Appl Microbiol Biotechnol (1990) 33:395-400

Applied am Microbiology Biotechnology © Springer-Verlag 1990

Improved production of heterologous protein from Streptomyces lividans Gregory F. Payne ~'2, Neslihan DelaCruz 1, and Steven J. Coppella ~'3 1 Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, Baltimore, MD 21228, USA 2 Center for Agricultural Biotechnology, University of Maryland, Baltimore County, Baltimore, MD 21228, USA 3 Medical Biotechnology Center, University of Maryland, Baltimore County, Baltimore, MD 21228, USA Received 14 August 1989/Accepted 30 January 1990

Summary. Protein-secreting procaryotic host organisms are currently being sought as alternatives to Escherichia coli for recombinant processing. In this study we examined how manipulation of the cultivation conditions can enhance heterologous protein production by Streptomyces lividans. The recombinant S. lividans used in this study expressed and excreted a Flavobaeteriutn enzyme capable of hydrolyzing organophosphates. Initial shake-flask studies demonstrated that supplementing Luria-Bertani medium with moderate amounts of glucose (30 g/l), led to improved enzyme production. In fermentor studies with controlled pH, a further twofold increase in production was observed when glucose was fed continuously as compared to batch cultivation. This improved production in the glucose-fed culture may be related to a reduced accumulation of acids. Continuous feeding of both glucose and tryptone led to a further sixfold increase in production. In addition to enhancing production 25-fold, the efficiency of enzyme production and the specific activity of the excreted enzyme were also improved by glucose and tryptone feeding. These results demonstrate that in addition to genetic manipulations, optimization of cultivation conditions can lead to significant improvements in the production of heterologous proteins from Streptornyces.

Introduction The most commonly used procaryotic host for recombinant processes is Escherichia coli. Although high levels of expression can be obtained from this bacterium, heterologous proteins are generally retained within the cells as an insoluble and often biologically inactive inclusion body (Wang 1988). As a result of these problems, alternative hosts capable of protein excretion are being investigated. An alternative, protein-excreting procaryotic host which is recently gaining attention is Streptornyces

Offprint requests to: G. F. Payne

(Crawford 1988). This procaryote is commonly used in the pharmaceutical industry, and appropriate vectors are being developed to permit genetic modifications. However, significant improvements in heterologous protein expression by Streptomyces will be required before production by this procaryote is competitive with E. coli. In our work, we are attempting to enhance heterologous protein expression by Streptornyees. In initial studies, a Flavobaeteriurn gene coding for the enzyme parathion hydrolase was cloned into the high copy number plasmid pIJ702 (Katz et al. 1983) and S. lividans was transformed with this new pRYE1 plasmid (Steiert et al. 1989). This transformant was observed to express and excrete parathion hydrolase. The goal of the work reported here was to examine the environmental conditions required to enhance production of extracellular enzyme activity. Results from this work indicate that parathion hydrolase production was improved by fedbatch cultivation in which both the carbon (glucose) and complex nitrogen (tryptone) sources were fed.

Materials and methods Culture, medium and conditions. Spores of transformed S. lividans (Steiert et al. 1989) were stored at -70°C in a 30% (v/v) glycerol solution. Inocula were prepared by transferring these spores into a culture tube containing 1-2 ml Luria-Bertani (LB) medium (tryptone, 10 g; yeast extract, 5 g; NaC1, 10 g; and thiostrepton, 30 mg; per liter distilled water). After incubating for 1 day, this culture was inoculated into 20 ml of the same medium in a 250-ml Erlenmeyer flask and incubated for 1 day more. This culture was then used as inoculum for experiments. The media used in this study were all modifications of LB, and the modifications are noted in the text. In experiments in which glucose was used, this sugar was autoclaved separately from the other ingredients. Although thiostrepton was used in preparing the inoculum, all experiments reported here were conducted without thiostrepton selection. Shake-flask studies were conducted in 500-ml Erlenmeyer flasks containing 150 ml liquid medium. Fermentor studies were conducted in a 1.6-1 BioFlow III fermentor (New Brunswick Scientific, Edison, NJ, USA). All experiments were performed at 30°C.

396

Analytical procedures. Parathion hydrolase activity was determined by measuring the rate of formation of the parathion hydrolysis product p-nitrophenol at 410 nm using a spectrophotometer

(Gilford Response, Oberlin, OH, USA). The reaction mixture for this enzyme assay consisted of: 3 ml TP(IS buffer (0.01 M) containing 0.005% Tween 80, pH 8.5; 9.9 p~l parathion solution containing 43 mM parathion in methanol; and culture broth or supernatant (typically 9.9 ~tl) containing the enzyme. An extinction coefficient of 16,500 M -1 cm -I was used (Serdar and Gibson 1985) and results are expressed as units/ml where 1 unit is equal to 1 ~tmol reacted/rain. Dry cell concentration was determined by filtering and washing cells on preweighed filters pads (Whatman GF/B, Maidstone, England). These filter pads were dried for 1 day at 60° C in a vaCallm oven. Glucose was determined by the glucose oxidase method using a glucose analyzer (Yellow Springs Instrument, Yellow Springs,

o~a, USA). Total extracellular protein was assayed by the Bio-Rad (Richmond, Calif, USA) method with bovine plasma gamma globulin as the standard. Tryptone and yeast extract were observed not to interfere with this assay.

Results and discussion

Culture stability Because of limited availability, thiostrepton cannot be used to maintain selective pressure in large-scale operations. Thus, for this transformant to be commercially practical, it is essential that hydrolase production be stable in the absence of thiostrepton selection. Using flask cultures to simulate a large-scale operation, this recombinant was cultured in the absence of thiostrept o n s e l e c t i o n for several generations. In the first trans: fer, hydrolase activities peaked at 24 h and, thus, subsequent transfers were made at 24 h. As shown in Table 1, variation in hydrolase activities between the four transfers was small despite the fact that over 20 generations o f cells were grown in the absence of thiostrepton selection. If a seed is grown with thiostrepton selection, it is unlikely that more than 20 generations would be required for larger scale appliTable 1. Stability of hydrolase production in the absence of thiostrepton selection a Transfer no.

Broth activityb at 24 h

Cell conc at 24 h

Generations No. in each

Total

(units/ml)

(gin/l)

transfer

no.

1

0,98

1.8

5

5

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1.07 0.92 0.89

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a Inoculum for this study was prepared by growing cells from spores in the presence of thiostrepton. Cells from this suspension were inoculated into flasks containing Luria-Bertani (LB) medium with glucose (30 g/l), and without thiostrepton. After 24 h, 4 ml of this suspension was transferred to 150 ml LB medium containing glucose (30 g/l) and this suspension was designated transfer no. 1. Subsequent transfers were made after 24 h to an initial dry cell concentration of 0.05 g/1. b Parathion hydrolase activity was measured using culture broth (i.e. without removing the cells)

cations, and thus we conclude that hydrolase production by this recombinant is stable. Others (Ghangas and Wilson 1987; Bertrand et al. 1989) have also observed this host vector system to be stable in the absence o f thiostrepton selection. In all subsequent studies, the inoculum was grown in the presence of thiostrepton, but all experiments were conducted without this antibiotic.

Effect of glucose on growth and hydrolase production The importance of a carbon and energy source on hydrolase production was investigated by supplementing LB medium with varying levels of glucose. Figure 1 shows that increasing the initial glucose concentration up to 30 g/1 led to increased growth and extracellular hydrolase activities. This stimulation probably results from the availability of a readily utilizable carbon and energy source. This conclusion is supported by the observation that in the absence of glucose, maximum cell concentrations and hydrolase activities were observed at 25 h while growth and production continued in the glucose-supplemented flasks until at least 47 h (the fermentation profiles for these studies are not shown). A higher initial glucose level (53 g/l) was observed to suppress both growth and hydrolase production and glucose consumption was observed to cease when 28 g/1 of this substrate remained in the culture medium. Glucose was completely consumed when lower initial levels were used. Although the reasons for suppressed metabolism in the presence of high initial glucose levels are unclear, it is possible that the effect is mediated by the acidification of the medium which occurred during glucose utilization. As shown in Fig. 1, increases in the initial glucose level led to reduced pH. The production of glycolytic and Krebs cycle acids by Streptomyces has been previously reported (Doskocil et al. 1959; Ahmed et al. 1984; Surowitz and Pfister 1985; Dekleva and Strohl 1987). Using gas chromatography, we did not observe the accumulation of significant amounts of pyruvate or a-ketoglutarate, the acidic products typically reported to be produced by Streptomyces. However, our chromatographic analysis did suggest that an acid of unknown structure accumulated at the time that medium acidification occurred. To prevent p H changes during cultivation, cells were grown in a fermentor with the p H controlled at 7.0. Since increased growth was observed in initial fermentor studies, LB medium was modified by increasing the levels of tryptone (from 10 to 15 g/l) and yeast extract (from 5 to 10 g/l). Further studies on the effects of these complex ingredients will be discussed later. Figure 2 shows that in a batch fermentor with 30 g/1 glucose, growth and hydrolase production were somewhat enhanced relative to cultivation in shake flasks. If the maximum extracellular activity of parathion hydrolase is divided by the cell concentration at that time, a specific production of 0.7 units/mg cells is attained. Since this value is similar to the specific production in shake flasks, the enhancement in production appears to be due to improved growth in the fermentor.

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Despite some deviations, Fig. 2 also shows that hydrolase was produced during periods of active growth. This observation is consistent with previous reports in which constitutive expression of parathion hydrolase was observed in Flavobacterium (Sethunathan and Yoshida 1973), Pseudomonas alcaligenes (Munnecke and Fischer 1979) and other natural isolates (Mulbry and Karns 1989). Also, in initial work with this recombinant (Steiert et al. 1989), growth-associated parathion hydrolase production was observed. Since the previous results indicated that glucose supplementation stimulated hydrolase production, but high initial glucose levels led to reduced production, a glucose-fed fermentation was studied. Figure 3 shows that in this glucose-fed culture, growth was similar, but hydrolase production was nearly doubled compared to the batch culture (Fig. 2). Thus specific hydrolase production was enhanced to 1.3 units/mg dry cells. It should be noted that at 50 h, when hydrolase activity peaked in the fed-batch culture, only 30 g/1 glucose had been consumed by the cells. This amount of sugar is similar to that supplied to, and consumed by, the batch culture. Thus the increased hydrolase production in the glucose-fed culture was not due to an increase in the total amount of sugar consumed; rather this substrate appeared to be more efficiently converted into hydrolase by the fed-batch culture. With respect to acid production, at the time that 30 g/1 glucose had been consumed, 7.2 mmol/1 and 1.2 mmol/1 NaOH were required to maintain a pH of 7.0 in the batch and the glucose-fed cultures respectively.

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Fig. 1. Effect of varying initial glucose levels on growth, hydrolase production and medium acidification.Cells were inoculated into flasks containing Luria-Bertani(LB) medium with varying levels of glucose Finally, despite glucose feeding and pH control, significant hydrolase inactivation was observed during the latter stages of this fermentation. Thus, a continuous supply of the carbon and energy source does not guarantee continued hydrolase production. With respect to hydrolase inactivation, our results indicate that inactivation is not mediated solely by low pH (preliminary studies have indicated that hydrolase is less stable at low pH). Further, protease activities could not be detected using an azocasein-based protease assay (data not shown). Thus, the loss of parathion hydrolase activities at the later stages of the fermentation are not understood at this time.

Effect of complex nitrogen source on 9rowth and hydrolase production Preliminary shake-flask studies were conducted to identify the role of the various components of LB medium. As can be seen from Table 2, when salt or yeast extract was deleted from LB medium, the amount of parathion hydrolase produced was unaffected. In the absence of tryptone however, the culture produced significantly less enzyme. Thus, a subsequent shake-flask study was conducted in which cells were inoculated into a medium containing only tryptone (20 g/l) and glucose (30 g/l). At 52 h, when the remaining glucose concentration was 18 g/l, hydrolase production was observed to cease. At that time the culture was split into a

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Table 2. Effect of LB medium components on parathion hydrolase production Component deleted

Maximum hydrolase (units/ml)

Maximum growth (g/l)

Minimum pH

None NaC1 Yeast extract Tryptone

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Summary of improvements Table 3 summarizes the improvements obtained through this work. As previously mentioned, by using a glucose and tryptone feeding scheme, parathion hydro-

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control and an experimental flask. No further additions were made to the control, and it can be seen that u p o n further incubation, hydrolase was inactivated in this control. At 52 h, 15 g tryptone/1 culture was added to the experimental flask, and u p o n subsequent incubation hydrolase production resumed. As indicated in the figure legend further glucose and tryptone additions were made to this experimental culture at various times. Figure 4 shows that these glucose and tryptone additions stimulated production with extracellular activities in this experimental flask reaching 20 units/m1 at 104 h. Figure 4 shows that tryptone feeding led to increased hydrolase production as c o m p a r e d to a culture which received the same initial (but not the same total) a m o u n t of tryptone. In further studies (data not shown) it was observed that tryptone and glucose feeding led to increased hydrolase production c o m p a r e d to a culture in which the same total level of tryptone (85 g/l) and glucose (60 g/l) were added initially (hydrolase activities reached 33 units/ml in the fed-batch culture while activities of 5.5 u n i t s / m l were observed in the batch culture). These results suggest that slowly supplying both the carbon and nitrogen source can significantly enhance production of parathion hydrolase. To examine the effect of tryptone feeding u n d e r a constant p H of 7.0, a fermentor was used in which both glucose and tryptone were fed. Figure 5 shows that in this fed-batch culture, both growth and hydrolase production were enhanced c o m p a r e d to previous batch or glucose-fed fermentor studies (Figs. 2, 3). The specific production o f the enzyme was calculated to be 2.6 units/rag cells which exceeds that for either the batch or glucose-fed cultures. In this glucose- and tryptonefed culture, hydrolase activities were observed to level off at the time when the dissolved oxygen was reduced to zero (not shown). Thus, further i m p r o v e m e n t s in productivity m a y require either an i m p r o v e m e n t in oxygen transfer, or a reduction in the culture's oxygen d e m a n d (e.g. by reducing cell growth).

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Fig. 5. Fe~entation profiles in a tffptone and glucose-fed batch fermentor. Cells were grown in a medium initially containing tryptone (17 g/l) and glucose (25 g/l). Glucose and tryptone feeding commenced at 22 h, and the total amount of glucose and tryptone fed is shown in the upper plot. A concentrated feed solution (133 g/1 glucose and 167 g/1 tryptone) was used such that 0.570 1 was added during the course of the experiment. ~ i s compares to the initial liquid volume of 1.1 1 lase production was enhanced c o m p a r e d to initial shake-flask cultures. This level of production represents a 25-fold increase c o m p a r e d to initial reports with this recombinant (Steiert et al. 1989), and also appears to be significantly higher than hydrolase production observed with wild-type organisms (Brown 1980; Munnecke and Fischer 1979). I f it is assumed that 1 unit of activity corresponds to 1 ~tg o f enzyme (B. M. Pogell, personal communication), then the glucose and tryptone feeding scheme resulted in an extracellular hydro-

lase level of 25 mg/1. Although this level is high relative to other reports of heterologous protein expression by Streptomyces (Illingsworth et al. 1989), it remains low c o m p a r e d to E. coli. However, efforts to obtain high levels of parathion hydrolase production in a recombinant E. coli resulted in the formation of inclusion bodies that a p p e a r e d to have reduced activity (J. S. Karns, personal communication). In addition to improving production, the fed-batch fermentation system was also observed to result in more efficient production of hydrolase with respect to nutrient consumption. As indicated in Table 3, fermentot and fed batch cultures were observed to enhance growth. However, the a m o u n t of enzyme produced per gram of cell or per gram of glucose consumed was further enhanced by fed-batch cultivation in the fermentor. This enhanced metabolic efficiency m a y be due to a decrease in the conversion of substrates to acid. Finally, Table 3 shows that the a m o u n t of extracellular activity per gram of extracellular protein was also enhanced relative to shake-flask cultures. This supports the idea that hydrolase production was enhanced relative to the production of other extracellular proteins. F r o m a processing standpoint, an increase in the specific activity of the heterologous protein will facilitate further downstream purification. It should also be noted that parathion hydrolase represents approximately 2% of the total extracellular protein. In conclusion, although expression is low relative to E. coli, excretion is a major advantage for using Streptomyces for heterologous protein production. In our case, where parathion hydrolase could be used as a crude cell-free preparation, excretion greatly facilitates downstream recovery. In fact, cell removal m a y be the only required recovery operation. I f this enzyme were produced in E. coli, cell breakage and possibly protein solubilization and protease inactivation steps would be required. Under the conditions studied, we could not detect extracellular protease activity with this S. lividarts transformant.

Acknowledgements. Considerable technical assistance was obtained from our collaborators Drs. B. M. Pogell, M. K. Speedie and J. S. Steiert. Laboratory support was provided by the Univer-

Table 3. Summary of improvements in parathion hydrolase production Expt.

Peak extracellular activity (units/ml)

Cell conc at peak activity (g/l)

Activity per cell (units/mg)

Activity per sugar consumed (units/mg)

Activity per extracellular protein (units/mg)

Batch fiaska Batch fermentorb Glucose-fed fermentorc Glucose and tryptone fed fermentora

0.5-3.0 4.5 8.8 25.7

3.2-4.5 6.0 7.0 10.0

0.2-0.7 0.7 1.3 2.6

0.17 0.14 0.29 0.33

5.4 ND e ND e 17.8

a LB medium with 30 g/1 glucose. Ranges are given to indicate significant variability observed in flask cultures: peak between 30 and 48 h b Batch fermentor (Fig. 2): peak at 45 h ° Glucose-fed fermentor (Fig. 3): peak at 51 h a Glucose and tryptone fed fermentor (Fig. 5): peak at 90 h e Not measured

400 sity of Maryland Engineering Research C~nter. Financial support for this work was provided by the University of Maryland Biotechnology Center. Also we wish to thank E. R. Squibb and Sons, Princeton, N J, USA, for their generous gift of thiostrepton and New Brunswick Scientific for the use of a BioFlow III fermentor.

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spora fusca cellulase gene in Streptomyces lividans and Bacillus subtilis. Appl Environ Microbiol 53:1470-1475 Illingsworth C, Larson G, Hellekant G (1989) Secretion of sweettasting plant protein thaumatin by Streptornyces lividans. J Ind Microbiol 4:37-42 Katz E, Thompson CJ, Hopwood DA (1983) Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol 129:2703-2714 Mulbry WW, Karns JS (1989) Purification and characterization of three parathion hydrolases from Gram-negative bacterial strains. Appl Environ Microbiol 55:289-293 Munnecke DM, Fischer HF (1979) Production of parathion hydrolase activity. Eur J Appl Microbiol 8:103-112 Serdar CM, Gibson DT (1985) Enzymatic hydrolysis of organophosphates: cloning and expression of a parathion hydrolase gene from Pseudomonas diminuta. Biotechnology 3:567-571 Sethunathan N, Yoshida T (1973) A Flavobacterium sp. that degrades diazinon and parathion. Can J Microbiol 19:873-875 Steiert JS, Pogell BM, Speedie MK, Laredo J (1989) A gene coding for a membrane-bound hydrolase is expressed as a secreted, soluble enzyme in Streptomyces lividans. Biotechnology 7:65-68 Surowitz KG, Pfister RM (1985) Glucose metabolism and pyruvate excretion by Streptomyces alboniger. Can J Microbiol 31 :702-706 Wang DIC (1988) Biotechnology: status and perspectives. American Institute of Chemical Engineers Monograph Series, vol 84. American Institute of Chemical Engineers, New York, pp 410