[J. Ferment. Techno!., Vol. 66, No. 2, 187-191. 1988]
`
`Fed-Batch Cultures of Recombinant Escherichia coli with
`Inhibitory Substance Concentration Monitoring
`
`NO RIO SHIMIZU*, SHINICHI FuKuzoNo, KIYOSHI FuJIMORI,
`NOBUKO NISHIMURA, and YOJI 0DAWARA
`
`Advanced Research Laboratory, Hitachi Ltd., 280, Higashikoigakubo 1-chome,
`Kokubunji-shi, Tokyo 185, Japan
`
`Fed-batch cultures of recombinant Escherichia coli HBIOl were investigated to obtain
`high cell density and large amounts of p-galactosidase (P-gal). E. coli HBIOl was
`transformed with a hybrid plasmid pTREZl, which contained a P-gal gene controlled
`by the trp promoter.
`In fed-batch cultures of recombinant E. coli, when the cell con(cid:173)
`centration reached around 13 g/1, the cell growth stopped and large amounts of inhibitory
`substances have accumulated in the broth. These inhibitory substances were isolated
`and identified. Acetate produced by the cells was evidently the main inhibitor of cell
`growth and p-gal production. Since the cells proved to assimilate acetate, the feed rate
`was controlled with acetate concentration monitoring in the fed-batch culture. As
`a result, the acetate concentration was maintained at a low level and cells grew smoothly
`without acetate-induced inhibition. Cell concentration and p-gal quantity reached
`high values of28 g/1 and 64 U/ml, respectively.
`
`technology allows
`Recombinant DNA
`large-scale production of valuable materials
`which otherwise might only be obtained in
`minute quantities
`from natural sources.
`And studies on highly-concentrated cell
`cultivation and high levels of expression for
`cloned genes are very important to obtain
`large amounts of valuable materials using
`host cells harboring hybrid plasmids.
`Highly-concentrated cultivation of recom(cid:173)
`binant Escherichia coli by fed-batch cultures
`was investigated to achieve overproduction
`of heterologous proteins. Fed-batch cultures 1>
`and dialysis cultures 2> for E. coli B have
`already yielded high cell densities of 125 gfl
`and 144 gjl, respectively.
`In
`this study,
`E. coli HBlOl harboring the hybrid plasmid
`pTREZl, which contained a p-galactosidase
`(p-gal) gene controlled by the trp promoter,
`was used as a
`recombinant. Fed-batch
`cultures were used to obtain high cell density
`and large amounts of p-gal.
`
`* Corresponding author
`
`This study deals with inhibitory substances
`produced by recombinant E. coli and fed(cid:173)
`batch cultures with acetate concentration
`monitoring
`for overproduction of gene
`products.
`
`Materials and Methods
`
`Bacterial strains and plasmid DNA
`E. coli
`HBlOl was used as a carrier for the recombinant
`plasmid pTREZJ.Sl pTREZl carries
`the coding
`sequence for a large part of P-gal, joined to the sequence
`for eight N-terminal amino acids of the trp E poly(cid:173)
`peptide.
`The E. coli strain harboring plasmid
`Cultivation
`pTREZ1 was cultivated in M9 medium containing
`2.5 gfl casamino acids, 5 g/l glucose, 1.5 gfl yeast
`extract, 0.1 g/l proline, 0.1 gfl thiamine HCl, 20 mgtl
`tryptophan, and 50 mg/l ampicillin. Cultures were
`placed in 0.5-l shake flasks closed with cotton plugs
`(working volume 0.05/). They were shaken on
`a
`reciprocal shaker at 115 oscillations/min with
`a 7-cm stroke at 37°C, Fed-batch cultures were grown
`in a 5-l jar fermentor with a 2-1 initial working volume,
`including a 200-ml overnight seed culture, and 0.2 m1
`of antifoam
`(1705-W, Lion Co. Ltd.). pH was
`
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`188
`
`SHIMIZU et a[.
`
`[J. Ferment. Technol.,
`
`c
`
`l6
`
`; ~~~\\\\\\\\1\\\\\\\\\~
`14 12
`' m ~4
`u
`
`maintained at 7, temperature at 37°C, gas flow rate at
`2 l(min, and agitation speed at 400--1000 rpm. Aque(cid:173)
`ous ammonia (6 N) was used to adjust pH. The
`dissolved oxygen (DO) level was manually controlled
`at 1-3 ppm by changing the agitation speed. When
`the agitation speed reached the maximum of 1000 rpm,
`50% oxygen gas was supplied to the fermentor instead
`of air. The feeding medium consisted of 200 gfl
`glucose, 100 g/l casamino acids, 60 gfl yeast extract,
`0.4 gfl tryptophan, and 0.1 gfl ampicillin, at pH 7.0.
`Analysis
`fl-Gal activity, glucose concentration,
`and cell concentration were assayed as described
`previously. 3 > Acetate concentration was measured
`with an isotachophoretic analyzer (Model IP-2A,
`Shimadzu, Ltd.) and a gas chromatograph (Model
`663, Hitachi, Ltd.).
`In isotachophoresis, the leading
`electrolyte was 10 mM HCl·fl-alanine at a pH of 3.1.
`The terminating electrolyte was 10 mM n-caproic acid.
`The electric current was 100 pA at 20°C. The gas
`chromatograph was used with a 2-m glass column
`packed with PEG 6000 10% Flusin P 30/60 (Gasukuro
`Kogyo, Inc.). Temperatures at the injection port,
`the column, and the FID detector were 150°C, 130°C,
`and 140°C respectively. The pH of culture super(cid:173)
`natant was adjusted to below 3 with 6 N HCJ before
`injection.
`
`Results and Discussion
`
`Inhibition of cell growth
`Highly-
`concentrated cell cultivation of E. coli
`harboring plasmid pTREZl by fed-batch
`culture was
`investigated. The DO con(cid:173)
`centration was used as a control indicator
`for medium feed, since the DO concentration
`increased when glucose in the broth was
`consumed.
`The results of cultivating the recombinant
`E. coli in the 5-l jar fermentor are shown in
`Fig. 1. When the cell concentration reached
`around 13 g/l, cell growth stopped despite
`medium feed. The cell concentration was
`gradually decreased by dilution with the
`broth by medium feed. Furthermore, /1-gal
`production could not be induced, though an
`inducer, 3-/1-indolylacrylic acid (IA) was
`added together with casamino acids after
`32 h of cultivation. Mori and colleagues 1>
`have reported a high cell concentration of
`125 gfl in a fed-batch culture of E. coli B.
`However, since Sinclair and Stokes 4 > and
`Landwall and Holme 2 > have described the
`
`c:'
`u
`c
`0 "8
`u
`
`0
`0
`
`.~- ~114~
`
`~- ~ 12-6:::::
`a ~ E
`o.'-
`.
`4 0 ~
`<.:>
`,---co~~-~-~-;,, 0
`I
`46 <n
`15
`20
`25
`30
`Time (h)
`
`Fig. I. Highly-concentrated cell cultivation of re(cid:173)
`combinant E. coli by fed-batch culture.
`tryptophan concentration was 10 mgfl.
`Initial
`the addition of 15 mgfl 3-fl(cid:173)
`Arrow indicates
`(lA) and 2.5 gfl casamino
`indolylacrylic acid
`acids.
`
`in
`accumulation of inhibitory substances
`E. coli B cultivation, these results seem to
`indicate that inhibitory substances accumu(cid:173)
`lating in the broth stopped cell growth.
`the
`The inhibition of cell growth by
`supernatant was investigated. Feeding medi(cid:173)
`um and fresh cells were added to a shake
`flask with the supernatant after 12 h of
`cultivation, when cell growth still continued.
`They were also added to a shake flask with
`the supernatant after 19 h of cultivation,
`when cell growth had stopped. The cells
`did not grow at all in the flask. Figure 2
`
`3
`
`......,
`.......
`0>
`
`c u
`
`c
`0
`u
`a;
`u
`
`Time
`Inhibition of cell growth by supernatant.
`Fig. 2.
`One and a quarter ml offeeding medium and 5 ml
`of fresh cells were added to shake flasks containing
`43.75 ml of the supernatant. A control flask
`contained M9 salt medium instead of the super(cid:173)
`natant. 0, control; ~c,., supernatant at 12 h of
`cultivation; 0, supernatant at 19 h of cultivation.
`
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`

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`Vol. 66, 1988]
`
`Fed-Batch Cultures of Recombinant Escherichia coli
`
`189
`
`shows these results. For the supernatant at
`12 h of cultivation,
`the specific growth
`rate of cells was about half that measured for
`a control without adding supernatant. For
`comparison, the frozen and stored cells were
`inoculated into fresh medium to examine
`growth. The specific growth rates of the
`cells at 12 h of cultivation, the cells at 19 h of
`cultivation, and fresh cells were 0.36, 0.32,
`and 0.45 1/h, respectively. The growth of
`the cells was not lowered as much. Further(cid:173)
`more, adding lA induced fJ-gal production.
`These results show that inhibitory substances
`accumulated in
`the broth as cultivation
`proceeded.
`identification of in(cid:173)
`Isolation and
`hibitory substances
`The supernatant
`at 19 h of cultivation, when cell growth had
`stopped, was used to isolate and identify
`inhibitory substances in the broth. Liquid
`fractions were obtained by fractionating the
`supernatant by molecular weight. The frac(cid:173)
`tion of molecular weight of 1000 or less
`inhibited cell growth. Next, anionic sub(cid:173)
`stances obtained from
`the fraction using
`ion-exchange regins inhibited cell growth.
`This
`indicates
`that substances
`inhibiting
`cell growth are low-molecular anionic sub(cid:173)
`stances such as organic acids, which E. coli
`cells are known to produce. 5>
`low-molecular
`The solution containing
`anionic substances (solution L) was analyzed
`
`A
`
`300
`
`> §
`
`~ 200
`
`~
`~ .e
`
`0
`(L
`
`100
`
`0 3o
`
`34 35 38
`32
`Time (min)
`
`40
`
`44
`42
`Time (min)
`
`46
`
`48 49
`
`for organic acids with an isotachophoretic
`analyzer. The isotachopherograms of the
`solution L and of authentic organic acids are
`shown in Fig. 3.
`It was found that com(cid:173)
`ponent A had accumulated in the solution L
`by comparison with the isotachopherogram
`of the feeding medium components (data not
`shown). The isotachopherogram of
`the
`authentic organic acids indicates that com(cid:173)
`ponent A
`is acetic acid. The isotacho(cid:173)
`pherogram of the solution L to which authen(cid:173)
`tic acetic acid was added supported this
`indication (data not shown). Acetic acid
`concentration in the supernatant at 19 h of
`cultivation was measured at a high value of
`0.55 M. Gaschromatograph analysis of the
`supernatant in the broth further confirmed
`acetic acid accumulation.
`Various amounts of ammonium acetate
`were added to a medium to investigate the
`influence of acetate on cell growth and fJ-gal
`production. Results are shown in Fig. 4.
`Both
`the specific growth rate and fJ-gal
`production decreased when the acetate con(cid:173)
`centration was 0.17 M and more.
`It can be
`concluded that acetate accumulated in the
`broth was the primary inhibitor of cell growth
`and fJ-gal production.
`Acetate production by recombinant E. coli
`is shown in Fig. 5.
`In the fed-batch culture
`with DO concentration monitoring, cell
`growth stopped at 14 h of cultivation, when
`acetate concentration reached 0.23 M. The
`cell concentration was 11.6 gfl. The growth
`yield was 0.26 g-cells producedfg-glucose
`
`~ 0.4
`16 :::
`c
`E
`'
`~
`~2
`~
`"0
`"'
`.c
`3 0.2
`8 ~
`e c.
`~
`4 0
`~ 0.1
`·u
`<.9
`I
`"'
`~ 0o!-----:'O'"-.I-~0.2_.::...:0::.:.3-.~0~.4--0.~5-~0.6-..J0.7° "'-
`Acetate concn. (M)
`
`Isotachopherograms of low-molecular anionic
`Fig. 3.
`solution and authentic organic acids.
`The low-molecular anionic solution was diluted
`ten times with distilled water. A mixture of
`0.02 M authentic organic acids solution was used.
`
`Inhibition of cell growth and fl-gal production
`Fig. 4.
`by acetate.
`Fifteen mgf/ lA and 2.5 gfl casamino acids were
`added after 15 h of cultivation.
`fl-Gal production
`was assayed after 24 h of cultivation.
`
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`

`
`190
`
`SHIMIZU et a/.
`
`[J. Ferment. Technol.,
`
`10
`
`c
`u
`c
`o~ u-.._,
`Cl>'-5
`If) 0>
`o -
`u
`:::J
`<:5
`
`0
`
`6 0.6
`c u
`c
`0 u
`Cl> 0.2
`+-
`0
`+-
`Cl> u
`<[
`
`0.4
`
`0
`
`12
`
`8
`
`4
`
`"
`" E'
`c
`u
`c
`0
`u
`
`Cl>
`u
`
`Time (h)
`
`Fig. 5. Acetate production by recombinant E. coli.
`
`consumed. Acetate production causes this
`very low growth yield. The cells continued
`to produce acetate after 14 h of cultivation.
`Smirnova6 > has reported that cell growth
`stopped at an average acetate concentration
`of 0.15 M in fed-batch cultures of E. coli
`Kl2. Furthermore, Yano and colleagues 7>
`have described that the cell growth of E. coli
`B was completely inhibited at 0.18 M of
`acetate. The variant
`inhibition of cell
`growth by acetate concentration seems to be
`due to differences in E. coli strains.
`These results show that acetate produced
`by E. coli was the main inhibitory substance.
`Recombinant E. coli cultivation with
`acetate concentration monitoring Re(cid:173)
`combinant E. coli produced acetate in the
`broth. This acetate inhibited cell growth.
`Therefore, acetate production by cells must
`be prevented to attain highly-concentrated
`cell cultivation. Since a high glucose con(cid:173)
`centration in the broth was considered to
`accelerate acetate production by expression
`of the Crabtree effect, 8> the feeding medium
`was fed continuously to the fermentor to
`maintain glucose concentration in the broth
`
`12
`
`E
`c
`0
`Lf)
`Lf)
`+-
`0
`0
`0
`
`~
`.§ 150
`c u
`
`100
`
`c
`0
`u
`Cl>
`+-
`0
`"t 50
`u
`<[
`
`2
`
`6
`4
`Time (h)
`
`8
`
`10
`
`Fig. 6. Acetate assimilation by cells.
`Ammonium acetate concentration: O, no addition
`(control); e, 17 mM; 0, 83 mM; •· 167 mM.
`
`under 1 gfl. However, cells produced a
`great deal of acetate in a manner similar to
`that indicated by Fig. 5.
`The assimilation of acetate by cells was
`investigated to decrease acetate concentration
`in the broth, as shown in Fig. 6. Ammonium
`acetate was added to M9 medium in amounts
`of 17, 83, and 167 mM. Glucose concen(cid:173)
`tration in the M9 medium was 1 gfl. Acetate
`concentration in the broth increased for
`glucose catabolism at
`the beginning of
`cultivation. After 2 h of cultivation, acetate
`concentration gradually decreased
`in all
`cases and cell concentration increased. This
`shows
`that cells assimilated acetate and
`converted it into cell mass.
`In fed-batch culture, the feed rate was
`controlled and acetate concentration was
`monitored. Medium feed was stopped or
`the feed rate was decreased when acetate
`accumulated in the broth. This allows cells
`to assimilate acetate. Medium feed was
`resumed or the feed rate was increased when
`the acetate concentration
`in
`the broth
`decreased. The results of fed-batch culture
`with acetate concentration monitoring are
`shown in Fig. 7. Acetate concentration in
`the broth was predetermined under 33 mM
`
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`

`
`Vol. 66, 1988]
`
`Fed-Batch Cultures of Recombinant Escherichia coli
`
`191
`
`.!: ~ 1.5
`
`is J rl ' /].r1oCl D nOD~~. ::::
`i;::l~,oo~
`
`50 ~
`:::::: ~ 10
`~
`~
`0 &
`~ 0
`~~40~ 6.
`c 20
`0
`~ u
`~ 8 °o
`
`5
`
`10
`
`15
`Time (h)
`Fig. 7. Fed-batch culture with acetate concentration
`monitoring.
`
`20
`
`25
`
`and measured with the gas chromatograph
`every 30 min. Medium feed was stopped
`when acetate accumulated in the broth.
`As a result, the acetate concentration level
`was kept
`low and cells grew smoothly
`without acetate-induced inhibition. At 26 h
`of cultivation, the cell concentration reached
`a high value of 28.1 gfl. Growth yield
`reached a high value of 0.53 gfg. Since the
`feeding medium used contained 0.4 gfl
`tryptophan, ,8-gal production was repressed.
`However, ,8-gal was produced by derepression
`of the trp promoter at the end of cultivation,
`since tryptophan in the broth was consumed
`as nutrient upon stoppage of the medium
`feed. These results indicate that high cell
`density could be obtained by using feed rate
`control with acetate concentration monitor(cid:173)
`ing.
`,8-Gal was produced using fed-batch culture
`,8-
`with acetate concentration monitoring.
`Gal production was induced by exchanging
`a feeding medium containing tryptophan
`for one containing no tryptophan, rather
`than by adding lA and casamino acids
`during cultivation.
`Iijima and colleagues 9>
`have reported that tryptophan in the broth
`was removed by the cross-flow filtration and
`,8-gal gene was expressed.
`In this work,
`tryptophan was consumed by cells.
`,8-Gal
`production results of feeding-medium ex(cid:173)
`change are shown in Fig. 8.
`,8-Gal pro(cid:173)
`duction started as soon as the feeding medium
`was exchanged at 12 h of cultivation. At
`the end of cultivation, 64 U /ml of ,8-gal were
`
`15
`
`10
`~""
`
`5
`
`-o
`c
`so B
`'-' c:-o , u,
`::J
`-o
`::' E
`40 0. ~
`a>-u
`0
`l!l
`0 ~
`~ 0
`"',540~
`t; c 20
`+- u
`~ §
`0o
`~ u
`
`5
`
`10
`
`15
`
`20 22
`
`Time (h)
`
`Fig. 8, P-Gal production by feeding-medium ex(cid:173)
`change.
`Arrow indicates when a feeding medium con(cid:173)
`taining tryptophan was exchanged for one con(cid:173)
`taining no tryptophan.
`
`produced, and the cell concentration was
`18.8 gfl. The glucose concentration in the
`broth ranged from 0.09 to 0.98 gfl. The
`growth yield was 0.49 gfg.
`,8-Gal per cell
`mass was 3.4 U/mg. This is a very large
`amount of ,8-gal compared with that previ(cid:173)
`ously reported.s>
`These results show that fed-batch culture
`with acetate concentration monitoring yielded
`high cell density and high ,8-gal production.
`
`References
`
`I) Mori, H., Yano, T., Kobayashi, T., Shimizu, S.:
`J. Chern. Eng. Jpn., 12, 313 (1979).
`2) Land wall, P., Holme, T.: J. Gen. Microbial.,
`103, 345 (1977).
`3) Shimizu, N., Fukuzono, S., Nishimura, N.,
`Odawara, Y., Fujiwara, K.: J. Ferment. Techno!.,
`65, 7 (1987).
`4) Sinclair, N. A., Stokes, J. L.: J. Bacteriol., 83,
`1147 (1962).
`5) Bergey's Manual of Systematic Bacteriology, Vol. 1,
`420, Williams & Wilkins, Baltimore/London
`(1984).
`6) Smirnova, G. V., Oktyabr'skii, 0. N.: Mikro(cid:173)
`biologiya, 54, 252 ( 1985).
`7) Yano, T., Mori, H., Kobayashi, T., Shimizu, S.:
`J. Ferment, Techno!., 58, 259 (1980).
`8) Crabtree, H. G.: Biochem. J., 23, 536 (1929).
`9) Iijima, S., Kawai, S., Mizutani, S., Taniguchi,
`M., Kobayashi, T.: Appl. Microbial. Biotechnol.,
`26, 542 (1987).
`
`(Received September 28, 1987)
`
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