Biotechnol. hog. 1992, 8,307-315
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Bioprocess Development To Improve Foreign Protein Production from Recombinant Streptomyces Neslihan DelaCruz,t,t Gregory F. Payne,*,?*s Jeffrey M. Smith,?and Steven J. Coppella?*llgL Department of Chemical and Biochemical Engineering, Center for Agricultural Biotechnology, and Center for Medical Biotechnology, University of Maryland Baltimore County, Baltimore, Maryland 21229
Bioprocessing strategies to improve production of the heterologous protein parathion hydrolase from recombinant S t r e p t o m y c e s l i v i d a m were investigated. Initial limitations to increased production were overcome by using large amounts of nutrients and feeding these nutrients throughout the fermentation. Batch addition of such large amounts of nutrients resulted in byproduct acid accumulation. Our data suggest that byproducts resulted from incomplete utilization of peptide medium ingredients and not from an overflow of glucose catabolism. Over extended fed-batch operation, oxygen transfer became limiting and these limitations were overcome by sparging oxygen-enriched gas. When cultivation was continued past about 90 h, we observed that despite nutrient feeding and oxygen enrichment enzyme activities no longer increased. Our results show that during such late cultivation periods the rates of enzyme synthesis and deactivation became balanced. If synthesis is prevented, either by a nutritional limitation or by the addition of the protein synthesis inhibitor chloramphenicol, enzyme activities were observed to decrease. Since deactivation rate constants in these experiments were similar to those observed in cell-free studies, and because extracellular protease activities were not detected in our fermentation, it appears that deactivation results from the inherent instability of the parathion hydrolase enzyme.
Introduction Because of their ability to secrete proteins, Streptomyces have been considered as an alternative host organism for producing recombinant proteins. Development of Streptomyces as an alternative host has been facilitated by the construction of a stable, high copy number plasmid (Katz et al., 1983). Initial studies focused on the identification, characterization, and use of Streptomyces promoters (Grayet al., 1984;Brawner et al., 1985; Pulido et al., 1986; Westpheling and Brawner, 1989; Koller and Riess, 1989). Since it has been suggested that the secretion rate may limit the extracellular appearance of foreign proteins [e.g., Bender et al. (1990a)l,several groups have examined strategies to improve secretion. Such strategies have generally involved the fusion of the desired gene to Streptomyces signal sequences (Lichenstein et al., 1988; Illingsworth et al., 1989; Bender et al., 1990b) or to genes coding for normally secreted Streptomyces proteins (Koller et al., 1989; Taguchi et al., 1989). It is also interesting to note that cloning of regulatory genes may result in enhanced production of extracellular proteins from Streptomyces (Daza et al. 1990). In addition to genetic strategies, bioprocessing strategies are also important for improved production from Streptomyces. Traditionally,bioprocessing strategies for Streptomyces have been developed to ensure that the physiological needs of the culture are satisfied. These strategies generally involve the use of improved media and fed-batch t
Department of Chemical and Biochemical Engineering.
t Current address: Arctech Inc., 5390 CherokeeAve.,Alexandria,
VA 22312. 5 Center for Agricultural Biotechnology. 11 Center for Medical Biotechnology. 1 Current address: Pure Carbon Co., 441 Hall Ave., St. Marys, PA 15857.
operation to obtain high productivities over extended fermentation periods (Bader, 1986). Although these strategies have been effective for the production of secondary metabolites, there have been few studies reported on the use of bioprocessing strategies to improve production of foreign proteins from Streptomyces. In our work, we are examining how bioprocessing strategies aimed at meeting the physiological needs of the culture can be used to enhance heterologous protein production in Streptomyces. It should be noted that for many recombinant microbes, bioprocessingstrategies, such as two staged chemostat operation, are often considered to address genetic, and not physiological, requirements [e.g., Park and Ryu (1990) and Bentley et al. (1990)l.We have not considered bioprocessing strategies aimed at addressing genetic issues for two reasons. First, Streptomyces transformed with pIJ702-derived plasmids have often been observed to be stable in the absence of selective pressure (Ghangas and Wilson, 1987;Bertrand et al., 1989; Payne et al., 1990a) and thus strategies to address genetic instabilities appear to be unnecessary. Second, since strong promoters which are also readily inducible are currently unavailable for Streptomyces, it is not possible to consider bioprocessing strategies to optimize the timing of induction. If strong and/or inducible promoters are developed for Streptomyces, the above two reasons may no longer hold. In previous studies (Payne et al., 1990a), we examined bioprocessing strategies to enhance the production of a heterologous protein (the parathion hydrolase enzyme) from a transformed Streptomyces lividam (Steiert et al., 1989). We observed that incorporation of a readily utilizable carbohydrate into a peptide-based complex medium resulted in enhanced growth and expression. Similar conclusions were obtained by Erpicum et al. (19901, who studied the production of extracellular enzymes from
8756-7938/92/3008-0307$03.00/0 0 1992 American Chemical Society and American Institute of Chemical Englneers
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various recombinant Streptomyces. In addition, we observed that slowly feeding both the carbohydrate and nitrogen source (tryptone)resulted in substantial increases in enzyme production. Nutrient feeding to industrial fermentations is common. Traditionally, glucose feeding has been used to prevent the accumulation of this nutrient in fermentations which are susceptible to glucose catabolite regulation. More recently, glucose feeding has been employed to prevent the accumulation of byproducts which can be inhibitory or toxic to the culture. Ethanol from yeast (Sonnleitner and Kappeli, 1986),acetate from Escherichia coli (Anderson and von Meyenburg, 1980; Majewski and Domach, 19901, and acetate and lactate from Bacillus (Snay et al. 1989)are believed to be produced under aerobic conditions due to an imbalance in the flux of metabolites through the glycolytic, TCA, and oxidative phosphorylation pathways. Glucose feeding has been observed to reduce the accumulation of these byproducts thus eliminating the associated toxicity and limiting the wasteful conversion of the carbohydrate to byproducts (Wang et al., 1979; Robbins and Taylor, 1989; Paalme et al., 1990). Acid production in our cultures was suggested by the observation that 6 times less base was required to maintain neutrality in a glucose-fed fermentation as compared to a batch fermentation which had consumed a comparable amount of glucose. Streptomyces have previously been reported to produce as byproducts, pyruvic and a-ketoglutaric acids (Hockenhull et al., 1954; Doskocil et al., 1959; Bormann and Hermann, 1968; Ahmed et al., 1984; Surowitz and Pfister, 1985;Dekleva and Strohl, 1987). If the acids which accumulate during batch cultivation of our S. liuidans suppress growth and enzyme production, then it is possible that the increased performance in glucose-fed systems is due to a reduction in byproduct accumulation. The first goal in the present study was to provide a better characterization of byproduct formation. In addition to ensuring an appropriate supply of the soluble nutrients, it is important to consider the culture's requirement for oxygen. Streptomyces are aerobic microorganisms, and it has commonly been observed that reductions in dissolved oxygen have significant adverse effects on Streptomyces secondary metabolite biosynthesis (Hostalek, 1964; Rollins et al., 1988; Chen and Wilde, 1991;Yegneswaran et al., 1991). The importance of oxygen can be understood by recognizing that oxygen is generally the limiting nutrient in industrial Streptomyces fermentations (Bader 1986). Interestingly, Magnolo et al. (1991) used a genetic approach to overcome oxygen limitations. These authors cloned a bacterial hemoglobin gene into Streptomyces and observed that these recombinants were able to grow and produce antibiotics even under oxygenlimiting conditions. Presumably, the hemoglobin protein conferred a higher oxygen affinity to the recombinant cells. In our study, the second goal was to explore if culture performance could be improved by avoiding oxygen limitations. Finally, we report here that despite meeting the nutritional needs of the culture continued accumulation of the parathion hydrolase enzyme is not observed after about 90 h when enzyme activities reached about 40 units/mL. Our results suggest that the limitation to further improvement is the inherent instability of the parathion hydrolase enzyme.
Materials and Methods The transformed Streptomyces liuidans used in this study was previously described by Steiert et al. (1989).
Inoculum was prepared by adding spores to LB medium containing 10 g of tryptone; 5 g of yeast extract; 10 g of NaC1; and 30 mg of thiostreptoniL of distilled water. All experiments were conducted in media containing only glucose and tryptone and without thiostrepton. To systematically investigate the effects of oxygen on enzyme production, we used two experimental approaches. To determine trends of how enzyme production was affected by varying levels of oxygen, a shake flask system in which gases of varying oxygen content were passed through the head space was studied (Payne et al., 1990b). In the second experimental approach, a fermentor (BioFlow 111,New Brunswick Scientific)was used and oxygenenriched air was sparged to maintain the dissolved oxygen levels above 30% air saturation. Oxygen enrichment was done either manually or by using computer control (Smith et al., 1990). The pH in all fermentor runs was maintained constant at 7.0. During cultivation, samples were taken intermittantly and various analyseswere performed. After centrifugation, the pellet was washed and dried to obtain the dried cell concentration (DCW). The supernatant was analyzed for glucose (Yellow Springs Instrument); ammonium (EktachemDT 60, Kodak); parathion hydrolase; and fermentation acids. Parathion hydrolase activity was determined by measuring the rate of formation of the parathion hydrolysis product, p-nitrophenol, using a spectrophotometer (Gilford Response) at 410 nm [see Payne et al. (1990a)l. The reaction mixture for this enzyme assay consisted of 3 mL of TRIS buffer (10 mM) containing 0.005% Tween 80, pH 8.5; 9.9 pL of parathion solution containing 43 mM parathion in methanol; and culture supernatant. A unit of enzyme activity is defined as 1 pmol reacted per min. Fermentation acids were determined by gas chromatography (Hewlett Packard). For analysis, the samples were first acidified with 10 N HC1 and then 1 pL was injected into a cross-linked FFAP column. A temperature gradient of 60 to 235 "C in 20 min was used. The hydrogen carrier gas flow was 2 mL/min. Acids were identified on the basis of comparison of their retention times with the retention times of pure acid standards.
Results and Discussion Byproduct Accumulation. In our previous work, we observed that expression of extracellular parathion hydrolase by recombinant Streptomyces lividans was improved by supplementing the amino acid-peptide based complex medium with moderate amounts ( ~ 3 g/L) 0 of glucose (Payne et al., 1990a). When higher initial glucose levels (>30 g/L) were used, we observed an increase in acid production on the basis of either the degree of pH reduction in shake flask cultures or the amount of caustic required to maintain neutrality in fermentor cultures. Cultures without glucose supplements, or after glucose depletion, became more basic with time. These results suggested that varying types and levels of byproducts were produced in response to differing initial glucose concentrations. Figure 1 shows our hypothesis for the assimilation of the peptide and glucose components under the extremes of excessive glucose (panel a) and insufficient glucose (panel b). Under the extreme of the excessive glucose,the carbohydrate appears to serve as the primary carbon source while the peptide components appear to be used as a source of nitrogen with the accumulation of deaminated acids. Support for this hypothesis is illustrated in Figure la, in which we compare the results from a batch culture
809
Biotechnol. Rog., 1992, Vol. 8, No. 4 High Glucose Levels
Glucose
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Figure 1. Hypotheses to account for byproduct accumulation under conditions of excess glucose (a) and insufficient glucose (b) conditions in complex media.
containing high levels of initial nutrients (100 g/L glucose and 67 g/L tryptone) to those from a fed-batch culture in which a large total amount of nutrients (a total of 90 g/L for both glucose and tryptone) were added throughout the fermentation, In both cases, oxygen limitations were avoided by either manually or automatically (Smith et al., 1990) enriching the sparged air stream with pure oxygen. As shown in Figure 2, the major acid accumulated during these fermentor runs was identified (by comparison of GC retention times with those for pure standards) as isovaleric acid with lesser amounts of acetic acid being observed. Although little is known about the production of isovaleric acid, isovaleryl-CoA is known to be produced from the deamination of leucine [e.g., Naglak et al. (1988)l. Thus, if our identification of isovaleric acid is correct, a significant amount of the acids may have resulted as byproducts from the deamination of the peptide medium components (i.e., tryptone), and not from an overflow of carbohydrate catabolism. As can be seen in Figure 2a, higher levels of isovaleric acid accumulated in batch cultures when excessive initial levels of glucose and tryptone were used. Accumulation of both isovaleric and acetic acid was reduced by nutrient feeding. Figure 2c shows dramatic differences in enzyme levels. In fed-batch cultures, enzyme accumulated throughout the fermentation reaching 31 units/mL. In the batch culture however, both growth and enzyme accumulation ceased at 70 h when isovalericacid levels peaked. The maximum enzyme level in this batch culture was only 8.3 units/mL. These observations suggest that the accumulation of acidic byproducts (as illustrated by isovaleric acid) may be responsible for suppressed enzyme synthesis in batch culture. It should be noted however, that when varying amounts of isovaleric acid (up to 2.5 g/L) were added to actively producing cultures, no adverse affects on enzyme production were observed (data not shown). Thus isovaleric acid by itself may not be inhibitory, but rather conditions in which isovaleric acid accumulates have been observed to be inappropriate for efficient parathion hydrolase production. It should be noted that numerous smaller peaks appeared on the chromatograms from the
batch experiment and that it is possible that alternative, unidentified byproducts are inhibitory. Figure 2 illustrates two additional features of the fermentation which have been commonly observed. First, when nutrient conditions are used which permit rapid initial growth (i.e., the batch culture), parathion hydrolase production is generally reduced. Although it may be necessary to avoid rapid initial growth, Figure 2 also illustrates that parathion hydrolase is produced during growth and that fed-batch operations are most successful when sufficient nutrients are supplied to permit a prolonged growth phase. Further support for the hypothesis that excessive glucose results in a reduced utilization of peptide carbon (Figure la) is provided by considering the off-gas carbon dioxide levels between a batch and a glucose-fed culture. When glucose was slowly fed, not only was acid accumulation reduced, but the molar ratio of carbon dioxide evolved to glucose consumed was observed to be between 8 and 9 (data not shown). This can be contrasted to the 3-4 mol of carbon dioxide evolved per mole of glucose consumed observed in batch cultures. Since only 6 mol of carbon dioxide can be evolved from the complete oxidation of a mole of glucose,then carbon from peptide ingredients (e.g., tryptone) must have been utilized when the glucose supply was restricted by slow feeding. Finally, indirect support for the hypothesis that excessive glucose limits peptide-carbon utilization (Figure la) is provided by Erpicum et al. (1990), who reported that recombinant Streptomyces were more productive in the presence of glucose if ammonium was added to reduce the utilization of amino acids. When ammonium was not included in their glucose-supplementedcomplex medium, large pH reductions were observed and foreign protein production was reduced. The other extreme of insufficient glucose is illustrated in Figure 1b. When insufficientglucose is available,carbon from the peptide medium components is preferentially utilized, while ammonium accumulates in the medium. This is illustrated by the batch cultures in Figure 3 in which moderate initial nutrient levels were used. When moderate nutrient levels were used batchwise, Figure 3 shows that ammonium accumulates to 150 mM and the pH increases during fermentations which lack glucose. Again, it is unclear whether ammonium accumulation is responsible for the poor performance of cultures lacking glucose. However, it is important to note that the ammonium level of 150 mM which was observed in the studies shown in Figure 3 is within a range which could be inhibitory. In Bacillus fermentations, Kole and Gerson (Kole et al., 1988; Kole and Gerson, 1989) observed that excessive ammonium levels reduced protease and amylase production, while significant improvements in production were observed when ammonium feeding was used to maintain ammonium levels at approximately 5 mM. In E. coli fermentations, Thompson et al. (1985) also observed that high ammonium concentrations (greater than 170mM) adversely affected growth. It is well-known that ammonium levels of about 40mM can adversely affect the production of some secondary metabolites in Streptomyces (Shapiro and Vining, 1985; Brana et al., 1985; Wallace et al., 1990). Also, Held and Kutzner (1990) recently reported that 10 mM ammonium repressed enzyme (i.e., tyrosinase) synthesis in Streptomyces michiganensis. With respect to the previously cited report of Erpicum et al. (19901, it should be noted that the ammonium supplement used to enhance recombinant enzyme production in Streptomyces was only 50 mM.
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In conclusion, our results indicate that high total amounts of nutrients (both glucose and tryptone) are required to achieve high levels of parathion hydrolase. However, to supply such high nutrient levels, we have observed that nutrient feeding is better than batch addition (Payne et al., 1990a). It is possible that improved performance in fed-batch cultures is due to a reduction in the accumulation of potentially inhibitory byproducts. Also, it is possible that the low initial nutrient levels in the fed-batch cultures support slower initial growth. We have observed that rapid initial growth is correlated to poor performance in this fermentation. Oxygen Requirements. In order to systematically study how enzyme production is affected by oxygen, we used a shake flask system in which gases of varying oxygen content were passed through the head space (Payne et al., 1990b). By using small media volumes (50 mL in a 500mL flask),high gassing rates (150mL/min), and high levels of agitation (320 rpm), we attempted to ensure that the oxygen transfer capacity of the flasks exceeded the consumption rates of the cultures such that the gases and liquids would be nearly in equilibrium. The advantage of this system is that since various oxygen levels can be studied simultaneously by using multiple flasks, batch to batch variability can be avoided. Such variability often results when studies are carried out sequentially using different inocula. The disadvantage of this system is that
because of the practical difficulties in maintaining gasliquid equilibrium, the actual dissolved oxygen levels will be somewhat less than equilibrium values predicted from the gas-phase oxygen content. Thus, although this system can be used to establish the trends of how the culture will respond to varying oxygen levels, these results cannot be taken to be quantitative. Using this shake flask system in which gases of varying oxygen content were passed through the head space, we observed that increases in the oxygen content of the gas resulted in a substantially greater production rate. This is shown in Figure 4 as the production rate of the enzyme is plotted as a function of the gas-phase oxygen content. The trends shown in Figure 4 are reproducible as evidenced by the agreement between three separate experiments. Since the heterologous parathion hydrolase enzyme is produced during growth, it is likely that the growth rate of the culture would vary with oxygen content in a manner similar to that observed in Figure 4 for enzyme production. Growth rates could not be determined due to the limited medium volume in the flasks. However, under conditions in which enzyme production was low (e.g., low oxygen levels), it was also observed that glucose consumption was small (not shown). Thus, the data obtained in this experiment (glucose consumption and enzyme production) suggest that oxygen-limiting conditions suppressed the culture's overall metabolic activity. A reduction of met-
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Figure 4. The effect of oxygen on parathion hydrolase production as studied in shake flasks. The productivity was calculated from the slopes obtained from enzyme activity vs time plots during the period when enzyme synthesis was maximal. As indicated by the various symbols, this experiment was conducted in triplicate using different inocula. Medium used for this study contained 20 g/L tryptone and 30 g/L glucose.
abolic activity under oxygen-limiting conditions is not unexpected since Streptomyces are classified as obligate aerobes, while oxygen-limiting conditions are known to have significant adverse effects on Streptomyces secondary metabolite biosynthesis (Hostalek, 1964; Rollins et al., 1988; Chen and Wilde, 1991; Yegneswaran et al., 1991). To avoid oxygen-limiting conditions in subsequent fermentor studies, either pure oxygen or oxygen-enrichedair was sparged to fermentorsto maintain the dissolved oxygen above about 30% air saturation. The importance of avoidingoxygen limitations can be seen in Figure 5, where the performance of fed-batch cultures with and without oxygen limitations are compared. It should be noted that
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Figure 5. Growth and parathion hydrolase production when oxygen limitations were avoided. The control was a glucose plus trypton fed culture in which dissolved oxygen was not controlled (e;(Tryp+ Gluc) Fed-batch). Enrichedgas was used induplicate cultures to avoid oxygen-limiting conditions (m and e;Fed-batch wloxygen supplement, 2 cultures). In culture I, changes in both total gas flow rate and oxygen content of the sparged gas were used to avoid oxygen limitations. In culture 11,the total gaa flow rate was maintained constant a t 1 wm and oxygen limitatione were avoided using oxygen-enriched air.
the total amounts of nutrients added and the feeding schedules were similar for these cultures. For the culture without oxygen enrichment, Figure 5 shows that the dissolved oxygen decreased linearly until about 80 h. After 80 h, the dissolved oxygen concentration remained below 5 % air saturation and the accumulation of parathion hydrolase ceased. In fact, during this oxygen-limiting condition, enzyme activities were observed to decrease
312
over time. When oxygen limitations were avoided by sparging pure oxygen (culture I) or oxygen-enriched air (culture 11), Figure 5 shows that parathion hydrolase activities were greater, reaching maxima of 40-43 units/ mL. These levels represent the highest we have observed for this transformant. Interestingly, Figure 5 shows that despite nutrient feeding and oxygen enrichment, the net accumulation of parathion hydrolase ceased after about 90 h. It should be noted that growth in all cultures of Figure 5 was observed to be similar. In summary, it appears that continued enzyme accumulation requires that both sufficient nutrients are supplied and adequate dissolved oxygen is available. However, as shown in Figure 5, these requirements are necessary but not sufficient to obtain further increases in enzyme levels. Despite nutrient feeding and oxygen supplements, we have not observed increases in enzyme levels past 90 h. Enzyme Deactivation during the Fermentation. Although a significant improvement in enzyme levels was achieved by eliminating nutrient (including oxygen) limitations, we have not observed further enzyme accumulation after 85-90 h in any of the cultures. In fact, as illustrated by the oxygen-limited culture in Figure 5, we have observed that enzyme activities are rapidly lost under conditions in which the physiological requirements of the culture are not met. Other researchers have observed significant losses in heterologous protein activities in recombinant Streptomyces fermentations (Lichenstein et al., 1988; Bender et al., 1990b) and have suggested that such losses may be due to proteolytic (Bender et al., 1990a) and nonproteolytic (Gardner and Cadman, 1990)reactions or to the production of low molecular weight enzyme inhibitors (Erpicum et al., 1990). In our studies, we could not detect protease activities using the azocasein method. However, we have observed nonproteolytic deactivation of this parathion hydrolase in unpurified (Coppella et al., 1990) and purified (Rowland et al., 1991) enzyme solutions. Quantitatively, parathion hydrolase deactivation has been observed to approximately follow first-order kinetics such that the rate of deactivation (rd) is described by rd = k d [El (1) where k d is the first-order rate constant and [El is the enzyme activity (Coppella et al., 1990). From studies with cell-free broth, we observed k d to be 0.03 h-' at the temperature and pH of the fermentation. To examine the hypothesis that enzyme is deactivated during the fermentation, it is useful to consider the data in Figure 5. For the fed-batch culture in which oxygen enrichment was not used, enzyme accumulation was observed to cease at 80 h when oxygen-limiting conditions were observed. From previous results (Figure4), we believe that under such oxygen-limiting conditions enzyme biosynthesis is greatly reduced. Thus if biosynthesis is neglected during this time, changes in enzyme activity would be due to deactivation and would be described by
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Time(hrs) Figure 6. The effect of chloramphenicol (CAP) addition on the accumulation of parathion hydrolase. The fed-batch culture shown in Figure 2 is used as a control. In the experimental culture, glucose feeding, tryptone feeding, and enriched gas were used to avoid nutritional limitations. Chloramphenicol (200 mg/L final concentration) was added to this experimental culture a t 68 h.
limitations which are expected to prevent enzyme synthesis, deactivation of parathion hydrolase was observed. The first-orderdeactivation rate constants for these studies ranged from 0.02 to 0.09 h-I. To further study deactivation in the presence of cells, we conducted a study in which enzyme synthesis was stopped by the addition of the protein synthesis inhibitor chloramphenicol. In these studies, the cultures were grown in a nutrient-fed fermentor where oxygen limitation was avoided by sparging oxygen-enriched air. As expected, Figure 6 shows that enzyme accumulated in the control culture until about 90 h and levels reached about 30 units/ mL. In the experimental culture, 200 mg/L chloramphenicol was added at 68 h. After the chloramphenicol addition, Figure 6 shows that parathion hydrolase activities decreased with a first-order rate constant of 0.02 h-l. Again these results are in agreement with cell-free enzyme inactivation studies. It should be noted that although details are limited, Western blots suggest that the deactivation of parathion hydrolase is associated with significant alteration of the enzyme's structure. Samples from the chloramphenicol study showed that the loss in parathion hydrolase activity in later samples was accompanied by a reduction in band intensities in Western blots. The above discussionsupports the contention that under conditions that are likely to eliminate enzyme synthesis (i.e., nutrient limitations or chloramphenicol additions) enzyme deactivation is observed. The similarity in the deactivation rate constants between cell-free and fermentation studies suggests that much of the deactivation results from the inherent instability of the parathion hydrolase enzyme and is not due to specific cell-mediated processes such as proteolytic degradation. Thus, it seems likely that the change in enzyme activity during the fermentation represents the net between synthesis and deactivation (Gardner and Cadman, 1990;Erpicum et al., 1990) such that drEl
As predicted from eq (2),enzyme deactivation is observed during this oxygen limitation. Using the data during the deactivation period, we estimated the first-order rate constant (kd) to be 0.07 h-l, which is of the same order of magnitude as values observed in cell-free studies. From an examination of all previous data (not shown), we observed that under conditions of nutrient or oxygen
(3)
where the left-hand term is the accumulation, or net production rate and rp is the rate of enzyme synthesis. It should be noted that since concentrated nutrient feeds were used in our studies, we have neglected the flow terms in the above material balance (Le., dilution of the enzyme due to nutrient addition was negligible).
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Figure 7. The time profile for parathion hydrolase production in repeated fed-batch operation. Glucose and tryptone feeding and oxygen enrichmentwere used to avoidnutritional limitations. Culture broth was harvested at 71.5 h by withdrawing 600 mL of the culture and adding 600 mL of fresh medium. A second harvest, similar to the previous one, was performed at 96 h.
To support the contention that deactivation occurs throughout the course of the fermentation, it is useful to consider conditions in which enzyme synthesis is expected to occur but net accumulation is not observed. For instance, even when nutrients are fed and oxygen-enriched air is sparged, Figure 5 and the control in Figure 6 show that net enzyme accumulation is not observed after 90 h. This observation can be explained if it is assumed that a steady state is established such that enzyme synthesis and deactivation are balanced rd (4) Substitution of eq 1 into eq 4 and rearrangement shows rp
that the steady-state enzyme level will be given by
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To estimate the steady-state enzyme activity, we performed the following calculations. Using data from initial fermentation periods, r p was estimated to be on the order of 0.3 to 1.0 unit/(mL.h), while cell-free studies suggest that kd is approximately 0.03 h-l. Using these values, it can be seen that the steady state enzyme level is expected to be approximately 10 to 35 units/mL which is similar to the activities observed in Figures 5 and 6. Further support for the hypothesis that the net production of the enzyme is a balance between synthesis and deactivation was provided by the following repeated fedbatch experiment. As illustrated in Figure 7, cells were cultured for the first cycle of this fermentation until 71.5 h when 20 units/mL of enzyme was observed. At this time, 60% of the culture was withdrawn, the same volume of fresh medium was added, and the fermentation was continued for a second cycle. During this second cycle, enzyme activities increased reaching 20 units/mL by 96 h. Again, at 96 h 60% of the culture was withdrawn, the same volume of fresh medium was added, and the fermentation was continued for a third cycle. During this third cycle, enzyme activities again increased eventually reaching 18units/mL at 121h. After this time, no further accumulation was observed, presumably due to the establishment of a steady state. The results from this experiment support the contention that enzyme accumulation is the net between synthesis and deactivation (eq 3). By harvesting and adding fresh medium, enzyme levels are reduced and therefore the rate of deactivation (rd) is proportionally reduced (equation 1). This reduction in rd below the synthetic rate ( r p )allows enzyme accumulation
to be observed past 90 h of the fermentation. It should also be noted that Figure 7 agrees with previous observations that S. liuidans transformed with pIJ702-derived plasmids are often stable (Ghangas and Wilson, 1987;Bertrandet al., 1989;Payne et al., 1990a). Despite the absence of selective pressure during this fermentation, the culture was observed to retain its ability to produce the heterologous enzyme over an extended period. In summary, the contention that enzyme accumulation is the net between synthesis and deactivation is supported by studies in which the synthetic rate ( r p )was reduced (by nutrient limitation or protein synthesis inhibitors) and by studies in which the degradation rate (rd) was reduced (repeated fed-batch operation).
Conclusions Our results indicate that to obtain high levels of heterologous protein from recombinant S. liuidans it is necessary to use large amounts of nutrients (Payne et al. 1990a). However, for improved performance, the nutrients should not be supplied at a single time but rather fed throughout the fermentation. In our studies with a glucose-tryptone medium, we observed acid production when high concentrations of glucose were present. Since the major acid identified in our studies was isovaleric, and not intermediates of the glycolytic or Krebs pathways, it appears that acid production under high glucose conditions resulted from the deamination of amino acids and not from an overflow of glucose catabolism. Under conditions of insufficient glucose, ammonium accumulated suggesting that under these conditions, the amino acids and peptides were deaminated and the carbon skeletons were preferentially utilized by the culture. Thus, these studies strongly suggest the peptide medium components as the source of potentially inhibitory byproducts. In addition to supplying adequate nutrients, oxygen limitations must be avoided if synthesis of this heterologous enzyme is to be maintained. It is important to note that since parathion hydrolase is produced in a growthassociated manner, maintaining synthesis for extended periods in fed-batch operation requires increasing rates of oxygen transfer to account for the increased cell concentrations. In our studies, we sparged oxygenenriched air during the later portions of these fermentations. Our studies indicate that enzyme accumulation is the net between synthesis and deactivation and that deactivation limits further improvements in parathion hydrolase production by this transformant. The deactivation of parathion hydrolase does not appear to be mediated by proteases but rather results from the inherent instability of the enzyme. Currently, this deactivation limits enzyme accumulation to approximately 40 units/mL which has been estimated to be equivalent to 40 mg/L (B. M. Pogell, personal communication). To further increase enzyme levels, it may be necessary to obtain more rapidly producing transformants. It is interesting to compare our observations with recombinant Streptomyces with those for other microbial hosts [see Zabriskie and Arcuri (1986) and Georgiou (1988)l. First, the accumulation of byproducts by Streptomyces appears to be less severe than for other microbial hosts. For instance, we observed acid accumulation and reduced performance only when high nutrient levels were used (e.g., glucose concentrations exceeding 30 g/L). In contrast, significantly lower glucose concentrations appear to be required to limit problems associated with acetic acid accumulation in E. coli fermentations [e.g., Konstanti-
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nov et al. (1990)l. Second, the observed problems of product deactivation reflect a limited availability of efficient promoter systems for S t r e p t o m y c e s . Although parathion hydrolase deactivation appears to result from the inherent instability of this enzyme, and not to cell mediated processes, it is important to recognize that this enzyme is not markedly unstable (the half-life is approximately 24 h). The significance of deactivation results because of the long fermentation times required for this culture. In fed-batch operation, we cultured S. lividans for 90 h while a typical E. coli fermentation is unlikely to last more than 24 h. Such long-term cultivation provides an excessive time for enzyme deactivation. If strong promoters were available, the fermentation time could be reduced with a corresponding reduction in deactivation. Also, if inducible promoters were available, alternative operating strategies (e.g., immobilized cell operation) could be considered.
Acknowledgment Partial support for this work was provided by the National Science Foundation Grant CBT-8707827 and by the Center for Agricultural Biotechnology of the University of Maryland. We thank E. R. Squibb and Sons for their generous gift of thiostrepton, Kodak for the use of the Ektachem system, and New Brunswick Scientific for the use of a BioFlow I11 fermentor. Our collaborators, Drs. M. K. Speedie and B. M. Pogell have also provided valuable assistance to our studies.
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Registry No. Parathion hydrolase, 58693-64-4; oxygen, 778244-7;isovaleric acid, 503-74-2; ammonia, 7664-41-7.
