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Physiological and Genetic Strategies for Enhanced Subtilisin Production by Bacillus subtilist Jeffrey A. Pierce and Channing R. Robertson* Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025
Terrance J. Leighton Department of Biochemistry and Molecular Biology, University of California at Berkeley, Berkeley, California 94720 Defined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains. Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed-batch cultures. Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE:lacZ gene fusion. The @-galactosidaseproduction driven by this fusion reflected subtilisin accumulation in the culture medium. Subtilisin gene expression was temporally extended in sporulation-deficient strains (spoIIG),relative to co-genic sporogenous strains, resulting in enhanced subtilisin production. Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population. Fed-batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production. The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments. At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed.
Introduction The molecular genetic and physiological mechanisms which regulate the production of subtilisin by Bacillus subtilis are of developmental and biotechnological interest. Subtilisins are among the most valuable enzymes manufactured by the biotechnology industry (Debabov, 1982; Priest, 19891, yet detailed information concerning the rational design of efficientB. subtilis subtilisin production systems is not generally available. The development of recombinant protein production systems using B. subtilis as the host organism, especially those driven by the subtilisin promoter (Vasantha and Filpula, 1989; Oyama et al., 1989),provides an additional impetus for research in this area. Although subtilisin synthesis is not required for sporulation (Stahl and Ferrari, 19841, its production is triggered (controlled) by mechanisms common to those responsible for the initiation of sporulation (Sonenshein, 1989) and hence it has served as a model for developmentally-associatedgene expression. From a physiological perspective,subtilisin production is known to be regulated by carbon (Schaeffer, 1969) and nitrogen catabolite repression (Hanlon et al., 1982; Wouters and Buysman, 1977) as well as by energetic (Frankena et al., 1986) and growth rate control mechanisms (Hanlon and Hodges, 1981). The molecular basis for these modes of regulation is poorly understood; regulation is not achieved through global carbon or nitrogen control mechanisms analogous to the CAMPor ntr systems of Escherichia coli (Epstein + Portions of this manuscript were presented at the American Institute of Chemical Engineers 1989 Annual Meeting, November 5-10,1989,San Francisco, CA. * Corresponding author.
8756-7938/92/3008-0211$03.00/0
et al., 1975; Magasanik, 1988). Studies of the transcriptional regulation of subtilisin expression,however, suggest that positive and negative control is exerted through the concerted action of multiple trans-acting elements (Valle and Ferrari, 1989). The development of more efficiently engineered subtilisin production systems depends on an understanding of the linkage between subtilisin gene regulation and cellular physiology. Critical parameters include cell genotype,culture medium composition,and environmental (reactor) conditions, as well as their interactions. Since subtilisin expression ceases during the later stages of sporulation (Doi, 1989),we hypothesized that the use of sporulation-deficient strains would be beneficial in temporally extending subtilisin productivity. To test this hypothesis, we have employed both sporulation-proficient and sporulation-deficient (spollG) backgrounds. spoIlG mutants were used since they were blocked at a stage of development beyond the initiation of subtilisin expression, but prior to the later nonproductive stages of sporulation. A chromosomal subtilisin-8-galactosidase gene fusion (Ferrari et al., 1986)was also exploited to monitor transcriptional control of subtilisin expression. This fusion construct, integrated in tandem with the resident subtilisin gene, permitted a direct comparison between the transcriptional activity of the subtilisin gene and the levels of secreted subtilisin. Proper medium formulation is a critical enabling technology for studies of this type. While complex media promote rapid growth, high titers of subtilisin,and efficient sporulation, they do not allow unambiguous associations between nutrient availability and gene expression to be made. Therefore, a simple, chemically defined culture
0 1992 American Chemical Society and American Institute of Chemical Engineers
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Table I. strain
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genotype
relevant phenotype asporogenous
source or derivation# 55.1 spoZZG55 leu8 tall NG12.12,168 trpC2 background (Piggot, 1973) Leighton laboratory RS2 168 trp+ metC3 lysl Leighton laboratory RS1527 metC3 RS2 td RS1527,1yst selection RS1520 RS8 168 prototrophic, sporogenous RS2 td RS1520, met+ selection spoZZG55 tall prototrophic spontaneous revertant of 55.1 RS907 spoZZG55 oligosporogenous 55.1 td RS1520, met+ selection RS908 spoZIG55 asporogenous 55.1 td RS1520, met+ selection RS909 BG2100 trpC2 pheAl (::pSG35)b (Ferrari et al., 1986) RS7008 (::pSG35) sporogenous BG2100 tf RS8 spoZZG55 tall (::pSG35) asporogenous BG2100 tf RS907 RS7907 RS7908 spoZZG55 (::pSG35) oligosporogenous BG2100 tf RS908 RS7909 spoZZG55 (::pSG35) asporogenous BG2100 tf RS909 a Abbreviations: tf, DNA-mediated transformation; td, PBS1-mediated transduction. pSG35 is an integrating plasmid containing the aprE::tacZ gene fusion and a chloramphenicol-resistancegene.
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medium containing a singlemetabolizable carbon (glucose) and nitrogen (ammonium chloride) source was designed for use in these studies. The details of our medium development studies are the subject of a forthcoming manuscript. These studies focused on the subtilisin production response of prototrophic strains of B. subtilis to the exhaustion of ammonium, or limitation of its availability, since nitrogen limitation is known to result in the greatest induction of subtilisin expression in both batch (Hanlon et al., 1982) and continuous culture (Wouters and Buysman, 1977). Batch culture was first explored to characterize the starvation response in our minimal medium, and then fed-batch culture methods were employed in an attempt to further extend subtilisin production beyond that possible in batch culture. The results obtained from the application of the described genetic, physiological, and engineering approaches suggest that asporogenous host strains and fedbatch culture techniques are beneficial in temporally extending subtilisin production, but that means must be developed to maintain the physiological and morphological integrity of nongrowing or slowly growing cells.
Materials and Methods Organisms. Bacillus subtilis strains used in this study are listed in Table I. The co-genic series (RS8, RS908, RS7008, and RS7908) have as a background the wild-type RS8 strain. Isogenic derivatives were constructed containing spoIIG55 mutations (Piggot, 1973) and/or chromosomal transcriptional aprE::lacZfusions (Ferrari et al., 1986) using techniques of DNA-mediated transformation and transduction (Shazand et al., 1990). The aprE::lacZ gene fusion originated from integrating plasmid pSG35 (Ferrari et al., 19861,which also carried a chloramphenicolresistance marker. An additional strain, RS7907, similar to RS7908 but in the original genetic background (168 trpC2),was obtained by reverting the spoIIG leucine auxotroph (55.1) to prototrophy. Cells were grown in the absence of chloramphenicol, the selective marker resident in the fusion constructs, to minimize tandem duplications (Ferrari et al., 1986). Strains containing the aprE::lacZ fusion were routinely tested for growth on minimal medium plates supplemented with 5 pg/mL chloramphenicolto verify that the construct remained in the strain before and after each experiment. No loss of chloramphenicol resistance was detected throughout the duration of these studies. Media. The chemically defined media for the culture of B. subtilis were based on the MOPS medium (Neidhardt et al., 1974) and contained 10% (v/v) of a MOPS
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1OX mixture 1400 mM MOPS, 40 mM Tricine, 0.1 mM FeSO4,2.76 mM K~S04,1.4mM CaC12,40 mM MgC12,l.O mM MnC12, 3 X mM (NH4)6Mo,024, 4 X mM mM CoC12, mM CuSO4, and lo4 mM H3B03,3 X ZnSOtI, 3 mM KzHP04,lO mM NH4Cl (reduced to 5 mM for fed-batch experiments), and 70 mM glucose. In shake flask culture experiments, the final concentration of MOPS buffer was increased to 80 mM to maintain the culture pH in the range of 6.8-7.6. All media were formulated to be ammonium limited. Independent experiments demonstrated that the components of the MOPS 1OX mixture were sufficient to support growth to at least twice the cell densities attained in these experiments. Furthermore, analytical procedures (see below) used to quantify the remaining media components (KzHP04, NHdCl, glucose) established that ammonium chloride was the only growthlimiting nutrient. The provision of 70 mM glucose was sufficient to ensure carbon and energy excess throughout the duration of all fermentations. All media were filter sterilized 10.2 pm (Sterivex GS, Millipore)]. Minimal medium plates were made by combining the above specified minimal medium with 1.5% (w/v) agar (Difco). Apparatus. A 2-L fermenter (Setric Genie) was maintained at 37 OC and equipped with dissolved oxygen and pH probes. Dissolved oxygen levels were kept above 40% saturation by sparging with 25 L of air/h and agitating at 600 rpm. Controlled additions of 4 M KOH or 4 M HCl maintained the pH at 7.00 f 0.02. An antifoaming agent (Sigma, Antifoam A) was used, as required, to control foaming. For fed-batch culture, a sterile ammonium chloride solution (of the appropriate concentration to allow approximately one biomass doubling over the feeding period) was added at a constant rate (on the order of 1-2 mL/h) with a peristaltic pump. The delivery of ammonium into the fermenter was initiated before its exhaustion from the medium, thus ensuring an ammonium downshift rather than ammonium exhaustion followed by resupply. For shake flask culture, the temperature was maintained at 37 "C and agitation was set a t 360 rpm in a reciprocal shaker (Queue Systems). The culture volume to flask volume ratio was maintained below 12% (v/v) to avoid oxygen limitation. Culture Preparation. Growth of cells was initiated by inoculation from minimal glucose plates into shake flasks. The cultures were transferred 3-4 times, over a period of approximately 24 h, into fresh media before experiments were initiated. Reinoculations were timed to ensure that the cultures were under conditions of excess nutrients, and thus in an environment allowing balanced exponential growth, throughout the process. Using this
Bbteehnol. Rog., 1902, Vol. 8, No. 3
preinoculation procedure, no observable lag phase was encountered upon the initiation of experiments. Analytical Procedures. All concentrations reported were corrected for volume changes due to nutrient addition or sample collection. Biomass concentrations, reported in grams of dry weight per liter (g of DW/L), were determined directly as dry weight (centrifugation method; Stewart and Robertson, 1988)or through the use of optical density (light scattering at 660 nm) - dry weight correlations (in the exponential phase only). Viability was assessed through serial dilutions of culture samples in growth medium lacking glucose and subsequent plating on TBAB (Difco) and/or minimal glucose plates. Spores were quantified as for viable cell determinations, except that 1 mL of culture was treated with 0.025 mL of chloroform prior to dilution and plating on TBAB plates. Subtilisin activity was determined on previously frozen (-70 "C) culture supernatant fractions, using azoalbumin as the substrate (Frankena et al., 1985). Phenylmethanesulfonyl fluoride was used to determine the percentage of the culture fluid proteolytic activity attributable to subtilisin (Hanlon and Hodges, 1981). Units of activity were those defined for the subtilisin reference standard (Sigma, P-5380). Sampling for the remaining assays was accomplished by adding 1.0 mL of cell culture to 0.5 mL NaN3 (1.2 g/L), centrifuging the mixture, retaining the supernatant fraction at 4 "C, washing the pellet with 1.0 mL of ice-cold Tris-HCl(O.05 M, pH 8.0), centrifuging again, discarding the wash liquid, and freezing the pellet at -70 "C. Concentrations of glucose (Sigma, HK-50 assay kit), phosphate (Van Veldhoven and Mannaerts, 1987) and ammonium (Weatherburn, 1967) were determined on the supernatant fraction. The concentrations of excreted organic intermediates (primarily acetate) in the supernatant fraction were measured by HPLC using a HewlettPackard Model 1082B HPLC with a Hamilton PRP-X300 column and dilute HzS04 (1-10 mM) as the mobile phase. The protein concentrations of the cell pellets were measured using a bicinchoninic acid method (Smith et al., 1985). Bovine serum albumin (BSA; Sigma, A-7511) was used as a protein standard. Cellular protein concentrations were therefore expressed in BSA equivalents per liter (g of BSA eq/L). Under exponential growth conditions, approximately 0.35 gram of BSA equivalents corresponded to 1gram of dry weight. @-Galactosidaseactivities were determined on the cell pellets as described (Donnelly and Sonenshein, 1982). Activities are reported either as units (nanomoles of 0-(nitrophenyl) 8-D-galactopyranoside (ONPG) hydrolyzed per minute) per milligram of cellular protein (milligrams of BSA equivalents) or per milliliter of culture (for shake flask culture where protein assays were not performed). Poly(@-hydroxybutyrate)(PHB) content was determined as described (Ward and Dawes, 1973). Transmission electron microscopy techniques were performed on fixed cell pellets (Hayat, 1981; Mascorro et al., 1976).
Results Construction of Co-genicB. subtilis Series. Introduction of the spoIIG mutation into the RS8 background resulted in two classes of sporulation-deficient recombinants: those which were oligosporogenous (ca. 1% sporulation; RS908) and those which were asporogenous (RS909). Our interpretation of these results is that the donor 55.1 strain, derived from a primary mutagenized background (Piggot, 1973),actually contained two closely linked sporulation mutations. Incorporation of both of these mutations in the RS8 strain would result in a
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completely asporogenous phenotype, while incorporation of only one of them would confer an oligosporogenous phenotype. The asporogenous construct would be preferable for studying the effects of sporulation proficiency on subtilisin production since all of the stationary-phase population would be blocked at the same point in the developmental process. With an oligosporogenous phenotype, the developmental heterogeneity of the stationaryphase population would be greater. Unfortunately, another important and less desirable property of the asporogenous construct was the relative susceptibility of the stationary-phase cell population toward autolysis. This nongrowing cell population lysed more readily than the oligosporogenous strain or the asporogenous strain containing the spoIIG55 mutation(@ in the original background. Postexponential autolysis effectively converts a chemically defined medium into a complex one, where little can be learned about the physiological control of subtilisin production. Therefore, the more robust oligosporogenous recombinants (RS908 and RS7908)were used in the co-genic study. The asporogenous spoIIG mutant (RS907) in the original background (168 trpC2; Piggot, 1973) was included in the co-genic study for comparison purposes and used in all fermenter studies. Effect of Sporulation Proficiency and aprEDirected @-GalactosidaseProduction on B. subtilis Cellular Physiology. The co-genic series (RSB, RS908, RS7008, RS7908; see Table I) was employed to assess the effects of sporulation proficiency and/or the presence of the aprE:lacZ gene fusion on B. subtilis physiology. All strains were cultured in ammonium-limited, glucosesufficient, shake flask culture. The growth characteristics of each strain and pH profiles in each culture medium were similar. Exponential phase mass doubling times were approximately 60 min. The pH of each culture medium decreased from 7.6 to approximately 6.8 at the end of growth (data not shown). Following ammonium starvation, the metabolism of glucose continued and media pH increased toward 7.0. The sporulation-deficient strains (RS908,RS7907, RS7908) maintained essentially constant optical densities throughout the postexponential phase, indicating minimal cell lysis (data not shown). After 24 h of glucose-sufficient ammonium starvation, the sporogenous strains (RS8 and RS7008) had sporulated at a frequency of 30-40% of the total viable cells present at the end of growth. Panels a and b of Figure 1 show @-galactosidaseand subtilisin accumulation profiles,respectively,for all strains. The strains lacking the aprE:ZacZ gene fusion (RSB and RS908) exhibited little endogenous @-galactosidaseactivity (Errington and Vogt, 1990). In the sporulation-proficient RS7008 strain, the net accumulation of @-galactosidase commenced shortly after the end of growth and continued for the next 5-6 h before it ceased. In the oligosporogenous RS7908 strain of the co-genic series, @-galactosidase production began at a rate similar to the RS7008 strain but enzyme levels continued to increase for 15-20 h of starvation. Subtilisin production commenced at a similar rate for all strains in the co-genic series shortly after starvation for ammonium (Figure lb). The accumulation of subtilisin continued essentially throughout the starvation period for the oligosporogenous strains (RS908 and RS7908), whereas accumulation ceased and subtilisin levels decreased in the sporulating strains (RS8 and RS7008). Subtilisin and @-galactosidaseproduction profiles were qualitatively similar for the two spoIlG strains containing the aprE:lacZgene fusion (RS7908 and RS7907), though the levels attained were higher for the
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asporogenous strain derived from the original (168 trpC2) genetic background (RS7907). This strain (RS7907) was used in the remainder of the studies described in this report. Fermenter-Based Physiological Studies of Asporogenous Strain RS7907. The ammonium-limited behavior of the asporogenous spoIIG (RS7907) strain was further investigated in the controlled environment of a fermenter. During the exponential growth phase, cell mass doubled every 50 min (Figure 2a). The cells excreted approximately 0.65 mol of acetatelmol of glucose consumed. This acid production, together with the acid contributed by ammonium assimilation (Kleiner, 1985), essentially balanced the base addition required to maintain the pH at the set point of 7.0. Once growth ceased, due to ammonium exhaustion, acetate production terminated and the cells then assimilated all of the acetate along with the majority of the remaining glucose (data not shown). This acetate uptake behavior suggests that the TCA cycle was induced (derepressed) upon ammonium exhaustion. Others (Heineken and O'Connor, 1972) have shown that nutritional conditions favoring the induction of the TCA cycle are similar to those leading to subtilisin production. Within 1 h of starvation, @-galactosidase(Figure 2b) activity increased above basal levels and accumulated for the next 20 h to a final level (now presented on a per protein basis) approximately the same as that found for culture in shake flasks (Figure la). Subtilisin production (Figure 2b) also commenced shortly after ammonium starvation and continued over a slightly longer time interval than that observed for @-galactosidase. An examination of the biomass and cellular protein profiles (Figure 2a) in the stationary phase demonstrated that the postexponential biomass concentration increased
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significantly above its level a t the end of growth, while the protein concentration remained relatively stable. This indicated that the cells were accumulating non-nitrogen components. Transmission electron microscopy (TEM) (Figure 3a-d) of exponential- and starvation-phase cells showed that postexponential cells acquired electron transparent inclusion bodies shortly after ammonium exhaustion. These inclusion bodies, however, were not evident in late postexponential-phase cells. On the basis of the nutritional conditions (carbon-richand nitrogen-deficient) under which the structures were produced (Dawes and Senior, 1973) and their electron transparency (Hayat, 19811, the inclusion bodies may have contained endogenous lipid-like storage materials. Chemical analysis (Ward and Dawes, 1973),however, established that these inclusion bodies were not PHB, which has been known to accumulate in other bacillus species (Slepecky and Law, 1960). The electron micrographs also demonstrated that the spoIIG55 mutation, in the original 168 trpC2 background, indeed blocked the sporulation process at stage 11, which is normally identified by the production of a single asymmetric septa. This developmental block, however, resulted in multiple septation events in a significant fraction of the cell population during prolonged postexponential-phase culture. A disporic (one septum near each cell pole) phenotype had been a reported (Piggot, 1973) consequence of this mutation. AmmoniumControl of Subtilisin Productionin Asporogenous StrainRS7907-Fed-Batch Experiments. To further extend the period of subtilisin production by the asporogenous spoIIG (RS7907) strain beyond that possible in batch culture, fed-batch experiments were designed and implemented. In these experiments, cells were downshifted from excess nutrients (and hence exponential growth) to a feedingregime where the constant provision of ammonium allowed only slow, linear growth
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Figure3. Transmissionelectron micrographsof "7907 cells,from the experimentdescribed in Figure 2 and the text, duringexponential growth (a) and after 5 (b), 13 (c), and 37 (d) h of ammonium starvation. The bar equals 1 pm. (Yamane and Shimizu, 1984). No accumulation of ammonium was found in the medium, indicating that ammonium uptake was essentiallymatched with delivery (data not shown). Ammonium feed rates and feeding durations were set to allow approximately one doubling of biomass (protein)over the period of the feeding regime. In the first fed-batch experiment, the linear phase of growth occurred over 11h (Figure 4a). During this linear growth phase, @-galactosidaseand subtilisin production (Figure 4b) exhibited partial responses as compared to batch culture ammonium starvation (Figure 2b). Once feeding was terminated, the subsequent subtilisin and @galactosidase production responses elicited were similar to that observed following ammonium starvation in batch culture (Figure 2b). In a second fed-batch experiment, the ammonium feed rate was set to limit the linear doubling time to approximately 36 h (Figure 5a). The downshift from exponential growth to this appreciably lower linear growth rate resulted in initial subtilisin and @-galactosidase production responses (Figure 5b) similar to those found after ammonium exhaustion in batch culture (Figure 2b). However, once ammonium provision was terminated, no further increase in the level of subtilisin or @-galactosidase was observed. In both fed-batch experiments,the yield of biomass from glucose decreased markedly once the cells entered the linear growth regime. During exponential growth, the yield was approximately 0.33 g of dry weight/g of glucose, while this yield decreased to 0.14 g/g and 0.10 g/g during the linear growth regime of the first and second fed-batch experiments (Figures4 and 5), respectively. This decrease in biomass yield at lower growth rates may be due to a larger proportion of glucose being used for maintenance requirements and the increased uncoupling of catabolism from anabolism (Tempest and Neijssel, 1984). Also, the production of acetate was severely attenuated in the feeding regime of the first fed-batch experiment, and acetate assimilation occurred during the feeding regime of the second fed-batch experiment (data not shown). TEM analysis (not shown) of cells harvested during the feeding periods indicated that the average number of septa formed per cell increased in step with the severity of the
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ammonium limitation. Both feeding conditions also resulted in the accumulation of electron transparent inclusion bodies, whose size and number diminished with extended fermentation times.
Discussion For the development of engineered subtilisin production systems, rational strain selection is essential. Host strain developmentshould includegeneticconstructsto enhance both the rate and duration of subtilisin production. We
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initially focused on extending the duration of production and therefore exploited the incorporation of developmental mutations to block the differentiation of B. subtilis into metabolically inactive endospores. Stage I1 blocked cells seemed to be ideal candidates for our purposes: mutants blocked at the only earlier identifiable morphological stage (stage 0) exhibit markedly decreased subtilisin production capabilities (Ferrari et al., 1986),while attainment of the morphological state (stage 111)beyond stage I1 is temporally associated with attenuation of production (Doi, 1989). Thus, by blocking development at stage 11, one can presumably lock the cells in a state of prolonged production. After evaluating the available stage I1blocked strains in the context of known spoII gene function, we chose to study those containing the spoIIG55 mutation as our model sporulation-deficient hosts. The gene product disabled by this mutation is the minor sigma factor, uE. This factor is one of a cascade of developmentally activated sigma factors that modify the transcriptional specificity of RNA polymerase (Stragier and Losick, 1990) and therefore program the cell toward development into an endospore. uE serves as a developmental switch, inactivating early adaptation genes such as the subtilisin gene (uprE),and directing events toward spore formation (Stragier et al., 1988). With this switch disabled, we anticipated prolonged production. Our demonstration of temporally extended subtilisin gene expression (@-galactosidase production) and subtilisin enzyme production from the sporulationdeficient spoIIG strains confirms the expected benefits of our host selection. One concern in our studies, however, was the stability of secreted subtilisin in the culture medium. While being a key issue, it is seldom considered. Data shown in Figure 1 demonstrate its importance. The termination of subtilisin and @-galactosidaseproduction by the sporulationdeficient strains, and the subsequent stability of each, suggests that subtilisin activity persisted once subtilisin
was exported into the extracellular milieu. In contrast, when sporulating strains were employed, subtilisin and @-galactosidaseaccumulation terminated after several hours of ammonium starvation and was followed by a decline in activity of both enzymes. While the decrease in @-galactosidaseactivity could be attributed to the cessation of subtilisin gene expression and the subsequent protein turnover required to complete the sporulation process, the decrease in subtilisin activity itself was unexpected. Since both the sporulation-proficient and sporulation-deficient strains were grown in equivalent culture media, the differences in subtilisin stability must have been linked to continued spore development. A hallmark of the latter stages of sporulation is the uptake of calcium from the culture medium. During growth and through stage I11 of the sporulation process, B. subtilis actively excludes calcium from its cytoplasm. Calcium uptake then commences at stage IV using a highaffinity active transport system (Marquis, 1989). Interestingly, subtilisin has customarily been characterized as a non-metal-requiring enzyme [for example, Priest (198911, yet others have suggested adding calcium salts to stabilize preparations of subtilisin (Keay et al., 1970) or have provided spectroscopic evidence implicating calcium as an essential element to ensure subtilisin stability (Pantoliano et al., 1988). We believe that the limited ability to include calcium in a culture medium without the formation of insoluble calcium phosphate salts (Pirt, 19751, coupled with the appreciable amount of calcium required for spore formation (approaching 0.9 mmol of calcium/g of dry weight of spores; Warth, 19781, establishes a competition for calcium between the sporulating cell and the excreted subtilisin, with sporulation sometimes occurring at the expense of subtilisin stability. This hypothesis will be discussed in detail in a forthcomingpaper. We have found (unpublished data) that at the cell densities employed (ca. 1g of DW/L), sporogenous strains require approximately 0.5 mM calcium, rather than the 0.14 mM provided in the media used in these studies, to ensure subtilisin stability in the presence of efficient sporulation, while stage 11blocked hosts require on the order of 10pM calcium. These results underscore the importance of understanding both subtilisin production and stability requirements in the context of host strain developmental potential and medium formulation. Batch culture findings demonstrated that subtilisin productivity could not be maintained, even in asporogenous B. subtilis strains, for protracted periods of ammonium starvation. Under such conditions, the only means to produce new proteins is from the turnover of endogenous nitrogen sources or the uptake of previously secreted sources. A portion of this turnover occurs at the expense of required cellular constituents, such as ribosomes (Kaplan and Apirion, 19751, causing cells to progressively lose metabolic integrity and hence productivity. Fed-batch culture techniques appeared to be a logical strategy to forestall this productivity loss. We attempted to impose an adequate metabolic stress,through the slow provision of ammonium, to trigger subtilisin production, and yet to provide sufficient ammonium to maintain cellular metabolic integrity. Using this approach, a graded response to restricted ammonium availability was established: lower feed rates (and hence lower linear growth rates) correlated with higher initial rates of subtilisin (or &galactosidase) production. Only partial success, however, was achieved in further extending production. Cell populations still became nonproductive after extended periods of fed-batch culture, especially at the
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lower linear growth rate. Cessation of protein synthetic potential is not a valid explanation, however, since biomass (and protein content) continued to increase long after subtilisin accumulation had ceased. The loss of productivity may have been, in part, a consequence of the multiple septation of a significant portion of the cell population, or our inability to completely inactivate the genetic system which eventually suppresses transcription of the subtilisin gene. Given the complex regulatory circuits governing the transcriptional regulation of subtilisin expression (Valle and Ferrari, 1989), it is possible that no single mutation would completely overcome the normal cessation of subtilisin gene activity. The multiple septation of spollG55 cells in ammoniumexhausted batch or fed-batch fermenter culture is intriguing from a morphogenetic viewpoint. This phenotype suggests that the developmentally blocked mutants were attempting to initiate the asymmetric septation process multiple times. Others (Illing and Errington, 1991) interested in the genetic regulation of morphogenesis in B. subtilis have also recentlyreported alarge fraction (2060 7%) of disporic and multiseptated cells arising from the culture of a strain containing this mutation. Their results and modeling suggest that other stage I1 blocked mutants, which may septate more normally, could be more attractive as hosta for subsequent subtilisin production studies. While strains with a complete uncoupling of the usual association between subtilisin production and septation would ultimately be preferred, the only developmental mutants approaching this idealism are the sp00 mutants which also possess significantly reduced subtilisin production capabilities (Ferrari et al., 1986). The subtilisin expression and production results, coupled with ultrastructural information, glucose assimilation data, and acetate production and uptake patterns, suggest that B. subtilis has the means to offer a graded response to downshifts in ammonium availability. The larger the downshift, the more the response resembles that of starvation and attempted sporulation. These results may be compared to those obtained by others for ammoniumlimited chemostat culture. Lower dilution (growth) rates result in increased frequencies of sporulation (Dawes and Mandelstam, 1970) and increased subtilisin production (Wouters and Buysman, 1977; Frankena et al., 1986).We suggest, however, that fed-batch culture techniques are more desirable than continuous culture, due to the frequent selection of non-subtilisin-producing variants in chemostat production systems (Heineken and O'Connor, 1972; Frankena et al., 1985). This study demonstrates the utility of employinggenetic, physiological, and engineering strategies concurrently to increase subtilisin production by B. subtilis. Subsequent studies to extend the duration of subtilisin productivity will be directed at identifyingmore morphologically normal asporogenous strains and manipulating physiological and genetic conditions to forestall the loss of productivity. These strains should then be excellent candidates for the incorporation of hyperproducing mutations (Valleand Ferrari, 1989)to increase the rate, as well as the duration, of subtilisin production.
Acknowledgment These investigations were supported by the National Science Foundation (Grant NSF ECE 86-13227). We thank Mr. Ehab El Helow (Department of Biochemistry and Molecular Biology, University of California at Berkeley) and Ms. Pam Louie for assistance in strain construction. We also thank Ms. Fran Thomas (Depart-
ment of BiologicalSciences, Stanford University) and Ms. Susie Matsuno for their assistance in preparing transmission electron micrographs. Manuscript revisions suggested by Mr. Joseph R. Tunner (Department of Chemical Engineering, Stanford University) were also appreciated.
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