Optimization of a cultivation process for recombinant protein production by Escherichia coli

Journal of Biotechnology, 23 (1992) 271-289 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00

271

BIOTEC 00734

Optimization of a cultivation process for recombinant protein production by Escherichia coli Xiao-Ming Yang R&D Lab, New Brunswick Scientific Co. Inc., Edison, New Jersey, U.S.A.

(Received 6 August 1991; revision accepted 13 November 1991)

Summary A single-stage f e d - b a t c h b i o p r o c e s s for t h e p r o d u c t i o n o f a r e c o m b i n a n t p r o t e i n , f l - g a l a c t o s i d a s e , by E. coli has b e e n d e v e l o p e d . T h e X L l - b l u e strain of E. coli which h a r b o r s a m u l t i - n u m b e r f o r e i g n p l a s m i d P T was c u l t u r e d in a r e f o r m u l a t e d m e d i u m . Critical m e d i u m c o m p o n e n t s w e r e s e l e c t e d a n d t h e i r r e s p e c t i v e c o n c e n t r a t i o n s w e r e o p t i m i z e d with t h e O r t h o g o n a l T a b l e m e t h o d . A n e x p o n e n t i a l s u b s t r a t e f e e d i n g s c h e d u l e was u s e d to m a i n t a i n o p t i m u m c o n d i t i o n s . I n h i b i t i o n of g r o w t h a n d p r o t e i n e x p r e s s i o n c a u s e d by excessive c o n c e n t r a t i o n s o f glucose a n d a c e t a t e was i n v e s t i g a t e d a n d s u b s e q u e n t l y m i n i m i z e d with an i n c r e m e n t a l n u t r i e n t f e e d i n g s c h e d u l e which l i m i t e d t h e specific growth r a t e o f a culture. T h e p r o g r a m n e c e s s a r y to facilitate t h e c o n t r o l of s u b s t r a t e a d d i t i o n is fully d e s c r i b e d . This p r o g r a m has b e e n u s e d with a 2.5 1 b i o r e a c t o r a n d a c o m m e r c i a l l y available s o f t w a r e p a c k a g e for o p t i m i z a t i o n w i t h o u t o n - l i n e o r off-line m e a s u r e m e n t o f o p t i c a l d e n s i t y ( O D ) , C O 2, .glucose o r a c e t a t e . T h e o p t i m i z e d f e d - b a t c h p r o c e s s l i m i t e d t h e a c e t a t e c o n c e n t r a t i o n to less t h a n 20 m M ; m a i n t a i n e d an e x p o n e n t i a l

Correspondence to: X.-M. Yang, R&D Lab, New Brunswick Scientific Co. Inc., 44 Talmadge Rd., Edison, NJ 08818-4005, U.S.A. Nomenclature: # = specific growth rate (h- i); Cs = concentration of glucose in stock solution (g ml- 1); F=glucose pumping rate (ml h-~); Ga=total glucose added during time t; Go=total glucose consumption (g) during time t; k = time constant or assumed growth rate; t = time (h) in fed-batch period; X = cell mass (g); X 0 = initial cell mass (g) in batch period; X/-= cell mass increase during fed-batch period; yg = glucose yield, cells per glucose (g g-~); Ybatch initial glucose yield, cells per glucose (g g- 1). =

272 growth phase for 50 h; and produced a cell density of 51 g 1-1 dry cell weight (DCW) or 154 OD60o with a /3-galactosidase activity of 990 U ml-1. Recombinant protein; E. coli cultivation; High cell density; Fed-batch bioprocess; ~-Galactosidase

Introduction

The successful commercial exploitation of recombinant DNA technology for the expression of heterologous proteins in microbial hosts requires the development of process strategies which are based on the genetics and physiology of the host-vector system (Zabriskie and Arcuri, 1986). During the past two decades there have been intensive studies on strategies for the optimization of biomass production of E. coli (Bauer and Shiloach, 1974; Landwall and Holme, 1977; Mori et al., 1979; Alien and Luli, 1987; Eppstein et al., 1989; Paalme et al., 1990). In the early work of Bauer and Shiloach (1974) the cell density was increased to 35 g 1-1. Currently reports of cell density in excess of 100 g 1-1 DCW are common (Allen and Luli, 1987; Eppstein et al., 1989). For recombinant E. coli, the desired outcome is not only high cell density but also high protein concentration. Generally these goals can only be achieved with an investigation which is dedicated to the optimization of each expression system (Zabriskie and Arcuri, 1986). In recent years, studies on recombinant E. coli which express foreign proteins were conducted to exploit the advances of genetic engineering technology (Zabriskie and Arcuri, 1986; Allen and Luli, 1987; Fieschko and Ritch, 1986; Jung et al., 1988; Shimizu et al., 1988; Meyer et al., 1984). A major concern of these studies has been the elimination of the growth inhibition by elevated concentrations of glucose and metabolic products of E. coli. Fieschko and Ritch (1986) obtained a cell density of 67.5 g 1-1 and a high expression level of human alpha consensus interferon using a manual two-stage fed-batch process in which the cell growth and protein expression were separated. In a single-stage process, in which cell growth and protein production were concomitant, Shimizu et al. (1988) adjusted glucose feeding in response to the off-line measurement of inhibitory substance concentration and reached a cell density of 28 g 1-1 with 64 U ml-1 of /3-galactosidase activity. Since a major cost component of recombinant protein production is contributed by the process operation, the main priority is the attainment of high productivity (U l-1 h-l). Thus, the goal is to obtain the optimum final concentration of product consistent with the minimum process time. Few studies (Zabriskie and Arcuri, 1986; Shimizu et al., 1988) have reported single-stage processes that achieved high cell density with high recombinant protein productivity, therefore this investigation focuses on a single-stage fed-batch mode process to illustrate the design and implementation of a supervisory control

273 strategy which overcomes the commonly encountered inhibition from acetate accumulation in recombinant protein production by E. coli.

Materials and Methods

Organism The host, E. coli XLl-blue (Bullock et al., 1987), (recAl,gyrA96,thi, hsdR17 (rk - ,mk + ),supE44,relA1, y-,lac-,[F',proAB, lac11ZAM15, TnlO(tet)]), was transformed with a multi-copy number plasmid PT. The plasmid was constructed from plNIII which contains the lpp promoter and ampicillin resistance gene, and pKM005 which contains the lac promoter-operator and full gene for/3-galactosidase (Inouye and Inouye, 1985; Masui et al., 1984). Its size is 12.6 kb. The lac promoter in the plasmid PT was inserted downstream of the lpp promoter which allows the expression of the lacZ gene to be induced by a lac inducer such as isopropyl-/3-D-thiogalactoside (IPTG) or lactose. (This strain was provided as a gift by Dr. M. Inouye at Robert Wood Johnson Medical School, New Jersey.)

Cultivations The strain was stored in a 2 ml frozen glycerol stock vial at -60°C. It was aseptically transferred into a 500 ml baffled shake-flask which contained 100 ml of a reformulated medium. This culture was grown overnight at 37°C and 250 rpm in a New Brunswick Scientific Co. (NBS) G-25 incubator shaker. A series of 500 ml shake-flasks which contained 100 ml of the reformulated medium were each inoculated with 5 ml of this overnight seed culture. These cultures were used for the medium optimization and inhibition studies. A 100 ml aliquot of this seed culture was used to inoculate the medium in a 2.5 1 reactor for the fed-batch studies. The conditions were maintained at 37°C with a pH of 7.4 controlled by the automatic addition of 10 N ammonium hydroxide.

Bioreactor system A 2.5 liter working volume BioFlo III R bioreactor (New Brunswick Scientific Co., Inc., NBS) was used. It was equipped with an internal multi-loop controller which regulates temperature, agitation, dissolved oxygen, pH, antifoam addition and nutrient addition. The elevated oxygen demand of the high density culture in this investigation was supplied by adding pure oxygen to air used as the sparge gas. The ratio of air to oxygen in the sparge gas was regulated by the duty cycle of air and oxygen supply solenoid valves shown in Fig. 1. These valves were operated on a regular periodic cycle of 1 s. The duty cycle was regulated with an ML4100 R general purpose controller (NBS). The Advanced Fermentation Software, AFS R (NBS) was used to make the operation of the internal controller in BioFlo III and the external controller ML4100 interactive and to control the nutrient addition

274 BIOFLO III o.o.

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I Fig. 1. Bioreactor and control system.

profile. This software was executed on a dedicated IBM R model P S / 2 30-286 personal computer with 2 megabytes of expanded memory and an Opto 22 RS-422 serial communication port. The integrated setup is schematically shown in Fig 1.

Analytical procedures The optical density was measured at 600 nm with a Perkin-Elmer Lambda 4B U V / V I S Spectrophotometer. Dry cell weight was determined with a 5 ml culture sample which was centrifuged at 5,000 rpm for 15 min, the pellet was collected, washed with deionized water, and dried at 80°C for 72 h in an oven. Glucose concentration was analyzed enzymatically with an A L P K E M hexokinase glucose kit. Acetic acid (acetate) concentration was assayed using an enzymatic analysis kit from Boehringer Mannheim. The assay of/3-galactosidase followed Miller's method (Miller, 1972). One unit of the enzyme activity was defined as the amount of enzyme which hydrolyses 1 ~mol O-nitrophenyl-/3-D-galactoside (ONPG) per min at 28°C (Shimizu et al., 1988).

Medium optimization Originally, M9 (Miller, 1972) medium supplemented with 2% casamino acid, 20 mg 1-1 L-proline and 20 mg 1-1 L-tryptophan were used as a medium since the

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synthesis genes for proline and tryptophan in the plasmid were next to the enzymatic working sites and their expression might be affected. Studies were conducted to compare a reformulated medium with M9 complex medium. The reformulated medium was initially composed as follows: KH2PO 4 2 g 1-1, K2HPO4 3 g 1-1, (NHa)2HPO 4 5 g 1-1, yeast extract 2.0 g 1-1, trace metal (Bauer and Shiloach, 1974) 0.5 ml 1-1, glucose 5 g 1-1, vitamin B1 1 mg 1-1, 1 M MgSO 4 • 7 H 2 0 2 ml 1-1, ampicillin 50 mg 1-1, pH 7.4. The cell growth and protein production obtained using the defined medium as well as the two modified M9 complex media are shown in Fig 2. The results show that the reformulated medium was more productive than M9 complex media. The reformulated medium was further optimized relative to nitrogen sources using the orthogonal table method as shown in Table 2 (Connor and Young, 1961). Selection of the nitrogen source and the determination of its concentration in the formulation of the medium is critical to the optimization of protein expression. Table 1 shows the matrix of the investigated components and their respective concentration levels.

276 TABLE 1 Investigated components and concentrations Concentration level

Yeast

(NH4)2HPO 4

L-Proline

L-Tryptophan

I II III

0 0.5 g 1-1 2 g l -~

0 0.5 g 1-1 2gl ~

0 10 mg l - I 50mg1-1

0 10 mg 1-1 50mg1-1

TABLE 2 Orthogonal table for investigation of nitrogen source Culture No.

Yeast extract

(NH4) 2 HPO 4

1 2 3 4 5 6 7 8 9

I I I II II II III III III

I II III I II III I II III

)21/3 F.II/3 ~]III/3 BEST Deviation

14.3 15.5 27.5 III 106.6

11.6 13.5 32.2 III 259.2

LProline

LTryptophan

I II III II III I III I II

I II II III I II II III I

14.0 24.8 18.5 II 58.9

22.9 21.1 13.2 I 53.3

Enzyme activity (U m l -

1)

5.5 16.4 20.9 7.9 13.2 25.5 21.4 10.9 50.1

Average 19.1

TABLE 3 Composition of the media Components

KH2PO 4 (g 1-1) K2HPO 4 (g 1-1) Yeast extract (g I 1) (NHn)2HPO 4 (g 1- i) Trace metal a (ml 1-1) MgSO4.7H20 a (g 1-1) Glucose a (g 1 1) Vitamin B1 b (mg 1-1) IPTG b (mg 1-1) Ampicillin b (mg 1-1) pH Volume ml

Concentration Initial medium

Feeding medium A

Feeding medium B

2.0 3.0 2.0 5.0 0.5 0.5 2.0 1.0 500 50 7.40 1300

10 400 312 125

100 150 -

800

300

These components were sterilized separately in an autoclave. b These components were sterilized separately by filtration. Concentrated ammonia was used to control pH. a

277

This matrix of experiments consisted of a series of flask cultures as described in the method section. The enzyme activities shown in Table 2 were assayed 12.5 h after inoculation to ensure that the cultures had reached the exponential growth phase. The results in Table 2 show that the optimum concentration of tryptophan is 0 and thus this component was eliminated from the medium. The optimum protein production occurred at the highest concentrations of yeast extract and (NHa)2HPO 4 and mid-range concentration of proline. The level of protein expression responded more sharply to variations of (NH4)2HPO 4 concentration than to the variations of the others. This indicated that (NH4)2HPO 4 is the most effective nitrogen source for the protein production. Therefore, (NHa)2HPO 4 was selected as the primary nitrogen source in the feeding medium for the fed-batch cultures. The elemental composition of E. coli ceils (Andersen and von Meyenburg, 1980) and the results of the above experiments were combined to formulate the initial and feeding media. The compositions of these media are shown in Table 3. The feeding media A and B were pumped in the culture with the ratio of their volumes.

Analysis and strategy Inhibition by acetate A series of experiments was conducted to characterize the response of the strain to elevated acetate levels at 37°C. A sharp response of cell growth to the addition of acetate is shown in Fig. 3. These data show that inhibition was independent of the activation of the foreign gene system for the protein overproduction. Similar results were observed in

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Fig. 4. The production profile of protein and biomass during the assimilation of glucose and acetate. Medium 1, 1 g 1-1 glucose and 10 mM acetate; medium 2, only 8.3 mM acetate; medium 3, only 1 g 1-1 glucose.

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Fig. 4 (continued). cultures at 30°C. Cell growth inhibition was not observed when the acetate concentration was limited to 10 mM as described in the next section. Assimilation of acetate E. coli cells are able to utilize acetate as a carbon source when glucose is absent (Andersen and von Meyenburg, 1980). The capability of the strain XLI-blue-PT to assimilate acetate was studied using three different medium compositions: medium 1, 1 g 1-1 glucose and 10 mM acetate; medium 2, only 8.3 mM acetate; medium 3, only 1 g 1-1 glucose. The remaining components were present in the amounts indicated for the initial medium in Table 3. The complete results of these experiments are shown in Fig. 4. As shown in Fig. 4a, acetate concentration increased during the first 2 h in all cases. In this period cells utilized the available carbon sources and produced acetate. Both glucose and acetate were eventually exhausted after 8 ~ 9 h. Although the total amount of carbon source for medium 1 was equal to the sum of the carbon sources in media 2 and 3, the production of biomass fell short of the combined biomass production of media 2 and 3. However, Fig. 4d shows the enzyme activity of medium 1 is approximately the sum of the enzyme activities of media 2 and 3. Thus when the ceils switched their carbon source from glucose to acetate, the total carbon yield for biomass was reduced while the carbon yield for the protein expression was maintained. This suggests that acetate is utilized preferentially to build /3-galactosidase. This observation was an important consideration in the subsequent development of an incremental glucose feeding schedule for the fed-batch mode. Control strategy Previous studies (Allen and Luli, 1987; Fieschko and Ritch, 1986; Shimizu et al., 1988) suggested that unrestricted growth of E. coli cultures resulted in the

280 accumulation of acetate, a glycolytic intermediate. Furthermore, these studies associate accumulation of acetate with adverse effects on the production of both biomass and recombinant proteins. Restricted growth rates have been successfully used to improve the productivity of recombinant E. coli cultivations (Allen and Luli, 1987; Fieschko and Ritch, 1986; Shimizu et al., 1988). Shimizu et al. (1988) reduced the growth rate of a recombinant strain by monitoring acetate concentration off line and discontinuing glucose addition whenever the acetate concentration exceeded a threshold. The frequent off-line measurements of acetate required to prevent inhibition make this technique extremely labor intensive. Fieschko and Ritch (1986) have shown that a manually adjusted substrate feeding schedule could be used to control the accumulation of acetate of a recombinant E. coli strain. These workers observed a significant improvement in the productivity of alpha consensus interferon when the growth rate was restricted by the substrate addition schedule. Allen and Luli (1987) have shown that an exponential substrate feed schedule was useful in restricting the specific growth rate, and was successful in considerably improving the biomass productivity of several strains. The results of Fieschko and Ritch (1986) demonstrate the problems encountered when the feeding schedule is regulated with coarse, infrequent steps. Their data clearly suggest more frequent and smaller addition increments would have allowed their culture to maintain a more constant growth rate and thus would have provided a significant reduction in process time. The results of these previous studies combined with the current results concerning acetate assimilation suggest that a delicate feeding schedule should provide improved productivity. This schedule requires glucose to be added stepwise with a sufficient time interval between addition steps to allow complete assimilation of glucose and acetate. In this investigation, an automatic nutrient addition program was developed to test the hypothesis. An exponential model has been used to predict the amount of glucose, G c, which would be consumed in a glucose-limited fed-batch cultivation. The model is derived from the growth dynamics and mass balance as follows: The specific growth rate is presented as

/x

dX

d(ln X )

X dt

at

(1)

w h e r e / z is the specific growth rate ( h - l ) , X is the total cell mass (g), and t is the growth time (h). Integrating Eqn. (1) yields

Xf=Xo[exp(txt ) - 1]

(2)

where Xf is the cell mass increase during the growth time t and X o is the total cell mass at t = 0. From the mass balance, relationship X f can be expressed as a function of the glucose yield (yg) and the total amount of glucose consumed (G c) X f ~ Gcyg

(3)

281 Combining Eqns. (2) and (3), we get G c =Xo/yg[eXp(~t

) - 1]

(4)

where X 0 is defined as the initial cell mass at the beginning of the fed-batch phase; t is the time elapsed during fed-batch operation; yg is the yield of ceils from glucose. The determination of the value of X 0 is subject to substantial experimental error. This error can be reduced if the seed culture is prepared in a batch cultivation with a medium which has an initial amount of glucose G 0. The batch phase is allowed to proceed until the glucose in the medium is exhausted. At this point X 0 can be calculated using the equation: X o = Go/Ybatc h

(5)

Eqn. (4) provides the prediction of the total glucose consumption profile in function of time t while the specific growth rate is/z. If we seek the growth rate to be limited at k (k < ~ma×), then the total glucose consumption becomes: Gc = X o / y g [exp(kt) - 1]

(6)

where k is a time constant for the glucose addition profile. The total amount of glucose actually added to a fed-batch process is obtained by simply integrating the pump flow rate, F (ml h-~), during the process. Thus Ga =

Csf F d t

(7)

where C s is the glucose concentration of the stock medium (g ml-1). In order to limit the growth rate at k, the glucose addition should be activated when G a is less than the target value of G~ and continues until G a exceeds G c. In this way, we expect to maintain the real specific growth rate of the culture at the selected value of k (k
Results and Discussion

A series of fed-batch experiments was conducted with various time constants, k, in the exponential feeding program. The bioreactor was inoculated and allowed to grow in the batch mode until the glucose in the initial medium was exhausted. Sharp increases of D O and pH in the culture were used as an indicator of glucose depletion. The exponential feeding program was started when glucose depletion was detected. Excellent reproducibility of the specific growth rate (0.2.5 h -1) in the batch phase was observed and is shown in Figs. 7a, 8a and 9a.

282

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283

In the first fed-batch experiment, an exponential time constant k of 0.3 was used in the feeding program. Since this value is greater than the growth rate (~max) observed in the batch phase of the process, we expected that glucose would z,

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284

I~e accumulated in the fed-batch phase of the cultivation. This prediction was confirmed by the data shown in Fig. 7. In the fed-batch phase, acetate was not accumulated to excessive values shown in Fig. 7c. As glucose was accumulated, the acetate concentration appeared to .5

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reach a steady state. This implies that the growth and protein production of this culture were inhibited by the accumulation of glucose. In the second fed-batch experiment, the time constant of the exponential addition program was set to 0.2. Since this value is less than the specific growth (a)

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rate observed in the batch phase, we did not expect glucose to accumulate. However, the data shown in Fig. 8 failed to confirm our prediction. These data also show that the specific growth rate during the fed batch phase was substantially lower than the expected value of 0.2. In this case, excessive accumulation of acetate occurred. After 23 h of fed-batch operation, the glucose concentration had increased to 12 g 1-1. At this point glucose addition was discontinued and the culture continued to produce acetate and protein until growth was essentially completely inhibited. In the third and fourth fed batch experiments, the time constant for the feeding program was set to 0.1. In both of these runs the specific growth rate agreed well with the expected value of 0.1 (h-1). The data in Figs. 9a and 12a show that under these conditions the cultures were maintained in the exponential growth state for over 53 h and 48 h, respectively. The data in Fig. 9c show that glucose and acetate were not accumulated until the end of the fed-batch phase. This allowed these cultures to produce high levels of biomass ( > 51 g 1-1 DCW) and high levels of enzyme activity (750-1000 U

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287

ml-1) as shown in Figs. 9b and 12b. The data in Figs. 9b and 12b show that the specific protein productivity was essentially stable through the entire fed-batch phase. When the ceils switched from using glucose to using acetate, their respiration rate was reduced and thus the DO level went up as shown in Fig. 10. The data in Fig. 10 also show that with k of 0.1 there was sufficient time in each nutrient addition cycle for the culture to completely consume the glucose added during the addition cycle. This cyclic pattern of DO was observed until the end of the experiment No. 3 and 4. The data in Fig. 9c show that in addition to depletion of the glucose, sufficient assimilation of acetate occurred following each step to delay the accumulation of acetate until 53 h of fed-batch operation had elapsed. This is in contrast to the data in Fig. 8c for the fed-batch run with an additional time constant k of 0.2 where glucose is not exhausted during the intervals between additions. This is clearly indicated in Fig. 11 by the disappearance of the cyclic pattern in the DO during the fed-batch process.

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In an attempt to simplify the process so that only a single feeding solution was required, (NH4)2HPO 4 and yeast extract (medium B), were replaced by adding (NH4)2SO 4 (110 g 1-1) in the stock medium A. The results in Fig. 12 show that this change also achieved long period of consistent exponential growth and high cell density, but reduced the specific protein productivity about 25%.

Conclusion The orthogonal table method used in the optimization of the medium was found to be an effective tool. Broader use of such statistical experimental method should reduce the burden of experimental work required in the medium optimization for other recombinant strains. The maximum growth rate of the recombinant strain XLI-blue-PT was quite low (0.25 h-t). This result was expected based upon the large size of the vector PT (12.6 kb). The growth and protein production of this strain were severely inhibited when acetate was accumulated to a concentration of 20 mM. In spite of these limitations, high cell densities with high specific protein expression levels were achieved. The productivity of this process was further enhanced by taking advantage of the preferential conversion of acetate to product during the second phase of each addition cycle during the fed-batch process. The data summarized in Fig. 13 clearly show the benefit of an extended exponential growth period even when a low specific growth rate is required to eliminate acetate accumulation.

Acknowledgements The author is highly grateful to Dr. M. Inouye and Dr. J. Cheng for providing the strain. I thank Mr. D. Freedman, Mr. E. Weisman and Dr. L. Eppstein for

289

their consistent support of this project, also Mrs. J. Ralph and Mr. H. Song for their expert advice on software, Mr. G. Wang for his helpful discussions and Mrs. A. Lubiak for her aid in preparing this manuscript.

References Allen, B.R.and Luli, G.W. (1987) A gradient-feed process for E. coli fermentation. Biopharm 1, 38-41. Andersen, K. and von Meyenburg, K. (1980) Are growth rates of Escherichia coli in batch cultures limited by respiration? J. Bacteriol. 144, 114-123. Bauer, S. and Shiloach, J. (1974) Maximal exponential growth rate and yield of E. coli obtained in a bench-scale fermentor. Biotechnol. Bioeng. 16, 933-939. Bullock, W.O., Fernandez, J.M. and Short, J.M. (1987) XL1-Blue: a highly efficient plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5, 376-379. Connor, W.S. and Young, S. (1961) Fractional factorial design for experiments with factors at two and three levels. Natl. Bur. Stan. (U.S.), Appl. Math. Ser. 58, 1-65. Eppstein, L., Shevitz, J., Yang, X. and Weiss, S. (1989) Increased biomass production in a benchtop fermentor. Biotechnology 7, 1178-1181. Fieschko, J. and Ritch, T. (1986) Production of human alpha consensus interferon in recombinant Escherichia coli. Chem. Eng. Commun. 45, 229-240. Inouye, S. and Inouye, M. (1985) Up-promoter mutations in the lpp gene of Escherichia coli. Nucleic Acids Res. 13, 3101-3110. Jung, G., Denefle, P., Bequart, J. and Mayaux, J.F. (1988) High-cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1/3. Ann. Inst. Pasteur/Microbiol. 139, 129-146. Landwall, P. and Holme, T. (1977) Influence of glucose and dissolved oxygen concentrations on yields of Escherichia coli B in dialysis culture. J. Gen. Microbiol. 103, 353-358. Masui, Y., Mizuno, T. and Inouye, M. (1984) Novel high-level expression cloning vehicles: 10-fold amplification of Escherichia coli minor protein. Bio/Technology 2, 81-85. Meyer, H., Leist, C. and Fiechter, A. (1984) Acetate formation in continuous culture of Escherichia coli K12 D1 on defined and complex media. J. Biotechnol. 1, 355-358. Miller, H.J. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York. Mori, H., Yano, T., Kobayashi, T. and Shimizu, S. (1979) High density cultivation of biomass in fed-batch system with DO-star. J. Chem. Eng. Jun. 12, 313-319. Paalme, T., Tiisma, K., Kahru, A., Vanatalu, K. and Vilu, R. (1990) Glucose-limited fed-batch cultivation of Escherichia coli with computer controlled fixed growth rate. Biotechnol. Bioeng. 35, 312-319. Shimizu, N., Fukuzono, S., Fujimori, K., Nishimura, N. and Odawara, Y. (1988) Fed-batch culture of recombinant Escherichia coli with inhibitory substance concentration monitoring. J. Ferment. Technol. 66, 187-191. Zabriskie, D.W. and Arcuri, E.J. (1986) Factors influencing productivity of fermentations employing recombinant microorganisms. Enzyme Microb. Technol. 8, 706-717.