Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli

Fed-Batch Fermentor Synthesis of3-Dehydroshikimic Acid UsingRecombinant Escherichia coli

Kai Li,1 Mark R. Mikola,2 K. M. Draths,1 R. Mark Worden,2 J. W. Frost1

1Department of Chemistry, Michigan State University, East Lansing,Michigan 48824; telephone: (517) 355-9715; fax: (517) 432-3873; e-mail:[email protected] of Chemical Engineering, 2527 Engineering Building,Michigan Sate University, East Lansing, Michigan 48824; telephone: (517)353-9015; fax: (517) 432-1105; e-mail: [email protected]

Received 8 August 1998; accepted 3 December 1998

Abstract: 3-Dehydroshikimic acid (DHS), in addition tobeing a potent antioxidant, is the key hydroaromatic in-termediate in the biocatalytic conversion of glucose intoaromatic bioproducts and a variety of industrial chemi-cals. Microbial synthesis of DHS, like other intermediatesin the common pathway of aromatic amino acid biosyn-thesis, has previously been examined only under shakeflask conditions. In this account, synthesis of DHS usingrecombinant Escherichia coli constructs is examined in afed-batch fermentor where glucose availability, oxygen-ation levels, and solution pH are controlled. DHS yieldsand titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) syn-thase. This enzyme’s expression levels, sensitivity tofeedback inhibition, and the availability of its substrates,phosphoenolpyruvate (PEP) and D-erythrose 4-phos-phate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of afeedback-insensitive isozyme of DAHP synthase and am-plified expression of transketolase, DHS titers of 69 g/Lwere synthesized in 30% yield (mol/mol) from D-glucose.Significant concentrations of 3-dehydroquinic acid (6.8g/L) and gallic acid (6.6 g/L) were synthesized in additionto DHS. The pronounced impact of transketolase overex-pression, which increases E4P availability, on DHS titersand yields indicates that PEP availability is not a limitingfactor under the fed-batch fermentor conditions em-ployed. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64:

61–73, 1999.Keywords: Escherichia coli; DHS; synthesis

INTRODUCTION

Microbe-catalyzed conversion ofD-glucose into bioprod-ucts such asL-phenylalanine andL-tryptophan (Fig. 1) areintimately related to similarly catalyzed syntheses of indus-trial chemicals such as adipic acid and catechol (Fig. 1)from D-glucose (Draths and Frost, 1994a,b, 1995). For ex-ample, the carbon flow which is directed into the commonpathway (Fig. 1) of aromatic amino acid biosynthesis is ofcentral importance to product yields and titers in each of

these syntheses. Carbon flow directed into this commonpathway can be conveniently measured by the accumulationof 3-dehydroshikimic acid (DHS, Fig. 1) in the culture su-pernatants of microbial mutants lacking shikimate dehydro-genase activity. DHS is of particular importance since it isthe most advanced common pathway intermediate shared byaromatic amino acid biosynthesis and biocatalytic synthesesof adipic acid and catechol. DHS synthesis is also importantin its own right given the potent antioxidant activity whichhas been reported for this hydroaromatic (Richman et al.,1996). Biocatalytic syntheses of common pathway interme-diates have been examined (Draths and Frost, 1990a,b;Draths et al., 1992; Patnaik and Liao, 1994) under shakeflask conditions where cultures typically begin in a glucose-rich environment and end in a glucose-deficient environ-ment. Oxygenation levels and pH are also difficult to con-trol under standard shake-flask conditions. This account ex-amines synthesis of DHS by recombinantEscherichia colibiocatalysts under fed-batch fermentor conditions whereoxygenation levels and pH are controlled and where glucoseavailability is maintained at a constant, limiting level (Kon-stantinov et al., 1990, 1991). Titers and yields for synthe-sized DHS are compared with previously reported synthesesof common pathway intermediates. Theoretical maximumyields for product DHS and cell mass formation are alsodiscussed.

As the first enzyme in the common pathway of aromaticamino acid biosynthesis, DAHP synthase (Fig. 1) activitydictates the amount of cellular carbon directed into DHSsynthesis. Historically, transcriptional repression and feed-back inhibition of DAHP synthase by aromatic amino acidshave been viewed as the regulatory mechanisms that controlthe in vivo catalytic activity of this enzyme. Several differ-ent strategies are compared in this account for fed-batchfermentor synthesis of DHS using amplified expression of amutant isozyme of 3-deoxy-D-arabino-heptulosonate7-phosphate (DAHP) synthase which is insensitive to feed-back inhibition by aromatic amino acids. More recently, theintracellular concentrations of substrates phosphoenolpyru-

Correspondence to:J. W. Frost or R. M. WordenContract grant sponsors: NSF; USDAContract grant numbers: CHE 9633368; 95-37500

© 1999 John Wiley & Sons, Inc. CCC 0006-3592/99/010061-13

vate (PEP, Fig. 1) andD-erythrose 4-phosphate (E4P, Fig. 1)have come under scrutiny as critical determinants of in vivoDAHP synthase activity. Intracellular E4P availability hasbeen reported (Draths and Frost, 1990b; Draths et al., 1992;Patnaik and Liao, 1994; Patnaik et al., 1995) to be markedlyinfluenced by expression levels of the enzyme transketo-lase. Previous research (Patnaik and Liao, 1994) has indi-cated that transketolase overexpression has only a modestimpact on yields of common pathway intermediates synthe-sized from glucose under shake flask conditions until intra-cellular PEP availability is increased by overexpression ofPEP synthase. By contrast, transketolase overexpression is

observed in this account to have a pronounced impact on theyields and titers of DHS synthesized from glucose underfed-batch fermentor conditions even in lieu of PEP synthaseoverexpression.

MATERIALS AND METHODS

Culture Medium

All solutions were prepared in distilled, deionized water. LBmedium (1 L) contained Bacto tryptone (10 g), Bacto yeastextract (5 g), and NaCl (10 g). M9 salts (1 L) containedNa2HPO4 (6 g), KH2PO4 (3 g), NH4Cl (1 g), and NaCl (0.5g). M9 medium containedD-glucose (10 g), MgSO4 (0.12g), and thiamine (0.001 g) in 1 L of M9 salts. M63 medium(1 L) contained KH2PO4 (13.6 g), (NH4)2SO4 (2 g),FeSO4 ? 7H2O (0.0005 g),D-glucose (2 g), MgSO4 (0.12 g),and thiamine (0.001 g). The pH of M63 inorganic salts wasadjusted to 7.0 with 1N KOH before autoclaving. Solutionsof inorganic salts, magnesium salts, and carbon sourceswere autoclaved separately and then mixed. Fermenta-tion medium (1 L) contained K2HPO4 (7.5 g), ammoniumiron(III) citrate (0.3 g), citric acid monohydrate (2.1 g), andconcentrated H2SO4 (1.2 mL). The culture medium wasadjusted to pH 7 by addition of concentrated NH4OH beforeautoclaving. The following supplements were added imme-diately prior to initiation of the fermentation:D-glucose (18or 23 g ), MgSO4 (0.24 g), aromatic amino acids includingphenylalanine (0.7 g), tyrosine (0.7 g), and tryptophan (0.35g), aromatic vitamins includingp-aminobenzoic acid (0.01g), 2,3-dihydroxybenzoic acid (0.01 g), andp-hydroxy-benzoic acid (0.01 g), and trace minerals including(NH4)6(Mo7O24) ? 4H2O (0.0037 g), ZnSO4 ? 7H2O (0.0029g), H3BO3 (0.0247 g), CuSO4 ? 5H2O (0.0025 g), andMnCl2 ? 4H2O (0.0158 g).D-Glucose, MgSO4, and aromaticamino acids were autoclaved while aromatic vitamins andtrace minerals were sterilized through 0.22-mm membranesprior to addition to the medium. Antibiotics were addedwhere appropriate to the following final concentrations:chloramphenicol (Cm), 20mg/mL; ampicillin (Ap), 50mg/mL; kanamycin (Kan), 50mg/mL; and spectinomycin (Sp),50 mg/mL. L-Phenylalanine,L-tyrosine, shikimic acid, his-tidine, isoleucine, valine, proline, arginine, and serine wereadded to M9 medium or M63 medium where indicated to afinal concentration of 0.04 g/L. Antibiotics, thiamine, andall amino acid supplements to M9 or M63 medium weresterilized through 0.22-mm membranes prior to addition tothe medium. Solid medium was prepared by addition of1.5% (w/v) Difco agar to medium solution.

Genetic Manipulations

Standard procedures (Sambrook, 1990) were used for con-struction, purification, and analysis of plasmid DNA.E. coliDH5a served as the host strain for all plasmid constructions.T4 DNA ligase and Klenow fragment were purchased from

Figure 1. PEP, phosphoenolpyruvate; E4P,D-erythrose 4-phosphate;AroF, tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP) synthase; AroG, phenylalanine-sensitive DAHP synthase; AroH,tryptophan-sensitive DAHP synthase; AroB, 3-dehydroquinate (DHQ)synthase; AroD, DHQ dehydratase; AroE, shikimate dehydrogenase;AroK, shikimate kinase I; AroL, shikimate kinase II; AroA, 5-enolpy-ruvylshikimate 3-phosphate (EPSP) synthase; AroC, chorismate synthase;AroZ, 3-dehydroshikimate (DHS) dehydratase; AroY, protocatechuate(PCA) decarboxylase; CatA, catechol 1,2-dioxygenase.

62 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 64, NO. 1, JULY 5, 1999

Gibco BRL. Calf intestinal alkaline phosphatase was pur-chased from Boehringer Mannheim. PCR amplificationswere carried out as described by Sambrook et al. (1990).Each reaction (0.1 mL) contained 10 mM KCl, 20 mMTris-Cl (pH 8.8), 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1%Triton X-100, dATP (0.2 mM), dCTP (0.2 mM), dGTP (0.2mM), dTTP (0.2 mM), template DNA, 0.5mM of eachprimer, and 2 units of Vent polymerase. Initial templateconcentrations varied from 0.02 to 1.0mg.

KL3

E. coli KL3 was prepared from AB2834 by homolo-gous recombination of thearoB gene into theserA locus(serA::aroB). Localization of theserA gene in pMAK705followed by insertion of thearoB into anEcoRI site internalto theserAdirected recombination of thearoB into theserAlocus of the genome. Plasmid pMAK705 contains a tem-perature-sensitive pSC101 replicon. Since derivatives ofpMAK705 replicate at 30°C but are unstable at 44°C, iso-lation of all pMAK705 derivatives required culturing ofcells at 30°C.

Digestion of pKAD63 withSphI liberated a 1.9-kbserAfragment, which was subsequently inserted into theSphI siteof pMAK705 to afford pKAD76A. ThearoB gene wasobtained as a 1.7-kb fragment following digestion of pJB14with EcoRI. Insertion of thearoB fragment into theEcoRIsite ofserAwas complicated by two additionalEcoRI sitesin pKAD76A. Following EcoRI partial digestion ofpKAD76A, the resulting DNA fragments were resolved onan agarose gel and the 7.4-kb fragment corresponding to thelinearized plasmid was isolated. Ligation of the linearizedplasmid to the 1.7-kbEcoRI fragment of aroB affordedpKL3.82A.

Conditions for homologous recombination were based onthose previously described (Hamilton et al., 1989; Ohta etal., 1991). The competent host strain was transformed withthe pMAK705 derivative. Following heat-shock treatment,cells were incubated in LB at 44°C for 1 h and subsequentlyplated onto LB plates containing Cm. Plates were incubatedat 44°C for approximately 20 h before colonies appeared.The resulting cointegrates were inoculated into 5 mL of LBcontaining no antibiotics, and the cells were grown at 30°C

Table I. Bacterial strains and plasmids used in this study.

Strain/plasmid Characteristics Reference/source

StrainDH5a lacZDM15 hsdR recA BRLAB2834 aroE353 Pittard and Wallace (1966a)

BachmannKL3 AB2834 serA::aroB This studyAB3248 aroF363, aroG365, aroH367 Zurawski et al., (1981)

YanofskyAB2.24 AB3248serA::(aroFkanR) This studyAC2-13A AB2.24aroFFBR This study

PlasmidpSU18 CmR, PlaclacZ8, p15A replicon Bartolome et al. (1991)pCL1920 SpR, PlaclacZ8, pSC101 replicon Bartolome et al. (1991)pJB14 ApR, aroB in pKK223-3 Frost et al. (1984)pJF118EH ApR, laclQ in pKK223-3 Furste et al. (1986)

BagdasarianpD2625 serAsource GClpMAK705 CmR, lacZ8, ts-pSC101 replicon Hamilton et al. (1989)

KushnerpKAD76A CmR, serA in pMAK705 Snell et al. (1996)pKL3.82A CmR, aroB in pKAD76A This studypKD10.186A CmR, aroFkanR in pKAD76A This studypCL2-13A SpR, aroFFBR in pCL1920 This studypMF51A ApR, tktA in pBR325 Farabaugh (1996)pKL4.20B CmR, aroFFBR in pSU18 This studypKL4.33B CmR, serA in pKL4.20B This studypKL4.66A CmR, aroFFBR in pKL4.33B This studypKL4.71A ApR, aroFFBR in pJF118EH This studypKL4.79B ApR, serA in pKL4.71A This studypKL4.124A ApR, tktA in pKL4.79B This studypKL4.130B CmR, tktA in pKL4.66A This studypKD11.291A CmR, ParoF in pKL4.33B This studypKL5.17A CmR, tktA in pKD11.291A This studypKD136 ApR, tktA, aroF, aroBin pBR325 Draths and Frost (1990a)pKAD62A ApR, kanR in pTrc99A-E Snell et al. (1996)pMF63A ApR, tktA, aroF, aroBin pBR325 Farabaugh (1996), M.S. thesis, MSUpKAD63 ApR, serA in p34E Snell et al. (1996)

LI ET AL.: SYNTHESIS OF 3-DEHYDROSHIKIMIC ACID 63

for 12 h to allow excision of the plasmid from the genome.Cultures were diluted (1:20,000) in LB without antibiotics,and two more cycles of growth at 30°C for 12 h were carriedout to enrich cultures for more rapidly growing cells thathad lost the temperature-sensitive replicon. Cultures werethen diluted (1:20,000) into LB and grown at 44°C for 12 hto promote plasmid loss from the cells. Serial dilutions ofeach culture were spread onto LB plates and incubated at30°C overnight. The resulting colonies were screened onmultiple plates to select the recombined ones.E. coli KL3was isolated based on the following growth characteristics:growth on M9 containingL-tyrosine,L-phenylalanine, shi-kimic acid, and serine; no growth on M9 containingL-tyrosine,L-phenylalanine, and shikimic acid; growth on LB;and no growth on LB containing Cm.

AB2.24

E. coli AB2.24 was constructed to facilitate mutagenesis ofaroF to a feedback insensitive isozyme of DAHP synthase

(aroFFBR). AB2.24 was derived from AB3248 by homolo-gous recombination of anaroFkanR locus into theserAgene. ThearoF locus was obtained as a 1.3-kbEcoRI/BamHI fragment following PCR amplification frompKD136, while a 1.2-kbBamHI/EcoRI kanR fragment wasobtained from pKAD62A. Ligation ofaroF and kanR topSU18 which had been linearized byEcoRI affordedpKD10.156. Digestion of pKD10.156 withEcoRI yieldedthe 2.5-kbaroFkanR fragment which was subsequently lo-calized in theEcoRI site internal to theserA gene inpKAD76A to afford pKD10.186A. Following homologousrecombination of theserA::aroFkanR locus of pKD10.186Ainto AB3248, E. coli AB2.24 was isolated based on thefollowing growth characteristics: no growth on M63 con-taining histidine, isoleucine, valine, proline, and arginine;no growth on M63 containing histidine, isoleucine, valine,proline, arginine, and shikimic acid; growth on M63 con-taining histidine, isoleucine, valine, proline, arginine, andserine; growth on LB containing Kan; and no growth on LBcontaining Cm.

AC2-13A

AB2.24 was subjected to in situ UV mutagenesis, and che-motactic selection was performed in a diffusion gradientchamber to select mutant strains bearing thearofFBR. thisprocess is reported elsewhere (Mikola et al., 1998). StrainAC2-13A was isolated using this method. In vitro assay ofDAHP synthase in the presence of 125mM tyrosine con-firmed that thearoF locus in AC2-13A was feedback in-sensitive. ThearoFFBR locus was amplified from AC2-13Awith its native promoter to yield a 1.3-kb fragment. Inclu-sion ofEcoRI recognition sequences at the 58- and 38- endsof the aroFFBR fragment facilitated its insertion into theEcoRI site of pCL1920 to afford pCL2-13A. Plasmid pCL2-13A was used to sequence thearoFFBR locus.

pKL4.20B

Plasmid pCL2-13A was digested withEcoRI and the 1.3-kbaroFFBR fragment was isolated and ligated into theEcoRIsite of pSU18 to create pKL4.20B. ThearoFFBR gene istranscribed in the opposite orientation relative to thelacpromoter in pKL4.20B.

pKL4.33B

This 5.5-kb plasmid was created by ligation of a 1.9-kbDraI/EcoRV fragment encodingserA obtained frompD2625 into theSmaI site of the pKL4.20B. TheserAgeneis transcribed in the same orientation asaroFFBR.

pKL4.66A

ThearoFFBR locus was amplified by PCR from pKL4.20Bwith XbaI ends. Localization of the 1.3-kbaroFFBR into theXbaI site of pKL4.33B resulted in pKL4.66A. Transcription

Table II. Restriction enzyme maps of plasmids.

Plasmid (replicon) Plasmid mapa

aRestriction enzyme sites are abbreviated as follows: E4 EcoRI, H 4

HindIII, X 4 XbaI, B 4 BamHI, Sm 4 SmaI, N 4 NcoI, S 4 SphI, P4 PstI. Parentheses indicate that the designated enzyme site has beendestroyed. — vector DNA. insert DNA.


of thearoFFBR locus in theXbaI site of pKL4.66A proceedsin the opposite orientation ofserA.

pKL4.130B

This 8.9-kb plasmid was created by inserting thetktA frag-ment from pMF51A into pKL4.66A. Plasmid pMF51A wasdigested withBamHI and the resulting 2.2-kbtktA fragmentwas treated with Klenow fragment. Plasmid pKL4.66A wasdigested withHindIII and treated with Klenow fragment.Subsequent ligation of thetktA fragment to pKL4.66A af-forded pKL4.130B. ThetktAgene is transcribed in the sameorientation as theserAgene.

pKL4.71A

This 6.4-kb plasmid was constructed by ligating the openreading frame (ORF) ofaroFFBR into the EcoRI site ofpJF118EH. ThearoFFBR ORF was amplified frompKL4.33B using following primers: 58-GGAATTCATG-CAAAAAGACGCGCTGA and 58- GGAATTCTTAAGC-CACGCGAGCCGT. Localization of the resulting 1.1-kbfragment into pJF118EH afforded pKL4.71A. The ORF ofaroFFBR is transcribed in the same orientation as thetacpromoter in pKL4.71A.

pKL4.79B

This 8.3-kb plasmid was created by ligation of a 1.9-kbDraI/EcoRV serAfragment obtained from pD2625 into theSmaI site of the pKL4.71A. TheserAgene is transcribed inthe opposite orientation relative to thearoFFBR gene.

pKL4.124A

This 10.5-kb plasmid was constructed by inserting thetktAfragment from pMF51A into pKL4.79B. Following diges-tion of pMF51A withBamHI, the 2.2-kbtktA fragment wasmodified to blunt ends using Klenow fragment. PlasmidpKL4.79B was digested withHindIII and treated with Kle-now fragment. Subsequent ligation of thetktA fragment topKL4.79B afforded pKL4.124A. ThetktA gene is tran-scribed in the same orientation as thearoFFBR gene.

pKD11.291A

This 5.6-kb plasmid was constructed by inserting a frag-ment encoding the promoter region ofaroF into the XbaIsite of pKL4.33B.ParoF was amplified from pMF63A usingfollowing primers: 58-GCTCTAGAGAATTCAAAGG-GAGTGTA and 58- GCTCTAGACCTCAGCGAGGAT-GACGT. Transcription fromParoF is in the same orientationas theserAgene.

pKL5.17A

This 7.8-kb plasmid was created by replacing the 1.0-kbNcoI/SphI fragment of pKD11.291A with a 3.2-kbNcoI/SphI fragment from pKL4.130B that includedtktA. Diges-tion of pKD11.291A withNcoI and SphI afforded a 4.6-kbfragment while similar digestion of pKL4.130B yielded a3.2-kb DNA fragment. Ligation of these two purified frag-ments resulted in pKL5.17A.

Fed-Batch Fermentation

Fed-batch culture was performed in a 2.0-L capacity BiostatMD B-Braun fermentor connected to a DCU system and aCompaq computer equipped with B-Braun MFCS softwarefor data acquisition and automatic process monitoring. Thetemperature, pH, and glucose feeding were controlled witha PID controller. The temperature was maintained at 37°C.pH was maintained at 7.0 by addition of concentratedNH4OH or 2 N H2SO4. Dissolved oxygen was measuredusing a Braun polarographic probe and it was maintained at20% air saturation throughout the fermentation process. An-tifoam (Sigma 204) was added manually as needed.

Inoculant was grown in 100 mL of LB medium (enrichedwith 2 g glucose) containing the appropriate antibiotic in a500-mL Erlenmeyer flask for 12–14 h at 37°C with agita-tion at 250 rpm and then transferred to the fermentor. Theinitial glucose concentration in the fermentation was either18 g/L (for KL3/pKL4.79B, KL3/pKL4.33B, andKL3pKL11.291A) or 23 g/L (for KL3/pKL4.124A, KL3/pKL4.66A, KL3/pKL4.130B, and KL3/pKL5.17A). Thefermentation process was divided into three stages accord-ing to three different methods used to maintain dissolvedoxygen (DO) level at 20% air saturation. In the first phase,the air flow rate was set to 0.06 L/min and dissolved oxygenconcentration was maintained by increasing the impellerspeed. Impeller speed ranged from 50 to 940 rpm. Depend-ing on the construct, the time required for the impeller toreach 940 rpm varied from 8 to 12 h. The second phasebegan after the impeller speed reached its maximum valueat which time the mass flow controller maintained DO lev-els at 20% saturation by increasing the airflow rate. Ap-proximately 2 h were needed for the airflow to increase toits maximum rate of 3.0 L/min. In the third phase, at aconstant impeller speed and a constant airflow rate, DOlevels were maintained at 20% saturation by oxygen-sensor-controlled glucose feeding. At the beginning of this stage,dissolved oxygen levels fell below 20% for approximately 1h while the initial charge of glucose was being consumed.Automatic glucose feeding started once the oxygen concen-tration rose back to the setpoint value. The glucose feedconcentration was 60% (w/v). The PID control parameterswere set to 0.0 (off) for the derivative control (tD), 999.9 s(minimum control action) for the integral control (tI), and950.0% for the proportional band (Xp). As needed, IPTGwas added every 6 h from 12 h into the fermentation untilthe end of the fermentation.


Analysis of Fermentation Broths

Samples (5 mL) of fermentation broth were taken at timedintervals. A portion (1 mL) was used to determine cell den-sities by measurement of absorbance at 600 nm (OD600).Dry cell weight (g/L) was calculated using a conversioncoefficient of 0.43 g/OD600L. This conversion factor was anaverage value obtained by centrifugation (16,000g for 10min) of the entire fermentor volume (approximately 1 L) at48 h of three separate runs and drying the washed, harvestedcells to constant weight at 100°C.

The remaining 4 mL of each fermentation broth samplewas centrifuged using a Beckman microfuge. A portion(0.5–3.0 mL) of the supernatant was concentrated to drynessunder reduced pressure, concentrated to dryness one addi-tional time from D2O, and then redissolved in D2O contain-ing a known concentration of the sodium salt of 3-(trimeth-ylsilyl)propionic-2,2,3,3-d4 acid (TSP) purchased from Lan-caster Synthesis Inc. Concentrations of metabolites in thesupernatant were determined by comparison of integralscorresponding to each metabolite with the integral corre-sponding to TSP (d 4 0.00 ppm) in the1H NMR. All 1HNMR spectra were recorded on a Varian VXR-300 FT-NMR Spectrometer (300 MHz).

DAHP Synthase Activity

A 20-mL aliquot of fermentation broth was taken at theindicated intervals. Cells were collected by centrifugation.DAHP synthase was assayed according to the proceduredescribed by Schoner (1976). Harvested cells were resus-pended in 50 mM potassium phosphate (pH 6.5) that con-tained 10 mM PEP and 0.05 mM CoCl2. The cells weredisrupted by two passages through a French press (16,000psi). Cellular debris was removed by centrifugation at48,000g for 20 min. Protein concentrations were determinedusing the Bradford (1976) dye-binding procedure. Proteinassay solution was purchased from Bio-Rad. Protein con-centrations were determined by comparison to a standardcurve prepared using bovine serum albumin. Cellular lysatewas diluted in potassium phosphate (50 mM), PEP (0.5mM), and 1,3-propanediol (250 mM), pH 7.0. A dilutedsolution of E4P was first concentrated to 12 mM by rotaryevaporation and neutralized with 5N KOH. Two differentsolutions were prepared and incubated separately at 37°Cfor 5 min. The first solution (1 mL) contained E4P (6 mM),PEP (12 mM), ovalbumin (1 mg/mL), and potassium phos-phate (25 mM), pH 7.0. The second solution (0.5 mL) con-sisted of the diluted lysate. After the two solutions weremixed (time 4 0), aliquots (0.15 mL) were removed attimed intervals and quenched with 0.1 mL of 10% trichlo-roacetic acid (w/v). Precipitated protein was removed bycentrifugation, and the product DAH(P) in each sample wasquantified using a thiobarbituric acid assay (Gollub et al.,1971). One unit of DAHP synthase activity was defined asthe formation of 1mmol of DAH(P) per min at 37°C.

RESULTS

Shared Genomic and Plasmid Elements

All of the DHS-synthesizing biocatalysts shared several ge-netic and recombinant elements including a mutation in thegenomicaroE locus, a secondaroB gene inserted into thegenomicserA locus, plasmid-localizedserA,and plasmid-localizedaroFFBR. E. coli AB2834, anaroE mutant lackingshikimate dehydrogenase activity, was the ancestral strainused to construct the KL3 host used in all of the DHSsyntheses. The absence of catalytically-active,aroE-encoded shikimate dehydrogenase, which catalyzes the con-version of DHS into shikimic acid (Fig. 1), resulted in theaccumulation of DHS in the culture supernatants ofE. coliKL3. Growth ofE. coli KL3 required supplementation witharomatic amino acids for protein biosynthesis along withsupplementation with aromatic vitamins for biosynthesis offolic acid, coenzyme Q, and enterochelin. Aromatic aminoacid supplements includedL-phenylalanine,L-tyrosine, andL-tryptophan while aromatic vitamin supplements consistedof p-aminobenzoic acid,p-hydroxybenzoic acid, and 2,3-dihydroxybenzoic acid.

Because of the increased carbon flow directed into thecommon pathway (Fig. 1) resulting from increased in vivoactivity of DAHP synthase, wild-type expression levels ofaroB-encoded DHQ synthase is inadequate inE. coliAB2834 for conversion of substrate DAHP into productDHQ at a rate which is sufficiently rapid to avoid substrateaccumulation. DAHP undergoes dephosphorylation to 3-de-oxy-D-arabino-heptulosonic acid (DAH) which accumu-lates in the culture supernatant resulting in reductions in thetiter, yield, and purity of synthesized DHS. An approxi-mately 2-fold increase in DHQ synthase activity is required(Dell et al., 1993) to eliminate DAH accumulation whichcan be accomplished (Snell et al., 1996) by introduction ofa second copy ofaroB into the genome ofE. coli AB2834.

The genomic modification inE. coli KL3 responsible forincreased DHQ synthase expression resulted from site-specific insertion of a cassette consisting ofaroB withflankingserAnucleotide sequences into theserAlocus ofE.coli AB2834. TheserA locus encodes 3-phosphoglyceratedehydrogenase which is an enzyme necessary for biosyn-thesis ofL-serine. DAH never accumulated during synthesisof DHS by any of the KL3 constructs under fed-batch fer-mentor conditions. Disruption of the genomicserA locuswas also the basis for plasmid maintenance. Growth in me-dium lackingL-serine supplementation required expressionof serA localized in all plasmids carried by KL3 biocata-lysts. Plasmid loss was not observed during synthesis ofDHS by KL3 biocatalysts under fed-batch fermentor con-ditions.

In E. coli, the most important regulation of DAHP syn-thase is feedback inhibition of the enzyme by aromaticamino acids (Ogino et al., 1982). All DHS-synthesizingconstructs therefore employed a mutant isozyme of DAHPsynthase, designated asaroFFBR, which was insensitive to


feedback inhibition by aromatic amino acids. This mutantisozyme was obtained by photochemical mutagenesis of anE. coli strain AB2.24 which expressed only the genome-encoded, tyrosine-sensitive isozyme (AroF) of DAHP syn-thase. ThearoFFBR gene was isolated from a mutant se-lected as a result of its more rapid growth in a diffusiongradient chamber (Mikola et al., 1998) against an increasingconcentration ofm-fluorotyrosine. Sequencing of the iso-lated aroFFBR gene revealed a Pro-148 to Leu-148 pointmutation which corresponds to a previously reported(Weaver and Herrmann, 1990) AroF mutant isozyme insen-sitive to feedback inhibition byL-tyrosine.

Fed-Batch Fermentor Runs

Three different methods (Konstantinov et al., 1990, 1991)were used to maintain dissolved oxygen (DO) levels at 20%saturation during the course of each fed-batch fermentorsynthesis of DHS. After inoculation of the fermentor solu-tion containing inorganic salts, aromatic amino acids, aro-matic vitamins, and a quantity of glucose, DO was main-tained by increasing the impeller stirring rate until a presetmaximum value was reached. Approximately 10 h wererequired before the impeller reached its maximum stirringrate. The mass flow controller then maintained DO levels at20% saturation at the constant impeller stirring rate by in-creasing the airflow rate until a preset maximum value wasreached. Approximately 2 h were needed for the airflow toincrease to its maximum rate. At a constant impeller stirringrate and constant airflow rate, DO levels were then main-tained at 20% saturation by oxygen-sensor-controlled glu-cose feeding for the rest of the fermentation. Oxygen sensorcontrol of glucose addition usually became too difficult tomaintain at about 48 h into a fermentor run. Loss of oxygensensor control was characterized by unregulated addition ofglucose to the fermentor culture.

Fermentor runs (Figs. 2a and 3a) typically entered loga-rithmic growth 6 h after inoculation. After approximately 24h, fermentor cultures (Figs. 2a and 3a) moved from a loga-rithmic to a stationary growth phase. Microbial cell densi-ties normally reached a maximum of 30–35 g/L dry cellweight (Figs. 2a and 3a). Over the course of the fermenta-tions, the culture solutions turned progressively darker. Bythe end of all of the fermentor runs, the culture solutionswere always a deep black color. Acetic acid accumulationwas observed at 6 h and at the conclusion of fermentor runswhich corresponds to early logarithmic and late stationarymicrobial growth phases (Figs. 2b and 3b). Concentrationsof acetic acid declined or were absent for most of the loga-rithmic and early stationary microbial growth phases (Figs.2b and 3b). Higher concentrations of acetic acid were ob-served when transketolase was overexpressed (Fig. 3b) rela-tive to when expression of this enzyme was not amplified(Fig. 2b). Maximum productivity in DHS synthesis gener-ally started at 12 h (Figs. 2a and 3a) and continued until 48h. DHS synthesis typically did not continue beyond 48 h.

DAHP Synthase Activity

One approach taken to manipulate the levels of DAHP syn-thase expression entailed replacement of the native pro-moter ofaroFFBR with a Ptac promoter along with inclusionof lacIQ in the same plasmid. AroF specific activity wasthen controlled by the amount and frequency of inducerisopropyl b-D-thiogalactopyranoside (IPTG) addition (en-tries 1–8, Table IIIa). Optimization of IPTG addition regi-mens led to several trends. During addition of IPTG at timedintervals (entries 1–5, Table IIIa), a maxiumum specificactivity of DAHP synthase was achieved (entry 5, TableIIIa) after which an additional increase in IPTG concentra-tion resulted in a decline (entry 6, Table IIIa) in DAHPsynthase specific activity. A high specific activity forDAHP synthase could also be achieved by a single addition(entry 7, Table IIIa) of a relatively large quantity of IPTGearly in the fermentor run.

AroF is precedented (Tribe and Pittard, 1979) to be labileto protease activity during the stationary phase ofE. coligrowth. However, DAHP synthase specific activities werestable over the course of most of the fed-batch fermenta-tions where IPTG was added at timed intervals (entries 2–4and 6, Table IIIa). By contrast, DAHP synthase specificactivity was not stably maintained over the course of manyof the fed-batch fermentations whenaroFFBR expressionwas under the control of its native promoter (entries 9–11,

Figure 2. KL3/pKL4.66A (a) DHS production and cell growth. (b) DHQ,gallic acid, and acetic acid production.


Table IVa). These differences may suggest that aPtac butnot aParoF promoter allows foraroFFBR transcription dur-ing stationary phase. Genes expressed from aPlac promoterand aPtrp promoter continue to be transcribed during sta-tionary phase inE. coli (Tanaka et al., 1993). There doesappear to be a ceiling above which AroF specific activitiescan not be maintained even whenaroFFBR expression isunder Ptac promoter control. This is evident in the pro-

nounced decline in enzyme activity observed for the twoIPTG addition regimens resulting in the highest DAHP syn-thase activities (entries 5 and 7, Table IIIa) measured at 12h into the fermentor runs. DAHP synthase specific activitiesalso declined over the course of the fed-batch fermentor runin the E. coli biocatalyst (entry 8, Table IIIa) containingplasmid-localizedtktA in addition toaroFFBR underPtaccontrol.

Because of the anticipated need to use strong promoterssuch asPtac for amplified expression of enzymes other thanDAHP synthase, options were explored for exploiting thenative promoter ofaroFFBR for amplified expression of thisgene. ThetyrRgene product represses transcription ofaroF.Upon plasmid localization, the increased copies ofaroFFBR

with its unmodified native promoter titrate away the cellularsupply of TyrR which binds to the operator region ofaroFFBR. Some percentage of thearoFFBR promoters thusevadetyrRbinding and are derepressed. This was the reasonfor comparing localization of onearoFFBR locus per plas-mid (entry 9, Table IVa), twoaroFFBR loci per plasmid(entries 10 and 11, Table IVa), and onearoFFBR locus ac-companied by oneParoF promoter per plasmid (entries 12and 13, Table IVa). The strategy (entries 12 and 13, TableIVa) where onlyParoF, the promoter region ofaroF, wasinserted into a multipcopy plasmid along with a single copyof aroFFBR was designed to achieve derepression of

Table IIIa. DAHP synthase activities (mmol/min/mg) whenaroFFBR ex-pression is controlled byPtac.

Entryno.

IPTGaddition

DAHP synthase specific activity

12 h 24 h 36 h 48 h

1a 0.0c 0.009 0.011 0.012 0.0072a 0.32c 0.11 0.098 0.13 0.0483a 1.6c 0.10 0.33 0.13 0.124a 4.8c 0.24 0.30 0.27 0.425a 8.0c 4.0 2.8 1.2 1.16a 40.0c 1.5 1.5 0.91 1.27a 1000d 3.0 2.2 0.29 0.218b 4.8c 0.27 0.12 0.092 0.062

aKL3/pKL4.79B.bKL3/pKL4.124A.cAmount of IPTG (mg) added at 12, 18, 24, 30, 36, and 42 h.dAmount of IPTG (mg) added at 4 h.

Figure 3. KL3/pKL4.130B (a) DHS production and cell growth. (b)DHQ, gallic acid, and acetic acid production.

Table IIIb. Product titers and yields whenaroFFBR expression is con-trolled by Ptac in the absence and in the presence of amplifiedtktA.

Entryno.

[DHS](g/L)

DHSyield

(mol/mol)[DHQ](g/L)

[GA}(g/L)

Totalyield

(mol/mol)

1a 24.3 13% 1.4 0.6 14%2a 29.1 16% 2.6 1.5 18%3a 34.0 21% 2.0 1.4 23%4a 52.0 20% 6.3 3.7 24%5a 26.8 12% 1.8 1.7 14%6a 22.0 11% 1.0 1.4 12%7a 17.4 12% 0.1 0.2 13%8b 66.3 28% 6.0 6.0 33%

aKL3/pKL4.79B.bKL3/pKL4.124A.

Table IVa. DAHP synthase activities (mmol/min/mg) whenaroFFBR ex-pression is controlled byParoF.

Entryno. Plasmida

DAHP synthase specific activity

12 h 24 h 36 h 48 h

9 pKL4.33B 0.118 0.228 0.014 0.01810 pKL4.66A 0.41 0.14 0.077 0.1111 pKL4.130B 0.23 0.11 0.08 0.07512 pKD11.291A 0.059 0.060 0.072 0.05413 pKL5.17A 0.037 0.031 0.033 0.025

aAll plasmids were carried by host strain KL3. See Table II for plasmidmaps.


aroFFBR while reducing the amount of DNA contained inthe multicopy plasmid. Use of two plasmid-localized copiesof aroFFBR (entry 10, Table IVa) resulted in significantlyhigher specific activities for AroFFBR relative to a biocata-lyst (entry 9, Table IVa) possessing only a single plasmid-localized copy ofaroFFBR. DAHP synthase specific activi-ties were significantly lower for the biocatalyst containing aplasmid carrying theParoF promoter and a single plasmid-localized copy ofaroFFBR (entry 12, Table IVa). However,this biocatalyst’s DAHP synthase specific activities re-mained reasonably constant over time. Expression oftktAhad only a modest impact on measured DAHP synthaseactivities for the biocatalysts containing plasmid-localizedaroFFBR underParoF promoter control (entry 11 vs 10; entry13 vs 12, Table IVa).

Product Titers and Yields

Incremental increases in added IPTG concentrations led tocorresponding improvements in DHS titers and yields (en-tries 1–4, Table IIIb). Beyond synthesis of 52 g/L of DHS in20% yield (mol/mol) from glucose (entry 4, Table IIIb),addition of higher IPTG concentrations resulted in a pro-nounced decrease in DHS titers and yields (entries 5–7,Table IIIb). This cut-off point appears to correspond to aceiling in DAHP synthase activity (entry 4, Table IIIa)above which further increases in DAHP synthase activitylevels (entries 5–7, Table IIIa and Table IIIb) diminish thebiocatalyst’s ability to synthesize DHS. A further improve-ment in DHS synthesis was possible at the optimized IPTGconcentration whentktA expression was amplified. Uponplasmid-localization oftktA, 66 g/L of DHS were synthe-sized in 28% (mol/mol) yield (entry 8, Table IIIb). Thissame biocatalyst (entry 8, Table IIIb) also synthesized thehighest concentrations of gallic acid (6 g/L) and DHQ (6g/L) observed foraroFFBR expression controlled byPtac.

Plasmid-localization of two copies ofaroFFBR resulted in39 g/L of synthesized DHS (entry 10, Table IVb) relative tothe 21 g/L titer associated with plasmid-localization of asingle aroFFBR (entry 9, Table IVb). The yield of DHS(mol/mol), in contrast to the titer, did not change signifi-cantly as a function of whether one or two copies of plas-mid-localizedaroFFBR were employed. For the biocatalystcontaining a single copy ofaroFFBR along withParoF, DHS

titers of 41 g/L (entry 12, Table IVb) were obtained whichwere similiar to the DHS titers associated (entry 11, TableIVb) with plasmid-localization of two copies ofaroFFBR.Amplified expression of transketolase also had a pro-nounced impact on synthesized DHS yields and titers whenaroFFBR expression was under the control of its native pro-moter. When two copies of plasmid-localizedaroFFBR wereutilized, DHS yields and titers went from a titer of 39 g/Land 16% yield (entry 10, Table IVb) to a titer of 69 g/L and30% yield (entry 11, Table IVb) upon plasmid-localizationof tktA. A DHS titer of 41 g/L synthesized in 18% yield(entry 12, Table IVb) improved to a titer of 58 g/L synthe-sized in 24% yield (entry 13, Table IVb) upon plasmid-localization oftktAalong witharoFFBR andParoF. The high-est concentrations of synthesized gallic acid (6.6 g/L) andDHQ (6.6 g/L) were again observed for the biocatalyst (en-try 11, Table IVb) which also synthesized the highest con-centrations of DHS.

DISCUSSION

Comparison of Titers and Yields

Determining (Fo¨rberg et al., 1988) the theoretical maximumyield for biocatalytic synthesis of DHS begins with balanc-ing (Eq. (1)) the PEP and E4P inputs with DHS product andbyproducts. The PEP and E4P inputs are then equated to theamount ofD-glucose which is required to form these sub-strates (Eq. (2a)). BecauseE. coli relies on the carbohydratephosphotransferase (PTS) system for glucose uptake, a py-ruvic acid term is included in Eq. (2a) to reflect the con-version of one molecule of PEP into pyruvic acid for eachmolecule of glucose transported into the cytoplasm as glu-cose 6-phosphate. Eq. (2a) reflects the effective absence ofpyruvic acid recycling back to PEP which would be cata-lyzed by the enzyme PEP synthetase (Patnaik and Liao,1994). PTS-generated pyruvic acid is considered to be theprimary carbon source for the anabolism and catabolismrequired for generation ofE. coli cellular biomass.

PEP+ E4P → 2 H3PO4 + H2O + DHS (1)

x glucose→ PEP+ E4P + x pyruvic acid (2a)

x 6~C! → 3~C! + 4~C! + x 3~C! (2b)

A coefficient is determined (Eq. (2b)) to balance the num-ber of carbon atoms in the glucose starting material with thetotal number of carbon atoms formed in PEP, E4P, andpyruvic acid. The determined coefficient leads to a maxi-mum theoretical yield of 43% (mol DHS/mol glucose) forsynthesis of DHS from glucose. KL3/pKL4.130B (entry 11,Table IVb), the construct producing the highest yields andtiters of DHS whenaroFFBR was expressed from its nativeParoF promoter, synthesized DHS at 70% of the theoreticalmaximum yield. DHS was synthesized at 66% of theoreticalmaximum yield by KL3/pKL4.124A (entry 8, Table IVb),which produced the highest titer and yield of DHS when

Table IVb. Product titers and yields whenaroFFBR expression is con-trolled byParoF in the absence of (Entries 9, 10, and 12) and in the presenceof (Entries 11 and 13) amplifiedtktA.

Entryno.

[DHS](g/L)

DHSyield

(mol/mol)[DHQ](g/L)

[GA}(g/L)

Totalyield

(mol/mol)

9 20.3 17% 1.0 0.5 18%10 38.5 16% 2.2 3.2 18%11 69.0 30% 6.8 6.6 36%12 41.2 18% 2.9 2.6 21%13 58.1 24% 3.6 4.6 27%


aroFFBR was expressed from aPtac promoter. In consideringthe performance of KL3/pKL4.130B and KL3/pKL4.124A,synthesis of DHQ and gallic acid must also be taken intoconsideration. DHQ is a metabolic precursor to DHS andgallic acid is a metabolite derived from DHS. IncludingDHQ and gallic acid synthesis, KL3/pKL4.130B and KL3/pKL4.124A channeled a total of 83% and 77%, respec-tively, of the theoretical maximum amount of carbon whichcan be directed into aromatic amino acid biosynthesis.

A similar balancing approach was used to estimate thetheoretical maximumE. coli cell yield from the pyruvicacid, ATP, and electron equivalents generated by this path-way. For every seven glucose molecules entering the path-way, 7 pyruvic acid, 10 reduced NADH, and 4 ATP mol-ecules are produced. The stoichiometries for production ofpyruvic acid from glucose via glycolysis (Eq. (3)) and cell-mass production from glucose (Eq. (4), from Papoutsakis,1984) can be summed to estimate the stoichiometry of cellmass production from pyruvic acid (Eq. (5)). A fraction ofthe pyruvic acid produced must be converted to acetyl CoAand then catabolized to CO2 in the

2 pyruvic acid+ 2 ATP+ 2 NADH → glucose (3)

glucose+ 0.87NADH + 14.8ATP→ 6 C-mole cells(4)

2 pyruvic acid+ 16.8ATP+ 2.87NADH → 6 C-mole cells(5)

Krebs cycle to provide energy and electrons needed for cellsynthesis. Each FADH2 produced in the Krebs cycle wasassumed to yield two ATP in the electron transport chain,and each NADH was assumed to yield three ATP. Balancedequations for pyruvic acid, cells, FADH2, NADH, and ATPwere written, based on Eq. (5) and the known stoichiometryof pyruvic acid oxidation via the Krebs cycle. The pseudo-steady-state assumption was invoked for non-secreted inter-mediates (Papoutsakis, 1984). Solving the balances simul-taneously resulted in a cell yield of 15 C-moles of cells perseven moles of glucose consumed. This number corre-sponds to 28% of the pyruvic acid derived from the PTSbeing catabolized, and 72% being used for cell synthesis.The mass of one C-mole ofE. coli cells is 24.6 g, based ona measurement of the cell’s elemental composition. Usingthis value, the theoretical maximum cell yield on a massbasis is 0.29 g cells/g glucose. Typically, the cell concen-trations obtained in these fermentations were about 25 g/L,and the glucose consumption levels were about 250 g/L.Thus, the experimental cell yields were about 0.10 g cells/gglucose which is well below the theoretical maximumvalue.

Microbial synthesis of DHS leading to a titer of 5.2 g/Land a yield of 54% (mol/mol) has previously (Draths andFrost, 1990a) been realized under shake flask conditions.These experiments employed overexpressed nativearoFandtktA. Accumulation in the culture supernatant of DAH,which results from dephosphorylation of DAHP (Fig. 1),has been more frequently employed as the measure of car-

bon flow directed into the common pathway of aromaticamino acid biosynthesis. DAH has been synthesized (Pat-naik and Liao, 1994) under shake flask conditions at a titerof 6.2 g/L and a yield of 63% (mol/mol) by anE. coliconstruct overexpressing the isozyme of DAHP synthaseencoded byaroGFBR which is insensitive to feedback inhi-bition by L-phenylalanine. Overexpression of transketolase(encoded bytktA) and PEP synthase (encoded bypps) inaddition to overexpression ofaroGFBR led to a titer (Patnaikand Liao, 1994) of 12.5 g of DAH synthesized in a yield of94% (mol/mol). All of these reported yields of DHS andDAH are substantially in excess of the theoretical maximumyield of 43% (mol/mol) and 86% (mol/mol) for DAH andDHS synthesis in the absence and presence, respectively, ofPEP synthase-catalyzed recycling of pyruvate back to PEP.Such yields reflect the method employed for DHS synthesiswhere theE. coli construct is initially grown in rich me-dium, harvested, and then resuspended in minimal salts me-dium containing glucose where synthesis of DHS proceeds.Using fed-batch fermentor conditions similar to those em-ployed in this account for DHS synthesis, anE. coli con-struct overexpressingaroFFBR synthesized (Konstantinovet al., 1990) 46.8 g/L ofL-phenylalanine in 20% (mol/mol)yield.

Intracellular E4P and PEP Availability

The key determinant of carbon flow directed into a biosyn-thetic pathway is often the in vivo activity of the first en-zyme in the pathway. For the common pathway of aromaticamino acid biosynthesis, the in vivo activity of DAHP syn-thase is dictated by feedback inhibition, transcriptional re-pression, and the availability of substrates E4P and PEP.E.coli uses three different isozymes of DAHP synthase en-coded by aroF, aroG, and aroH which are feedback-inhibited, respectively, byL-tyrosine,L-phenylalanine, andL-tryptophan. Feedback inhibition was circumvented in theDHS-synthesizing strains of Tables III and IV by use ofaroFFBR which was obtained via photochemical mutagen-esis of aroF. Choice of aroFFBR, as opposed to use offeedback-insensitive mutants of other DAHP synthase iso-zymes, followed from previous employment (Konstantinovet al., 1990, 1991) ofaroFFBR to achieve the highest titersthus far reported for microbial synthesis ofL-phenylalanineunder fed-batch fermentor conditions. Several differentstrategies were explored for circumventing transcriptionalrepression including placing plasmid-localizedaroFFBR un-der Ptac control and employing two plasmid-localized cop-ies of aroFFBR or ParoF.

However, higher DAHP synthase activity did not neces-sarily translate into higher DHS yields or titers as evidencedby the relationship between DAHP synthase specific activ-ity and DHS synthesized when plasmid-localizedaroFFBR

was underPtac control (Table III). Even though DAHP syn-thase specific activity and DHS synthesis initially increased(entries 1–5, Table IIIa,b) with increasing IPTG concentra-tion, a point was reached (entry 4, Table IIIa,b) where fur-


ther increases in IPTG concentrations and associated im-provements in DAHP synthase activity did not result inincreased DHS titers or yields. Indeed, the IPTG additionregimens leading to the three highest DAHP synthase spe-cific activities (entries 5–7, Table IVa,b) resulted in DHStiters which were no more than 52% of the DHS titersobserved for the best DHS synthesis (entry 4, Table IIIa,b)whenaroFFBR was underPtac control in the absence oftktAoverexpression.

A reasonable explanation for the observed ceiling in syn-thesized DHS concentrations with increasing DAHP syn-thase activity is that intracellular PEP and E4P availabilitybecomes a limiting factor. Historically, examinations ofsmall molecule limitations to aromatic amino acid biosyn-thesis have focused on PEP availability (Chen et al., 1997;Flores et al., 1996; Miller et al., 1987; Mori et al., JP62,205,782, 1987). Competition between PTS-mediatedglucose uptake and DAHP synthase for PEP is an obviousproblem during biocatalytic synthesis of aromatics and hy-droaromatics like DHS. Other enzymes which employ PEPas a substrate such as PEP carboxylase, pyruvate kinase,PEP carboxykinase, and 3-deoxy-D-manno-octulosonate8-phosphate synthase add to this intracellular competitivefray. More recently, E4P availability has been discovered(Draths and Frost, 1990b; Draths et al., 1992; Patnaik andLiao, 1994) to be an important limiting factor in aromaticamino acid biosynthesis. Steady-state concentrations of E4Pare low (Williams et al., 1980) even in the absence of am-plified DAHP synthase activity. E4P is prone to dimeriza-tion, trimerization, and polymerization (Williams et al.,1980). Dissociation of these E4P forms back to monomericE4P is quite slow. By closely matching the rate of E4Psynthesis with the rate of E4P utilization, the resulting low,steady-state concentration of E4P may be Nature’s in vivostrategy favoring the monomeric form of E4P.

Although various strategies have been examined for in-creasing in vivo PEP availability, amplified expression ofPEP synthase is particularly effective (Patnaik and Liao,1994). PEP synthase catalyzes the conversion of pyruvicacid into PEP at the expense of both of ATP’s high-energyphosphodiesters. Overexpressed PEP synthase enables PTS-generated pyruvate to be recycled back into PEP therebyameliorating the competition between glucose uptake andaromatic amino acid biosynthesis for intracellular PEP con-centrations. Amplified expression of either transketolase(Draths and Frost, 1990b; Draths et al., 1992; Patnaik andLiao, 1994) or transaldolase (Lu and Liao, 1997; Farabaugh,1996) has been demonstrated to increase the in vivo avail-ability of E4P inE. coli.Transketolase and transaldolase areenzymes in the nonoxidative pentose phosphate pathwaywhich facilitate the interconversion of C-7, C-6, C-5, andC-4 aldoses and ketoses.

The impact of both E4P and PEP availability on the invivo activity of DAHP synthase has been examined by Liaoand co-workers (Patnaik and Liao, 1994) using amplifiedexpression oftktA-encoded transketolase andpps-encodedPEP synthase activities. These experiments (Patnaik and

Liao, 1994) demonstrated that, whenaroGFBR, pps,andtktAwere all overexpressed, yields close to the theoretical maxi-mum value were achieved for a common pathway interme-diate used to gauge carbon flow directed into aromaticbiosynthesis. Overexpression of feedback-insenstivearoGFBR andpps-encoded PEP synthase in lieu oftktAover-expression did not increase the flow of carbon directed intothe common pathway of aromatic amino acid biosynthesisrelative to overexpression of justaroGFBR (Patnaik andLiao, 1994). Only a modest increase in common pathwaycarbon flow was detected whenaroGFBR and tktA wereoverexpressed in lieu of amplifiedpps expression. Theseobservations suggested that even in a biocatalyst environ-ment possessing ample PEP concentrations and overex-pressed, feedback-insensitive DAHP synthase, E4P avail-ability was a critical factor which limited aromatic aminoacid biosynthesis.

DHS synthesis under fed-batch fermentor conditionsclearly demonstrates the pronounced effect (entry 8 vs 7,Table IIIb; entry 11 vs 10, Table IVb; entry 13 vs 12, TableIVb) of tktA overexpression even in the absence of PEPsynthase overexpression. This contrasts with the report byLiao (1994) that carbon flow directed into the commonpathway was only modestly increased by transketolaseoverexpression in the absence of amplified PEP synthaseexpression. Discovering that the full impact oftktA overex-pression can be realized without PEP synthase overexpres-sion is an important observation since overexpression ofPEP synthase inhibits (Patnaik and Liao, 1994) growth ofE.coli. In actively growingE. coli cultures, this growth inhi-bition negates the positive impact PEP synthase has on titersand yields of synthesized DAH.

An important difference between this study and the pre-vious Liao (1994) study may be DO control of glucoseaddition during fed-batch fermentor synthesis of DHS rela-tive to the shake flask culturing conditions employed byLiao (1994). The steady-state concentration of glucose wasmaintained at approximately 200mM during most of thefed-batch fermentor synthesis of DHS as a result of em-ploying DO to control glucose concentration. By contrast,glucose concentrations are not maintained at a constantlevel during shake flask cultures. Such cultures begin in aglucose-rich environment and end in a glucose-deficient en-vironment. DO levels are also maintained at a constantvalue over the entire course of the fed-batch fermentor runs,whereas DO levels are uncontrolled in shake flasks. Titersand yields of DHS synthesized in Tables IIIb and IVb maytherefore highlight a fundamental difference in the in vivoavailability of PEP inE. coli cultured under fed-batch fer-mentor conditions relative to culturing ofE. coli undershake flask conditions.

DHQ and Gallic Acid Formation

Substantial concentrations of 3-dehydroquinic acid (DHQ,Fig. 1) and gallic acid (Fig. 1) accumulated during DHSsynthesis. Various common pathway metabolites have been


reported (Dell and Frost, 1993; Snell et al., 1996) to accu-mulate in the culture supernatants ofE. coli constructs inwhich carbon flow directed into the common pathway ofaromatic amino acid biosynthesis has been substantially in-creased. These metabolites result from rate-limiting com-mon pathway enzymes which can not catalyze the conver-sion of substrate into product at a sufficiently rapid rate toavoid substrate accumulation and subsequent export ofthese metabolites into the culture supernatant. However,DHQ accumulation has not previously been reported forE.coli biocatalysts despite the detailed scrutiny this microbehas received for rate-limiting, common pathway enzymes(Dell and Frost, 1993; Snell et al., 1996). Gallic acid for-mation during DHS synthesis is also surprising. Beyond itsoccurrence (Gross, 1992) in plants and reported (Haslam etal., 1961: Rivero and Cerda´-Olmedo, 1994) biosynthesis inthe filamentous fungusPhycomyces blakesleeanus,gallicacid synthesis has not previously been observed inE. coli orany other bacterium. For constructs KL3/pKL4.130B (entry11, Table IVb) and KL3/pKL4.124A (entry 8, Table IIIb)which synthesized DHS in the highest yields and titers,substantial quantities of DHQ (6–7 g/L) and gallic acid (6–7g/L) were produced.

Metabolite accumulation has typically been attributed toimpeding, rate-limiting enzymes when the common path-way (Fig. 1) is operating in the forward direction towardsbiosynthesis of aromatic amino acids. An alternative expla-nation may be that synthesized DHS accumulating in thefermentor culture medium is transported back into the cy-toplasm where DHQ dehydratase catalyzes the conversionof the DHS back into DHQ. DHS uptake byE. coli may bemediated by protein(s) encoded by theshiA locus (Brownand Doy, 1976; Pittard and Wallace, 1966b) which catalyzeuptake of the structurally similar shikimic acid (Fig. 1). Inaddition, DHQ dehydratase is known to catalyze DHS hy-dration (Davis et al., 1955; Mitsuhashi and Davis, 1954).Further conversion of DHQ is unlikely since the next com-mon pathway enzyme, DHQ synthase (Fig. 1), catalyzes anirreversible reaction which precludes conversion of DHQback into DAHP. Equilibration of DHS with DHQ is sup-ported by the molar ratio of [DHS]/[DHQ] synthesized bythe constructs summarized in Tables IIIb and IVb. These[DHS]/[DHQ] molar ratios are similar in magnitude to theknown (Davis et al., 1955; Mitsuhashi and Davis, 1954)equilibrium constant (Keq 4 15) for the reversible DHQdehydration/DHS hydration catalyzed by DHQ dehydratase.

Several different mechanisms can be offered for forma-tion of gallic acid. Abiotic, inorganic phosphate-catalyzedconversion of DHS into gallic acid is precedented (Richmanet al., 1996). Alternatively, DHS could be enzymaticallyconverted into gallic acid by DHS dehydratase-catalyzedconversion of DHS into protocatechuic acid (PCA) fol-lowed by hydroxylation of the PCA to form gallic acid (Fig.1). Oxidoreductase-catalyzed dehydrogenation (Fig. 1) ofeither the C-4 or C-5 hydroxyl group of DHS could alsolead to gallic acid formation since subsequent aromatizationwould be expected to be spontaneous and rapid. Despite

repeated attempts, no conversion of DHS into gallic acidwas observed in cell-free extracts to which a variety ofdifferent cofactors were added. This lack of assayable en-zyme activity could be consistent with either an abioticroute (Richman et al., 1996) for gallic acid formation orindicative of an enzymatic process where the responsibleenzymes do not survive cell lysis. Abiotic, inorganic phos-phate-catalyzed conversion of DHS into gallic acid is al-ways accompanied by PCA formation (Richman et al.,1996). The absence of PCA when gallic acid is formedduring microbial synthesis of DHS is thus not consistentwith an abiotic conversion. Furthermore, much higher con-centrations of inorganic phosphate are required (Richman etal., 1996) to catalyze abiotic conversion of DHS into gallicacid than were employed during the fed-batch fermentorsyntheses of DHS. The absence of PCA formation appearsto be more consistent with oxidoreductase oxidation of theC-4 or C-5 alcohol of DHS rather than dehydration of DHSand hydroxylation of the intermediate PCA. However, thepossibility remains that PCA might be present in undetectedlow, steady-state concentrations.

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Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli

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