously than high tocochro-manol seed harboring only the combined At-HPPD, Eh-
TYRA, and Syn-VTE2 expression constructs. LC-MSanalysis of such seed had provided some evidence forincreased 2M6PBQ- and 2M6GGBQ-levels in Arabi-dopsis and canola. In WT and transgenic soybean seedthese compounds remained below or at the limit ofdetection, making it impossible to decide if theseintermediates increased or decreased as a result of transgene expression. The recent characterization of At-
VTE3 (Motohashi et al., 2003; Cheng et al., 2003)revealed that this enzyme is required for both themethylation of 2M6PBQ, and 2-methyl-6-solanylbenzo-quinol (2M6SBQ), the precursor of plastoquinone.Plastoquinones are essential for plant metabolism. It istherefore possible that the shift in metabolite poolsdisrupts the balance between 2M6PBQ, 2M6GGBQ,and 2M6SBQ. Vastly increased 2M6PBQ- and2M6GGBQ-pools may inhibit plastoquinol formation,and thus result in poor seed vigor.
The work presented here demonstrates that oilseedtocochromanol levels can be increased substantially.The 4800 ppm seed tocochromanol obtained in soybeantranslates to a concentration of 2.4% tocochromanol inthe oil. Under these conditions the vast majority oftocochromanols were accumulated as d-tocotrienol,suggesting limitations by the availability of PDP andby VTE3 enzyme activity. The limitation by VTE3 issupported by our observation of increased totaltocochromanols obtained in soybean seed from thecrossing experiment of high tocochromanol soybeanlines with all a-tocopherol soybean lines. To which levelseed tocochromanols can be increased when theselimitations are removed remains to be tested in futureexperiments.
Acknowledgments
We wish to thank our colleagues in plant transforma-tion, and the plant growth facilities for their expert
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assistance and support. We also wish to thank ourcolleagues at Renessen for their support and advice.
Appendix A. Supplementary Materials
The online version of this article contains additionalsupplementary data. Please visit doi:10.1016/j.ym-ben.2005.05.005.
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