Review High Cell-density Fed-batch Fermentation Using Escherichia Coli

Abstract

For recombinant protein production in Eastward. coli fed-batch cultures, post-induction conditions have great influence in the quantity and quality of the production. The nowadays paper covers the effect of different factors affecting the cellular environment in recombinant aldolase (rhamnulose-1-phosphate aldolase, RhuA) production. An operational mode employing an exponential addition profile for constant specific growth charge per unit has been analyzed, in social club to understand and ascertain possible modifications with influence on post-consecration cellular beliefs. A abiding improver profile has been demonstrated to return college specific aldolase product than the exponential addition profile, probably due to a more constant environs for the cells. On the other hand, amino acrid (leucine) supplementation has proven to increment poly peptide quality in terms of action units (U) per unit mass of RhuA (U mg⁻¹ RhuA), alleviating metabolic overload. Based on the above, a production process was set upwards and scaled up to pilot constitute. Resulting production was double that of a standard laboratory operation, 45,000 U Fifty^−ane, and almost all the protein retained the 6xHis-tag with the highest quality, 11.iii U mg⁻¹ RhuA.

Introduction

Microbial protein production is i of the battle horses of modern Biotechnology. For relatively simple proteins with lack of post-translational modifications, East. coli is ane of the preferred expression systems, especially for the production of industrially relevant enzymes [10, 23].

From a productivity bespeak of view, high-jail cell density cultures can be obtained by fed-batch functioning. Exponential feeding has been commonly employed to obtain a desired specific growth rate (μ) before induction of expression [10, 28]. In that case, the process is operated under glucose (or other essential substrate) limiting weather, which has the reward of continuously providing the required corporeality of nutrients to sustain growth and achieve high productivities [nineteen, 28]. Even so, during induction of the target protein, overexpression under carbon source limiting conditions (like exponential feeding) tin can lead to cellular stress situations conducive to proteolysis, with the objective to obtain additional resources for growth and maintenance [7, viii, 11, 16], supporting both overexpression of recombinant protein and heterologous Deoxyribonucleic acid replication. On the other mitt, the high amino acid demand during protein synthesis imposes a metabolic overload to the microbial prison cell [4, xx]. This is peculiarly important if the target protein has an amino acid composition profile significantly different that the mean of the Eastward. coli cytoplasmic proteins, conducive to disturbances in the amino acrid synthesis network [9, 15]. On the other hand, the shortage of precursors during induction of overexpression, as well as the abnormal accumulation of proteins, atomic number 82 to stringent response [2, 6, 22, 26].

Thus, from a process development signal of view, it is essential to ascertain proper operation conditions that could alleviate the in a higher place-mentioned furnishings, by ensuring an appropriate surroundings for jail cell growth and expression. Unlike publications have dealt with the definition of operational conditions to be scaled upwards, and minimization of the negative result of foreign poly peptide overexpression on growth and overall yields [10, 21, 23]. The suggested alternatives involve, among others, medium optimization past selective amino acid supplementation earlier consecration [ix, 13], and use of unlike feeding policies subsequently pulse induction, such equally linear or constant feeding [3, 27].

Our research group has been working on the production of recombinant aldolases using an auxotrophic strain during the terminal years, covering aspects such every bit production strategies [17], modeling and control [xviii]. As a consequence, a single isopropyl β-D-i-thiogalactopyranoside (IPTG) pulse induced fed-batch civilization methodology was established. Starting from the knowledge of the system, the master objective of the present work was to develop an operational methodology for improving production that could be translated to pilot plant and industrial calibration. The growth and induction strategies volition exist postulated, taking into account ease of implementation, moderate consumption of resource and ability to continue a favorable cellular environment. Improvements tested at bench scale will later exist scaled to a pilot (l L) production procedure.

Materials and methods

Bacterial strain and plasmids

A M-12 derived strain E. coli M15∆glyA [pREP4] harboring the vector pQEαβrham was used for rhamnulose 1-phosphate aldolase (RhuA) overexpression. The organisation is based on glycine auxotrophy, avoiding the need of antibiotic supplementation to ensure plasmid stability [25]. Transcription of the rhaD gene is under the control of the stiff T5 promoter in a low re-create number plasmid derived from pQE-xl (Qiagen, Hilden, Germany), and aldolase is expressed as a fusion protein to a 6xHis tag, assuasive further easy affinity purification.

Reagents

A stock solution of kanamycin (Sigma, St. Louis, Mo, USA) 100 mg mL^−one was prepared in milliQ water, filter sterilized and stored at iv °C. Stock solutions of ampicillin (Sigma) 100 mg mL⁻¹ were prepared in 50 % (v/v) ethanol, filter-sterilized, and stored at −twenty °C. IPTG was purchased from Sigma and a 100 mM stock solution was prepared using milliQ water as solvent, filter-sterilized and stored at −20 °C.

Media limerick

LB medium, with a limerick of ten g 50⁻¹ peptone, 5 m L^−one yeast extract and 10 yard 50^−one NaCl, was used for the preinoculum preparation.

A divers mineral medium (DM), utilizing glucose equally the sole carbon source, was used for inocula, for milkshake flask experiments and for all bioreactor cultivation experiments.

The medium for shake flask cultures was composed of 5 g Fifty⁻ⁱ glucose, 2.97 g 50^−one 1000₂HPO_iv, 0.59 chiliad Fifty⁻¹ KH_twoPO₄, 0.46 yard L⁻ⁱ NaCl, 0.75 grand L⁻¹ (NH₄)_twoAnd then_four, 0.11 chiliad L^−ane MgSO_iv·7H₂O, 0.006 m L^−ane FeCl_three, 0.025 grand 50⁻¹ thiamine, 0.001 chiliad L^−one CaCl₂·2H_twoO and 0.eight mL L⁻¹ of trace elements solution.

The batch stage of bioreactor cultivations was composed of twenty g 50⁻¹ glucose, 11.ix g L^−one Chiliad₂HPO₄, 2.iv g L⁻ⁱ KH₂PO₄, one.8 yard L⁻¹ NaCl, iii grand L⁻¹ (NH₄)₂And so_four, 0.45 grand L⁻¹ MgSO₄·7H₂O, 0.02 g L⁻¹ FeCl₃, 0.1 1000 L⁻¹ thiamine, 0.004 1000 50⁻¹ CaCl₂·2H₂O and ii.86 mL L⁻ⁱ of trace elements solution.

The feed medium for demote scale high-cell-density fermentations consisted of 487 chiliad L⁻¹ glucose, 9.56 g 50⁻¹ MgSO₄·7H₂O, 0.5 thou L^−one FeCl₃, 0.34 g L^−one thiamine, 0.089 g L⁻¹ CaCl₂·2H₂O, 63 mL L⁻ⁱ trace element solution and 0.5 mL L⁻¹ of antifoam (Sigma).

The feed medium for airplane pilot institute civilization independent: 300 1000 L⁻¹ glucose, 6.75 1000 Fifty⁻¹ MgSO₄·7H₂O, 0.three g L⁻ⁱ FeCl_three, 0.3 thou L^−one thiamine, 0.06 g L⁻ⁱ CaCl_ii·2H₂O, 40 mL L⁻¹ trace element solution and 0.5 mL Fifty⁻¹ of antifoam (Sigma).

The trace element solution composition contained (g L⁻¹): 0.042 AlCl_iii·6H₂O, 1.74 ZnSO₄·7H_twoO, 0.sixteen CoCl₂·6H₂O, 1.half-dozen CuSO₄, 0.01 H₃BO₃, 1.42 MnCl₂·4H₂O, 0.01 NiCl₂·6H₂O, 0.02 Na_twoMoO₄ [18].

Phosphates were non included in the feeding solution in order to avoid co-precipitation with magnesium salts. Instead, a full-bodied phosphate solution containing 500 g L⁻¹ Chiliad_iiHPO₄ and 100 thou 50⁻¹ KH₂PO₄ was pulsed during the fed-batch stage when necessary to avert their depletion.

Cultivation conditions

Inocula preparation

Pre-inocula were grown from glycerol stocks in 10 mL LB medium with ampicillin 100 μg mL^−ane and kanamycin 100 μg mL^−ane for 14–xvi h at 37 °C, 150 rpm. Inocula were grown in 2 × 100 mL milk shake flask cultures at 37 °C, 150 rpm in defined medium (DM) until OD_600nm was 1.2 (4.5 h).

Standard fed-batch cultures

For standard bioreactor fed-batch experiments, eighty mL of inoculum culture were transferred to the fermentor containing 720 mL of divers medium. Cultures were carried out using a Biostat^® B bioreactor (Sartorius, Goettingen, Deutschland) equipped with a 2 50 fermentation vessel. The pH was maintained at 7.00 ± 0.05 by adding 15 % NH_fourOH solution to the reactor. The temperature was kept at 37 °C. The pO2 value was maintained at fifty % of air saturation past adapting the stirrer speed between 350 and 1,120 rpm and supplying air (enriched with pure oxygen when necessary) at a space velocity of 2 vvm. The end of the batch stage was identified by a reduction in the oxygen consumption rate and an increment in pH.

Once the substrate was consumed, a fed-batch phase started supplying the feedstock solution with a microburette MICRO BU 2030 (Crison Instruments, Alella, Catalunya, Kingdom of spain) equipped with a 2.5 mL syringe (Hamilton, Reno, NV, USA). The specific growth rate (μ) was kept abiding using detached feed additions as an approximation to a predefined exponential feeding profile [five], according to:

$$ \Updelta 5\; = \;\frac{{X_{0} \cdot V_{0} }}{{S_{f} \cdot Y_{Ten/S\;ap} }} \cdot \left( {\exp \left( {\mu_{set} \cdot t} \right) - 1} \right) $$

where: ∆V, volume to be added at fourth dimension t; X₀ and V₀, biomass concentration and liquid volume in the fermentor at time of the previous addition; S_f, glucose concentration in the feed solution; Y_{Ten/S ap}, apparent yield biomass/glucose; μ_set, desired specific growth rate; t, fourth dimension since previous add-on. The value of μ_set used was 0.22 h⁻¹.

Induction of the cultures was carried out with an IPTG pulse. The exponential feeding profile was maintained until glucose started to accumulate in the bioreactor. To avoid glucose concentrations higher than 0.v g L⁻¹, medium feeding was manually and periodically interrupted and restarted as a role of glucose off-line measurements.

Modifications of the standard process

• Model based culture. A modification of the standard performance was to use predictions of a mathematical model to anticipate glucose accumulation acting on the feeding flow rate. The procedure was like the standard, but feeding was stopped and restarted according to model predictions instead of from off-line analysis. This strategy allows a closer approximation to exponential feeding than the standard i, due to more than frequent suspension sequences, improving procedure automation and reducing nutritional requirements (35 % less nutrients) [xviii].

• Constant feeding flow rate. Some other modification of the post-induction standard performance was to utilize a abiding feeding flow rate during the consecration stage.

• Amino acid supplemented cultures. Standard cultures with amino acid supplementation were performed by adding the required amounts of each of the post-obit amino acids: histidine (22 mg L⁻ⁱ), tryptophan (17 mg L^−one), threonine (50 mg L^−ane), leucine (70 mg L⁻¹) and glycine (60 mg L^−ane).

Fed-batch fermentation at pilot scale

For airplane pilot establish fermentations, a Biostat^® UD50 bioreactor (Sartorius) with a total capacity of 50 L was employed. 200 mL of a 1.5 50 laboratory calibration civilisation were inoculated into 30 L of divers medium. Media composition, too as batch and fed-batch functioning mode, was similar to laboratory cultures. The airplane pilot plant-specific conditions were as follows: (ane) a programmable pump was employed for fed-batch feeding; (ii) after reaching an OD of threescore, a pulse of 2.7 m of leucine was added (representing twenty % of the necessary for the expected aldolase titer), and the culture was induced past an IPTG pulse of 476 mg (3 μmol IPTG g⁻¹ DCW); (3) an approximately abiding feeding profile was implemented later on induction, corresponding to 0.5–0.4 g glucose one thousand⁻¹DCW h⁻ⁱ.

Downstream processing

The fermentation broth of laboratory scale cultures was centrifuged at 10,000 rpm for 20 min at 4 °C using a Beckman J2-21 M/Due east centrifuge. Harvested cells were resuspended in lysis buffer: 43 mM Na_twoHPO₄, vii mM NaH₂PO_iv, 20 mM Imidazol, 300 mM NaCl (pH = eight) at a ratio of 1 mL buffer :0.3 g harvested cells. Resuspended cells were lysed past one-shot high-pressure level disruption (Constant Systems LTD I Shot) at 2.57 kbar and at constant temperature of 4 °C. The rough cell lysate was centrifuged at xiv,000 rpm for 35 min at 4 °C and cell debris was rejected. Enzymatic activity of the supernatant was measured, and sodium azide was added to proceed a concentration of 0.02 % (west/w) to avoid biological deposition of the clear lysate.

For pilot constitute production, culture broth was centrifuged at 10,000 rpm in a CSA-ane Gea Westfalia Separator. Harvested cells were resuspended in lysis buffer to reach an optical density (OD) of 100, and lysed in a continuous high pressure level cell disruption system (Constant Systems TS5) at 2.57 kbar and a abiding temperature of 4 °C.

RhuA purification-immobilization

Aliquots (10 mL) of clear lysate at the appropriate activity concentration were employed for one-step purification immobilization on Co-IDA support (Chelating Sepharose FF Amersham Biosciencies-GE Healthcare with Co²⁺ chelated onto it). One mL sample was used every bit reference and was kept nether balmy horizontal agitation at 4 °C. Its action was measured both at the beginning and finish of the immobilization process. The second sample (ix mL) was added to 1 mL of Co-IDA support, and the residual activity of the break and supernatant was measured until the adsorption–desorption equilibrium was reached.

Analytical procedures

Monitoring bacterial growth

Growth was followed by optical density measurements at 600 nm (OD_600nm). The samples were diluted in deionised water until the measurement was inside the linear range of the spectrophotometer. The dry cell weight (DCW) was measured by centrifugation of aliquots of the goop. The pellets were washed twice with deionised water and dried at 110 °C until abiding weight. As a consequence of a calibration bend, 1 OD_600nm was found to be equivalent to 0.three chiliad L^−one DCW.

Total cell number was adamant using period cytometry. All measurements were performed with a Guava EasyCyte Mini cytometer. Culture broth samples were diluted to a cellular concentration between 10⁴ and 10⁵ cell mL⁻¹ (OD 10⁻⁵–10^−four) to ensure proper counting. Samples were candy by quadruplicate and results expressed equally the arithmetic mean.

For other determinations, one milliliter of civilisation was centrifuged. The supernatant was then used for glucose, organic acids, and ammonium and phosphate measurements. Glucose and organic acids were analyzed past HPLC (Hewlett Packard 1050) on an Aminex HPX-87H (Bio-Rad, Berkeley, CA, USA) cavalcade at 25 °C with IR detector (HP 1047) using xv mM H₂And so₄ (pH = 3.0) as eluent at a menstruation rate of 0.6 mL min⁻¹. Ammonium and phosphates were determined past colorimetric kit assays (Dr. Lange, Basingstoke, Uk) post-obit the supplier instructions.

To quantify the product concentration during cultures, broth samples were withdrawn and centrifuged. The pellet was resuspended in 100 mM Tris·HCl (pH = vii.five) to a last OD_600nm = 3 for enzyme conclusion. Cell suspensions were placed in water ice and sonicated using a Vibracell^® model VC50 (Sonics & Materials, Newtown, CT, Usa), with four 15 s pulses and 2 min intervals in water ice between each pulse,. Cellular debris was removed by centrifugation and the clear supernatant was collected for product analysis.

Product quantification

Total protein content was determined using a Coomassie^® Protein Assay Reagent Kit (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). To make up one's mind the percentage of RhuA amongst the rest of intracellular soluble proteins, 12 % polyacrylamide SDS-Page gels were performed in a Miniprotean^® Two musical instrument (Bio-Rad) according to the manufacturer'due south instructions and quantified past Kodak Digital Science^® densitometry software.

Conclusion of RhuA activity was carried out every bit described previously [24]. I unit of measurement of RhuA activeness was defined as the amount of enzyme required to convert one μmol of rhamnulose i-phosphate in DHAP per infinitesimal at 25 °C under the analysis conditions.

Results and give-and-take

Standard fed-batch cultures for aldolase production (in the present work rhamnulose-1-phosphate aldolase, RhuA) were performed as indicated in the "Materials and methods" section.

Their chief characteristics were: (1) fed-batch growth using a predefined exponential profile; (2) pulse IPTG consecration; and (three) after induction, once glucose was detected at levels effectually 0.5 g L⁻¹, feeding policy was interrupted and restarted according with off-line glucose analyses. This operational procedure avoids glucose aggregating, which could favor partial proteolysis of the target protein with loss in protein quality [17]. A modification of the above procedure was to employ predictions of a mathematical model to conceptualize glucose accumulation acting on the feeding flow rate. As previously reported [xviii], protein production was similar, with the added value of improving process automation and reducing nutritional requirements (35 % less nutrients).

The ratio inducer/biomass at the induction moment affects protein quality, measured in terms of activity units (U) per unit mass of RhuA, U mg^−oneRhuA. Figurei presents this dependency. The bodily selected value of 3 μmol IPTG g⁻¹ DCW corresponds to a maximum of around 8.8 U mg^−oneRhuA. Under the indicated conditions, standard cultures yielded an average of 950 U g⁻¹DCW and 108 mg RhuA g^−aneDCW.

Fig. ane

Effect of the inducer-biomass ratio I/10 (μml IPTG g⁻ⁱ DCW) on RhuA aldolase quality (U mg⁻¹RhuA) in fed-batch standard cultures

Fig. 1

Consequence of the inducer-biomass ratio I/Ten (μml IPTG g⁻¹ DCW) on RhuA aldolase quality (U mg⁻¹RhuA) in fed-batch standard cultures

As pointed out in the Introduction section, knowledge of the system will exist used to propose modifications at both operational and nutrient requirements level, with the aim of setting an operational procedure to exist standardized and translated to higher calibration production.

Post-consecration behavior

The analysis of post-induction cultures led to some conclusions about the applied feed policy during protein overexpression. The first one was observed when growth was analyzed both in terms of optical density and flow cytometry. Figure2 shows an instance of both measurements for non-induced stage (a) and induced stage (b). In the non-induced flow, both measurements are coincident, meaning that OD increment is due to an increase in the number of cells. Even so, after induction, cell concentration measured by period cytometry is much lower than the value obtained from OD measurements. The same result was observed for all the cultures, indicating that the OD increase is not just due to changes in the number of cells, merely besides to other factors similar changes in shape or other properties. One effect of the above observation is that the nutritional requirements tin be causeless to be different for induced and non-induced cells.

Fig. ii

Jail cell concentration from OD measurements (filled circle) and jail cell concentration from period cytometry (foursquare). (a) Evolution along feeding time for a non-induced culture; (b) Evolution with time subsequently induction for civilization induced at I/X = 3

Fig. 2

Jail cell concentration from OD measurements (filled circle) and cell concentration from menstruation cytometry (square). (a) Evolution forth feeding time for a non-induced civilization; (b) Development with time later on consecration for culture induced at I/X = 3

A second experimental finding can be seen in Fig.3. After induction, specific growth rate, calculated from OD data, had an initial increment, although nutrients flow charge per unit did non take substantial changes from the last moments of non-induced stage. After, at that place was a sustained decrease of specific growth rate during protein expression until growth arrest, and therefore nutritional requirements likewise changed.

Fig. 3

Specific growth charge per unit (μ) before and after consecration for a standard fed batch culture

Fig. iii

Specific growth charge per unit (μ) before and after induction for a standard fed batch civilisation

In summary, an exponential feeding afterwards induction implies an excess of nutrients, considering the nutrients supply is not consistent with culture growth. Every bit a result, glucose starts to accrue and feeding must be stopped several times to maintain low glucose levels as desired. The alternate start and stop of feeding produces sudden changes in the cellular environment so that cells must readapt continuously, and is thus an boosted factor for cellular stress during poly peptide overproduction.

Culling mail service-induction feeding policies

The proposed alternative for post-induction policy was to replace exponential add-on profile by a constant flow rate. The aim is avoiding frequent feed stop-start sequences as a upshot of the command of glucose concentration bellow 0.5 g Fifty^−one and to keep a more than homogeneous environment for the cells.

At the terminate of the non-induction stage, the glucose fed per gram of dry cell weight and hour was calculated from the glucose menstruum rate and the biomass concentration according to the following equation:

$$ F_{specific} \left( {\frac{{g_{gluc} }}{{g_{DCW} \cdot h}}} \correct) = \frac{{F_{gluc} \left( {\frac{{g_{gluc} }}{h}} \correct)}}{{X\left( {\frac{{g_{DCW} }}{L}} \right)\cdot V \left( L \right)}}, $$

where F_specific is the specific flow rate of glucose to be fed; F_gluc is the glucose period charge per unit at the end of the non-induced stage; and X and 5 are the dry out cell weight concentration and the liquid volume, respectively, at the finish of the non-induction phase.

The value for standard cultures was 0.55 chiliad glucose k⁻¹ DCW h⁻¹, and this was assumed, as a first approach, to be enough for poly peptide overexpression and cell growth and maintenance during consecration stage. Working with a feeding flow rate of 0.55 g glucose g⁻¹ DCW h^−one, glucose did non start to accumulate until the terminate of the induction phase and after the maximum production of RhuA was attained, showing that this feeding rate was quite equilibrated between excess and defect of nutrients.

Four experiments were compared: (1) a standard experiment; (2) a model based experiment; (3) a abiding feeding flow rate (0.55 g glucose g⁻¹ DCW h⁻¹) civilization; and (4) a constant feeding menstruation rate (0.27 g glucose one thousand^−one DCW h⁻¹, l % of the value of experiment 3) culture.

Experiments 1–3 are different in the interruption frequency of the feed: model based control led to college frequency of interruptions than standard operation, and the constant flow rate experiment has no interruptions of the feed. The fourth experiment is presumed to take a shortage of nutrients because of the low value of flow rate used.

The corporeality of RhuA produced per unit mass of DCW is presented in Fig.4, equally a percentage of the maximum obtained in a standard civilisation. It seems clear that reducing feed to one half (0.27 g glucose g^−ane DCW h⁻ⁱ) has a negative effect on poly peptide production. Apropos the results of the other 3 experiments, information technology can be seen in Fig.4 that the highest amount of RhuA produced is in the constant catamenia rate experiment (no interruptions of the feed), and that the everyman production is in the model based experiment (feeding more oft interrupted). Standard culture showed a product in betwixt, because feeding was non interrupted as oft as in the model based culture.

Fig. 4

Intracellular aldolase profiles for unlike feeding policies during induction phase expressed as percentage of maximum in a standard culture. (circle, spaced hyphen) standard civilisation; (diamond, hyphen with dots) model-based; (triangle, solid line) constant feeding at 0.55 thousand glucose one thousand⁻¹ DCW h⁻¹; (square, dots)abiding feeding at 0.27 thou glucose grand⁻¹DCW h⁻ⁱ

Fig. four

Intracellular aldolase profiles for different feeding policies during induction phase expressed as pct of maximum in a standard civilisation. (circle, spaced hyphen) standard civilization; (diamond, hyphen with dots) model-based; (triangle, solid line) constant feeding at 0.55 thou glucose g⁻¹ DCW h⁻¹; (square, dots)abiding feeding at 0.27 thousand glucose one thousand⁻¹DCW h⁻¹

This is a event in favor of the hypothesis that cell environs is more than constant when constant feeding is used, reducing the stress of the cell and yielding a college product of RhuA per unit of measurement mass of DCW. Chen et al. [3] reported a similar behavior for glucosamine synthase production.

Amino acid supplementation for protein quality comeback

As pointed out in Introduction section, the loftier amino acid demand during protein overexpression constitutes a metabolic overload. The coordinated addition of amino acids during consecration phase, based in a pick of the candidate amino acids from the target protein composition, has been suggested every bit an effective tool to alleviate cellular stress [ix, 14, fifteen]. In the previous section, information technology was shown that maintaining medium homogeneity for a long time had a positive result on aldolase quantity. In order to improve the enzyme quality (Units per mg of aldolase), medium supplementation with required essential amino acids was explored.

The amino acid composition of a subunit of the rhamnulose-1-phosphate aldolase tetramer was obtained from NCBI (National Center for Biotechnology Information) data bank [12]. Effigyfive shows the relative frequency of each amino acrid in the master structure of the aldolase, together with the mean E. coli cytoplasmic proteins composition [one].

Fig. 5

Amino acid relative frequency (%) in the main structure of RhuA and the average cytoplasmic proteins of Due east. coli

Fig. 5

Amino acid relative frequency (%) in the primary structure of RhuA and the average cytoplasmic proteins of Eastward. coli

The selected amino acids for supplementation were histidine (H), tryptophan (Westward), threonine (T), due to the biggest differences in relative corporeality; leucine (L), highly present in RhuA, and glycine (G), due to auxotrophy. According to the literature [14], the amount of every amino acid to be added was calculated to be twenty % of that necessary for the synthesis of the expected aldolase in a standard civilisation (around iii g of poly peptide). This xx % is estimated with the aim of avoiding possible inhibitions in the amino acids synthetic pathway. Supplementation was performed in fed-batch cultures but earlier induction. Combinations of the above (or other) amino acids could also be investigated.

The obtained results are summarized in Tableane. Final biomass concentration was very like in all cases. The highest quality of protein equally activity units per unit mass of RhuA was obtained for the leucine addition case. Leucine improved the amount only slightly, 5 %, but the combination of amount and quality, thirty %, yielded the highest specific activity, 35 % more than standard civilisation.

Amino acrid supplemented cultures: Aldolase quality, amount and specific activeness expressed every bit percentage of maximum in a standard culture

	Protein quality U mg−one RhuA (%)	RhuA amount mg RhuA g−1 DCW (%)	Specific activity U g−ane DCW (%)
Standard culture	100	100	100
Histidine add-on	fourscore	150	105
Leucine addition	130	105	135
Threonine improver	110	130	125
Triptophane addition	120	105	115
Glycine addition	80	140	120

	Protein quality U mg−1 RhuA (%)	RhuA corporeality mg RhuA thou−ane DCW (%)	Specific activity U thou−1 DCW (%)
Standard culture	100	100	100
Histidine addition	80	150	105
Leucine addition	130	105	135
Threonine improver	110	130	125
Triptophane add-on	120	105	115
Glycine addition	lxxx	140	120

Amino acrid supplemented cultures: Aldolase quality, amount and specific action expressed every bit per centum of maximum in a standard culture

	Poly peptide quality U mg−1 RhuA (%)	RhuA amount mg RhuA thou−1 DCW (%)	Specific activity U chiliad−ane DCW (%)
Standard culture	100	100	100
Histidine improver	80	150	105
Leucine addition	130	105	135
Threonine addition	110	130	125
Triptophane addition	120	105	115
Glycine addition	80	140	120

	Protein quality U mg−one RhuA (%)	RhuA amount mg RhuA g−1 DCW (%)	Specific activity U thou−i DCW (%)
Standard civilisation	100	100	100
Histidine addition	80	150	105
Leucine addition	130	105	135
Threonine addition	110	130	125
Triptophane addition	120	105	115
Glycine addition	80	140	120

Pilot plant functioning

From the above experiments, it can exist seen that a more than constant cell environment led to an comeback of the corporeality of protein produced per 1000 DCW, and that the improver of leucine led to a ameliorate quality of the protein than in the standard cultures. These aspects should be taken into account to improve the product processes, mainly at larger scales, such every bit airplane pilot and industrial.

Aldolase product was translated to a airplane pilot scale (50 L fermentor), operating in fed-batch way as indicated in "Materials and methods". The operational strategy was based on all the above-presented results. Consequently, before induction, leucine was added at a concentration of 70 mg L⁻¹. Moreover, an approximately abiding feeding profile was implemented afterward consecration. In this example, it was between 0.5 and 0.4 g glucose chiliad^−ane DCW h⁻¹, slightly lower than at laboratory calibration, in order to prevent possible glucose accumulation at the end of the induction stage.

The time profiles of biomass, glucose and aldolase concentration and aldolase activity are presented in Fig.vi. Biomass growth was maintained until 3.5 h after consecration without glucose accumulation, and reached biomass concentrations similar to those in standard laboratory experiments. On the other hand, specific production of RhuA reached high levels in both mass and activity units: 160 mg RhuA 1000^−one DCW and 1800 U g⁻¹DCW, which ways a quality of 11.3 U mg RhuA^−ane.

Fig. 6

Fourth dimension profiles for the pilot plant RhuA product. The dashed pointer indicates the induction moment. (filled circumvolve) Optical density; (square) Glucose concentration; (filled triangle) Specific activity; (inverted triangle) RhuA mass specific amount

Fig. 6

Time profiles for the pilot plant RhuA production. The dashed pointer indicates the induction moment. (filled circle) Optical density; (square) Glucose concentration; (filled triangle) Specific activity; (inverted triangle) RhuA mass specific corporeality

The results can be better understood if the pilot institute behavior is compared with the different production strategies: standard culture, model-based feeding, and leucine addition. These are presented in Fig.7a, b, c. Quality of the protein enhanced as a consequence of leucine addition. The obtained levels, eleven.3 U mg⁻ⁱ RhuA, are similar to the laboratory culture with leucine improver (Fig.7a). This is an improvement of xxx % with respect to standard operation. On the other paw, nutrient addition at an almost constant rate maintained a more homogeneous surround for the cells, thus assuasive for higher protein corporeality per g DCW (Fig.7b). The l % increment over standard culture is college than the 20–25 % observed at laboratory scale for abiding feeding. To explain this fact, one can hypothesize near the differences in feeding organisation. Feeding was performed employing a programmable pump in the instance of the pilot found, which could ensure a more abiding menses (and thus, homogeneous surround for the cells) than the microburette used at the laboratory scale, with alternate accuse and feed steps. Another factors could also be taken into account. Although the global homogeneity was proven to be good in previous studies, some local gradients may exist, influencing the behavior of the arrangement.

Fig. 7

Performance of the different operational strategies and the pilot plant operation in terms of (a) protein quality, (b) mass RhuA amount and (c) specific activity

Fig. 7

Functioning of the different operational strategies and the pilot found operation in terms of (a) protein quality, (b) mass RhuA amount and (c) specific activeness

The combination of both factors gives the activity per g of DCW presented in Fig.7c. A 100 % increase of specific activity (U g⁻ⁱ DCW) over the standard culture was obtained in pilot institute functioning.

From the global perspective of the process, the integrity of the protein produced (maintenance of the six-histidine tag) was tested past submitting prison cell lysate to metal-chelate affinity chromatography, every bit explained in the "Materials and methods". RhuA was nearly totally attached to the support, giving recovery yields higher than xc %. This yield allows both recovery of the produced aldolase, likewise as its direct utilise as immobilized biocatalyst.

Conclusions

In the nowadays work, different factors affecting recombinant aldolase production in E. coli fed-batch cultures accept been assessed.

The ratio of the inducer concentration to the biomass concentration affected the quality of the protein produced. In the case of this study, a value around 3 μmol of IPTG per chiliad of DCW gave the all-time results.

Information technology was experimentally noted that the increases in OD later induction were the result of an increment in number of cells and changes in cell morphology. This finding, together with the decrease of specific growth rate calculated from OD measurements, indicated that exponential feeding after induction was non the best operational strategy. A more than constant feeding contour after induction has been demonstrated to have a favorable effect on the amount of aldolase produced per unit mass of DCW. Temporal homogenization of the jail cell environment seems to be the reason for less cellular stress leading to higher protein yields.

On the other paw, among the amino acids studied for supplementation before consecration, leucine improver was shown to positively affect the quality of the protein as activeness units per unit mass of RhuA.

The combination of both improvements was employed to design a proper operational procedure for airplane pilot plant production. In this case, the obtained protein was of the highest quality, 11.3 U mg RhuA⁻¹ with a production of 45000 U 50⁻ⁱ, double that of the standard one. The obtained aldolase has been near quantitatively purified (> xc %) by affinity chromatography.

The principles and methodologies developed here could be extended to the product of other proteins employing similar expression systems.

Acknowledgments

This piece of work has been supported by the Spanish MICINN, projects CTQ2008-00578 and CTQ2011-28398-C02-01 and by DURSI 2009SGR281 Generalitat de Catalunya. The Department of Chemic Engineering of UAB constitutes the Biochemical Engineering Unit of the Reference Network in Biotechnology of the Generalitat de Catalunya (XRB). Alfred Fernández-Castané acknowledges UAB for a predoctoral grant.

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