Studies on the expression of recombinant fuculose-1-phosphate aldolase in E. coli

Process Biochemistry 39 (2004) 1677–1684

Studies on the expression of recombinant fuculose-1-phosphate aldolase in E. coli Olga Durany, Glòria Caminal, Carles de Mas, Josep López-Sant´ın∗ Departament d’Enginyeria Qu´ımica, Escola Tècnica Superior d’Enginyeria, Unitat de Biocatàlisi Aplicada Associada al IIQAB (UAB-CSIC), Universitat Autònoma de Barcelona, Edifici Cn, Bellaterra 08193, Cerdanyola del Vallès, Spain Received 26 March 2003; received in revised form 14 July 2003; accepted 19 July 2003

Abstract Fuculose-1-phosphate aldolase (Fuc-1-PA) is a dihydroxyacetone phosphate (DHAP) dependent aldolase with potential application in chiral synthesis. The influence of the growth medium on the expression of the enzyme in Escherichia coli has been studied. Complex LB medium, a defined medium (MD) and a semi-complex medium (MSC) have been compared in order to maximize aldolase production. The defined medium produced highest expression levels (700 activity units (AU)/g of dry cell weight (DCW)). The optimal induced isopropyl-␤-thiogalactopyranoside (IPTG) concentration of 100 ␮M produces in the MD medium of 41 ␮mol/g dry cell weight of enzyme. © 2003 Elsevier Ltd. All rights reserved. Keywords: Recombinant aldolases; Fuc-1-PA; Growth medium; IPTG induction; E. coli

1. Introduction The potential utilization of aldolases as biocatalysts for the formation of C–C bonds of defined chirality has been widely reported as a suitable alternative to conventional strategies in the synthesis of carbohydrate derivatives [1–3]. Four dihydroxyacetone phosphate (DHAP) dependent aldolases have been proposed as a battery of biocatalysts able to catalyze the synthesis of diols of complementary stereochemistry [4,5]. These enzymes have been employed in the synthesis of dioxysugars, fluorosugars, azo and thio-sugars [6–9]. One of the main limitations for the industrial development of such processes is the commercial availability of the DHAP dependent aldolases. Consequently, it is necessary to develop economically feasible processes for the production of this family of aldolases. The present work has been focused on the overproduction of fuculose-1-phosphate aldolase (Fuc-1-PA) in recombinant Escherichia coli as a model member of this family. This enzyme catalyzes the selective formation of R–R diols (d-eritro compound) by aldol condensation of DHAP with a wide variety of acceptor aldehydes [4,5].

∗

Corresponding author. Tel.: +34-935811018; fax: +34-935812013. E-mail address: [email protected] (J. L´opez-Sant´ın).

0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00302-9

For the expression of intracellular recombinant proteins, in order to obtain high product yields, cultures with high biomass concentrations which exhibit at the same time high specific productivity are required. In both aspects, the culture media composition has been widely reported to play a significant role [10–12]. A well designed fermentation medium must contain all the necessary elements required for biomass synthesis. To ensure that, two extreme approaches have been commonly employed: complex media and defined media based on bacterial composition as well as nutrient to biomass yield coefficients. Working with defined medium (MD) allows easier control of culture evolution (aspects as limiting substrate availability and accumulation of toxic metabolic products accumulation) and makes it easy to employ growth strategies and obtain predictable and reproducible results. It should be mentioned that, in relation to high cell density cultures achievement, the best reported results correspond to controlled growth strategies employing defined media. Concentrations around 100 g dry cell weight (DCW)/l have been reported by different research groups [13–15] and have been raised to 150 g DCW/l with optimization of process control [16]. Recently, employing dialysis fermentation systems with defined media, cell densities in recombinant E. coli cultivation as high as 190 g DCW/l have been reported [17]. Defined media are also of interest

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in industry when robust fermentation control is required [18]. When considering specific yields of recombinant proteins, complex media that contain free amino acids from hydrolyzed proteins, vitamins and other products of cellular metabolism must be taken into account. It has been widely reported that this kind of media not only allows higher microbial growth rates thus improving process productivity by reducing fermentation time, but also often improve productivity in recombinant protein production processes as they alleviate the metabolic burden caused by recombinant expression [19,20]. A common intermediate approach consists in working on the basis of a properly designed defined medium but supplemented with extra nutrients, trying to maintain defined medium process control advantages but, at the same time, a faster growth and higher specific productivity. The provision of additional nutrients by supplementation of the medium with a complex source as casamino acids, peptones or yeast extract, to produce semi-synthetic or semi-complex media has found application in controlled strategies to achieve high cell density cultures for recombinant protein production [21–23]. A more time consuming but elegant approach, also based on the above described idea, is the enrichment of defined media with defined amounts of some amino acids considered of relevance in some routes of cell metabolism or largely required for the synthesis of the recombinant product [24–27]. On the basis of the reported literature, the selection of the optimal medium required in a recombinant production process will depend, basically, on the recombinant strain employed, the target protein to be produced and the growth strategy, making it difficult to generalise. Fuc-1-PA has been produced in complex LB medium by a recombinant strain of E. coli under control of the isopropyl-␤-thiogalactopyranoside (IPTG) inducible promoter trc [28]. In the present paper, employing the same microbial strain, the selection of a growth medium both to maximize specific Fuc-1-PA productivity and to be suitable for high cell density cultures is presented. The influence of inducer concentration is also studied.

2. Materials and methods 2.1. Strain and plasmid Escherichia coli XL1 Blue MRF’ [
(mcrA)183
(mcr CB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac (F’ proAB lacIq Z
M15 Tn10 (Tetr ))] harbouring plasmid pTrcfuc, for the expression of fuculose-1-phosphate aldolase, was a generous gift of Dr. E. Garc´ıa-Junceda of the Instituto de Qu´ımica Orgánica (CSIC, Madrid) [28]. Escherichia coli XL1 Blue MRF’ (Stratagene Co., San Diego, CA) is a thiamine auxotroph and requires its addition for growth in minimal media. Plasmid pTrcHis (Invitrogen,

San Diego) contains the trc promoter to allow a high expression of the target protein. This is a hybrid derived from promoters trp and lac, regulated by the lacO operon and the expression product of the gene lacIq . It carries the resistance gene against ampicillin, gene bla, as selection marker. Under this expression system proteins are expressed as fusion proteins to a six histidine tag allowing their easy one-step purification. 2.2. Media and growth procedures Ampicillin has been supplemented in all media as a selection marker, to a final concentration of 100 ␮g/ml. Cultivation conditions for the recombinant strain have been fixed at 37 ◦ C on a rotary shaker at 200 rpm. 2.2.1. Stock maintenance and inocula preparation The strain was conserved at −80 ◦ C in glycerol stocks, prepared using commercial Cryobilles (AES Laboratoire, France), from aliquots of exponential phase cultures of the strain in Luria–Bertani (LB) medium. Inocula for all shake flask comparison experiments were prepared by transferring one single glycerol ring to a 100 ml flask containing 15 ml of LB growth media and incubated overnight. 2.2.2. Growth media Luria–Bertani medium composition (g/l): peptone, 10; yeast extract, 5; NaCl, 10. As defined medium, a modification of that described by Yee and Blanch for shake flasks experiments has been employed [29]. Defined medium composition (g/l): K2 HPO4 , 13.23; KH2 PO4 , 2.65; NaCl, 1.33; (NH4 )2 SO4 , 5.47; MgSO4 ·7H2 O, 0.09; CaCl2 ·2H2 O, 0.06; FeSO4 ·7H2 O, 0.04; glucose, 10; ampicillin, 0.1; thiamine, 0.1 and trace elements solution, 16 ml. Trace elements solution composition (mg/l): Cl3 Al·6H2 O, 110; ZnSO4 ·7H2 O, 100; CoCl2 ·6H2 O, 160; CuSO4 ·H2 O, 40; H3 BO3 , 20; MnCl2 ·4H2 O, 400; NiCl2 ·6H2 O, 20; Na2 MoO4 , 100. Two mineral salt solutions were prepared and sterilized in an autoclave (120 ◦ C, 30 min): the trace elements solution and another one containing the required macro elements in a concentrated solution. Glucose was autoclaved separately (120 ◦ C, 15 min) in 100 g/l stock solution and also individual 100 mg/ml stock solutions of ampicillin and thiamine were prepared and sterilized through a 0.22 ␮m filter (Millex-GS, Millipore). When addition of yeast extract was required as a supplement in MD, it was added to the macro elements solution before sterilization. 2.2.3. Comparative experiments in shake flask Medium comparison experiments were performed in 1 l flasks containing 200 ml of growth media and, in all cases, inoculated to 0.3–0.35 U of optical density (OD) at 600 nm. For the induced cultures, it was made at the beginning of the exponential phase. Isopropyl-␤-thiogalactopyranoside was

O. Durany et al. / Process Biochemistry 39 (2004) 1677–1684

used as inducer at the concentrations indicated in the text, starting from a concentrated 100 mM solution. 2.2.4. Analytical procedures Cell growth during fermentation was monitored by measuring the absorbance of adequately diluted samples at 600 nm (KONTRON Uvicon 941plus Espectophotometer). In addition, dry cell weights were determined from aliquots of culture medium collected at different growth times. Cells were centrifuged (10 min, 4 ◦ C, 4000 × g) and resuspended in an equal volume of 0.9% NaCl solution. Finally, water washed cells were dried at 105 ◦ C until constant weight. A standard curve to convert OD experimental values to dry cell weigh conducted to a straight relationship of one absorbance unit equal to 0.27 g DCW/l. Fuc-1-PA soluble production levels in E. coli cell intracellular extracts were estimated by laser densitometric scanning of denaturing 12% polyacrylamide gels prepared basically as described by Laemmli [30]. Cell extracts were prepared by sonication (maintained on ice) of comparative cell suspensions corrected by biomass in order to contain 2.2 mg DCW/ml of homogenization buffer using four 30 s pulses (at 50 W) with a Vibra-cellTM Sonicator (Sonic & Materials Inc., Danbury, CT) and a microtip probe. This biomass sample correction allowed a comparison of the evolution of protein production and accumulation in soluble form during growth in different media. In addition, Fuc-1-PA activity levels were estimated by specific enzymic assay from E. coli cell intracellular extracts prepared by direct 0.5 ml culture sample sonication in the same conditions described above. The activity assay was a modification of the coupled enzymic system reported by Garc´ıa-Junceda et al. [28]. 1.972 mM of the natural substrate, fuculose-1-phosphate as dicyclohexy-

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lamine salt (Fuc-1-P (CHA)2 ), 0.136 mM NADH, 1.7 U/ml of glycerolphosphate dehydrogenase (GDH), 86.4 mM triethanolamine and 129.6 mM KCl are incubated at pH 7.5 and 25 ◦ C. After temperature stabilization, the enzymic reaction was initiated by addition of the corresponding aldolase sample, and the decrease in the absorbance at 340 nm was monitored. Results were corrected by blank assay measurement when no GDH was included. One unit of enzyme activity (AU) is defined as the quantity that catalyses the formation of 1 mmol DHAP/min. Fuc-1-PA activity was measured at different cultivation times after IPTG induction, allowing the determination of the recombinant enzyme production rate and specific yield (aldolase units per gram of DCW). The dicyclohexylamine salt of fuculose-1-phosphate was synthesized according to reported procedures [31].

3. Results and discussion 3.1. Growth in complex LB medium Fig. 1 shows growth in complex LB medium of the wild type E. coli XL1 Blue MRF’ and the recombinant strain transformed with the IPTG inducible vector carrying the fucA gene. Cultures were performed in shake flasks and induced with the indicated amounts of IPTG in the early exponential phase. In all cases the recombinant strain cultures showed a maximum specific growth rate (0.6 h−1 ) that was the same as the plasmid-free wild type one. Neither the maintenance of the antibiotic-selection plasmid or the extra expression of the target protein when induced cause a metabolic burden high sufficient to influence the maximal growth rate or the

OD 600nm

10

1

Wild type strain Recombinant strain (non-induced) Recombinant strain ( induction 50 µM IPTG) Recombinant strain (induction 500 µM IPTG) 0,1 0

5

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25

time (h) Fig. 1. Growth profile of the wild type and recombinant strains in LB medium.

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Table 1 Maximum aldolase activity for non-induced and induced cultures in LB medium

Wild type RS (non-induced) RS (50 ␮M IPTG) RS (500 ␮M IPTG)

Table 2 Maximum specific growth rates for the recombinant strain in defined medium supplemented with different amounts of yeast extract

Fuc-1-PA activity (U/g DCW)

Growth medium

µmax (h−1 )

Not detected 108 200 290

MD MD MD MD MD

0.345 0.560 0.654 0.740 0.725

growth profile. These results seem to indicate that the recombinant expression levels should be moderate in spite of the induction [19]. Intracellular Fuc-1-PA activity was measured and the maximum obtained values, corresponding to the end of the growth phase, are summarized in Table 1. These results indicate that the amount of aldolase obtained by IPTG induction represents a moderate over-expression level and also that recombinant expression in the absence of inducer must be taken into account. 3.2. Growth in defined (MD) and semi-complex (MSC) medium With the aim of evaluating the influence of medium type in bacterial growth and aldolase expression, an adaptation of a previously reported defined medium was included in the study [29]. This defined medium was supplemented with different amounts of yeast extract in a first approximation to evaluate the effect of complex supplementations in a defined medium. Yeast extract supplementation was chosen because it has been widely suggested as the most favourable for the expression and stability of recombinant proteins in E. coli when compared with the addition of other complex sources [32–34]. In addition, it has been reported to be more effective for improving cell yields in studies realized with an E. coli

+ 2 g/l yeast extract + 4 g/l yeast extract + 8 g/l yeast extract + 10 g/l yeast extract

XL1 Blue strain similar to that employed in this work [35]. On the other hand, some works have shown yeast extract to increase non-induced background expression from the lac promoter [36,37]. The time profile of the recombinant strain growth in the different media is presented in Fig. 2 and Table 2 summarizes the corresponding maximum specific growth rates. Growth in the defined medium was slower than in complex LB medium, and an addition of 4 g/l of yeast extract to the MD medium was necessary to obtain a specific growth rate similar to the complex medium. No significant improvements of growth rate were observed with higher yeast extract supplementations. The selection of a suitable growth medium for Fuc-1-PA production has to be done after determining its influence on specific recombinant aldolase expression. Three growth media were selected for aldolase expression studies: complex LB medium, defined MD medium and MD medium supplemented with 4 g/l of yeast extract (a semi-complex medium indicated as MSC). The addition of EDTA has been reported in the literature, allowing solubilization of higher amounts of medium components, a significant point when the final application of studied media could to be achieve high cell density cultures for optimized production processes [15,34]. As Fuc-1-PA is a metalloenzyme dependent of Zn2+ [38],

OD 600 nm

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1 MD medium MD medium+ 2 g/litre YE MD medium + 4g/litre YE MD medium + 8g/litre YE MD medium+ 10g/litre YE 0,1 0

5

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15

20

25

30

time (h) Fig. 2. Growth profile of E. coli XL1 Blue MRF’(pTrcFuc) in the defined medium supplemented with different amounts of yeast extract.

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All cultures were induced with 500 ␮M IPTG and, simultaneously, the temperature was reduced from 37 to 30 ◦ C as it has been previously reported that, at 37 ◦ C and working with LB media, overexpressed Fuc-1-PA is accumulated in inclusion bodies [28]. Although these aggregates were not observed in previously described work in LB media at 37 ◦ C neither by SDS-PAGE analysis of the insoluble intracellular fraction, nor by microscopic analysis of cell morphology (data not show), it was decided to reduce the working temperature to avoid their formation in all media included in this study [42]. The last intention was to compare total recombinant protein accumulation in terms of specific activity in the soluble intracellular fraction. No evidence of insoluble aggregates was detected in any media (data not shown). The growth profile and time evolution of intracellular recombinant protein accumulation after induction for each medium is presented in Fig. 3. No differences in growth were recorded when comparing the non-induced (Figs. 1 and 2) and induced profiles in any medium. As observed in the previous works in LB, no additional metabolic burden

3.3. Effect of the growth medium on the expression of Fuc-1-PA Once the host strain and recombinant protein expression system are determined, the optimal conditions for inducing recombinant expression under control of lac derived promoters depend not only on the quality of the growth media but also on other factors including the extent of the metabolic load caused by the overexpressed product under growth conditions, the growth time when inducer is added and the concentration of inducer employed [10,39–41]. Here, common conditions of work have been selected to compare the suitability of the different media on the expression of Fuc-1-PA and the required concentration of inducer to achieve maximal productivity in each case.

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Fig. 3. Biomass profile (䊉) and specific Fuc-1-PA activity () for the growth of the recombinant strain in the different media.

Specific activity AU/g DCW

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induction

Specific activity AU/g DCW

in order to evaluate the effect of such a sequestering agent on the stability and activity of the expressed protein, an additional fourth medium has been added: MD medium with addition of EDTA (0.033 g/l of Na2 -EDTA·2H2 O).

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affecting the strain growth was observed after induction. The target protein was accumulated as intracellular active protein during the growth phase but no extra expression was detected later in the stationary phase. It seems that, in all cases, the time profile of the accumulated specific Fuc-1-PA was correlated with bacterial growth. The accumulated aldolase specific activity at the end of the growth was clearly higher in the defined medium MD than in semi-complex (MSC) and complex (LB) media. It has been widely reported that growth rate influences recombinant production, the accumulation of well folded active recombinant proteins being higher at slow growth rates. The slower growth in defined media in comparison with the media containing complex sources could suggest an advantage for Fuc-1-PA specific activity accumulation. The volumetric activity led to the following values: 400 AU/l for LB medium, 808 AU/l for MSC medium and 1616 AU/l for MD medium. These data confirm the influence of the medium in Fuc1PA production, and the higher expression in MD medium. The densitometer scanning of SDS-PAGE of samples at the end of each growth (Fig. 4) confirms the results obtained by quantification of intracellular activity. Fuc-1-PA was accumulated to a level of approximately 30% total intracellular soluble protein content in MD media (both when EDTA was included and not), of 25% in MSC media and just of 15% in LB. In LB medium it is difficult to appreciate the increase of expression when compared with the non-induced control, showing that the expression system employed, based on pTrcHis plasmids (Invitrogen Co.), is difficult to regulate in complex media with significant levels of basal recombinant transcription. The lack of total repression observed in all media is an often reported behaviour when lac and de-

rived promoters are employed because the operator site is not continuously occupied by the lac repressor and there is generally a basal level of transcription of the lac genes [10]. The maximum specific activity was obtained in MD medium, around 700 AU/g DCW. The supplementation of MD media with EDTA neither influences the growth profile nor the intracellular soluble Fuc-1-PA accumulation (according to the densitometric analysis results) but, as it could be suspected, it affects the activity of the produced enzyme reducing the intracellular Fuc-1-PA activity to around one half. The addition of EDTA seems to limit the availability of Zn2+ for the synthesis of active Fuc-1-PA by the microorganism. As the total recovery of the metalloenzyme activity after treatment with sequestering agents seems to be difficult to achieve (if not impossible) and needs of additional steps in the downstream process [43], supplementation with EDTA will be avoided in future works. 3.4. IPTG concentration influence The effect of the IPTG concentration in each media has been studied by inducing the cultures with increasing IPTG concentrations. A comparison for the different media is presented in Fig. 5 plotting the Fuc-1-PA activity per gram of dry cell weight versus IPTG concentration. According to the accumulation profile in Fig. 3, the enzymic activities have been measured at the end of growth in order to attain maximal values. Although the optimal IPTG concentration when working with tac or other lac derived promoters for the expression of soluble cytoplasmatic recombinant proteins has been widely reported to be around 1 mM [10], our results show that, in all media, an aldolase activity near the maximum is obtained at

Fig. 4. SDS-PAGE of E. coli XL1 Blue (pTrcFuc) intracellular extract samples at the end of the growth in the different media. Lanes description: (a) M: MW markers, 1: LB non-induced, 2: LB 500 ␮M IPTG; (b) M: MW markers, 1: MSC non-induced, 2: MSC 500 ␮M IPTG, 3: MD non-induced, 4: MD 500 ␮M IPTG, 5: MD-EDTA non-induced, 6: MD-EDTA 500 ␮M IPTG.

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800 750

Specific activity (AU/g DCW)

700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 0

100

200

300

400

500

IPTG concentration (µΜ) Fig. 5. Effect of inducer concentration on specific Fuc-1-PA activity in the different media. LB medium (䉱), MSC medium (䊏), MD medium (䊉), MD + EDTA medium (䉬).

Table 3 Maximum specific Fuc-1-PA activity and optimal specific inducer amount for the different media Growth medium

Specific Fuc-1-PA activity (U/g DCW)

Specific IPTG (␮mol/g DCW)

LB MSC MD MD + EDTA

200 460 700 375

70 52 41 33

tively, the amounts of active protein per gram of biomass at the end of the growth. From the point of view of enzyme productivity, the defined medium can be chosen as the more suitable, with an expected production of 700 AU/g DCW when induced with 41 ␮mol IPTG/g DCW.

Acknowledgements relatively low inducer concentrations. IPTG concentrations higher than 100 ␮M have a reduced effect on Fuc-1-PA active protein production under this expression system. For production purposes, the amount of IPTG has to be reduced to the minimum in order to ensure the economical feasibility. In high cell density cultures, the amount of IPTG to induce the recombinant protein expression has to be selected according to the expected amount of biomass to be obtained [34]. These data are presented in Table 3, showing the aldolase activity in each medium induced at 100 ␮M IPTG, and the corresponding amount of IPTG per gram of cells at the end of the induced growth.

4. Conclusion The results obtained in this work emphasize the significant effect of the growth medium on the production of Fuc-1-PA by E. coli. The behaviour of the recombinant strain in the formulated MD and MSC media seems to be more suitable for the production of fuculose-1-phosphate aldolase than in complex LB medium increasing 3.5 and 2.3 times, respec-

This work has been supported by the Spanish Plan Nacional de I + D, project numbers BIO99-1219-C02-01 and PPQ2002-04625-CO2-01. The authors wish to thank Dr. Eduardo Garc´ıa-Junceda (Instituto de Qu´ımica Orgánica, CSIC, Madrid) for supplying the recombinant strain. The Department of Chemical Engineering of the Universitat Autònoma de Barcelona is the Unit of Biochemical Engineering of the Centre de Referència en Biotecnologia de la Generalitat de Catalunya.

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