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NOTES Effect of Modulated Glucose Uptake on High-Level Recombinant Protein Production in a Dense Escherichia coZi Culture Chih-HsiungChou3 George N. Bennett? and Ka-Yiu Sari',? Department of Chemical Engineering and Department of Biochemistry and Cell Biology, Institute of Biosciences and Bioengineering, Rice University, P.O. Box 1892, Houston, Texas 77251-1892
Methyl a-glucoside (a-MG) is a metabolically inert glucose analog sharing the same phosphotransferase system with glucose. The potential of using this compound, which acts as a nontoxic competitive inhibitor, to modulate glucose uptake and subsequently reduce the acetate accumulation rate was investigated. In a complex medium, no significant effect on the growth rate was observed when the a-MG to glucose ratio was low. The effect of a-MG supplementation on the production of a model recombinant protein, CadA-/3-galactosidase, under the regulation of a pH-inducible promoter in a batch culture was also examined. It was observed that the amount of acetate accumulation was drastically reduced in the presence of a-MG. More importantly, recombinant protein productivity was significantly improved. A very high volumetric productivity of approximately 1.6 g/Lrecombinant protein in a dense culture with a n OD600 of 35 was obtained in a simple batch fermentation. Even at this high cell density, the specific protein productivity was maintained at a high level and was estimated to account for about 40% of the total cellular protein.
Introduction Escherichia coli has been used widely to produce highvalue recombinant proteins. One challenging technical problem in most of the recombinant protein production processes is to achieve a high volumetric productivity. However, the goals of high gene expression and high cell density seldom can be obtained simultaneously in a dense culture. This is often due to the accumulation of harmful waste products that inhibit both cell growth and protein production. One of the major byproducts is acetate, which builds up rapidly in batch and dense cultures in which nutrient-rich environments are frequently provided. Traditional process improvement strategies to overcome byproduct formation or accumulation include fed-batch cultivation (Shimizu et al., 1988) and in situ waste product removal (MacDonald and Neway, 1990). A different strategy to reduce acetate accumulation based on modulated glucose uptake rate was examined in this study. The proposed approach builds on the following observations: (1)cells grown in complex medium derive most building blocks for macromolecule synthesis from enriched sources, such as yeast extract and casamino acids, and thus glucose is mainly used for energy supply (Ingraham et al., 1983);(2) glucose uptake by E. coli is loosely regulated (Holms, 1986); and (3) uptake of glucose, when present in excess amounts, normally exceeds the need for proper cell functions and subsequently leads to waste product formation, particularly acetic acid (El-Mansi and Holms, 1989). On the basis of these observations, it is speculated that acetate excretion may be reduced by properly modulating the
* Author to whom all correspondence should be addressed. t
Department of Chemical Engineering. Department of Biochemistry and Cell Biology.
glucose uptake rate. The objective of this work is to test this argument. Methyl a-glucoside is a glucose analog sharing the same phosphotransferase system with glucose (Postma, 1987). Because a-MG accumulates in the cell as the metabolically inert a-MG 6-phosphate to a high concentration without being toxic to the cell, it can be used as a nontoxic competitive inhibitor to modulate cellular glucose uptake. The effect of supplementing a-MG in rich medium on recombinant protein production was investigated. The results suggest that modulated glucose uptake in the presence of a-MG delays the onset of acetate excretion. An extremely high volumetric recombinant protein productivity of more than 1.6 g/L can routinely be attained in simple batch fermentations using the modified medium.
Materials and Methods Bacterial Strains and Plasmids. GJT-001 is a spontaneous S-(2-aminoethyl)-~-cysteine (SAEC)-resistant mutant of MC4100, a Alac strain ((arg-lac)U169 rspL150 refill (Tolentino et al., 1992). The construction of the plasmid pSM552-545C- is described in detail elsewhere (Meng and Bennett, 1992). The expression of the CadA-/3-galactosidase fusion protein under the regulation of a pH-inducible promoter system was used as a model system. The host cell GJT-001 was used in the growth characterization, and GJT-001 carrying the plasmid pSM552-545C- was used in the study of recombinant protein expression. Cultivation in Shake Flasks. Media. LB medium (Miller, 1972) buffered with phosphate salts (8.2 g& NazHP04.7Hz0 and 2.7 g/L KHzP03 a t pH 7 was used as the base medium. Glucose at 10 g/L was supplemented
8756-7938/94/3010-0644$04.50/00 1994 American Chemical Society and American Institute of Chemical Engineers
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Table 1. Composition of Media Used in This Study media component SB RSB-1 RSB-la RSB-2 RSB-2a RSB-3 RSB-3a NaCl(g/L) 5 5 5 5 5 5 5 yeastextract 20 40 40 50 50 50 50 (&)
tryptone(g/L) 32 glucose(&) 10 a-MG( g G M9saltsa lx
32 15
32 15
lx
5 lx
32 15
32
32
15
20
32 20 6.67
lx
lx
5 lx
lx
l x : 7 g/L Na2HP04,3 g L KH2PO4,l g/L NHdCl, and 0.5 g/L NaCl.
as an extra carbon source. Various amounts of a-MG (2 and 10 g L ) were added to investigate its effect on cell growth. Seed Culture Preparation. Cells were reactivated by transferring 10 pL of frozen cell stock culture into 50 mL of LB medium. The culture was grown in an orbital shaker (Lab-Line) at 300 rpm and 37 "C for 12 h. Shake Flask Conditions. Batch cultivations were carried out in the flasks containing 50 mL of the appropriate medium. The cultures were grown in an orbital shaker at 300 rpm and 37 "C. Samples from each flask were redrawn periodically for analysis. Cultivation in Bioreactors. Media. Superbroth (Ausubel et al., 1988) supplemented with M9 salts (Miller, 1972) and 10 g/L glucose was used as the base medium recipe (SB). Three types of richer media, RSB1, RSB-2, and RSB-3 were also prepared by increasing the yeast extract and glucose concentrations. For those media containing a-MG, an appropriate amount of a-MG was added (Table 1). All of the media were supplemented with 100 mg/L ampicillin, 50 mg/L kanamycin, and 100 pLIL antifoam 289 (Sigma). Preparation of Large Inocula. The inoculum for the protein production study was prepared by growing cells in a bioreactor since a large amount of culture was required (San et al., 1994). About 10-20 mL of the seed culture was transferred into a 1-L fermentor (Virtis) containing 600 mL of SB medium. The reactor pH, temperature, and agitation rate were 7.00 f 0.05,27 "C, and 800 rpm, respectively. Oxygen was supplied by sparging filter-sterilized air at 2 Umin. After 12 h, 80100 mL of stationary culture (ODs00 of about 35) was collected and centrifuged. The cell pellet was resuspended with 50 mL of LB medium and was ready to be transferred into another fermentor for the protein production study. Bioreactor Conditions for the Protein Production Study. Batch fermentations were carried out in a 1-L fermentor containing 500 mL of the appropriate medium. The reactor pH, temperature, and agitation rate were 6.00 f 0.05, 30 "C, and 1000 rpm, respectively. Oxygen was supplied by sparging filter-sterilized air at 2 Umin. The fermentor was inoculated with an initial optical cell density of 5.0. All process variables were interfaced to a microcomputer for monitoring and data logging. Analysis. Enzyme Assay. Cell extracts for the enzyme assay were prepared by sonicating (Heat Systems) the properly diluted fermentation samples for 6 min. P-Galactosidase was assayed at 28 "C using onitrophenyl b-D-gdadopyranoside as the substrate (Miller, 1972). The unit of volumetric protein activity was defined as the product of specific activity (Miller unit) and optical cell density measured at 600 nm. Acetate Concentration. One milliliter of extracellular culture medium was acidified by adding 20 pL of 50% sulfuric acid. Five microliters of acidified medium was injected into a Varian 3300 GC equipped with a 6 ft
Table 2. Effect of a-MGSupplementation on Apparent Growth Rate a-MG concentration (g/L) 0 2 10 apparent growth rate papp,~ P~PP,~ P~PP,~ run 1 1.50 1.54 1.40 run 2 1.47 1.47 1.37 run 3 1.44 1.51 1.40 av 1.47 1.51 1.39 null hypothesis Pam1 = P ~ P PPaw2 , ~ = P ~ P PP, S~P P= , ~Pam1 50 99 98 confidence level to reject the null hypothesis (%)
100/120 Chromosorb-101-packed column (Alltech) and a flame ionized detector.
Results and Discussion Effect of a-MG on Cell Growth. Previous studies (Ingraham et al., 1983) showed that a-MG can be used as a nontoxic and metabolically inert competitive inhibitor for glucose uptake. Subsequently, the addition of a-MG to glucose minimal medium can be used to modulate the culture specific growth rate. However, no experiment has been conducted to examine the effect of a-MG on the cellular growth rate in a complex medium. The growth characterization studies in a complex medium (LB supplemented with 10 g/L glucose) were carried out in the flasks with two a-MG concentrations at 2 and 10 g/L, respectively. Three parallel runs for each a-MG concentration were performed to allow a statistical analysis. A logistic model (Zwietering et al., 1990)given by the following equation was used to estimate the apparent specific growth rate from the experimental data:
where x is the optical density measured at 600 nm, xm is the estimated final cell density, and pa, is the apparent specific growth rate. The sigmoidal function was chosen to fit growth curves because it can simulate the growth rate changes after the exponential phase. The apparent can be treated as an average specific growth rate, pappr growth rate indicator, in which all of the growth-rateaffecting factors, excluding the amount of unused medium, are lumped for the whole growth curve (Zwietering et al., 1990). The estimated apparent specific growth rates are listed in Table 2. It was shown that the growth rate decreased, with at least a 98% confidence level when subjected to the t-test, by adding 10 g/L a-MG. However, no significant effect on the growth rate could be detected when the a-MG concentration was only 2 g L . These results agree with the common observations that the major role of glucose in complex medium is as an energy supply (Ingraham et al., 1983) and that glucose uptake by E.coli normally exceeds the need for proper cell functions (El-Mansi and Holms, 1989). These observations, coupled with the speculation that byproduct formation is a consequence of excessive glucose uptake, suggest that an appropriately modulated glucose uptake rate will reduce the extent of waste product formation without impairing cell growth in enriched media. Effect of Enriched Medium with a-MG Supplementation. The control fermentation using SB reached a relatively high optical cell density of 30 and a high volumetric recombinant protein productivity of 1.8 million units/mL. The specific gene expression increased to a maximum value of 60 000 Miller units (MU), which
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Table 3. Effect of a-MGSupplementation on Recombinant Protein Production media SB RSB-1 RSB-la RSB-2 RSB-2a RSB-3 RSB-3a 46 35 34.5 39 40 46 OD6ooU 30 VPb 1.8 1.7 2.1 1.2 1.9 1.2 1.9 SPC 61.4 35.8 59.5 39.2 48 33.1 45.9 A& 2 2.7 2.9 4.8 2.3 5 2.7 Final optical cell density measured at 600 nm. Final volumetric P-galactosidase productivity (million units/mL). Final specific @-galactosidaseproductivity (thousand Miller units, kMU). Highest acetate concentration observed during the experiment (gfu.
accounts for approximately 40% of total cellular protein, that was maintained to the end of the batch (data not shown). For a richer medium composition, RSB-1, the specific gene expression was reduced by 40% while the final biomass was increased by almost 50%(Table 3, rows 1 and 2). This led to a lower volumetric productivity compared to that in the SB control. The results appear to suggest that most of the available resources are preferentially converted to biomass instead of the desired product, recombinant protein. This observation is consistent with previous reports that the inhibitory effect of acetate on recombinant protein synthesis is stronger than that on cell growth (Bauer et al., 1990). Further deterioration in both specific recombinant protein expression and cell growth was obtained for richer media (RSB-2 and RSB-3) due to a higher level of acetate accumulation (Table 3, columns 5 and 7). Effect of a-MG Supplementation. A significant improvement in culture performance was obtained by adding a-MG to the medium. Experiments with the glucose analog supplement consistently outperformed those without (Table 3, rows 2 and 3). The glucose analog, which serves as a modulator for glucose uptake, appears to be capable of reducing culture sensitivity to medium composition. Recombinant protein as high as 1.6 g/L can be obtained in a simple batch fermentation with RSB-la. The higher recombinant protein productivity in the presence of a-MG may be attributed to the reduction in acetate accumulation: the maximum acetate levels dropped to about one-half for RSB-2a and RSB3a compared to those of RSB-2 and RSB-3, respectively (Table 3, row 4). Comparable levels of maximum acetate were detected for the RSB-1 medium with and without a-MG supplementation. However, the experiment with a-MG had a substantially lower initial acetate concentration. The reduction in acetate accumulation during this protein production period is critical to the final productivity. The delay in the onset of acetate accumulation enables the specific protein expression to increase by more than 60%, representing a jump from 36000 to almost 60 000 MU (Table 3, row 3). It should be recalled that 60 000 MU corresponds to about 35-40% of total cellular proteins. Effect of Medium Composition. The effect of a-MG supplementationon culture performance in a wider range of medium compositions was examined further. Several types of enriched medium formulation were prepared by varying yeast extract and glucose concentrations. Batch fermentations instead of fed-batch ones, in which culture performance frequently depends on the optimized feeding strategies, were chosen in this study to provide a better basis for comparison. A summary of culture performances for 16 experiments is shown in Figure la,b. One general observation is that, by increasing yeast extract (Y) and glucose (G) concentrations, both the specific and volumetric recombinant protein productivities decreased
Figure 1. Culture performance with various media. Results from a superbroth (Y20, G10, AO) are also included. The symbols Y, G, and A denote yeast extract, glucose, and a-MG, respectively; the number following the symbols indicates the quantity used in g/L. Results are summarized as (a) final optical cell density (ODsoo) and (b) final volumetric P-galactosidase productivity in three-dimensional bar charts.
due to higher acetate accumulation. For example, enrichment of Y G = 20:lO to 50:20 results in a sharp decrease from 61 400 to 33 100 MU. The volumetric activities also declined by more than 30%, from a value of 1.8 million units for Y:G = 20:lO to 1.2 million units for Y:G = 50:20. Similar to previous results, the addition of a-MG is capable of improving culture performance, even in these much enriched conditions. The volumetric productivities were improved from 1.7 to 2.1 million units and from 1.2 to 1.9 million units/mL for two medium formulations of Y G = 40:15 and Y:G = 50:20, respectively, by the addition of a-MG. However, no significant effect was observed by increasing the a-MG dosage (Figure lb, Y G = 50:15 and 60:20).
Conclusion A systematic study was performed to examine the effect of a-MG supplementation on cell growth and recombinant protein production in E . coli. The results show that no significant effect on the specific growth rate can be detected in complex medium when the ratio of a-MG to glucose is low, and only a slight decrease is detected when the ratio is 1. The results suggest that the glucose uptake rate is not a limiting factor for cells growing in complex medium. Experiments with various medium compositions demonstrated that a-MG can be used as a potent and nontoxic competitive inhibitor for modulating the cellular glucose uptake rate. The addition of a-MG effectively and consistently reduces acetate production during the growth and protein production phases. The lower level of acetate accumulation in the
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bioreactor results in a significant increase in recombinant protein productivity. The results u s i d various medium compositions with and without a-MG supplementation also indicate that a-MG is capable of minimizing culture sensitivity to an enriched environment. Since overfeeding is one of the many technicd'pqoblems frequently encountered during fed-batch cultivations (Shimizu et al., 19881, results from the current study suggest that a strategy of adding a-MG can be adopted to alleviate the overfeeding problem and widen the operating window.
Acknowledgment This material is based in part upon work supported by the Texas Advanced Technology Program under Grant Nos. 003604-011 and 003604-035. Literature Cited Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. J., Smith, J. A., Struhl, K., Eds. Current protocols in molecular biology; John Wiley & Sons: New York, 1988. Bauer, K. A.; Ben-Bassat, A.; Dawson, M.; de la Puente, V. T.; Neway, J. 0. Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant. Appl. Environ. Microbiol. 1990, 56, 1296-1302. El-Mansi, E. M. T.; Holms, W. H. Control of carbon flux t o acetate excretion during growth of Escherichia coli in batch and continuous cultures. J . a n .Microbiol. 1989,135,28752883. Holms, W. H. The central metabolic pathways of Escherichia coli: relationship between flux and control a t a branch point, efficiency of conversion to biomass, and excretion of acetate. In Current topics in cellular regulation; Horecker, B. L., Stadtman, E. R., Eds.; Academic press, Inc.: Orlando, FL, 1986; Vol. 28, pp 69-105.
647 Ingraham, J. L.; Maaloe, 0.; Neidhardt, F. C. Growth ofthe bacterial cell; Sinauer Associates Inc.: Sunderland, MA, 1983. MacDonald, H. L.; Neway, J. 0. Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12. Appl. Environ. Microbwl. 1990, 56, 640-645. Meng, S.-Y.; Bennett, G. N. Regulation of the Escherichia coli cad operon: Location of a site required for pH induction. J . Bacteriol. 1992, 174, 2670-2678. Miller, J. H. Experiments in Molecular Genetics; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY,1972. Postma, P. W. Phosphotransferase system for glucose and other sugars. In Escherichia coli and Salmonella typhimurium-cellular and molecular biology; Neidhardt, F. C., et al., Eds.; American Society for Microbiology: Washington, D.C., 1987; Vol. 1, pp 127-141. San, K.-Y.; Bennett, G . N.; Chou, C.-H.; Aristidou, A. A. An optimization study of a pH-inducible promoter system for high level recombinant protein production in Escherichia coli. Ann. N.Y. Acad. Sci. 1994, 721,268-276. Shimizu, N.; Fukuzono, S.; Fujimori, R;Nishimura, N.; Odawara, Y. Fed-batch cultures of recombinant Escherichia coli with inhibitory substance concentration monitoring. J . Ferment. Technol. 1988, 66, 187-191. Tolentino, G. J.; Meng, S.-Y.; Bennett, G. N.; San, K.-Y. A pHregulated promoter for the expression of recombinant proteins in Escherichia coli. Biotechnol. Lett. 1992, 14, 157-162. Zwietering, M. H.; Jongenburger, I.; Rombouts, F. M.; van't Riet, K. Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 1990, 56, 1875-1881. Accepted July 6, 1994.@ @Abstractpublished in Advance ACS Abstracts, August 15, 1994.
