APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2005, p. 5678–5684 Vol. 71, No. 100099-2240/05/$08.00�0 doi:10.1128/AEM.71.10.5678–5684.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Application of the Synechococcus nirA Promoter To Establish anInducible Expression System for Engineering the

Synechocystis Tocopherol PathwayQungang Qi,1 Ming Hao,2 Wing-on Ng,3† Steven C. Slater,3‡ Susan R. Baszis,1

James D. Weiss,1 and Henry E. Valentin1*Monsanto Co., 700 Chesterfield Parkway West, Chesterfield, Missouri 630171; Monsanto Co., 800 N. Lindbergh Blvd.,

St. Louis, Missouri 631672; and Cereon Genomics, 45 Sidney St., Cambridge, Massachusetts 024923

Received 30 January 2005/Accepted 28 April 2005

Tocopherols are important antioxidants in lipophilic environments. They are synthesized by plants and somephotosynthetic bacteria. Recent efforts to analyze and engineer tocopherol biosynthesis led to the identificationof Synechocystis sp. strain PCC 6803 as a well-characterized model system. To facilitate the identification of therate-limiting step(s) in the tocopherol biosynthetic pathway through the modulation of transgene expression,we established an inducible expression system in Synechocystis sp. strain PCC 6803. The nirA promoter fromSynechococcus sp. strain PCC 7942, which is repressed by ammonium and induced by nitrite (S.-I. Maeda et al.,J. Bacteriol. 180:4080–4088, 1998), was chosen to drive the expression of Arabidopsis thaliana p-hydroxyphe-nylpyruvate dioxygenase. The enzyme catalyzes the formation of homogentisic acid from p-hydroxyphenylpyru-vate. Expression of this gene under inducing conditions resulted in up to a fivefold increase in total tocopherollevels with up to 20% of tocopherols being accumulated as tocotrienols. The culture supernatant of thesecultures exhibited a brown coloration, a finding indicative of homogentisic acid excretion. Enzyme assays,functional complementation, reverse transcription-PCR, and Western blot analysis confirmed transgene ex-pression under inducing conditions only. These data demonstrate that the nirA promoter can be used to controltransgene expression in Synechocystis and that homogentisic acid is a limiting factor for tocopherol synthesisin Synechocystis sp. strain PCC 6803.

Vitamin E is a collective term that refers to the biologicalactivity of a group of eight natural amphipathic compounds: �-,�-, �-, and �-tocopherol and �-, �-, �-, and �-tocotrienol (Fig.1). Tocotrienols can be distinguished from their correspondingtocopherols by the presence of three isolated double bonds intheir prenyl side chains. These compounds are synthesized byplants and certain photosynthetic bacteria and are well recog-nized as effective oxygen radical scavengers in lipophilic envi-ronments such as oils and the lipid bilayer of biological mem-branes (3, 4). In photosynthetic organisms, tocopherols aresuggested to function as membrane-associated antioxidantsand as constituents of the chloroplast membranes (17, 18, 27,41). �-Tocopherol is an essential component in the mamma-lian diet and has the highest vitamin E activity among theisomers described above (3). Because of these health benefitsand biological functions, there is considerable interest in en-gineering tocopherol biosynthesis to increase tocopherol levelsand optimize their composition in agricultural crops.

The regulation and rate-limiting reactions in tocopherol bio-synthesis are currently poorly understood. The first committedreaction of tocopherol biosynthesis is the prenylation of ho-mogentisic acid (HGA), derived from the shikimate pathway,

with phytyldiphosphate (PDP) derived from the 2C-methyl-D-erythritol 4-phosphate (MEP)-pathway, for the formation of2-methyl-6-phytylbenzoquinol (Fig. 1). Tocotrienols are syn-thesized when geranylgeranyl diphosphate replaces PDP in theprenylation reaction of HGA, resulting in the formation of2-methyl-6-geranylgeranylbenzoquinol. Therefore, the avail-ability of HGA has an impact on tocopherol and tocotrienolbiosynthesis. The cyanobacterium Synechocystis sp. strain PCC6803 has been used as a model organism for elucidating themechanisms of critical biological processes such as photosyn-thesis (14, 16), metabolic engineering of fatty acid saturation,and zeaxanthin biosynthesis (2, 21). A common strategy todefine the rate-limiting steps of a biosynthetic pathway exper-imentally is to modulate expression of specific pathway en-zymes and then monitor the corresponding changes of inter-mediates and end products. In this setting, it is advantageous touse inducible promoters for the controlled expression of targetgenes.

Cyanobacteria are prokaryotic organisms that perform oxy-genic photosynthesis through the operation of a photosyntheticsystem very much like that present in the chloroplasts of higherplants. They preferentially use inorganic carbon, nitrogen, andmineral salts for growth. Nitrate and ammonium are excellentnitrogen sources for cyanobacteria (11). Nitrate is taken upinto the cells and reduced to ammonium by sequential actionof nitrate reductase and nitrite reductase. The genes encodingthe nitrate transporter (nrtABCD) (29), nitrate reductase(narB) (32), and nitrite reductase (nirA) (22) form the niroperon (nirA-nrtABCD-narB) in Synechococcus sp. strain PCC7942 (38). In all cyanobacteria tested to date, transcription of

* Corresponding author. Mailing address: Monsanto Co., CalgeneCampus, 1920 5th St., Davis, CA 95616. Phone: (530) 792-2136. Fax:(530) 792-2454. E-mail: henry.e.valentin@monsanto.com.

† Present address: Civil and Environmental Engineering, 318 Cam-pus Dr., E250, Stanford University, Stanford, CA 94305.

‡ Present address: The Biodesign Institute, Arizona State Univer-sity, Mail Zone 4501, Tempe, AZ 85287.

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the nir operon is repressed by ammonium (20, 38). In contrast,the presence of nitrate in the culture medium enhances mRNAlevels of the nir operon (12, 23, 24). This is further evidencedby discovering the requirement of the nitrate-promoted NtcBtranscription factor for regulation of nir operon transcriptionin cyanobacteria (1, 12, 13, 19, 24, 38, 39). We hypothesizedthat the nirA promoter of Synechococcus can be used as a tightinducible system for controlling transgene expression in Syn-echocystis. Furthermore, by substitution of ammonium withnitrate for activation of the promoter this system would pro-duce a minimal perturbation on the cellular processes in Syn-echocystis. Several studies have provided evidence that p-hy-droxyphenylpyruvate dioxygenase (Hpd) limits tocopherolbiosynthesis in plants (10, 15, 31, 42, 43). We reasoned, there-fore, that transgenic expression of Arabidopsis thaliana hpd(hpdAt) under nirA promoter control would be an excellentmodel for developing a controlled Synechocystis expressionsystem and to demonstrate tocopherol pathway engineering inSynechocystis.

MATERIALS AND METHODS

Construction of a nirAp-based expression plasmid. Custom multiple cloningsites (MCS) were made by annealing the primers MCS2 (5�-AAGGCCTGACATATGTCGCGGCCGCTCTAGATTTAAATACTAGTCCCGGGCTAGCGAGCTCT-3�) and MCS2-rev.comp (5�-AGAGCTCGCTAGCCCGGGACTAGTATTTAAATCTAGAGCGGCCGCGACATATGTCAGGCCTT-3�). The an-nealed primers were cloned into the EcoRV site of pBluescript SK(�) (Strat-agene, La Jolla, CA), yielding the plasmid pCER7.

A 166-bp fragment upstream of the nirA operon was amplified by PCR fromthe genomic DNA of Synechococcus sp. strain PCC 7942 by using the primer pairnirA1 (5�-TAGGCCTTCCCTCTCAGCTCCAAAAAGT-3�) and nirA3 (5�-CTTGAGCCATATGTCCATCTGCCTAACA-3�). Underlined bases in the primersequence indicate StuI and NdeI restriction sites that were added at the 5� and3� termini of the nirA promoter element, respectively. The PCR product harbor-ing nirAp was digested with StuI and NdeI, gel purified, and ligated into StuI- and

NdeI-digested pCER7, resulting in the formation of pCER11. Promoter se-quence integrity was confirmed by DNA sequence analysis. The amplified regionpossesses an NtcB-binding site (an inverted repeat with a LysR motif,TGCAN5TGCA), an NtcA-binding site (a palindromic structure with a con-served sequence signature, GTAN8TAC), and a TAN3T sequence fitting the �10box of the Escherichia coli �70 consensus promoter. This DNA segment has beenshowed to retain full responsiveness to nitrogen and transcription activity (24).To provide a selection marker for the transcription unit (nirAp plus MCS), thegentamicin resistance cassette (Gmr) from pUCGM (35) was cleaved as a SmaI-fragment, gel purified, and ligated into HindIII-digested and T4 DNA poly-merase-blunt-ended pCER11. The resulting plasmids were screened and thosethat carried the Gmr cassette in the opposite orientation to the nirA promoterwere selected and designated pCER12. Transcription terminators for the aboveexpression cassettes were cloned from pHP45 (30). They were PCR amplifiedfrom pHP45 by using the primer pHP45-1 (5�-TAGGCCTGGATGACCTTTTGAATGACC-3�). The Omega cassette is flanked by two identical transcriptionterminators. The terminators are inverted relative to each other. The primeranneals to both terminators and extends outward during PCR. The final PCRproduct has the two terminators at the ends plus the rest of the plasmid (minusthe spectinomycin cassette). The resulting PCR product was ligated to the blunt-ended XhoI- and EcoRI-digested Gmr::nirAp::MCS fragment of pCER12. Thisyielded the plasmid pCER18, which carries the final transcription module(Gmr::nirAp::MCS). The transcription module was excised with EcoRI, and theends were filled in with T4 DNA polymerase. This DNA fragment was ligated tothe 5.8-kb HincII-HincII fragment of plasmid RSF1010 (34), resulting in the newexpression vector pCER20 (Fig. 2). RSF1010 and its derivatives replicate auton-omously in cyanobacteria of the genera Synechococcus and Synechocystis (25, 28).All enzymes used were purchased from New England Biolabs (Beverly, MA).

Construction of a Synechocystis hpdAt expression vector. For convenience ofsubsequent cloning steps, a DNA fragment containing multiple cloning sites(5�-GCGGCCGCGGGCCCTGATCATCTAGAGTCGACGGCCGGCCGAATTCGCGGCCGCTCTAGA-3�) was inserted into NotI- and XbaI-digestedpCER20. The resulting plasmid, designated pMON36546, was used as a vectorcontrol. To generate a nirAp::hpdAt expression vector, the full-length of hpdAt wasPCR amplified from pMON36596 (43) (forward primer 5�-TGCTCTAGAACACAGGAGGACAGCCATGGGCCACCAAAACGCC-3� and reverse primer 5�-ATAAGAATGCGGCCGCAAGCTTGTCGACTTCATCCCACTAACTGTTTG-3�). The Shine-Dalgarno sequence and ATG start codon are underlined in theforward primer. The resulting PCR fragment was XbaI and NotI digested and

FIG. 1. Schematic drawing of the tocopherol biosynthetic pathway. Abbreviations: DMAPP, dimithylallyldiphosphate; ChlP, geranylgeranyl-diphosphate reductase; Hpd, p-hydroxyphenylpyruvate dioxygenase; IPP, isopentenyldiphosphate; MEP, methylerythritol phosphate; TyrA, bi-functional chorismate mutase and prephenate dehydrogenase; Vte1, tocopherol cyclase; Vte2, homogentisate phytyltransferase; Vte3, 2-methyl-6-phytylbenzoquinol methyltransferase; Vte4, �-tocopherol methyltransferase.

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cloned into XbaI- and NotI-digested pMON36546, resulting in the formation ofplasmid pMON36547.

Generation of Synechocystis sp. strain PCC 6803 �slr0090. The open readingframe slr0090 had been identified to encode the Synechocystis sp. strain PCC 6803hpd gene (6). To create a hpd mutant, a knockout construct, pMON29153, wasgenerated. This construct harbors the Synechocystis hpd interrupted by insertionof the nptII marker. Plasmid pMON29153 was constructed by digestingpMON29138 (43) with BstXI, filling in the sticky ends using Klenow fragment,and inserting the nptII expression cassette (blunted EcoRI fragment) frompUC4K (40). The resulting plasmid contained the nptII cassette inserted 647 bpdownstream of the ATG start codon. This recombinant vector was transformedinto wild-type (WT) Synechocystis sp. strain PCC 6803 by using the transforma-tion procedure described by Williams (44). Homologous recombination led tothe replacement of the WT slr0090 gene. Complete segregation of the mutantgenome was obtained by restreaking single colonies several times on BG11 agarplates supplemented with kanamycin (25 g/ml). PCR and Southern analysis ofthe Synechocystis sp. strain PCC 6803 �slr0090 mutant were performed to con-firm complete segregation of the mutant genome (data not shown).

Strains, growth conditions, and cell sample preparation. Synechocystis sp.strain PCC 6803 (ATCC 27184) was obtained from the American Type CultureCollection. WT and recombinant cells of Synechocystis sp. strain PCC 6803 werecultivated photoautotrophically at 30°C under continuous illumination providedby fluorescent lamps (70 E m�2 s�1). Liquid cultures were shaken at 225 rpmon a rotary shaker. The basal medium (BG110) was a nitrogen-free mediumobtained by replacing NaNO3, Co(NO3)2, and ferric ammonium citrate in me-dium BG11 (37) with equimolar fractions of NaCl, CoCl2, and ferric citrate,respectively. Ammonium-containing medium (BG110NH4

�) and nitrate-con-taining medium (BG110NO3

�) were prepared by addition of 17.6 mM NH4Cl or17.6 mM NaNO3, respectively, to the basal medium. Both media were bufferedwith 10 mM N-Tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)–

NaOH (pH 8.0). For growth on solid media, BG110NH4� medium containing

1.5% (wt/vol) agar (Difco) was used. Plasmids replicating autonomously in Syn-echocystis were transformed into Synechocystis sp. strain PCC 6803 via triparentalmating (9), and transformants were selected on medium supplemented withkanamycin at 25 g ml�1 and/or gentamicin at 10 g ml�1. Conjugated cells werespread on a 0.45-m-pore-size cellulose nitrate membrane filter (Whatman) andplaced on nonselective solid ammonium medium, incubated for 24 h as describedabove, and transferred to selective ammonium medium plates. Resistant colonieswere used to inoculate 2 ml of liquid BG110NH4

� medium supplemented withgentamicin (and/or kanamycin) and incubated for 2 days. These cultures servedas precultures for the final 150-ml liquid cultures. Cell density in the 150-mlcultures was monitored spectrophotometrically (SpectraMAX; Molecular De-vices) at 730 nm (A730). Cells were harvested when the A730 of the cell culturereached 0.4 to 0.5 by 10 min of centrifugation at 25°C and 3,500 � g. The cellpellet was washed with 20 ml of BG110 and resuspended in 150 ml of fresh nitrate(BG110NO3

�) or ammonium (BG110NH4�) medium. For promoter activation,

cells were subsequently grown under the light and temperature conditions de-scribed above. Cell samples were harvested at various time intervals to measuretocopherol and tocotrienol content, gene expression, and enzyme activity.

Analysis of hpdAt transcript by reverse transcription-PCR (RT-PCR). Nitrate(BG110NO3

�) or ammonium (BG110NH4�) medium-grown Synechocystis har-

vested from three representative cultures was ground in liquid nitrogen. TotalRNA was isolated as previously described (26), and any contaminating DNA wasremoved by treatment with RQ1-RNase-free DNase (Promega, Madison, WI).Three micrograms of total RNA for each sample was reverse transcribed togenerate cDNA in two 50-l reactions by using an Omniscript RT kit accordingto the manufacturer’s recommendations (QIAGEN, Inc., Valencia, CA). Analiquot of cDNA corresponding to 200 ng of total RNA was subjected to 23cycles of PCR with primers 5�-TTCCTTCGTCGCCTCCTATCG-3� (forward)and 5�-ACTCCTTGATCTGATCATCGC-3�) (reverse), resulting in the ampli-fication of a 600-bp fragment internal to the hpdAt gene. The reaction was doneunder the following thermocycle conditions: 5 min of incubation at 95°C, fol-lowed by 23 cycles of 1 min at 95°C, 1 min of annealing at 56°C, and a 2-minextension at 72°C. The amount of amplified DNA was estimated by DNA gelelectrophoresis with ethidium bromide staining.

Tocopherol and tocotrienol analyses. Tocopherols for WT and recombinantSynechocystis strains were measured by a normal-phase high-pressure liquidchromatography (HPLC) as described previously (33), but lyophilized cell pelletswere used instead of fresh harvested cells. Tocotrienol content was analyzed byusing the same procedure with tocotrienol standards purchased from Calbio-chem (La Jolla, CA). Tocopherol content was normalized to the dry cell mass.

Protein analysis. The presence of HpdAt protein in recombinant strains wasdetermined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis andWestern blot analysis with peptide-directed antibodies obtained by immunizationof a rabbit with a synthetic peptide (CRTLREMRKRSSIGG). Peptide synthesisand antibody generation were performed by Sigma-Genosys, St. Louis, MO.Synechocystis sp. strain PCC 6803 cell extracts were prepared by six passagesthrough a French press (Sim-Aminco Spectronic Instruments) at 20,000 lb/in2

with cells suspended in 50 mM Tris-HCl (pH 7.6) containing 5 mM dithiothreitol,0.1% Triton X-100, 50 U of DNase I, and protease inhibitor cocktail tablets(Boehringer-Mannheim). The supernatant fraction from a 15-min centrifugationat 10,000 � g was used as a crude extract for protein analysis and enzyme assays.For protein gel analysis, 25 g of total protein per lane was loaded on a 4 to 20%sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Invitrogen), andpolypeptides were separated according to the manufacturer’s protocol. Immu-nodetection was performed with a 1:500 dilution of rabbit polyclonal peptidespecific antibodies raised against a synthetic HpdAt peptide. An anti-rabbit im-munoglobulin G–alkaline phosphatase conjugate was used as a secondary anti-body (Sigma). The blots were developed by using nitroblue tetrazolium dye inconjunction with the alkaline phosphatase substrate BCIP (5-bromo-4-chloro-3-indolylphosphate; Sigma). Proteins were quantified with Bradford reagent (Bio-Rad) using bovine serum albumin as a standard.

Hpd assay. Hpd assays were performed according to a modified procedurefrom Secor (36). Radiolabeled [U-14C]p-hydroxyphenylpyruvate ([U-14C]HPPA)was prepared enzymatically from [U-14C]tyrosine (449 mCi/mmol; AmershamLife Science) as described by Secor (36). The Hpd reaction mixture contained 50mM potassium phosphate (pH.7.4), 0.1 mM unlabeled HPPA (freshly preparedand equilibrated for 2 h at room temperature), 0.5 Ci of [U-14C]HPPA, 5,000U of catalase (Sigma), 100 l of a freshly prepared 1:1 (vol/vol) mixture of 150mM reduced glutathione (Sigma), 3 mM dichlorophenolindophenol (Sigma),and 250 g of protein extract in a total volume of 500 l. The reaction wasincubated for 30 min at 30°C and terminated by the addition of 150 l of 20%(wt/vol) perchloric acid. Precipitated protein was removed by 5 min of centrif-

FIG. 2. Plasmid map of the inducible cyanobacterial expressionvector pCER20. The expression of target gene(s) is under the tran-scriptional control of the nitrite reductase promoter, nirAp. The aacCgene, which confers resistance to gentamicin, serves as the selectionmarker. Bracketing nirAp and the selection marker are two transcrip-tional terminators (TT) from the plasmid, pHP45. The origin ofreplication (oriV) and replication proteins (repABC), as well as theorigin of transfer (oriT) are derived from the broad-host-range plasmidRSF1010 (34).

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ugation at 14,000 rpm in an Eppendorf centrifuge. The analysis was performedon an HP1100 series HPLC system consisting of an HP G1329A automaticsampler, an HP G1311A quaternary pump, an HP G1315A diode array detector,and an HP G1321A fluorescence detector (Agilent Technologies, Englewood,CO) and a Packard Radiomatic 500TR flow scintillation analyzer (Hewlett-Packard, Meridien, CT). Hpd reaction products were separated by reversed-phase HPLC using a Vydac model 201HS54 C18 HPLC column (4.6 by 250 mm,5 m) coupled with an All-tech C18 guard column (P. J. Cobert Associates, Inc.,St. Louis, MO) and a solvent system consisting of buffers A (0.1% H3PO4 inwater) and B (0.1% H3PO4 in methanol). The solvent gradient raised buffer Bfrom 0 to 15 min from 25 to 50% and held buffer B from 15 to 20 min constantat 50%. Compounds of interest were detected by diode array detector set at 210and 254 nm and by the flow scintillation analyzer set at 156 KeV of ULD todetect 14C compounds. The sample injection volume was 20 l, and the flow ratewas set at 1.0 ml/min.

RESULTS AND DISCUSSION

Functional complementation of Synechocystis sp. strain PCC6803 �slr0090 with hpdAt driven by the nirA promoter. A tar-geted mutation in Synechocystis sp. strain PPC 6803 was cre-ated by homologous recombination (see Materials and Meth-ods), and the resulting mutant, Synechocystis sp. strain PPC6803 �slr0090, was analyzed for changes in tocopherol contentcompared to WT cultures. Tocopherol levels in the mutantwere below the limit of detection ( 5 ng/mg dry cell mass)(Fig. 3) (6), indicating an essential role of Hpd in tocopherolbiosynthesis. This result is consistent with the loss of Hpdactivity in this mutant (data not shown). The mutant cellgrowth rates were comparable to that of WT cells, indicatingthat photosynthesis was not affected (6). Subsequently, themutant was used to test the functionality of nirAp::hpdAt incomplementation experiments.

For complementation, Synechocystis sp. strain PCC 6803�slr0090 was transformed with pMON36547. This plasmid har-bored a nirAp::hpdAt expression cassette. As shown in Fig. 3,complemented strains grown on BG110NO3

� medium con-tained �3.5-fold more tocopherol than WT cells grown on the

same medium. As expected, Synechocystis sp. strain PCC 6803�slr0090(pMON36547) failed to accumulate tocopherol whengrown in BG110NH4

� medium (Fig. 3). To confirm hpdAt

expression in BG110NO3�-grown cells, RT-PCR amplifica-

tions were performed. The hpdAt transcript was detected inSynechocystis sp. strain PCC 6803 �slr0090(pMON36547) at2 h after nitrate induction and continued to accumulate at 8and 16 h postinduction (Fig. 4). In contrast, the transcript wasnot detected in Synechocystis sp. strain PCC 6803 �slr0090-(pMON36547) cultivated in BG110NH4

� medium. These re-sults support the induction of nirAp by nitrate in Synechocystissp. strain PPC 6803 and the functionality of hpdAt complement-ing the �slr0090 mutation.

Nitrate-responsive expression of hpdAt under nirA promotercontrol in Synechocystis sp. strain PCC 6803. To further dem-onstrate nitrate-dependent activation of the SynechococcusnirA promoter and assess its efficacy in induction of heterolo-gous gene expression in Synechocystis, the expression vectorpMON36547 and the empty vector control pMON36546 wereconjugated into WT Synechocystis sp. strain PCC 6803. In or-der to analyze gene expression and tocopherol levels, trans-genic and WT cells were grown in parallel and sampled at 2, 4,10, and 12 days after inoculation. As shown in Fig. 5, utilizationof different nitrogen sources in the culture media resulted insubstantial differences in HpdAt polypeptide level and enzymeactivity. Using anti-HpdAt peptide antibody, a single immuno-reactive band was detected in an extract of Synechocystis sp.strain PCC 6803(pMON36547) expressing hpdAt when grownin BG110NO3

� medium. Extracts of this recombinant straingrown in BG110NH4

� did not contain detectable immunore-

FIG. 3. Complementation of Synechocystis sp. strain PCC 6803�slr0090 with Arabidopsis hpd driven by the nirA promoter. Totaltocopherol content was normalized to dry cell mass. Cells used fortocopherol analysis were harvested after an incubation period of 10days. Tocopherol levels shown for strains Synechocystis �slr0090 andSynechocystis �slr0090(pMON36546) were below the limit of detection( 5 ng mg–1). Abbreviations: �slr0090, Synechocystis hpd insertionalinactivation mutant; pMON36546, empty vector control; pMON36547,nirAp::hpdAt.

FIG. 4. RT-PCR expression analysis of hpdAt in Synechocystis sp.strain PCC 6803 �slr0090(pMON36547). Three micrograms of totalRNA prepared from BG110NO3

�- or BG110NH4�-grown Synechocys-

tis was subjected to RT-PCR (see Materials and Methods). The aga-rose gel shows a 1-kb DNA ladder (lane MW; Invitrogen Life Tech-nologies), PCR of pMON36547 (nirAp::hpdAt) plasmid control, RT-PCR from Synechocystis sp. strain PCC 6803 �slr0090 or wild-typeSynechocystis sp. strain PCC 6803 incubated for 16 h in BG110NO3

�

medium, and that of pMON36S47-harboring Synechocystis sp. strainPCC 6803 �slr0090 incubated for 2, 8, and 16 h in BG110NH4

� orBG110NO3

� medium.

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active proteins against anti-HpdAt peptide antibody (Fig. 5A).These results confirmed the induction of hpdAt in BG110NO3

�

and its repression by ammonium. The immunoreactive bandwas also not observed in extracts of WT cells growing in eitherBG110NH4

� or BG110NO3� medium. This result was consis-

tent with the lack of cross-reactivity with the E. coli-expressedSynechocystis Hpd (data not shown).

To evaluate whether the increase in polypeptide levels cor-responds with increased enzyme activity, Hpd enzyme assayswere carried out with crude extracts of the Synechocystisstrains. As shown in Fig. 5B, the extract derived from nitrate-induced Synechocystis sp. strain PCC 6803(pMON36547) had�5-fold increased Hpd activity compared to extracts derivedfrom the vector control [Synechocystis sp. strain PCC6803(pMON36546)] or WT cell extracts. Such an increase inHpd activity was not observed with cell extracts derived fromSynechocystis sp. strain PCC 6803(pMON36547) incubated inammonium-containing medium. In addition, the culture super-natant of hpdAt-expressing Synechocystis sp. strain PCC6803(pMON3654) incubated in BG110NO3

� medium turned adark brown color after 10 days of incubation (Fig. 6). A similarphenomenon was observed in recombinant E. coli culturesexpressing Arabidopsis or Synechocystis hpd (data not shown)and has been reported in other systems as well (7, 8). Thebrown color is thought to be the result of HGA accumulationand excretion into the culture medium (7). In aqueous mediumHGA can be oxidized and polymerized to form a brown mel-anin-like pigment. Western blotting experiments, enzyme assaydata, and color changes of the culture supernatant are consis-tent with transgenic hpdAt expression in Synechocystis sp. strainPCC 6803(pMON36547) incubated in BG110NO3

�. Similarly,Western blot and enzyme assay data obtained with crude ex-tracts of the same strain after incubation in BG110NH4

� didnot provide any evidence for hpdAt expression, suggesting thatthe nirA promoter is inactive under such growth conditions.

Transgenic expression of hpdAt led to increased tocopherollevels. To determine whether the tocopherol content and com-position had been affected by transgenic expression of hpdAt,aliquots of the cell culture were taken at various time pointsfor tocopherol analysis. Cultures of Synechocystis sp. strain

PCC 6803 harboring pMON36547 that were incubated for 12days in BG110NO3

� had fivefold-increased tocopherol levelscompared to the WT control, whereas cells from the samestrain incubated in BG110NH4

� and cultures from the vectorcontrol had tocopherol levels comparable to the WT control(Fig. 7). Thus, tocopherol accumulation showed a positive cor-relation with the HpdAt polypeptide level and enzyme activity.These results confirm functionality of the nirA promoter forregulation of transgene expression via nitrate availability, dem-onstrate its utilization for engineering of the tocopherol met-abolic pathway in Synechocystis, and support the hypothesisthat Hpd catalyzes a rate-limited reaction in tocopherol bio-synthesis (10, 15, 31, 42, 43).

More interestingly, Synechocystis cells expressing hpdAt ac-cumulated up to 20% of their total tocopherols as tocotrienols

FIG. 5. Induction of nirAp in BG110NO3� media. (A) Immunoblot analysis of HpdAt peptide in Synechocystis sp. strain PCC 6803 harboring

pMON36547 (nirAp::hpdAt) or pMON36546 (empty vector control) incubated for 4 days in BG110NO3� medium (lane 1, 4dNO3

�) or for 2 daysin BG110NO3

� or BG110NH4� medium (remaining lanes). (B) Hpd enzyme activity detected in the corresponding crude cell extracts. Hpd enzyme

activity in Synechocystis sp. strain PCC 6803(pMON36547) incubated in BG110NH4� medium and in Synechocystis sp. strain PCC

6803(pMON36546) as well as in the WT control originates from endogenously expressed Synechocystis Hpd. Hpd activity in E. coli expressing hpdAtwas typically 30 to 50 nmol min�1 mg of protein�1.

FIG. 6. Synechocystis culture supernatants. Culture supernatantsshown above were collected by 25 min of centrifugation at 4,000 � gafter 10 days of incubation at 30°C. The culture supernatant of Syn-echocystis sp. strain PCC 6803(pMON36547) incubated in BG110NO3

�

medium exhibits a characteristic brown color that is typical for bacteriaexcreting HGA (7).

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(Fig. 7). None of the WT or vector control Synechocystis sam-ples contained detectable tocotrienol levels. As describedabove, the tocotrienol precursor, 2-methyl-6-geranylgeranyl-benzoquinol, is formed via the condensation of HGA andgeranylgeranyl diphosphate catalyzed by the homogentisatephytyltransferase (Vte2), whereas tocopherol synthesis utilizesHGA and PDP. The formation of tocotrienols by SynechocystisVte2 is consistent with data provided by Collakova and Della-Penna (5) and suggests that 2-methyl-6-phytylbenzoquinolmethyltransferase (Vte3), tocopherol cyclase (Vte1), and�-methyltransferase (Vte4) in Synechocystis sp. strain PCC6803 can utilize tocotrienol precursors as substrates as well. Inaddition, the formation of tocotrienols and accumulation ofHGA provide evidence for a limitation of endogenous PDP,suggesting that experiments to engineer further tocopherolincreases in Synechocystis should focus on this part of thepathway.

Conclusions. A 166-bp DNA fragment from the 5� untrans-lated region of the nirA operon was isolated from the genomeof Synechococcus sp. strain PCC 7942. Sequence analysis re-vealed the presence of the consensus sequence (GTAN8TAC)for cyanobacterial NtcA-binding sites, an L1-like inverted re-peat containing a LysR motif (TN11A), and the putative �10element, showing a typical structure for a nitrogen-regulatedpromoter in cyanobacteria (12, 24, 38). By monitoring theexpression level of hpdAt and its impact on tocopherol levels inSynechocystis sp. strain PCC 6803 or Synechocystis sp. strainPCC 6803 �slr0090 harboring pMON36547, a nirAp::hpdAt ex-pression construct, we were able to show that the nirA pro-moter is active in BG110NO3

� medium and inactive inBG110NH4

� medium. More importantly, the results presentedhere are the first, to our knowledge, to demonstrate that thenitrate-inducible nirA promoter can be used to drive and reg-ulate the transcription of transgenes in Synechocystis sp. strainPCC 6803 and that this regulation can lead to changes inmetabolite flux for production of target chemicals, such astocopherols. Therefore, the nirA promoter system provides a

suitable tool for metabolic engineering in Synechocystis. Itsability to be inactivated in ammonium-containing mediummakes it particularly suitable for the expression of potentiallytoxic genes or for engineering metabolic pathways that mayproduce toxic products or intermediates. In the present study,we obtained evidence that the upregulated hpdAt expressionresults in an increased synthesis of HGA, tocopherols, andtocotrienols in Synechocystis in vivo, indicating that Hpd playsan important role in tocopherol production and that additionalenzymes such as geranylgeranyl diphosphate reductase may berequired to overcome additional constraints for further en-hancement of the tocopherol accumulation in this strain. Theresults obtained from this photosynthetic model system pro-vide useful information for tocopherol metabolic engineeringin other organisms.

ACKNOWLEDGMENTS

We are grateful to Mylavarapu Venkatramesh, Dusty Post-Beitten-miller, and Kenneth J. Gruys (Monsanto Company) for helpful discus-sions during the experimental course of this study and the preparationof the manuscript. We thank Jeffrey M. Staub and Lisa M. Weaver(Monsanto Company) for critical reading of the manuscript. We alsothank Yanping Zhu for skillful technical assistance with Synechocystiscell culture and Tatsuo Omata (Nagoya University) for Synechococcussp. strain PCC 7942.

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FIG. 7. Tocopherol content and composition in Synechocystis sp.strain PCC 6803 cultures grown in BG110NH4

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