WO2008037265A2

WO2008037265A2 - Method of fed-batch cultivation and recombinant protein expression in escherichia coli bl21(de3) in deuterated medium

Info

Publication number: WO2008037265A2
Application number: PCT/EE2007/000017
Authority: WO
Inventors: Kalju Vanatalu; Katrin Tomson
Original assignee: Tallinn University Of Technology
Priority date: 2006-09-29
Filing date: 2007-10-01
Publication date: 2008-04-03
Also published as: WO2008037265A3; EE200900028A

Abstract

A method for cultivation of E.coli BL21 and its derivatives in deuterated media in fed-batch culture and expression of recombinant DNA products is described. The method is based on the usage of inexpensive deuterated carbon sources succinate and acetate together. The simultaneous supply of these substrates at molar ratio of 4-8 (mole/mole) acetate/succinate to the culture enables good growth to high cell densities and expression in fed-batch culture. A specific fed-batch control algorithm is applied to automatically adjust the feeding rate to the rate of the utilization of carbon sources based on dissolved oxygen control.

Description

METHOD OF FED-BATCH CULTIVATION AND RECOMBINANT PROTEIN EXPRESSION IN ESCHERICHIA COLI BL21(DE3) IN DEUTERATED MEDIUM

Background of the invention

Technical field

The invention relates to a fed-batch method of cultivation of E. coli strain BL21(DE3) in deuterated growth media and expression of recombinant biomolecules by using a mixture of acetate and succinate as carbon sources and controlling the concentration of acetate and succinate for providing suitable conditions for such growth.

Background art

The gram-negative bacterium Escherichia coli (E. coli) is the most commonly used organism for heterologous protein production. E. coli systems are also most commonly used for industrial and pharmaceutical protein production. E. coli strain BL21 and its derivatives are the most, frequently used (Terpe, 2006). E. coli BL21(DE3) (Studier and Moffatt, 1986) is a host bearing the T7 RNA polymerase gene (λDE3 lysogen) for expression of target proteins typically under control of the IPTG-inducible /αcUV5 promoter (Baneyx, 1999). The T7 polymerase system enables tight control of expression. The other big advantage of the strain BL21 is being deficient in both Ion (Phillips et al, 1984) and ompT proteases. Several expression systems have been developed for the expression of different types of proteins in E. coli BL21 ( pET vectors, commercialized by Novagen, Madison, WI).

For structural studies the proteins, peptides or other recombinant DNA products are isotopically labeled in vivo by growing the cells in media enriched with the corresponding isotope: mainly ¹³C, ¹⁵N and ²H (deuterium). Isotopic labeling with ²H (deuteration) of the biomolecules is advantageous while applying various methods of nuclear magnetic resonance (NMR) (Gardner and Kay, 1998), neutron scattering (Stuhrmann and Nierhaus, 1996) or mass spectrometry (MS) (Schuker and Bartch, 1994). The enrichment of the cells and recombinant products with deuterium is achieved by feeding the cells with perdeuterated (i.e., fully deuterated) carbon sources in the environment of heavy water (²H₂O). In order to obtain fully deuterated biomass both the heavy water and all the substrates used should also be fully deuterated (Lederer et al, 1986). Labeling of cells and the recombinant products with deuterium is a very complicated task because deuterated environment has negative effect on the growth and severely distorts the physiology (Katz and Crespi, 1966; Gardner and Kay, 1998), and the deuterated substrates and heavy water are very expensive. This in turn necessitates to select the deuterated substrates according to their price and to apply high cell density fermentation methods as this helps to economize the use of very expensive heavy water. Even in cases of production of non-labeled heterologous proteins or in labeling with isotopes other than deuterium it is usually preferred to achieve high cell density for which different methods of fed-batch cultivation have been developed (Lee, 1996). Up to now there are a couple of publications about cultivating E. coli in heavy water in fed-batch culture (Vanatalu et al, 1993 , Jϋnemann et al, 1996) using the strain E. coli MRE600 and applying the feed- forward type of fed-batch cultivation (Paalme et al, 1990) where the overfeeding is avoided by periodically analyzing the growth medium with high performance liquid chromatograpy (HPLC) which is a laborious task as the cultivations usually last several days. The authors of this invention have demonstrated the possibility to apply a . special growth control algorithm which enabled to achieve maximum growth rates in continuous cultivation (Vanatalu, 1997) including the growth of E. coli MRE600 on succinic acid in a fully deuterated medium (Tomson et al, 2006).

On the other hand the perdeuterated carbon sources are also very expensive. Therefore, there is a need for a suitable composition of carbon sources that are not prohibitively expensive and still enable satisfactory growth.

Strains of E. coli have been grown in heavy water with different deuterated carbon sources like algal hydrolysate (Seeholzer et al, 1986), glucose (Mann and Moses, 1971), succinate (Tomson et al, 2006), acetate (Mann and Moses, 1971) or mixture of succinate and acetate (Vanatalu et al, 1993).

Strain E. coli BL21 has been cultivated in highly deuterated media either on commercial deuterated hydrolyzates (Fiaux et al, 2004), or H-acetate(Venters et al, 1995; Sosa-Peinado, 2000; Fiaux et al, 2004). Of these only ²H-acetate can be taken as cheap carbon source. In all cases the authors denote the poor growth while acetate being the carbon source.

In a recent publication (Paliy and Gunasekera, 2006) the authors conclude that the growth of E. coli BL21 is very poor on the gluconeogenic substrates acetate and succinate. Thus, there is a need for a new method to gain reasonable growth rate of E. coli BL21 in highly deuterated growth media on reasonably priced deuterated substrates enabling to achieve high biomass densities and recombinant protein expression.

Disclosure of the invention

One aspect of the invention is a method of cultivation of E. coli BL21 in a fermentation medium with deuterated media in fed-batch and expression of recombinant DNA products, comprising the steps of feeding deuterated acetate and deuterated succinate into the fermentation medium; and controlling concentrations of said acetate and said succinate in the fermentation medium below the predetermined critical levels.

Another aspect of the invention is a method of cultivation of E. coli BL21 in a fermentation medium with deuterated media in fed-batch and expression of recombinant DNA products, comprising the stages of introducing a culture of said E. coli BL21 into said fermentation medium, containing a predetermined low amount of acetate and succinate; feeding a solution of deuterated acetic acid into said fermentation medium in pH-stat regime until predetermined pH level is reached; monitoring the growth of the culture by measuring the optical density of said culture; changing the feeding algorithm from pH-stat regime to adaptastat regime when the optical density reaches a predetermined level, said adaptastat regime comprising the steps of pumping of said solution into said fermentation medium for a predetermined pumping time at a predetermined first pumping speed, corresponding to a low growth rate of said culture; halting said pumping and monitoring the change of the pθ₂ of said culture until sharp rise of the pθ₂ occurs; resuming pumping of said solution into said fermentation medium at a modified pumping speed, said modified pumping speed being increased by a predetermined first factor compared to said first pumping speed if the sharp rise of the pθ₂ occurred within a predetermined time limit and said modified pumping speed being decreased by a predetermined second factor compared to said first pumping speed if the sharp rise of the pθ₂ occurred after said predetermined time limit; and repeating the steps according to adaptastat regime until the end of the fermentation process.

The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the drawings, specification, and claims. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposed, and not to limit the scope of the inventive subject matter.

Brief description of the drawings

The technical essence of the invention is described in details by following figures:

Fig. 1 depicts the kinetics of fed-batch culture, namely growth of E. coli BL21(DE3) in fully deuterated medium in the mixture of succinate and acetate and expression of recombinant protein, where A600- optical density; FLUO- fluorescence of GFP measured in cell suspension (relative units per equal cell density); pmpl- speed of feeding pump (arbitrary units);

Fig. 2 depicts the polyacrylamide gel electrophoresis (PAGE) pattern of the protein, expression levels from the fermentation, where M - Molecular weight marker (66, 45, 36, 29, 24, 20.1, 14.2 kDa); BC- subculture to a shaker flask from the 87th h of fermentation and induction for 43 h; C - Control, E. coli BL21(DE3) grown in shaker flask, uninduced.

Modes for carrying out the invention

According to one embodiment of the invention, a method of cultivation of E. coli BL21 in a fermentation medium with deuterated media in fed-batch and expression of recombinant DNA products, comprises the steps of feeding deuterated acetate and deuterated succinate into the fermentation medium; and controlling concentrations of said acetate and said succinate in the fermentation medium below predetermined critical levels.

According to another embodiment of the invention, a method of cultivation of E. coli BL21 in a fermentation medium with deuterated media in fed-batch and expression of recombinant DNA products, comprising the stages of introducing a culture of said E. coli BL21 into said fermentation medium, containing a predetermined low amount of acetate and succinate; feeding a solution of deuterated acetic acid into said fermentation medium in pH-stat regime until predetermined pH level is reached; monitoring the growth of the culture by measuring the optical density of said culture; changing the feeding algorithm from pH-stat regime to adaptastat regime when the optical density reaches a predetermined level, said adaptastat regime comprising the steps of pumping of said solution into said fermentation medium for a predetermined pumping time at a predetermined first pumping speed, corresponding to a low growth rate of said culture; halting said pumping and monitoring the change of the pθ₂ of said culture until sharp rise of the pθ₂ occurs; resuming pumping of said solution into said fermentation medium at a modified pumping speed, said modified pumping speed being increased by a predetermined first factor compared to said first pumping speed if the sharp rise of the pθ₂ occurred within a predetermined time limit and said modified pumping speed being decreased by a predetermined second factor compared to said first pumping speed if the sharp rise of the pθ₂ occurred after said predetermined time limit; and repeating the steps according to adaptastat regime until the end of the fermentation process.

The invention is based on our studies of the growth of E. coll BL21 on succinate and acetate and on the following phenomena:

in ordinary protiated media the cells grow slowly on acetate or succinate as single carbon sources;

cells grow much faster in the mixture of acetate and succinate than either of them supplemented as single carbon source;

at pH 6.5-7.5 acetic acid inhibits growth already starting from concentrations above 0.5 g/1. The succinic acid was not found remarkably inhibiting at 2 g/1;

the molar ratio of utilisation acetate/succinate by the cells depended on the growth rate being approximately 4 (mole/mole) in fast growing culture and approximately 8 (mole/mole) during slow growth.

The main prerequisites for the fed-batch culture are that both the succinate and acetate should be supplied in a manner that neither of them accumulates in the fermentation medium, e.g., in a fermentor, above the critical values- about 0.5-1 g/1 for acetic acid and 2-4 g/1 for succinic acid. This task is even more complicated because the ratio of the two carbon sources was dependent on the growth rate. Disbalance of the ratio of the carbon sources can lead to overfeeding by one of the components which can be detrimental to the whole cultivation process causing irreversible stop of growth and death of cells. According to the invention, the growth rate is defined by the acetate/succinate ratio in the feeding solution. The portion of succinic acid in the feeding mixture is slightly increased to avoid accumulation of acetic acid but allowed slight accumulation of succinate which is not so inhibiting to the cells. From these considerations the ratio between 4 and 8 (mole/mole) of acetic acid per succinic acid was chosen for fed-batch cultivation.

It is well known that the overall growth rate usually decreases in fed-batch cultures with the increase of cell density, presumably because of several concurrent factors, but mainly due to the accumulation of excreted metabolites and disbalance of different nutrients. Therefore a feeding algorithm similar to the adaptastat (Tomson et al, 2006) was applied, which periodically automatically checked the substrate limitation and accordingly lowered or increased the feeding rate in fed-batch culture. Thus the optimal feeding rate was secured irrespective of the changes in growth rate of the culture. Automatic adjustment is especially critical during the induction of recombinant protein expression which is very often accompanied by sharp decrease of growth rate.

EXAMPLE

The method according to the invention was tested in several fermentations where E. coli BL21(DE3) was grown to optical densities (A600 nm) above 20 (about 10-15 g/1 biomass dry weight) and the recombinant proteins were expressed at that cell density.

Preparation of the inoculum for fermentation

Because deuterium confers stress to the cells the culture has to be adapted to growth in deuterated media. We have used a scheme for the strain E. coli BL21(DE3) where the cells were transferred directly from Luria Bertani (LB) broth to fully deuterated minimal medium supplemented with deuterated acetate and succinate. But principally the cells can be adapted more smoothly by gradually increasing the deuteration level in every subsequent subculture.

LB broth contained per liter: Bacto Tryptone- 1O g, Bacto Yeast Extract- 5 g, NaCl- 1O g.

The deuterated minimal medium for adaptation and cultivation of the inoculum were prepared as following. For the preparation of 100 ml of media into 85 ml Of D₂O were added 10 ml of 10xM9 stock solution, 0.1 ml of 20%(w/v) MgSO₄*7H₂O, 0.1 ml of 2%(w/v) CaCl₂*2H₂O, 0.5 ml of 10%(w/v) deuterated acetic acid, 4 ml of 5% deuterated succinic acid and 0.1 ml of kanamycin stock solution(50 mg/ml). After addition of all the components the pH of the solution was adjusted 6.8 with the addition of 4N NaOD. The 10xM9 stock solution contained per 100 ml: KH₂PO₄- 3 g, Na₂HPO₄- 6 g, NH₄Cl- 2 g. D₂O and the stock solutions except those of acetic acid and kanamycin were autoclaved in tightly capped bottles 30 min. 105° C. All the stock solutions were prepared in D₂O. Kanamycin was filter sterilised (pore size 0.2 micrometers) which is an alternative to autoclaving also for the other components.

The deuterated growth medium for the fermentation contained per 100 ml of final volume (including 25 ml of inoculum): 64 ml of D₂O, 7.5 ml of 10xM9 stock solution, 0.2 ml of 20% (w/v) MgSO₄*7H₂O, 0.1 ml of 2%(w/v) CaCl₂*2H₂O, 0.5 ml of 10%(w/v) deuterated acetic acid, 2 ml of 5% deuterated succinic acid and 0.1 ml of kanamycin stock solution (50 mg/ml). Principally the proportion of the inoculum may be bigger or smaller and accordingly the proportion of D₂O should be adjusted.

The feeding solution was prepared in D₂O and contained per liter: deuterated acetic acid- 120 g, deuterated succinic acid- 50 g, MgSO₄- 1.5 g, trace element solution- 5 ml. The trace element solution was prepared in D₂O and contained per liter: FeSO₄-7H₂O - 4 g, CaCl₂-2H₂O- 3.2 g, CuSO₄-5H₂O- 0.8 g, H₃BO₃ - 0.8 g, MnSO₄-5H₂O- 0.4 g, CoCl₂-2H₂O- 0.32 g, ZnSO₄-7H₂O- 0.16 g, NaMoO₃-2H₂O- 0.16 g in 0.1 M DCl solution.

The titrant was the solution of saturated ND₄OD in D₂O diluted in proportion 1:1 with D₂O.

The fermentation system was supplied with the following sensors which were also connected with the computer: pH, pθ₂, stirrer speed, air flow into the system, temperature. The computer algorithm enabled to control the following devices: stirrer speed, titrant pump, speed of substrate feeding pump, a valve controlling the flow rate into the fermentor. Temperature was controlled autonomously by a controller independant of the computer.

The system could be used with an Applikon 2 liter fermentor vessel or with a custom made minifermentor vessel with working volume of about 100 ml.

The time interval was 1 minute for both the computer control and data collection using the program BioXpert. After inoculation with the seed culture the feeding was started in pH-stat regime as following. The fermentation medium initially contained low amount of acetate and succinate (usually 0.5 g/1 and 1,0 g/1 correspondingly) to guarantee that the cells are not starving shortly after inoculation. Then by using the feeding pump solution of acetic acid is added to bring the pH to about 6.7 i.e. 0.1-0.2 units below the setpoint of titration. This starts the automatic addition of acetic acid to replenish the substrates utilized by the cells because the utilization of acetate and succinate brings along the rise of pH.

After the optical density (OD) had increased to a value of about 2 to 3 the feeding algorithm was changed from pH-stat to adaptastat and the above described feeding solution was applied. The algorithm works as following. Initially the feeding pump is switched on for a certain period (25 min.) at speed corresponding to a low growth rate (0.03-0.06 h^"1) which is presumably below the maximum growth rate of the culture as observed in preliminary shaker flask cultivations (0.07-0.1 h^'1). After that the feeding is stopped and the behaviour of the pθ₂ is checked. If the pθ₂ is rising sharply within 3 min. after the pump has stopped the next pumping cycle is started at elevated speed (usually 110% of the previous pumping speed. Alternatively, if the sharp pθ₂ rise occurs later than 3 min. after the pause of pumping then in the next feeding cycle the speed of the pump is decreased (usually to 90% of the previous level). Such an algorithm is applied until the end of fermentation and it adjusts the rate of feeding addition to the rate of its utilization by the culture.

When the necessary biomass density is achieved the recombinant product expression is switched on usually by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to final concentration of 0.05-2.0 niM depending on the system. Very often this will cause to drain away the metabolic resources in the cellular metabolism causing the growth to significantly slow down. Accordingly, at this point the acetate/succinate ratio in the feeding may be increased. But we have also used the same initial acetate/succinate ratio to feed the cells throughout the cultivation including the period of recombinant product induction.

It is evident that the adaptastat feeding algorithm is indispensable especially during the period of induction avoiding the possible overfeeding if feed-forward type of algorithms were used. Nevertheless, once the main growth parameters are estimated by the preliminary experiments using adaptastat principally the feeding patterns can be repeated in future cultivations simply by using feed-forward type of fed-batch. E. coli BL21(DE3) bearing plasmid pET42 coding a green fluorescent protein (GFP) fusion with glutathione-S-transferase (GST) was used. The transfected cells were maintained as glycerol stock cultures at -2O⁰C. The stock cultures were used to get single cell colonies on Petri dishes with Luria Bertani (LB) broth. Here and in all the next steps of subcultures 50 micrograms/ml of kanamycin was added as selective marker. The liquid cultures were grown on a rotary shaker and cultivation temperature was all ways held at 3O⁰C. From a single colony a LB liquid culture was inoculated and grown overnight to optical density (OD) at 600nm about 3. From the LB culture a new culture was inoculated on M9 mineral medium supplemented with 2 g/1 succinate and 0.5 g/1 acetate and grown for 8 h during which the OD increased from 0.03 to 0.20. In the next step the cells from 1.2 ml of culture were pelleted by centrifugation in sterile tubes (2 min. x 10000 rpm) and resuspended in 5 ml of fully deuterated M9 medium supplemented with deuterated carbon sources (2 g/1 succinate and 0.5 g/1 acetate). After the cells had adapted to deuterium and grown in 26 h to OD of 0.25 they were transferred to a flask containing 25 ml of fresh medium of the same composition and grown for 47 h during which the OD reached 0.37 and acquired a growth rate 0.07 h^"1. This culture served as inoculum for the fermentation.

Fermentation was carried out in a custom made fermentor vessel with working volume of 70 to 200 ml. The fermentor was sterilised by autoclaving 20 min. at 121° C, the pH and pθ₂ sensors were sterilised by rinsing with 70% ethanol and incubating in a laminar flow bench under UV illumination for 1 h.

The fermentor was first filled with 75 ml of deuterated M9 media supplemented with deuterated carbon sources- 1 g/1 succinate and 0.5 g/1 acetate. After temperature and pθ₂ values had stabilised the fermentation was started by adding the above described inoculum (25 ml, OD 0.37). The pθ₂ was kept automatically above 20% of saturation with the help of regulating the stirrer speed and the air flow rate into the fermentor. The course of the fermentation is depicted in Fig. 1. In the beginning the feeding was started as a pH-stat culture which kept pH at 6.7 by adding a solution of 5 g/1 of deuterated acetic acid automatically by the feeding pump. By the 38th h of fermentation the OD had reached 3 and according to approximate estimations most of the carbon sources were used up by the cells. Therefore the feeding solution was changed to a new one containing deuterated acetic and succinic acid in the predetermined ratio of 2.4:1.0 (g/g) correspondingly as described above. On the 42nd Ii the feeding control algorithm was changed from pH-stat to adaptastat. When OD had reached 21 by the 87th h of cultivation the recombinant protein expression was induced by addition of IPTG to final concentration of 0.5 mM. As it was known from preliminary experiments, that induction causes significant decrease of growth rate then the content of acetic acid was increased in the feeding solution from 120 g/1 to 190 g/1. Thus the ratio of acetic acid to succinic acid in the feeding was increased to 3.8 (g/g), corresponding to the utilization of these substrates at slow growth rate. The cells were cultivated further and samples taken to characterize the expression both by PAGE (Fig.2) and direct measurement of the recombinant GFP fluorescence (Fig.l). Both methods evidence of remarkable expression level of fully deuterated protein. The biomass yield was 80 g wet weight per liter.

Although this invention is described with respect to a set of aspects and embodiments, modifications thereto will be apparent to those skilled in the art. The foregoing description of the embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of this disclosure. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

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Claims

1. A method of cultivation of E. coli BL21 in a fermentation medium with deuterated media in fed-batch and expression of recombinant DNA products, comprising the stages of:

introducing said E. coli BL21 into said fermentation medium;

5 feeding deuterated acetate and deuterated succinate into said fermentation medium; and

controlling concentrations of said acetate and said succinate in said fermentation medium ** below predetermined critical levels.

2. A method as in claim 1, supplying said acetate so that an accumulation of acetic acid in said fermentation medium is below 1 g/1 and an accumulation of succinic acid in said

10 fermentation medium is below 4 g/1.

3. A method as in claim 1, wherein said accumulation of said acetic acid is below 0.5 g/1 and an accumulation of said succinic acid is below 2 g/1.

4. A method as in claim 1, wherein controlling said concentrations of said acetate and said succinate by keeping acetate to succinate molar ratio in feeding solution between 4.0 to 8.0.

15 5. A method as in claim 1, wherein said succinate and acetate are introduced separately into said fermentation medium and are mixed together in said fermentation medium.

6. A method as in claim 1, mixing said succinate and said acetate into a solution before introducing said solution into said fermentation medium.

7. A method as in claim 1, said succinate and said acetate are used in the form of salt.

20 8. A method as in claim 1, said succinate and said acetate are used in the form of acid.

9. A method of cultivation of E. coli BL21 in a fermentation medium with deuterated media in fed-batch and expression of recombinant DNA products, comprising the stages of:

introducing a culture of said E. coli BL21 into said fermentation medium, containing a predetermined low amount of acetate and succinate; feeding a solution of deuterated acetic acid into said fermentation medium in pH-stat regime until predetermined pH level is reached;

monitoring the growth of the culture by measuring the optical density of said culture;

changing the feeding algorithm from pH-stat regime to adaptastat regime when the optical density reaches a predetermined level, said adaptastat regime comprising the steps of

pumping of said solution into said fermentation medium for a predetermined pumping time at a predetermined first pumping speed, corresponding to a low growth rate of said culture;

halting said pumping and monitoring the change of the pθ₂ of said culture until sharp rise of the pCh occurs;

resuming pumping of said solution into said fermentation medium at a modified pumping speed, said modified pumping speed being increased by a predetermined first factor compared to said first pumping speed if the sharp rise of the pθ₂ occurred within a predetermined time limit and said modified pumping speed being decreased by a predetermined second factor compared to said first pumping speed if the sharp rise of the pθ₂ occurred after said predetermined time limit; and

repeating the steps according to adaptastat regime until the end of the fermentation process.

10. As in claim 9, wherein said predetermined pH level is about 6.7, said predetermined level of the optical density is between 2 and 3, said predetermined pumping time is about 25 minutes, said predetermined time limit is 3 minutes, said predetermined first factor is 1.1 and said predetermined second factor is 0.9.