l dopa vitreoscilla

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2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1

Biotechnol. J. 2009, 4 DOI 10.1002/biot.200900130 www.biotechnology-journal.com

1 IntroductionL-DOPA (3,4-dihydroxyphenyl- L-alanine) anddopamine [2-(3,4-dihydroxyphenyl)ethylamine or3,4-dihydroxytyramine] are two high-value com-pounds of pharmaceutical interest. Both are de-rived from the amino acid L-tyrosine and are im-portant therapeutic agents used in many neurode-generative diseases, especially for the treatment of Parkinsons disease, a condition caused by a defi-ciency in the action and formation of dopamine by the dopaminergic neurons of the brain [1,2]. Of 250tons of L-DOPA produced per year, approximately

half is produced by the enzymatic method involv-ing tyrosine phenol-lyase (TPL, EC 4.1.99.2), apyridoxal phosphate-requiring enzyme of bacteri-al origin [3, 4]. The Ajimato Co. Ltd. (Japan) began

using Erwinia TPL for enzymatic L-DOPA produc-tion in 1993 by a simple one-step method, one of the most economical processes to date [5]. Al-though a large number of bacterial isolates havebeen reported to produce TPL, the enzyme forcommercial purposes ( e.g ., synthesizing L-tyrosineor L-DOPA) has mainly been isolated from two en-teric bacteria ( Citrobacter freundii and Erwinia her-bicola ) [6, 7].This enzyme normally catalyzes the -elimination of L-tyrosine to produce pyruvate, am-monia, and phenol, thereby enabling bacteria toutilize L-tyrosine as a carbon and a nitrogen source.

This reaction is reversible, and when catechol issubstituted for phenol, L-DOPA is produced [8].

Research Article

Production of L-DOPA and dopamine in recombinant bacteriabearing the Vitreoscilla hemoglobin gene

Asli Giray Kurt1, Emel Aytan1, Ufuk Ozer 1*, Burhan Ates2 and Hikmet Geckil 1

1 Department of Biology, Inonu University, Malatya, Turkey2 Department of Chemistry, Inonu University, Malatya, Turkey

Given the well-established beneficial effects of Vitreoscillahemoglobin (VHb) on heterologous or-ganisms, the potential of this protein for the production of L-DOPA and dopamine in two bacte-ria, Citrobacter freundiiand Erwinia herbicola, was investigated. The constructed recombinantsbearing the VHb gene ( vgb+) had substantially higher levels of cytoplasmic L-DOPA (112 mg/L forC. freundiiand 97 mg/L for E. herbicola) than their respective hosts (30.4 and 33.8 mg/L) andthevgb control strains (35.6 and 35.8 mg/L). Further, the vgb+ recombinants of C. freundiiand E. her-bicola had 20-fold and about two orders of magnitude higher dopamine levels than their hosts,repectively. The activity of tyrosine phenol-lyase, the enzyme converting L-tyrosine to L-DOPA, waswell-correlated to cytoplasmic L-DOPA levels. As cultures aged, higher tyrosine phenol-lyase ac-tivity of the vgb+ strains was more apparent.

Keywords: L-DOPA Dopamine Erwinia herbicola Tyrosine phenol-lyase Vitreoscillahemoglobin

Correspondence: Professor Hikmet Geckil, Department of Biology,Inonu University, Malatya 44280, TurkeyE-mail:[email protected]: +90-422-341-0037

Abbreviations: TPL, tyrosine phenol-lyase; VHb, Vitreoscillahemoglobin;vgb, Vitreoscillahemoglobin gene

Received 25 May 2009Revised 9 June 2009Accepted 15 June 2009

*Present address:University of South Carolina, Department of BiologicalSciences, Columbia, SC 29208, USA


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and grown at 37C (200 rpm) until an A 600 readingof about 0.4.Aliquots of 1 mL of each culture (in du-plicates or triplicates) were then transferred intoEppendorf tubes and left on ice for 10 min. Cells

were centrifuged (5000 rpm) for 5 min at 4C, andthe pellet was re-suspended in 0.5 mL ice-cold0.1 M CaCl 2 and left on ice for 20 min. After cen-trifugation as previously, 0.5 mL ice-cold 0.1 MCaCl 2 was added to pellet, gently mixed,and left onice 20 min. The cell suspension was centrifuged(5000 rpm, 5 min at 4C) and 150 L ice-cold 0.1 MCaCl 2 was added to the pellet. DNA (0.11 g) in1 L 10 mM Tris-HCl pH 8.0 with 1 mM EDTA wasadded to these suspensions of competent cells,

which were placed on ice for 1 h. The cells werethen heat shocked by placing the tubes into a 42C

water bath for 2 min and then on ice for 10 min. LBmedium (1 mL) was added to mixtures and these

were incubated at 37C for 1 h. From each, a 200-Laliquot was plated on ampicillin (100 g/mL)-con-taining LB agar plates, which were incubated for atleast 24 h until the main colony, with satellitecolonies on its periphery, was distinctly visible.This

method of transformation revealed five to ten pre-sumptive colonies and plasmid isolation from thesecolonies was carried out as described above.

2.3 Bacterial strains and culture conditions

The bacterial strains used in this study were C. fre-undii (NRRL B-2643), E. herbicola (NRRL B-3466)and their vgb + and vgb recombinants constructedin this study. Wild-type bacterial strains (NRRL B-2643 and NRRL B-3466) known for TPL activity and L-DOPA and dopamine production were pro-

vided by Dr. Alejandro Rooney, the curator of bac-terial stock cultures at the United States Depart-ment of Agriculture (USDA, Peoria, IL, USA). E.herbicola was transformed with plasmids pUC8 andpUC8:15, the vgb -carrying recombinant plasmid of pUC8 (Fig. 1), while C. freundii was transformed

with pMK57 and its vgb -carrying recombinantplasmid (pMK79) (Fig. 2). The plasmid presence

was confirmed by restriction minipreparation of asatellite-forming colony on LB ampicillin agar

Biotechnol. J. 2009, 4 www.biotechnology-journal.com

Figure 1. Physical maps of plasmidspUC8 and pUC8:15. pUC8 is a deriv-ative of plasmid pBR322 and fila-mentous phage M13. PlasmidpUC8:15 bears a 2.3-kb Vitreoscillachromosomal fragment ( Hind III-Hind III) containing the Vitreoscillahemoglobin gene ( vgb). ori, origin of replication of plasmid pMB1; Amp R,ampicillin resistance gene; lacZa, -galactosidase gene encoding alphamonomer (peptide) of the enzyme;plac, -galactosidase gene promoter.

Figure 2. Physical maps of plasmidspMK57 and pMK79 (re-drawn from[41]). Plasmid pMK57 is derived fromplasmid pUC8 through the insertion

of -amylase gene (the Pvu II-HindIII fragment) of Bacillus stearother-mophilus. A derivative of pMK57,plasmid pMK79 bears the Vitreoscillahemoglobin gene on a 2.3-kb frag-ment insert (the Hind III-Hind IIIfragment). ori, origin of replication; Amp R, ampicillin resistance gene; Amy, -amylase gene; lacZa, -galac-tosidase gene encoding alphamonomer (peptide) of the enzyme;plac, -galactosidase gene promoter;vgb, Vitreoscillahemoglobin gene.


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BiotechnologyJournal Biotechnol. J. 2009, 4

4 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

plate (Fig. 3). Cells were maintained on LB agar(wild types) and ampicillin plates (recombinants)at 4C with transfers at monthly intervals. Thegrowth medium used for TPL activity and L-DOPA and dopamine production was LB broth (pH 7.0):10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl. A 1/100 inoculum of overnight culture grown in LBmedium was transferred into the fresh medium(20 mL in 125-mL flasks) and cells were harvestedafter 12 and 24 h of incubation (37C at 200 rpm)phases, keeping both cell-free medium and cellpellets. In experiments for TPL induction, L-tyro-sine was added to this medium at 0.1% concentra-tion after 6 h of incubation for 12-h cultures and af-ter 12 h of incubation for 24-h cultures. L-DOPA and dopamine levels determined in the cell-freemedium were recorded as extracellular, whiletheir levels in cell-free extracts were indicated ascytoplasmic.The cell-free extracts were also uti-lized for determination of TPL activity at 12- and24-h culture phases.

2.4 L-DOPA and dopamine levels of cultures

The intracellular (cytoplasmic) and extracellular L-DOPA and dopamine levels of bacteria and theirvgb and vgb + recombinants were determined only for 24-h cultures, as the production of both com-pounds only occurred at post-stationary phases of growth. Cells were harvested by centrifugation(10 000 rpm for 5 min) at room temperature. Su-pernatants (1 mL each) were transferred into tight-ly capped colored tubes and, if not assayed imme-

diately, were kept at +4C for not more than 24 h be-fore measurement of the extracellular L-DOPA anddopamine contents. The pellets were used forpreparation of cell-free extracts to determine thecytoplasmic levels of L-DOPA and dopamine andalso TPL activity. The pellets were washed once

with 0.05 M potassium phosphate buffer (pH 8.6),and re-suspended in the same buffer to an equaldensity, A 600 =10.0. The suspensions were sonicatedon ice for 3 min (four cycles of 40 s with 20-s paus-es between each cycle) using an ultrasonic cell dis-ruptor (Sanyo 150, Palisades, NJ, USA).

2.4.1 AssayingL-DOPA and dopamine by HPLC The extracellular and cytoplasmic concentration of L-DOPA and dopamine was determined by HPLC(Agilent 1100; Santa Clara, CA, USA); column, SGEC18 .The elution buffer was 20 mM potassium phos-phate (pH 3.7) with 6% methanol at a flow rate1 mL/min. HPLC chromatograms (270 nm) of L-DOPA and dopamine were obtained from columnat temperature 25C using commercial HPLC gradeL-DOPA and dopamine as the standards at1500 ppm, which resulted an excellent linear be-havior.

2.4.2 TPL activityThe enzyme activity was measured by the methodof Wriston [83]. The method is based on the deter-mination of ammonia liberated from L-tyrosine by the Nessler reaction. Reaction was started by adding 0.2 mL cell-free extract into 0.8 mL pre-

warmed 0.005 M L-tyrosine and 0.1 mM pyridoxal5-phosphate prepared in 0.05 M KPi buffer(pH 8.6) and incubated for 30 min at 37C. The re-action was terminated by the addition of 0.1 mL1.5 M TCA.The reaction mixture was centrifuged atroom temperature (10 000 rpm for 5 min) to removethe precipitate and the ammonia released in thesupernatant was determined colorimetrically (A 480 ) by adding 0.25 mL Nessler reagent to tubescontaining 0.5 mL supernatant and 1.75 mL dH 2O.The content in the tubes was vortexed and incu-bated at room temperature for 10 min, and the A 480readings were read against the blanks that receivedTCA before extract addition. One TPL unit is de-

fined as the amount of enzyme that liberates 1 molammonia/min at 37C.Specific activity is expressedas U/mg protein released.The ammonia concentra-tion produced in the reaction was determined onthe basis of a standard curve obtained with ammo-nium sulfate. The limit of detection of ammonia by this method was about 10 M.Total protein was de-termined colorimetrically [84] using BSA as thestandard.

Figure 3. Restriction minipreps of plas-mids from vgb+ and vgb recombinantsof C. freundiiand E. herbicola. In bothpanels: 1, Hind III digest of DNA.(a) 24, Hind III digest of pMK79; 5-7,Hind III digest of pMK57. (b) 24, HindIII digest of pMK79; 57, Hind III digestof pMK57; 810, Hind III digest of pUC8:15. , 2.3-kb Vitreoscillachromo-somal fragment containing vgbgene; ,plasmid pMK57 (5.7-kb); , plasmidpUC8 (2.7-kb).


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3 Results and discussion

The production of L-DOPA and dopamine wasstudied in two bacteria ( C. freundii and E. herbico-la ) and in their recombinants harboring the vgb .Where indicated, L-tyrosine was included in themedium (LB) as the inducer. L-Tyrosine was addedafter 6 h of growth to cultures harvested at 12 h,

whereas for 24-h cultures L-tyrosine was added af-ter 12 h. The intracellular (cytoplasmic) and extra-cellular (cell-free growth medium) L-DOPA anddopamine levels were determined in cultures har-

vested at 24 h. Our preliminary studies showed thatthese compounds were produced during the late(post-stationary) secondary phase of growth. Onfurther incubation ( e.g ., 48 h), a substantial de-crease in L-DOPA and dopamine production wasobserved. The effect of various conditions on theproduction of TPL, the enzyme converting L-tyro-sine to L-DOPA, was also investigated.

In LB-grown cultures, the extracellular L-DOPA levels of wild-type strains of both bacteria ( C. fre-undii and E. herbicola ) were substantially higherthan that of their respective recombinants, the vgb and vgb + strains (Fig. 4). C. freundii showed a morethan fivefold higher L-DOPA production (about180 mg/L) compared to its vgb (Cf[pMK57]) andvgb + (Cf[pMK79]) recombinants, which showedsimilar L-DOPA profiles (about 33 mg/L). There

was a similar trend for E. herbicola and its vgb (Eh[pUC8]) and vgb + (Eh[pUC8:15]) recombinants,and the value here was about fourfold higher in the

wild-type strain (171 mg/L). Although to a lesserextent, the extracellular levels of dopamine for the

wild-type strains of both bacteria were also higherthan for their recombinant counterparts (Fig. 4). C.

freundii had slightly more than twofold higherdopamine levels, and E. herbicola about threefold.

The two species ( C. freundii and E. herbicola )showed a different correlation with respect to totalcell mass (recorded as A 600 values) and the productformation. C. freundii and its recombinants showedan almost linear correlation between cell mass andextracellular L-DOPA and dopamine levels (Fig. 4).The same three strains, however, had an inversedtype of correlation for cell mass and cytoplasmic L-DOPA and dopamine. In E. herbicola and its recom-binants, there was an inverse relationship betweenthe total cell mass and extracellular product level;the highest product formation was observed in cul-tures with lowest total cell mass (Fig. 4). In contrastto C. freundii and its recombinants, the highest cy-toplasmic L-DOPA and dopamine levels in E. her-bicola and its recombinants were recorded in cul-tures with highest total cell mass, suggesting thatthe relation between L-DOPA and dopamine for-mation (extra- or intracellular) and total cell mass

was species dependent. These results show that agrowth characteristic determined for higher prod-uct formation in one bacterium may not be appliedto another.Thus, the conditions for the product for-mation should be independently investigated on acase-to-case basis.

Inclusion of L-tyrosine to LB medium after 12 hof growth resulted about a twofold induction of L-DOPA production in wild-types of both bacterialstrains. The effect on the recombinants, however,

was lower (1560% increase). The lower levels of extracellular L-DOPA and dopamine in recombi-nant bacteria ( vgb or vgb + strains) may be due toplasmid burden on cells.These cells are thought tokeep an internal reserve of their amino acids andtheir derivatives ( e.g ., L-DOPA) in the cell insteadof releasing them into the growth medium, given ahigher demand for metabolism and growth.The ef-fect of plasmid-mediated metabolic burden on the


Figure 4. Total cell mass and extra-cellular L-DOPA and dopamine levelsof C. freundiiand E. herbicolaandtheir vgb+ and vgb harboring recom-binants grown in LB medium for24 h. Each data point is the averageof three independent experimentsmade in duplicates. Error bars indi-cate SDs ( n-1). For clarity no errorbars given for L-DOPA and dopamineexperiments but they are less than10% of mean for each data point.


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expression of the host genes has been the subjectof some studies [85, 86], in which biosynthetic bur-den associated with plasmid presence was shownto limit the cell growth, the metabolites studied andthe recombinant protein production. Thus, in thecurrent study, the burden that the presence of plas-mid exerts on the cells may well exceeded the ben-eficial effect of VHb. Lower extracellular L-DOPA levels of vgb + and vgb recombinants of both bacte-ria may also be due to a higher metabolic burdeninflicted by the presence of these high copy-num-ber plasmids. This, however, was the case for only L-DOPA, the immediate product from L-tyrosine.The reaction is readily reversible under appropri-ate conditions ( i.e ., at high growth demand).

In contrast to L-DOPA levels, the extracellulardopamine level in L-tyrosine-supplemented medi-um was substantially higher in the strains bearingvgb than that of in their counterparts (Fig. 5). Thevgb + strain of C. freundii (i.e ., Cf[pMK79]) had abouttwofold higher extracellular levels of dopaminethan both the wild-type strain and Cf[pMK79], thevgb strain. There was a similar trend for the vgb +strain of E. herbicola (i.e., Eh[pUC8:15]), which

showed 22% and 54% higher extracellular dopa-mine levels than the wild-type and the vgb strain,Eh[pUC8], respectively. Apparently, vgb -bearingstrains of both bacteria preferred to secretedopamine, a secondary metabolite, into the growthmedium.

The intracellular (cytoplasmic) level of L-DOPA and dopamine was significantly higher in vgb + re-combinants of both bacteria (Fig. 6).The vgb + strainof C. freundii had 3.7-fold and 3.2-fold higher lev-els of L-DOPA than the host and vgb strain, re-spectively. For the vgb + recombinant of E. herbicola

these values were 2.9- and 2.7-fold higher.The dif-ference in dopamine levels were even higher forthe vgb + recombinants. Cf[pMK79] (the vgb + re-combinant of C. freundii ) had an about 20-fold andEh[pUC8:15] (the vgb + recombinant of E. herbicola )had about two orders of magnitude higherdopamine levels than their respective hosts. What

was puzzling, although not at the level of vgb + re-combinants, was that the vgb strains of both bacte-ria also had high dopamine levels.The cytoplasmiclevels of the compounds ( L-DOPA and dopamine)in L-tyrosine-supplemented medium were gener-ally more or less within the same range (Fig. 7),

with the exception that the wild-type hosts of bothbacteria had higher L-DOPA levels (in the range of 3080 %), while the vgb + strains produced moredopamine (from 30% to about onefold).The low ex-tracellular level of L-DOPA and dopamine invgb +/vgb recombinants of both bacteria and sub-stantially higher cytoplasmic level of these com-pounds in vgb + recombinants may be due to theirdifferential use in cell metabolism. It is known thatL-DOPA and dopamine differently affect thegrowth and metabolic properties of bacteria

[8789].TPL, a pyridoxal phosphate-requiring enzyme,is of particular interest because of its capacity forsynthesizing L-DOPA (or L-tyrosine) from the sub-strates phenol (or pyrocatechol), pyruvate, and am-monia. About half of the L-DOPA in the market to-day is produced enzymatically using TPL.Althoughthis enzyme is expressed in a number of phyloge-netic groups, most studies have focused on the useof E. herbicola and C. freundii , two bacterial species

with high TPL activities [37]. The study hereshowed that TPL activity in LB medium was well-

Figure 5. Extracellular L-DOPA anddopamine levels of C. freundiiand E.herbicolaand their vgb+ and vgb re-combinants grown in LB mediumwith L-tyrosine for 24 h. Each datapoint is the average of three inde-pendent experiments made in dupli-cates.


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correlated with cytoplasmic L-DOPA levels of cellsin the same medium (Fig. 8). At the 12-h culturephase, the vgb + strain of C. freundii showed a TPL-specific activity 0.08 U/mg, while the values for thehost and vgb strain were 0.032 and 0.058 U/mg, re-spectively. At 24 h of cultivation, these values were0.371,0.031,and 0.067 U/mg for the vgb +, wild-type,and vgb strains of C. freundii , respectively. E. her-bicola and its vgb and vgb + recombinants showedsimilar TPL activity at the 12-h culture phase(Fig. 8), whereas at 24 h, the vgb -bearing strain hadsubstantially higher TPL activity than the host bac-terium ( E. herbicola ) and its vgb strain (Eh[pUC8]).At this culture phase, TPL activity was 0.017 and0.098 U/mg for the wild-type and the vgb strains of

E. herbicola , respectively, while the activity ofthe enzyme in vgb + recombinant strain was0.513 U/mg.As cultures aged, the effect provided by presence of vgb was more apparent. The vgb -bear-ing C. freundii and E. herbicola after 24 h of culturehad 5- and about 20-fold higher TPL activity thanthe same cells harvested at 12 h, respectively, whileother strains (the hosts and vgb control strains)showed more or less similar level of enzyme activ-ity in both phases of culture. These findings are inagreement with those from previous studies show-ing that the dramatic effect of VHb on cell metabo-lism and product formation is found under limitedoxygen conditions where an elevated expression of VHb occurs [44, 76, 79]. In induction experiments,


Figure 6. Total cell mass and cyto-plasmic L-DOPA and dopamine lev-els of C. freundiiand E. herbicolaandtheir vgb+ and vgb harboring recom-binants grown in LB medium for24 h. Each data point is the averageof three independent experimentsmade in duplicates. These are thesame cultures given in Fig. 4. Errorbars indicate SDs ( n-1). For clarityno error bars given for L-DOPA anddopamine experiments but they areless than 10% of mean for each datapoint.

Figure 7. Cytoplasmic L-DOPA anddopamine levels in C. freundiiand E.herbicolaand in their vgb+ and vgb

recombinants grown in LB mediumwith L-tyrosine. Each data point isthe average of three separate experi-ments made in duplicates.


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L-tyrosine was added to 12-h grown cultures after6 h of incubation, and after 12 h for 24-h cultures(Fig. 9). L-Tyrosine addition only had a positive ef-fect in terms of TPL induction for cell cultures har-

vested after 12 h. This was especially apparent forvgb -bearing strains of both bacteria. Compared totheir TPL activity in LB medium, in LB + L-tyrosinemedium Cf[pMK79] and Eh[pUC8:15] had about60% and fourfold higher activity, respectively. TheTPL activity of 24-h cultured vgb + strains in LB

with L-tyrosine supplement was about threefoldhigher than their counterparts at the 12-h incuba-tion phase, while other strains showed similar lev-el of TPL activity at both phases.

4 Conclusion

White (or industrial) biotechnology is becoming in-creasingly important as the unique metabolic ca-pacities of microbes offer new synthetic routes fora broad range of valuable products from fuels andmaterials to fine chemicals [90].The purpose of thisstudy was to determine whether an efficient oxy-gen-uptake system, the VHb, provides an advan-tage for production of L-DOPA and dopamine, twodrugs with high importance in health industry. Toachieve a sustainable source of these compounds,studies with biotechnological approaches havebeen conducted aiming at the development of al-ternative production systems [39]. The findingshere show that, although the effect of VHb on prod-

Figure 8. Time course of TPL activityof C. freundii, E. herbicolaand theirvgb+ and vgb recombinants grown inLB medium. Each data point is theaverage of at least two independentexperiments in duplicates. SDs ( n-1)are less than 10% of mean for eachdata point. For clarity no error barsare given.

Figure 9. Time course of TPL activityof C. freundii, E. herbicolaand theirvgb+ and vgb recombinants grown inLB medium with L-tyrosine. Each datapoint is the average of at least two in-dependent experiments in duplicates.SDs (n-1) are less than 10% of meanfor each data point. For clarity no er-ror bars are given.


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uct ( L-DOPA and dopamine) formation cannot bereduced to a single factor for the two strains of bac-teria studied ( C. freundii and E. herbicola ), the mostbeneficial effect of this protein was on the culturesat the post-stationary growth phase ( i.e ., 24-h cells)

where the oxygen restriction is the main rate-lim-iting factor. At this culture phase, the vgb + recombi-nants of C. freundii had about 20-fold higher, and E.herbicola about two orders of magnitude higher, cy-toplasmic dopamine levels than their respectivehosts.The levels of L-DOPA were within the rangeof 3.2- and 3.7-fold higher for the vgb + strains.Theextracellular level of both compounds, however,

was generally higher in wild-type cells.Thus, to as-sign a real function to VHb on product formation inthis and similar studies, a case-by-case-based in-

vestigation is needed, taking into account the typeof bacterium, its respiratory metabolism, and theculture conditions. TPL activity correlated well tocytoplasmic L-DOPA levels of cells.The vgb + strainshad substantially higher TPL activity than the re-spective wild-type hosts or the strains bearing acomparable plasmid without vgb (i.e ., the vgb strains). This difference was even more profound(up to 30-fold) in post-stationary phase ( i.e ., 24 h)cultures. These results are all in agreement withprevious studies, showing a late-culture-phase de-pendence of VHb expression and thus its effects oncell growth, metabolism and products formation[30, 44, 51, 76, 79, 91].

The authors are grateful to the Scientific and Techni-cal Research Council of Turkey (TUBITAK) for the fi-nancial support (107T478) which culminated in thiswork. A.G.K. gratefully acknowledges a generous fel-lowship provided by TUBITAK during the course of the study. Our special thanks go to Prof. Benjamin C.Stark (Illinois Institute of Technology) from whoselaboratory the plasmids pMK57 and pMK79 origi-nated and to Dr. Alejandro Rooney, the curator of bacterial stock cultures at the United States Depart-ment of Agriculture (USDA, Peoria, IL), for supply-ing us with Citrobacter freundii (NRRL B-2643) andErwinia herbicola (NRRL B-3466).

The authors have declared no conflict of interest.

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