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Fed-Batch Operation of RecombinantEscherichia coli Containing trp Promoterw ith Controlled Specific Grow th Rate

Sung Kwan Yoon and Whan Koo KangLucky Biotech Research Institute, P.O. ox 10, Daejeon, Korea

Tai Hyun Park*Department of Genetic Engineering, Sung Kyun K wan University,300 Chunchun-Dong, Suwon, 440-746, Korea

Received June 15, 1993/Accepted December 5, 1993

A feb-batch operation for the product ion of bovine soma-totropin (bST) under the control of tryptophan promoterin Escherichia coli was investigated. The plasmid usedcontains a two-cistron system and altered codon usagefor higher expression of bST. Specific growth rate isan impor tant parameter in the fermentation, because itaffects the producti on of growth-inh ibitory organic acidsand the expression of recombinant protein. The feedingrate was adjusted to keep the specific growth rate con-stant in this study. The variable growth yield expressedas a funct ion of tim e was used for the calculation of thefeeding rate. The grow th yie ld decreases during the fer-mentation as product expression is induced. The specificgrowth rate was well controlled; however, intracellularbST concentration decreased at high cell concentrations.This is considered t o be due to degradation by proteases.The decrease was prevented by an exponential feedingof the yeast extract as an organic nitro gen source. 0 1994John Wiley 84 Sons, Inc.Key words: fed-batch operation recombinant E. colitr p promoter grow th rate, controlled

INTRODUCTION

Fed-batch operation has been widely used for Escherichiacoli fermentation. Various feeding strategies for the fed-batch operation have been proposed to obtain high cellconcentration and high expression of recombinant pro-tein. These include the feeding strategies using pH-stat,16DO-stat," measurement of glucose co n~ en tr at io n~ ,' ~ ndacetic acid concentration18 in the medium, and control ofspecific growth rate.'*12*15 he specific growth rate is animportant parameter because the production of growth-

inhibitory organic acids such as acetic acid and the ex-pression of recombinant protein depend on the specificgrowth rate.9,14,20

Control of the specific growth rate at an appropriatevalue can give desired cell growth unless the dissolvedoxygen in the medium is limited. However, intracellularconcentration of the recombinant protein decreases at highcell concentration, even if the product exists as an inclusionbody. This is considered to be due to degradation by

* To whom all correspondence should be addressed.

proteases. Protease is induced under stressful conditionssuch as the depletion of carbon or organic nitrogen source,high temperature, and exposure to e t h a n ~ l . ' ~ , ~ ~ roteaseactivity can be repressed and, consequently, the productivityis improved by feeding organic nitrogen."

Fed-batch operation is applied to the production ofbovine somatotropin (bST) in this study. The bST is apolypeptide consisting of 190 or 191 amino acids6 Tran-scription of the bST gene is under the control of thetryptophan promoter in our system. The tryptophan pro-moter is regulated by tryptophan and tryptophan analog,such as 3P-indoleacrylic acid (J.AA).l3 The cloned geneexpression is automatically induced in a fed-batch operationby feeding tryptophan-deficient medium.

In this article, we study a feeding strategy with whichthe specific growth rate is controlled at the set point andthe intracellular concentration of recombinant bST does not

decrease even at high cell concentration.

MATERIALS AND METHODS

Bacterial Strain and Growth Condition

Escherichia coli W3110/ptrphsBST was used for this re-search. The plasmid, ptrphsBST, contained a two-cistronsystem for higher expression of bST.17 Transcription wasregulated by the trp promoter, and codon usage was alteredin the first 54 nucleotides to reflect the bias of highlyexpressed E. coli genes.2

The M9 medium (100 mL) with 5 g/L yeast extract and10 g/L glucose was inoculated with 0.2 mL of glycerolstock, and was incubated overnight. This seed culture wastransferred to 5 L or 6 L of medium in a 14-L fermentor(Chemap CF 2000) for fed-batch operation. The mediumcomposition is shown in Table I. Temperature and air flowrate were maintained at 37C and 1 vvm, respectively.NH40H solution was used to keep pH at 7.0. Dissolvedoxygen in the medium was maintained at concentrationsabove 20% by regulating the stirrer speed and the pressureinside the fermentor.

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Table I. Medium com position in dual feed fed-batch fermentation.

Com position Starting medium (g/L) Feeding medium 1 (g/L) Feeding medium 2 (g/L)

Glucose 2 400 400K H 2 m 4 15MgSO4 . 7H2O 4 - 15.5Yeast extract 1 - 10 0

CaClz . 7 H 2 0 - 1.35 1.35M n S 0 4 * 5 H 2 0 - 0.075 0.075C o C l z . 6 H z O - 0.013 0.013Z n S 0 4 . 7 H 2 0 - 0.075 0.075C u c 1 2 . 5 H 2 0 - 0.013 0.013NazMoO4 . 2 H 2 0 - 0.013 0.013

- -

FeS04 . 7 H 2 0 - 0.2 0.2

Fermentation and Control

In the early phase of the fermentation, feed was not addeduntil the glucose in the medium was depleted and theconcentration of dissolved oxygen started to increase. Glu-cose concentration was maintained at zero to prevent aceticacid formation. Specific growth rate ( p ) was controlledat a constant value with an exponential feeding of theglucose solution. The feeding rate was determined by amass balance equation of the cell and the substrate, andis represented by the following equation when S = 0 andd s / d t = 0.

F = (pXoVo/YSF)epLt (1 )

where p is the set point of the specific growth rate andSF is the substrate concentration in the feeding medium.The growth yield (Y) may be constant or variable. Thecalculated value of the flow rate was converted to ananalog signal which operated a feeding pump (Cole-Parmer,Ismatec Reglo 100).

Analytical Procedure

Cell density was measured by a spectrophotometer (Perkin-Elmer, Coleman 575) at 600 nm. Glucose concentrationwas measured using a Sigma Glucose Kit (procedure no.510) or a strip (Glucopat, Kyoto Daiichi Kagaku Co.).Tryptophan concentration was determined by the method ofKupfer and Atkin~on.~ oncentration of bST per unit cellwas determined by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and Coomassie blue staining. A densitometer(Bio-rad, Model 620) with an interference filter was usedto scan the bST band on the gel for quantitative analysis.The standard curve, which is the correlation between theamount of bST and the peak area read by the densitometer,was obtained in every SDS-PAGE and showed a goodlinear correlation. The contribution of the native E . coliprotein, which may overlap with the bST band, to the signalmeasured by the densitometry, was negligible.

RESULTS AND DISCUSSION

Fed-batch operation was carried out using the startingand feeding medium 112 shown in Table I and the flow

rate described by Eq. (1). The set point of the specificgrowth was 0.15 h-' and the growth yield (Y) was assumedto be 0.714 (ODL/g glucose) which was chosen by thetotal amount of cell produced per glucose consumed inprevious fed-batch experiments. The specific growth rateis well controlled at 0.15 h-l, as shown in Figure 1.However, the growth yield decreases as the fermentation

is carried out. The difference of the assumed Y rom thereal value causes the calculated cell concentration, indicatedby the solid line, to deviate from the experimental values.But the final cell concentration and the final predictionmatch well, because the overall growth yield, which wasexperimentally obtained, was used for the constant growthyield. It should be noted that the specific growth rate waswell controlled even though incorrect growth yield wasused. Mathematical simulation confirms that the controlledspecific growth rate approaches the set point in this control

a

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0

0/ A A

0 5 10 15 20 25 30 35

Tlme(hrs)

Figure 1. Control of specific growth rate in fed-batch operation usingconstant growth yield: ( 0 ) ell concentration, (A) growth yield (r), ( 0 )specific growth rate ( p ) , nd set point of /I ( . . . ) Solid lines representthe calculated values.

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strategy even with the use of an incorrect growth yield.The specific growth rate and the growth yield decreaserapidly in the late phase of the fermentation. This iscaused by a depletion of the dissolved oxygen in themedium because the dissolved oxygen was not controlledin this experiment. A drastic decrease in the dissolvedoxygen was observed toward the end of the fermentation.The low values of the growth yield in the late phasecaused an underestimation of the overall growth yield of

0.714 (ODL/g glucose). The glucose concentration wasmaintained well at zero throughout the fermentation. Thegrowth yield is not constant as shown in Figure1. Toexpress the growth yield a s a function of time, it is assumedthat the decreasing rate of the growth yieldis linearlyproportional to the difference between the grow th yield andthe minimum growth yield(Y,). The decreasing rate of thegrowth yield is expressed by the following equation

dY/df = (l/ty)(Ym - Y ) Y(to) = Yo (2)

( 3 )= -(y, - Yo)e-('-'O)/'Y + y m

where t y is a time constant. This is an empirically fittedequation. Rearrangement of this equation gives the follow-ing equation, which is linear with respect to time.

ln(Y - Y,) = - ( t - tO)/ty + ln(Y0 - Ym) (4)According to the experimental data, the parameter valuesare Y, = 0.95 an d YO = 1.65. By plotting ln(Y- Y,) versust , other parameter values, fy = 8 and to = 8, are obtainedfrom the slope and the intercept. Eq.( 3 ) becomes

Y = 0.7e-('-')/' + 0.95 ( 5 )where t is the time after starting the fermentation. Ift is

the time after starting the feeding, Eq.(5) becomes thefollowing equation

(6)= 0.7e-('-W/' + 0.95

This equation is useful when the dissolved oxygen is no tlimited. The solid line forY in Figure 1 represents a resultcalculated by Eq. (5) .

The same experiment as in Figure1 was carried outusing variable Y expressed by Eq. (6). The set point of thespecific growth rate was 0.14 h-' and the gluc ose feed ingwas started 4 h after starting the fermentation. Resultsare shown in Figure 2. The solid lines for X and Y arethe calculated results. The specific growth rate was wellcontrolled at the set point and the calculated cell densityagreed well with the experimental data. The productionof bST is shown in Figure 3 . bST produced in E . coliexists as an inclusion body. The specific bST concentrationbased on cell mass reaches a maximum atOD = 40 thendecreases. Optical density can be used a s a valid indicatorofincrease in cell mass during induction, because the linearrelationship between dry cell weight and optical densityis stable following a relatively brief transition phase aftert h e i n d ~ c t i o n . ~he bST concentration based o n the culture

1 2 0

D

0 5 10 15 20 25 SO

Tlme(hrs)

Figure 2. Control of specific growth rate in fed-batch operation using

variable growth yield: (0) ell concentration, (A) rowth yield (Y),( 0 ) pecific growth rate (p ) , and set point of p ( . . . . ) Solid linesrepresent the calculated values.

volume increases continuously as the cell density increasesexponentially.

The decrease of the specific bST concentration at highcell density can be d ue to plasmid instability, product degra-dation by protease, intervening processes of transcriptionand translation, as well as dilution by growth. However,

h

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4

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u8

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' 2 0 " 1\

E

4?t,a

E0

v

4

2c

C8UE0

V

5U

U8

IarA

4u

Time(hrs)

Figure 3. Profile of cell concentration and spec ific bST concentration inspecific growth rate-controlled fed-batch operation: (0) ell concentra-tion, and ( 0 ) pecific bST concentration.

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the plasmid stability test showed that the plasmids werestable. A protease inhibitor, PMSF (phenyl methane sul-fonyl fluoride) was added to the medium to examinewhether the decrease of the specific bST concentrationwas due to the degradation by protease. The experimentwas carried out in a shaker flask containing M9 mediumwith 2 g/L casamino acid. When the OD reached 0.5,20 ,ug/mL of 3P-indoleacrylic acid (IAA) was added toinduce the expression. Cell growth curve and specific

bST concentration are shown in Figure 4. The specificbST concentration decreases rapidly when the cell growthreaches a stationary phase. The PMSF was added 7 hafter starting the experiment. This is a late exponentialphase. The specific bST concentration profiles are shownin Figure 4 when the PMSF concentrations in the mediaare 0.5 m M and 2 mM. The decreasing rate of the specificbST concentration was slower with the addition of 0.5 mMPMSF than without the addition. With the addition of 2 mMPMSF, the specific bST concentration could be maintainedconstantly. These results imply that the decrease of thespecific bST concentration was due to product degradationby the protease. It can be more clearly demonstrated ifwe take samples before and after the decrease in thebST production and immunoblot with polyclonal anti-bSTantiserum in an effort to identify degradation products.At the stationary phase caused by the depletion of thecarbon source in the batch operation, protease is inducedand degrades the intracellular bST. The degradation can beprevented by the addition of the protease inhibitor, PMSF.However, this process is not practical because PMSF isexpensive.

The temperature effect on the degradation was observednext to examine whether the protease activity could beinhibited by lowering the temperature. The temperature was

shifted from 37C to 30C, 2 5 T , and 20"C, respectively,7 h after starting the experiment. The growth temperaturedid not affect the stability of the bST (data no t shown).As described earlier, the degradation of the recombinantprotein by protease is known to be caused by culturingthe cell under stressful conditions, such as the depletion ofcarbon or organic nitrogen source, high temperature, andexposure to ethanol. It is considered that the decrease ofthe specific bST concentration in the fed-batch operationshown in Figure 3 is due to the deprivation of the organicnitrogen source at high cell density, whereas that in batchoperation is due to the deprivation of the carbon source inthe stationary phase.

The same experiment as shown in Figures 2 and 3 wascarried out with the exponential addition of yeast extractas an organic nitrogen source to prevent the decrease ofthe specific bST concentration at high cell concentration.Initial culture volume was 6 L and the set point of thespecific growth rate was 0.14 h-l. Medium composition isshown in Table I. Feeding medium 1, containing glucoseand trace metal, was supplied in the first feeding period;while feeding medium 2, containing all the components infeeding medium 1 together with yeast extract and MgS04,

.-.40

cv

-41

Ec

cV

0V

0

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' 1 4 0I

3 -

2 -

'!#0

0 5 10 15 20 25 SO

Time(hrs)

Figure 4. Effect of PMSF on decrease of specific bST concentration.Closed symbol represents the cell concentration and open symbol repre-sents the specific bST concentration.

was supplied when the cell concentration reached OD = 3 5 .The growth yield was constantly maintained at Y = 1.28(OD L/g glucose) with the feeding of the yeast extract.Thus, constant growth yield was used to calculate thefeeding rate in this range and the specific growth rate waswell controlled at the set point. Cell growth and specific

10 15 20 25 305

Time(hrs)

Figure 5. Profile of cell concentration and specific bST concentrationin fed-batch operation with exponential feeding of yeast extract: ( 0 ) el lconcentration, and ( 0 ) pecific bST concentration.

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bST concentration is shown in Figure 5. The specific bSTconcentration does not decrease at high cell concentration,whereas it decreases at higher cell concentrations thanOD = 30 - 40 in Figure 3. The addition of yeast extract,an organic nitrogen source, prevents the induction of theprotease and, consequently, prevents the degradation ofthe bST. The overall rate of the protein degradation wasobserved to increase many-fold when a carbon or organicnitrogen source such as amino acids was depleted.8 Tryp-

tophan concentration was zero and repression of the trppromoter was not observed during the exponential additionof the yeast extract. A similar experiment was carried outwith the addition of ammonium phosphate, an inorganicnitrogen source; however, the decrease of the specific bSTconcentration could not be prevented.

References

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13. Park, T.H., Seo, J.-H., Lim, H.C. 1989. Analysis of kinetic pa-rameters for the 3P-indoleacrylic acid effect on trp promoter inEscherichia cof i. Biotechnol. Lett. 11 : 87-92.

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15 . Riesenberg, D., Schulz, V., Knorre, W.A., Pohl H.-D., Korz, D.,Sanders, E.A., Ross, A,, Deckwer, W.-D. 1991. High-cell densitycultivation of Escherichia coli at controlled specific growth rate. J.Biotechnol. 20: 17-28.

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