Biotechnol. Prog. 1990, 6, 277-282
277
X Vectors for Stable Cloned Gene Expression N. Padukone,*S. W. Peretti, and D. F. Ollis Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905
The bacteriophage X offers a unique opportunity concurrently to minimize segregational instability in recombinant systems by chromosomal integration of the cloned gene and t o achieve high cloned gene expression during a n abortive lytic phase. Lysis leads approximately to a 100-fold amplification of the cloned gene. Cell lysis in the lytic state is blocked by a specific mutation (Sam),allowing the cell t o maintain its integrity, and X DNA packaging is blocked by other mutations ( Wam,Eam) that keep cloned genes open t o transcription. In the presence of these mutations, extremely high levels of cloned 0-galactosidase (more than 15%of total cell protein) have been obtained during abortive lysis from vectors found to be essentially 100% stable for over 75 generations in the lysogenic phase.
Introduction One of the more formidable challenges in the scale-up of recombinant DNA processes has been the instability of the expression vector, most often a plasmid. Such instability, both structural and segregational, must be counteracted in order to maintain cloned genes within a growing population. Structural instability refers to the occurrence of point mutations, gross deletions, or rearrangements that lead to loss of synthesis of active product. This behavior occurs most often with large plasmids. Segregational instability arises when one of the newly divided cells does not receive even a single copy of the vector. Cells freed from the metabolic burden of synthesizing heterologous protein grow more rapidly than those so burdened (Yamaguchi and Yamaguchi, 1983; Alldrick and Smith, 19831, and even a low-frequency generation of vector-free segregants can quickly lead to a population composed primarily of such cells. A number of approaches have been taken to solve the problem of segregational instability. Some studies have employed loci on the plasmid conferring a growth advantage upon vector-bearing cells. For example, antibiotic resistance markers have been widely studied and employed (Imanaka and Aiba, 1981;Schwartz et al., 1988). However, it has been shown that in some cases the resistant population can “clear” the medium of such selective compounds (Dennis et al., 1985), thereby allowing copropagation of nonresistant mutants. In addition, cost makes antibiotic usage uneconomical in large-scale production. Others have placed genes onto the vector that complement an auxotrophy exhibited by the host (Dibiasio and Sardonini, 1986). This procedure is generally effective but suffers from the shortcoming that the growth rate of the culture cannot be augmented by media supplements without running the risk of losing the selection pressure. A genetic approach to minimizing plasmid instability has been the insertion of a partition (pur) locus onto the plasmid, thereby stabilizing its inheritance (Meacock and Cohen, 1980; Skogman et al., 1983; Gerdes, 1989). Studies of stability using vectors with pur loci have been inconclusive. In chemostat culture, Nilsson et al. (1986) have reported a low plasmid stability in purA or parB systems under conditions required for cloned gene
expression, whereas Wood et al. (1990) have observed absolute stability under such conditions with a parB locus on the recombinant plasmid. For solving the problem of segregational instability, we have studied an alternate genetic approach that previously has received little serious consideration: the integration of the cloned gene into the chromosome by use of the phage X system. Earlier work in chromosomal integration has focused on plasmids, involving homologous recombination or transposition of the product gene to the host chromosome (Haas and Holloway, 1978; Breton et al., 1985). In organisms other than Escherichia coli,however, such integrated sequences have been found to be prone to structural instability. In E. coli, enhanced segregational stability has been reported for integrated sequences (Ashby and Stacey, 1984). However, integration causes a substantial reduction in the copy number of the gene, from as much as several hundred to one. Even with absolute stability, therefore, the integrated sequence would lead to decreased productivity due to a paucity of genes available for transcription. The bacteriophage X offers an unique opportunity in this regard. It is an obligate parasite that infects the host and utilizes its biosynthetic machinery either (1)to integrate into the chromosome and replicate passively (lysogeny) or (2) to replicate extrachromosomally to high copy number while suppressing certain aspects of cellular metabolism and then finally to lyse the cell t o release infectious phage (lysis). In this single system, therefore, reside the capabilities of maintainance at low copy number for cloned gene segregational stability, high level of expression through a shift to high copy number, and propagation mechanisms to offset segregational instability. In the lytic state, the replication of X DNA to a high copy number within the cell offers gene amplification as a distinct advantage over plasmid systems that are capable of integration. However, to achieve expression in X vector systems, the packaging of X DNA has to be prevented to enable accessibility of X DNA to host transcriptional components. Furthermore, the lysis of the cell in the lytic state has to be avoided in order to sustain cloned gene expression as long as possible. The key features of a potentially useful X expression system are therefore (i) an
8756-7938/90/3006-0277$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6,No. 4 Super-infection
xgt11 -
P
t Stable lysogen
&,+,+l&j
lac
I . I I I NM1070
phage per cell
.
P
.
Re-infection Replication
t I
Wam Eam
lac
I laCZ I
clts
nin5
Sam1 00
Figure 2. X vectors used.
Figure 1. Events occurring during multiplication of a stable lyso-
gen.
ability to switch from lysogeny to the lytic state, (ii) a deficiency in X DNA packaging, and (iii) a deficiency in the ability to lyse the cell in the lytic state. These features may be provided by special mutations in the X vectors used for expression. The lytic state in which the actual lysis of the cell is prevented will be referred to as the abortive lytic state. Two components of the X genome have been used extensively in the past to develop plasmid expression systems. The X p~ promoter, which is about 10 times stronger than the trp and lac promoters (McKenney et al., 1981; Franklin, 1971), is negatively regulated by the CIrepressor. A combination of a temperature-sensitive repressor protein (cIts) and the p~ promoter is commonly used on plasmids (Simons et al., 1984; George et al., 1985) to obtain induction through an increase of cultivation temperature. The capability of X vectors to make large amounts of protein by induction of a lysogen has been demonstrated by several researchers (Moir and Brammar, 1976; Kelley et al., 1977; Panasenko et al., 1977; Murray and Kelley, 1979). Induction of X from lysogeny to the lytic state has been found to give product yields at least 2 orders of magnitude higher than the expression from the native gene promoter for tryptophan (Moir and Brammar, 1976), DNA ligase (Panasenko et al., 1977), or DNA polymerase I (Murray and Kelley, 1979). A recent study of expression in lysogeny in batch and fed-batch operations shows that production of T4 DNA ligase can be enhanced by operating a fermentation in fed-batch mode (Whitney et al., 1989). Figure 1 charts the events occurring during the multiplication of a lysogen. A lysogen can take one of three routes upon cell division: it can (i) divide to produce stable lysogens or (ii) produce abortive lysogens in which the phage is extrachromosomal and nonlytic or (iii) enter the lytic state and release mature, infectious phage upon eventual cell lysis. Abortive lysogeny results from excision of the prophage in the repressed state. Since the phage cannot multiply in this state, only one of the daughter cells retains the phage. Upon division, the abortive lysogens give rise to cells devoid of phage ("cured" cells). Abortive lysogeny, therefore, would be the cause for segregational instability of the product gene in X expression systems. A low level of cell lysis occurs even a t 30 "C during lysogenic growth. The free phage thus released into the culture medium may reinfect cured cells present in the culture to form lysogens, or they may superinfect existing lyso-
gens. The presence of free phage, therefore, is advantageous for maintenance of high segregational stability in a lysogenic culture. A superinfecting phage may remain extrachromosomal (but nonlytic due to the presence of the X repressor protein, cI), or it may integrate into the host chromosome either at specific secondary attachment sites or a t random sites. In this work, we have studied the segregational stability and product activity in two X expression systems and have compared these systems to a plasmid system containing the parB locus for enhanced stability.
Materials and Methods Bacterial and Phage Strains: (1) A. (a) NM1070 (Wam403 EamllOO lacZ+ cZ857 nin5 Sam100) (Midgley and Murray, 1985). (b) Xgtll (lacZ+ cI857 nin5 Sam100) (Huynh et al., 1984). (2)E. coli. (a) JM109 (recAl endAl gyrA96 thi hsdRl7 (rk-,mk+) supE44 r e l A l A(lac-proAB), F', proAB lacIqZAM15) (Yanisch-Perron et al., 1985). (b) Y1090 (r-) (A lacU169 proA+ Alon araD139 strA s u p F [trpC22:: TnlOl(pMC9)) (Huynh et al., 1984). (c) Y1089 (r-) ( A lacU169 proA+ Alon araD139 strA hflA150 [chr::TnlO](pMC9)) (Huynh et al., 1984). (d) BK6 (recA- A(lacP0Z)c29 lacy+ X74 leuB6 trpC9830 hsdR+ galU galK strAr spc') (Wood et al., 1990). The vectors NM1070 and Xgtll are shown in Figure 2. A temperature-sensitive mutation in gene cZ of the vectors enables them to remain in lysogeny at 32 "C or lower and to be induced into lysis by a shift of temperature to 37 "C or higher. Both vectors are abortive phage, Le., they are incapable of cell lysis in the lytic state due to an amber mutation in gene S. In addition, the vector NM1070 is deficient in packaging functions resulting from amber mutations in genes W and E. These mutations can be suppressed by a host containing supF such as E. coli Y 1090. Both vectors contain a lacZ insert with its native promoter Plac. The strains Y 1089 and JM109 of E. coli have been used here as s u p P hosts for studies of stability and expression, and Y 1090 has been used for propagation of the vectors and for phage titer. The strain Y1089 is a high-frequency lysogen resulting from the hfl mutation, and it carries the repressor gene, lacIq, on a plasmid, pMC9. In contrast, the strain JM109 contains the same repressor gene on the episome, F'. Plasmids. (1) pMJR1750 (lacZ lacIq a m p 3 (Stark, 1987). (2) pTKWlO6 (lacZ lacIqparB kanramps) (Wood et al., 1990). T h e plasmid pMJR1750 contains t h e lacZ gene transcribed from the hybrid promoter ptac. Plasmid pTKWlO6 is a derivative of pMJR1750, having the parB locus and the kan gene inserted into the amp region of
Biotechnol. Prog., 1990,Vol. 6, No. 4
pMJR1750. The plasmid systems BK6::pMJR1750 and BK6::pTKWlOG (Wood et al., 1990) were available from other studies in our laboratory. Growth Media. Luria broth (LB) (Maniatis et al., 1982) was used as a complex growth medium. The minimal medium used was M9CA (Maniatis e t al., 1982). The M9CA medium was supplemented with appropriate amino acids when needed: 164 pg of proline/mL and 1 pg of thiamine/mL for the strain JM109, and 18 pg of tryptophan/mL and 41 pg of leucine/mL for BK6. Lysogens were counted on LB agar plates supplemented with 2 mg of X-gal and 0.3 mg of IPTG. Stable lysogens produce blue colonies, and cured cells or abortive lysogens grow into white colonies on these plates. The plasmidbearing strain BK6::pTKWlOG showed poor growth with concomitant gene expression on plates containing X-gal and IPTG. MacConkey agar was therefore used as the differential medium. Stability Measurements. To obtain a lysogen, host cells grown overnight in LB medium were infected with the phage a t a multiplicity of about 0.1, The infected cells were streaked on LB agar plates supplemented with X-gal and IPTG and then grown a t 30 "C. Restreaking of a blue colony (indicative of a stable lysogen) produced only blue colonies, thereby showing early promise of high segregational stability. A single blue colony was used as a 100 % lysogenic inoculum for batch cultures. T h e batch experiments were performed in Erlenmeyer flasks in a Queue shaker. For stability studies, sequential batch cultures of the lysogen were grown at 30 "C in about 25 mL of medium, with each batch being inoculated with a small sample (about 2 7% of culture volume to minimize lag phase) from the previous batch. Each batch was allowed to reach late exponential phase before it was used for inoculation of the next batch and plating cell samples for lysogen count. The segregational stability of the lysogen was measured as the percentage of stable lysogens in the culture. The lysogen Y1089-Xgtll was grown in the presence of 100 pg of ampicillin/mL to ensure retention of the plasmid pMC9, which carries the repressor lad? The stability of a plasmid system was measured by growing cultures at 37 "C and then plating cell samples on MacConkey agar. Plasmid-bearing cells were distinguished from plasmid-free cells by production of red colonies. Production of j3-Galactosidase. The production of P-galactosidase from the X systems was achieved in three steps: (i) Growth. The lysogen was grown a t 30 "C to an OD600 of about 0.2. (ii) Induction. The lysogen was induced into the lytic state by exposure of the culture to 42.5 "C for 30 min with vigorous aeration. (iii) Expression. IPTG was added to various final concentrations and the culture was incubated a t 37.5 "C. The optimal concentration of IPTG was found to be 0.1 mM for JM109-NM1070 and 1.0 mM for Y1089-Xgtll. A volume of 25 mL of the final culture was then centrifuged at 8000 rpm for 20 min in a Sorvall RC5C centrifuge. The cell pellet was resuspended in 750 pL of buffer (200 mM Tris, pH 7.4,20 mM EDTA, 250 mM NaC1,lO mM 6-mercaptoethanol, and 5% glycerol). The final cell suspension (800-850 pL) was sonicated for 2 min with a Fisher sonic dismembrator (Model 300), and the resulting crude cell homogenate was assayed for P-galactosidase. Expression in lysogeny was achieved by growing the lysogen at 30 " C in the presence of IPTG and harvesting the cells for product. The protocol for expression in the lytic state was optimized for induction time, IPTG concentration, and sonication time by our own experiments. Production of @-galactosidasein the plasmid system
279
c
m
0
m
0
105
1 0
.
lysogen non-lysogen
.
. 2
4
6 8 1 0 1 2 1 4 time (hours)
Figure 3. Comparison of the growth of the lysogen Y1089hgtll and the nonlysogenic host strain Y1089.
BK6::pTKWlOG was carried out by growing the cells at 37 "C to an ODem of about 0.2, followed by addition of IPTG and further incubation for 1h. Harvesting of cells for product was performed as with the X systems. @-GalactosidaseAssay. The cell homogenate samples were assayed for production of P-galactosidase. The activity of 0-galactosidase was measured by its rate of reaction with o-nitrophenyl P-D-gdactopyranoside (ONPG) (from Sigma Chemical Co.), which was monitored by the change in absorbance a t 410 nm in a spectrophotometer (Shimadzu Model UV 160U). One unit of enzyme activity is defined as 1 pmol of ONPG converted/min. T h e reaction mixture for the P-gal assay contained 2.7 mL of 0.1 mM sodium phosphate, 0.1 mL of 3.36 M 0-mercaptoethanol, 0.1 mL of 30 mM MgC12, and 0.1 mL of 34 mM ONPG. The specific enzyme activity was expressed as units/cell. The enzyme yield in the abortive lytic state was expressed as units/lysogenic cell existing prior to induction, because once induced into the lytic state, cells lose viability.
Results Growth i n Lysogeny. We studied two X expression systems, Y1089-hgtll and JM109-NM1070, for segregational stability and product activity. The system JM109NM1070 denotes a lysogen of the bacterial strain JM109 by the phage vector NM1070. The growth rate of Y1089Xgtll was the same as that of the nonlysogen, Y1089, in LB medium (Figure 3), which is in agreement with the finding of Clark et al. (1986) for complex media. There was a low level of cell lysis at 30 "C for the lysis-deficient strain Y1089-Xgtl1, as indicated by the presence of free phage in the medium (Figure 4). The number of free phage/cell reached a steady level in the exponential phase of growth. Assuming a lysis burst size of 100 phage/cell and no phage attachment to cells, the frequency of lysis in lysogeny for the mutated vector Xgtll is estimated to be about 5 X Segregational Stability. The lysogen Y1089-Xgtll grown in LB medium at 30 "C was found to be 100% stable for the entire monitored period of 75 generations in the exponential phase. Strictly speaking, we can only infer that the stability is greater than 99 % , since the dilutions prepared for plating will obscure cured cells that are present at a level 2 or more orders of magnitude below that for lysogens. A similar study carried out for the lysogen JM109-NM1070 in supplemented MSCA showed 1005% stability for the period of 85 generations under study. The stability in lysogeny with concomitant gene expression was examined in LB by growing a lysogen in the presence of IPTG a t 30 "C. Both the lysogens showed complete stability with gene expression during a period of 20
Biotechnol. Prog., 1990, Vol. 6, No. 4
280
-
120 I
I
Table I. Minimum Number of Stable Generations. medium complex minimal
system
Y 1089-Xgtll JM109-NM1070 Y1089-Xgtll (0.1 mM IPTG) JM109-NM1070 (0.1 mM IPTG) BK6::pTKWlOG
75
85 20
20 20 75
75
The stability experiment was terminated in each case at the generation number indicated. 0
2
4
6 8 1 0 1 2 1 4 time (hours)
$ ." 9
Figure 4. Phage released from cell lysis during growth of Y1089h g t l l at 30 "C. generations. The frequency of abortive lysogeny was observed to be less than 0.01 per lysogenic cell. The system BK6::pMJR1750 has been observed to be highly unstable in the presence of up to 400 pg of ampicillin/mL in both batch and continuous cultures (Wood et al., 1990). We observed that the same plasmid was lost in less than 10 generations in supplemented M9CA at 200 and 800 pg of ampicillin/L. We then studied the stability of BK6::pTKWlOG that contains the parB locus. The inoculum was grown in the presence of 50 pg of kanamycin/ mL and was verified t o be 100% plasmid-bearing. Sequential batches were then grown in the absence of kanamycin in LB and supplemented MSCA. The plasmid pTKWlO6 was found to be 100% stable in both the complex and minimal media for the monitored period of 75 generations. Table I summarizes the results of our segregational stability studies. Measurement of &Galactosidase Production. The studies of @-galactosidaseproduction were carried out in LB. In lysogeny, both h systems produced about 1.0 unit of @-galactosidase/cellwhen grown at 30 "C with 0.1 mM IPTG in the culture medium. Figure 5 is a comparison of the time course of @-galactosidaseproduction in the two h systems. The abscissa in the plot is the time after IPTG addition in the expression phase. Production in Y1089hgtll reached a steady level of 8.0 (X10+? units/lysogenic cell after 60 min. On the other hand, JM109NM1070 continued to make increasing levels of @-galactosidase until a peak of 57.1 (XIO-s) units/lysogenic cell was reached at 120 min. Figure 6 is a detailed representation of expression in the system JM109-NM1070. There was partial lysis in the culture despite the mutation carried on NM1070 to prevent cell lysis in the lytic state. The cell lysis is indicated in Figure 6 by a decline in total culture intracellular @-galactosidase activity and a corresponding increase in total culture extracellular activity after 60 min of expression. The production of @-galactosidasein the plasmid system BK6::pTKWlOG was observed to be 10.1 (X10-s) units/ final induced cell a t 5.0 mM IPTG after 60 min of expression. Table 11 is a summary of @-galactosidase production levels in the expression systems studied.
Discussion Since the method of integration of the phage vector into the host chromosome is identical in each of our X lysogenic systems, the stability results of one are believed to be valid for the other in the same growth medium. We conclude that the product gene in a X vector is stable for at least 75 generations in lysogeny, in both complex and minimal media in batch culture. Other genetic approaches to enhancement of segrega-
0
x
Y1089-gill
0
JM109-NM1070
30
60 90 120 Time (minutes)
150
180
Figure 5. Total @-galactosidaseactivity vs time in Y1089Xgtll and JM109-NM1070. Time is measured in the expression phase after heat induction and IPTG addition. One unit of enzyme activity represents 1 @molof ONPG converted/min. Activity is expressed as units/lysogeniccell before heat induction of culture.
0
30
60 90 120 Time ( m i n u t e s )
150
180
Figure 6. Activity of @-galactosidasevs time in JM109NM1070. The units are the same as in Figure 5. Extracellular activity is that measured in the supernatant after spinning down cells in a centrifuge. Intracellular activity represents that in the cell homogenate.
Table 11. Production of &Galactosidase" system
JM109-NM1070 lysogen Y1089-Xgtll (abortive lysis) JM109-NM1070 (abortive lysis) BK6::pTKW 106
P-gal IPTG yield, 108 X expression concn, units/cell time, h mM 0.9 8.1 57.1
10.2
6.0 1.0
2.0 1.0
0.1 1.0 0.1
5.0
a The enzyme activity represents the peak level reached in the X expression system and the duration of expression to reach this level. The IPTG concentrations used were found to be optimal. The expression level in the plasmid system was not verified to be the peak level.
tional stability have been mainly restricted to insertion of stability loci on the plasmid. Nilsson and Skogman
28 1
Biotechnol. frog., 1990, Vol. 6, No. 4
(1986) observed a complete loss in 75 generations of plasmid-bearing cells carrying a parA or a p a r B locus in continuous culture. They constructed a plasmid carrying a ualS gene t h a t was stably retained under similar conditions for 150 generations. However, Wood et al. (1990) have reported complete stability of a parB+ plasmid in chemostat culture for a t least 75 generations. In other studies, the cos ends of X were shown to stabilize a plasmid for 330 generations (Edlin et al., 1984), and the presence of the cZ repressor gene on a plasmid resulted in its stable inheritance in a cI- lysogenic host strain for 132 generations in batch culture (Rosteck and Hershberger, 1983). A recent study has shown that the ssb gene present on the plasmid in a ssb- host strain prevents takeover of the reactor by plasmid-free cells (Porter et al., 1990). Our X vectors have a built-in stability mechanism due to their ability to integrate into the host chromosome, and they do not require either an external locus or a specific mutation in the host strain for stable inheritance. The 6-galactosidase activity in JM109-NM1070 shows about a 60-fold amplification in the abortive lytic state, whereas Y1089-Xgtll shows only an 8-fold higher activity over lysogeny (Table 11). Since the copy number of X DNA in the lytic state is about 100/cell, we should expect a high amplification of product activity in the abortive lytic state over lysogeny. The roughly 7-fold difference in the product activity of the two systems may be attributed to the packaging mutations carried on NM1070 that prevents packaging of X DNA in the lytic state. To investigate the possibility that the presence of the plasmid pMC9 in the host strain Y 1089 might negatively affect final product synthesis, we measured P-galactosidase production in JMlO9-Xgtll. The activity in JMlO9-Xgtll was found to be 6.3 (XIO-s) units/lysogenic cell, which is comparable to that obtained in Y1089-Xgtll. We conclude that the low product activity in the Xgtll systems is due to the absence of packaging mutations on the vector. The abortive lytic state of JM109-NM1070 exhibits partial cell lysis and releases some product into the extracellular medium. This may be attributed to two factors. First, the presence of a supE gene in JM109 may result in a low level of suppression of the amber mutations in NM1070, thereby leading to cell lysis. Second, the X gene R encodes an endolysin that, in a normal lytic state, breaks the cell wall along with the product of gene S. In the defective phage NM1070, the individual action of R may still weaken the cell wall and lysis may occur late in the expression phase or in the subsequent steps involved in harvesting of cells for product. The observed total product activity in JM109-NM1070 is close to that expected from a 100-fold amplification in product gene copy number. Analysis by SDS-polyacrylamide gel electrophoresis of cellular protein samples obtained after 120 min of expression from the JM109NM1070 system showed 6-galactosidase t o be approximately 20% of total intracellular protein. The extracellular samples were too dilute to show bands of protein on the gels. Plasmid expression systems commonly use the combination of either XPL or XPR with the temperaturesensitive CI repressor to achieve high cloned gene expression. A PL-CIplasmid system that is regulated by tryptophan instead of cultivation temperature produced 0-galactosidase as 21 % of total protein (Mieschendahl et al., 1986). Other systems with PL-CI~S have also shown high production levels depending on the nature of the product. Tryptophan synthetase A was produced as 40% of total protein in batch culture (Remaut et al., 1981) and 1015% in two-stage continuous culture (Siege1 and Ryu,
1985). Expression systems using PR-CI~S have produced 20% of total cell protein as chloramphenicol transacetylase (Wright et al., 1986) and 15% as active bovine growth hormone (George et al., 1985). The production level observed in our A expression system compares well with those of the plasmid systems above. It should be noted, however, that expression of the lacZ gene in our case was under the control of the much weaker lac promoter. The product yields from our X system may be further enhanced by using a stronger promoter for transcription of the cloned gene and by optimization of the X vector to minimize production of the nonessential head and tail proteins in the abortive lytic state. The high productivity in JM109-NM1070 may be also due to other aspects of the lytic state that are advantageous for overproduction of cloned gene product. The interactions between phage and host include association of phage proteins with host genes or proteins. If such interactions result in the cellular biosynthetic capacity being directed largely to the expression of the cloned gene product, then A-based vector systems have the potential for extremely high, controlled gene expression and excellent segregational stability. A defective phage system such as JM109-NM1070 offers an excellent vehicle for study of such interactions by monitoring the expression of a marker gene carried on the phage.
Conclusions The X expression systems studied here have produced two main findings. First, from the evaluation of segregational stability, the product gene carried on the X vector is 100% stable in batch culture for at least 75 generations in lysogeny. Second, the switch to an abortive lytic state, in which both packaging of X DNA and cell lysis are prevented, can be used to achieve a very high protein productivity. Although our study has been restricted to the E . coli protein P-galactosidase, this product gene may be easily replaced by another product gene. The use of specialized X vectors for expression shows strong promise for large-scale production of recombinant proteins.
Acknowledgment We thank Professor T. R. Klaenhammer for providing the strains Xgtll, Y1089, and Y1090 and for his technical assistance in this project. We also thank Professor Noreen Murray for donating the strain NM1070, Robert Kuhn for providing the strain BK6, and Tom Wood for providing the plasmid pTKWlO6.
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Edlin, G.; Tait, R. C.; Rodriguez, R. L. A Bacteriophage h Cohesive Ends (cos) DNA Fragment Enhances the Fitness of PlasmidContaining Bacteria Growing in Energy-Limited Chemostats. BiolTechnology 1984, 2, 251. Franklin, N. C. The N Operon of Lambda. In T h e Bacteriophage Lambda; Hershey, A. D., Ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1971; pp 621-638. George, H.; L’Italien, J. J.; Pilacinski, W. P.; Glassman, D. L.; Krzyzek, R. A. High Level Expression in Escherichia coli of Biologically Active Growth Hormone. D N A 1985, 4, 273. Gerdes, K. The ParB (Hok/Sok) Locus of Plasmid R1: A General Purpose Plasmid Stabilization System. Biol Technology 1989, 6 , 1402. Haas, D.; Holloway, B. W. Chromosome Mobilization by the R Plasmid R68.45 A Tool in Pseudomonas Genetics. MGG, Mol. Gen. Genet. 1978,158, 229. Huynh, T. V.; Young, R. A.; Davis, R. W. In D N A Cloning Techniques: A Practical Approach; Glover, D., Ed.; IRL Press: Oxford, England, 1984. Imanaka, T.; Aiba, S. A Perspective on the Application of Genetic Engineering: Stability of the Recombinant Plasmid. A n n . N.Y. Acad. Sci. 1981, 369, 1-14, 1981. Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1982; p 440. McKenney, K.; Shimatake, H.; Court, D.; Schmeissner, U.; Brady, C.; Rosenberg, M. In Gene Amplification and Analysis, Vol. ZI: Structural Analysis of Nucleic Acids; Chirikjian, J. G., Papas, T. S., Eds.; ElsevierINorth Holland: New York, 1981; pp 383-415. Meacock, P. A.; Cohen, S. N. Partitioning of Bacterial Plasmids During Cell Division: A Cis-Acting Locus t h a t Accomplishes Stable Plasmid Inheritance. Cell 1980,20,529. Midgley, C. A.; Murray, N. E. T4 Polynucleotide Kinase; Cloning of the Gene (pseT) and Amplification of its Product. EMBO J . 1985, 4 (lo), 2695. Mieschendahl, M.; Petri, T.; Hanggi, U. A Novel Prophage Independent t r p Regulated Lambda PL Expression System. BiolTechnology 1986,4, 802. Moir, A.; Brammar, W. J. The Use of Specialized Transducing Phages in the Amplification of Enzyme Production. MGG, Mol. Gen. Genet. 1976, 149, 87. Murray, N. E.; Kelley, W. S. Characterization of hpolA Transducing Phages; Effective Expression of the E. coli em polA Gene. MGG, Mol. Gen. Genet. 1979, 175, 77. Nilsson, J.; Skogman, S. G. Stabilization of Escherichia coli Tryptophan Production Vectors in Continuous Cultures. Bio/ Technology 1986,4, 901-903.
Panasenko, S. M.; Cameron, J. R.; Davis, R. W.; Lehman, I. R. Five Hundredfold Overproduction of DNA Ligase After Induction of a Hybrid Lambda Lysogen Constructed in vitro. Science 1977, 196, 188. Porter, R. D.; Black, S.;Pannuri, S.; Carlson, A., Use of the E s cherichia coli ssb Gene to Prevent Bioreactor Takeover by Plasmidless Cells. BiolTechnology 1990,8, 47. Remaut, E.; Stanssens, P.; Fiers, W. Plasmid Vectors for HighEfficiency Expression Controlled by the p~ Promoter of Coliphage Lambda. Gene 1981,15,81. Rosteck, P. R., Jr.; Hershberger, C. L. Selective Retention of Recombinant Plasmids Coding for Human Insulin. Gene 1983, 25, 29. Schwartz, L. S.; Jansen, N. B.; Ho, N. W. Y.; Tsao, G. T., Plasmid Instability Kinetics of the Yeast S228C pUCKm8 [cir+] in Non-selective and Selective Media. Biotechnol. Bioeng. 1988, 32, 733. Siegel, R.; Ryu, D. D. Y. Kinetic S t u d y of Instability of Recombinant Plasmid pPLc23trpAI in E . coli Using TwoStage Continuous Culture System. Biotechnol. Bioeng. 1985, 27, 28. Skogman, S. G.; Nilsson, J.; Gustafsson, P. The Use of a Partition Locus to Increase Stability of Tryptophan-Operon-Bearing Plasmids in Escherichia coli. Gene 1983, 23, 105. Stark, M. J. R. Multicopy Expression Vectors Carrying the lac Repressor Gene for High-Level Expression of Genes in E s cherichia coli. Gene 1987, 51, 255. Whitney, G. K.; Glick, B. R.; Robinson, C. W. Induction of a T4 DNA Ligase in a Recombinant Strain of Escherichia coli. Biotechnol. Bioeng. 1989, 33, 991. Wood, T. K.; Kuhn, R. H.; Peretti, S. W. Enhanced Plasmid Stability Through Post-Segregational Killing of PlasmidFree Cells. Biotechnol. Tech. 1990, 4, 39. Wright, E. M.; Humphreys, G. O.,; Yarranton, G. T. Dual Origin Plasmids Containing an Amplifiable ColEl ori; TemperatureControlled Expression of Cloned Genes. Gene 1986,49, 311. Yamaguchi, K.; Yamaguchi, M. Copy Number Mutations (cop-) of the Plasmid Containing the Replication Origin (oriC) of the E. coli Chromosome: Lethal Effect of the cop Region onto a High-Copy-Number Vector on Host Cells. J . Bacteriol. 1983, 153, 550. Yanisch-Perron, C.; Vieira, J.; Messing, J. Improved M13 Phage Cloning Vectors and Host Strains: Nucleotide Sequences of the M13mp18 and pUC19 Vectors. Gene 1985,33, 103. Accepted June 13, 1990.
277
X Vectors for Stable Cloned Gene Expression N. Padukone,*S. W. Peretti, and D. F. Ollis Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905
The bacteriophage X offers a unique opportunity concurrently to minimize segregational instability in recombinant systems by chromosomal integration of the cloned gene and t o achieve high cloned gene expression during a n abortive lytic phase. Lysis leads approximately to a 100-fold amplification of the cloned gene. Cell lysis in the lytic state is blocked by a specific mutation (Sam),allowing the cell t o maintain its integrity, and X DNA packaging is blocked by other mutations ( Wam,Eam) that keep cloned genes open t o transcription. In the presence of these mutations, extremely high levels of cloned 0-galactosidase (more than 15%of total cell protein) have been obtained during abortive lysis from vectors found to be essentially 100% stable for over 75 generations in the lysogenic phase.
Introduction One of the more formidable challenges in the scale-up of recombinant DNA processes has been the instability of the expression vector, most often a plasmid. Such instability, both structural and segregational, must be counteracted in order to maintain cloned genes within a growing population. Structural instability refers to the occurrence of point mutations, gross deletions, or rearrangements that lead to loss of synthesis of active product. This behavior occurs most often with large plasmids. Segregational instability arises when one of the newly divided cells does not receive even a single copy of the vector. Cells freed from the metabolic burden of synthesizing heterologous protein grow more rapidly than those so burdened (Yamaguchi and Yamaguchi, 1983; Alldrick and Smith, 19831, and even a low-frequency generation of vector-free segregants can quickly lead to a population composed primarily of such cells. A number of approaches have been taken to solve the problem of segregational instability. Some studies have employed loci on the plasmid conferring a growth advantage upon vector-bearing cells. For example, antibiotic resistance markers have been widely studied and employed (Imanaka and Aiba, 1981;Schwartz et al., 1988). However, it has been shown that in some cases the resistant population can “clear” the medium of such selective compounds (Dennis et al., 1985), thereby allowing copropagation of nonresistant mutants. In addition, cost makes antibiotic usage uneconomical in large-scale production. Others have placed genes onto the vector that complement an auxotrophy exhibited by the host (Dibiasio and Sardonini, 1986). This procedure is generally effective but suffers from the shortcoming that the growth rate of the culture cannot be augmented by media supplements without running the risk of losing the selection pressure. A genetic approach to minimizing plasmid instability has been the insertion of a partition (pur) locus onto the plasmid, thereby stabilizing its inheritance (Meacock and Cohen, 1980; Skogman et al., 1983; Gerdes, 1989). Studies of stability using vectors with pur loci have been inconclusive. In chemostat culture, Nilsson et al. (1986) have reported a low plasmid stability in purA or parB systems under conditions required for cloned gene
expression, whereas Wood et al. (1990) have observed absolute stability under such conditions with a parB locus on the recombinant plasmid. For solving the problem of segregational instability, we have studied an alternate genetic approach that previously has received little serious consideration: the integration of the cloned gene into the chromosome by use of the phage X system. Earlier work in chromosomal integration has focused on plasmids, involving homologous recombination or transposition of the product gene to the host chromosome (Haas and Holloway, 1978; Breton et al., 1985). In organisms other than Escherichia coli,however, such integrated sequences have been found to be prone to structural instability. In E. coli, enhanced segregational stability has been reported for integrated sequences (Ashby and Stacey, 1984). However, integration causes a substantial reduction in the copy number of the gene, from as much as several hundred to one. Even with absolute stability, therefore, the integrated sequence would lead to decreased productivity due to a paucity of genes available for transcription. The bacteriophage X offers an unique opportunity in this regard. It is an obligate parasite that infects the host and utilizes its biosynthetic machinery either (1)to integrate into the chromosome and replicate passively (lysogeny) or (2) to replicate extrachromosomally to high copy number while suppressing certain aspects of cellular metabolism and then finally to lyse the cell t o release infectious phage (lysis). In this single system, therefore, reside the capabilities of maintainance at low copy number for cloned gene segregational stability, high level of expression through a shift to high copy number, and propagation mechanisms to offset segregational instability. In the lytic state, the replication of X DNA to a high copy number within the cell offers gene amplification as a distinct advantage over plasmid systems that are capable of integration. However, to achieve expression in X vector systems, the packaging of X DNA has to be prevented to enable accessibility of X DNA to host transcriptional components. Furthermore, the lysis of the cell in the lytic state has to be avoided in order to sustain cloned gene expression as long as possible. The key features of a potentially useful X expression system are therefore (i) an
8756-7938/90/3006-0277$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6,No. 4 Super-infection
xgt11 -
P
t Stable lysogen
&,+,+l&j
lac
I . I I I NM1070
phage per cell
.
P
.
Re-infection Replication
t I
Wam Eam
lac
I laCZ I
clts
nin5
Sam1 00
Figure 2. X vectors used.
Figure 1. Events occurring during multiplication of a stable lyso-
gen.
ability to switch from lysogeny to the lytic state, (ii) a deficiency in X DNA packaging, and (iii) a deficiency in the ability to lyse the cell in the lytic state. These features may be provided by special mutations in the X vectors used for expression. The lytic state in which the actual lysis of the cell is prevented will be referred to as the abortive lytic state. Two components of the X genome have been used extensively in the past to develop plasmid expression systems. The X p~ promoter, which is about 10 times stronger than the trp and lac promoters (McKenney et al., 1981; Franklin, 1971), is negatively regulated by the CIrepressor. A combination of a temperature-sensitive repressor protein (cIts) and the p~ promoter is commonly used on plasmids (Simons et al., 1984; George et al., 1985) to obtain induction through an increase of cultivation temperature. The capability of X vectors to make large amounts of protein by induction of a lysogen has been demonstrated by several researchers (Moir and Brammar, 1976; Kelley et al., 1977; Panasenko et al., 1977; Murray and Kelley, 1979). Induction of X from lysogeny to the lytic state has been found to give product yields at least 2 orders of magnitude higher than the expression from the native gene promoter for tryptophan (Moir and Brammar, 1976), DNA ligase (Panasenko et al., 1977), or DNA polymerase I (Murray and Kelley, 1979). A recent study of expression in lysogeny in batch and fed-batch operations shows that production of T4 DNA ligase can be enhanced by operating a fermentation in fed-batch mode (Whitney et al., 1989). Figure 1 charts the events occurring during the multiplication of a lysogen. A lysogen can take one of three routes upon cell division: it can (i) divide to produce stable lysogens or (ii) produce abortive lysogens in which the phage is extrachromosomal and nonlytic or (iii) enter the lytic state and release mature, infectious phage upon eventual cell lysis. Abortive lysogeny results from excision of the prophage in the repressed state. Since the phage cannot multiply in this state, only one of the daughter cells retains the phage. Upon division, the abortive lysogens give rise to cells devoid of phage ("cured" cells). Abortive lysogeny, therefore, would be the cause for segregational instability of the product gene in X expression systems. A low level of cell lysis occurs even a t 30 "C during lysogenic growth. The free phage thus released into the culture medium may reinfect cured cells present in the culture to form lysogens, or they may superinfect existing lyso-
gens. The presence of free phage, therefore, is advantageous for maintenance of high segregational stability in a lysogenic culture. A superinfecting phage may remain extrachromosomal (but nonlytic due to the presence of the X repressor protein, cI), or it may integrate into the host chromosome either at specific secondary attachment sites or a t random sites. In this work, we have studied the segregational stability and product activity in two X expression systems and have compared these systems to a plasmid system containing the parB locus for enhanced stability.
Materials and Methods Bacterial and Phage Strains: (1) A. (a) NM1070 (Wam403 EamllOO lacZ+ cZ857 nin5 Sam100) (Midgley and Murray, 1985). (b) Xgtll (lacZ+ cI857 nin5 Sam100) (Huynh et al., 1984). (2)E. coli. (a) JM109 (recAl endAl gyrA96 thi hsdRl7 (rk-,mk+) supE44 r e l A l A(lac-proAB), F', proAB lacIqZAM15) (Yanisch-Perron et al., 1985). (b) Y1090 (r-) (A lacU169 proA+ Alon araD139 strA s u p F [trpC22:: TnlOl(pMC9)) (Huynh et al., 1984). (c) Y1089 (r-) ( A lacU169 proA+ Alon araD139 strA hflA150 [chr::TnlO](pMC9)) (Huynh et al., 1984). (d) BK6 (recA- A(lacP0Z)c29 lacy+ X74 leuB6 trpC9830 hsdR+ galU galK strAr spc') (Wood et al., 1990). The vectors NM1070 and Xgtll are shown in Figure 2. A temperature-sensitive mutation in gene cZ of the vectors enables them to remain in lysogeny at 32 "C or lower and to be induced into lysis by a shift of temperature to 37 "C or higher. Both vectors are abortive phage, Le., they are incapable of cell lysis in the lytic state due to an amber mutation in gene S. In addition, the vector NM1070 is deficient in packaging functions resulting from amber mutations in genes W and E. These mutations can be suppressed by a host containing supF such as E. coli Y 1090. Both vectors contain a lacZ insert with its native promoter Plac. The strains Y 1089 and JM109 of E. coli have been used here as s u p P hosts for studies of stability and expression, and Y 1090 has been used for propagation of the vectors and for phage titer. The strain Y1089 is a high-frequency lysogen resulting from the hfl mutation, and it carries the repressor gene, lacIq, on a plasmid, pMC9. In contrast, the strain JM109 contains the same repressor gene on the episome, F'. Plasmids. (1) pMJR1750 (lacZ lacIq a m p 3 (Stark, 1987). (2) pTKWlO6 (lacZ lacIqparB kanramps) (Wood et al., 1990). T h e plasmid pMJR1750 contains t h e lacZ gene transcribed from the hybrid promoter ptac. Plasmid pTKWlO6 is a derivative of pMJR1750, having the parB locus and the kan gene inserted into the amp region of
Biotechnol. Prog., 1990,Vol. 6, No. 4
pMJR1750. The plasmid systems BK6::pMJR1750 and BK6::pTKWlOG (Wood et al., 1990) were available from other studies in our laboratory. Growth Media. Luria broth (LB) (Maniatis et al., 1982) was used as a complex growth medium. The minimal medium used was M9CA (Maniatis e t al., 1982). The M9CA medium was supplemented with appropriate amino acids when needed: 164 pg of proline/mL and 1 pg of thiamine/mL for the strain JM109, and 18 pg of tryptophan/mL and 41 pg of leucine/mL for BK6. Lysogens were counted on LB agar plates supplemented with 2 mg of X-gal and 0.3 mg of IPTG. Stable lysogens produce blue colonies, and cured cells or abortive lysogens grow into white colonies on these plates. The plasmidbearing strain BK6::pTKWlOG showed poor growth with concomitant gene expression on plates containing X-gal and IPTG. MacConkey agar was therefore used as the differential medium. Stability Measurements. To obtain a lysogen, host cells grown overnight in LB medium were infected with the phage a t a multiplicity of about 0.1, The infected cells were streaked on LB agar plates supplemented with X-gal and IPTG and then grown a t 30 "C. Restreaking of a blue colony (indicative of a stable lysogen) produced only blue colonies, thereby showing early promise of high segregational stability. A single blue colony was used as a 100 % lysogenic inoculum for batch cultures. T h e batch experiments were performed in Erlenmeyer flasks in a Queue shaker. For stability studies, sequential batch cultures of the lysogen were grown at 30 "C in about 25 mL of medium, with each batch being inoculated with a small sample (about 2 7% of culture volume to minimize lag phase) from the previous batch. Each batch was allowed to reach late exponential phase before it was used for inoculation of the next batch and plating cell samples for lysogen count. The segregational stability of the lysogen was measured as the percentage of stable lysogens in the culture. The lysogen Y1089-Xgtll was grown in the presence of 100 pg of ampicillin/mL to ensure retention of the plasmid pMC9, which carries the repressor lad? The stability of a plasmid system was measured by growing cultures at 37 "C and then plating cell samples on MacConkey agar. Plasmid-bearing cells were distinguished from plasmid-free cells by production of red colonies. Production of j3-Galactosidase. The production of P-galactosidase from the X systems was achieved in three steps: (i) Growth. The lysogen was grown a t 30 "C to an OD600 of about 0.2. (ii) Induction. The lysogen was induced into the lytic state by exposure of the culture to 42.5 "C for 30 min with vigorous aeration. (iii) Expression. IPTG was added to various final concentrations and the culture was incubated a t 37.5 "C. The optimal concentration of IPTG was found to be 0.1 mM for JM109-NM1070 and 1.0 mM for Y1089-Xgtll. A volume of 25 mL of the final culture was then centrifuged at 8000 rpm for 20 min in a Sorvall RC5C centrifuge. The cell pellet was resuspended in 750 pL of buffer (200 mM Tris, pH 7.4,20 mM EDTA, 250 mM NaC1,lO mM 6-mercaptoethanol, and 5% glycerol). The final cell suspension (800-850 pL) was sonicated for 2 min with a Fisher sonic dismembrator (Model 300), and the resulting crude cell homogenate was assayed for P-galactosidase. Expression in lysogeny was achieved by growing the lysogen at 30 " C in the presence of IPTG and harvesting the cells for product. The protocol for expression in the lytic state was optimized for induction time, IPTG concentration, and sonication time by our own experiments. Production of @-galactosidasein the plasmid system
279
c
m
0
m
0
105
1 0
.
lysogen non-lysogen
.
. 2
4
6 8 1 0 1 2 1 4 time (hours)
Figure 3. Comparison of the growth of the lysogen Y1089hgtll and the nonlysogenic host strain Y1089.
BK6::pTKWlOG was carried out by growing the cells at 37 "C to an ODem of about 0.2, followed by addition of IPTG and further incubation for 1h. Harvesting of cells for product was performed as with the X systems. @-GalactosidaseAssay. The cell homogenate samples were assayed for production of P-galactosidase. The activity of 0-galactosidase was measured by its rate of reaction with o-nitrophenyl P-D-gdactopyranoside (ONPG) (from Sigma Chemical Co.), which was monitored by the change in absorbance a t 410 nm in a spectrophotometer (Shimadzu Model UV 160U). One unit of enzyme activity is defined as 1 pmol of ONPG converted/min. T h e reaction mixture for the P-gal assay contained 2.7 mL of 0.1 mM sodium phosphate, 0.1 mL of 3.36 M 0-mercaptoethanol, 0.1 mL of 30 mM MgC12, and 0.1 mL of 34 mM ONPG. The specific enzyme activity was expressed as units/cell. The enzyme yield in the abortive lytic state was expressed as units/lysogenic cell existing prior to induction, because once induced into the lytic state, cells lose viability.
Results Growth i n Lysogeny. We studied two X expression systems, Y1089-hgtll and JM109-NM1070, for segregational stability and product activity. The system JM109NM1070 denotes a lysogen of the bacterial strain JM109 by the phage vector NM1070. The growth rate of Y1089Xgtll was the same as that of the nonlysogen, Y1089, in LB medium (Figure 3), which is in agreement with the finding of Clark et al. (1986) for complex media. There was a low level of cell lysis at 30 "C for the lysis-deficient strain Y1089-Xgtl1, as indicated by the presence of free phage in the medium (Figure 4). The number of free phage/cell reached a steady level in the exponential phase of growth. Assuming a lysis burst size of 100 phage/cell and no phage attachment to cells, the frequency of lysis in lysogeny for the mutated vector Xgtll is estimated to be about 5 X Segregational Stability. The lysogen Y1089-Xgtll grown in LB medium at 30 "C was found to be 100% stable for the entire monitored period of 75 generations in the exponential phase. Strictly speaking, we can only infer that the stability is greater than 99 % , since the dilutions prepared for plating will obscure cured cells that are present at a level 2 or more orders of magnitude below that for lysogens. A similar study carried out for the lysogen JM109-NM1070 in supplemented MSCA showed 1005% stability for the period of 85 generations under study. The stability in lysogeny with concomitant gene expression was examined in LB by growing a lysogen in the presence of IPTG a t 30 "C. Both the lysogens showed complete stability with gene expression during a period of 20
Biotechnol. Prog., 1990, Vol. 6, No. 4
280
-
120 I
I
Table I. Minimum Number of Stable Generations. medium complex minimal
system
Y 1089-Xgtll JM109-NM1070 Y1089-Xgtll (0.1 mM IPTG) JM109-NM1070 (0.1 mM IPTG) BK6::pTKWlOG
75
85 20
20 20 75
75
The stability experiment was terminated in each case at the generation number indicated. 0
2
4
6 8 1 0 1 2 1 4 time (hours)
$ ." 9
Figure 4. Phage released from cell lysis during growth of Y1089h g t l l at 30 "C. generations. The frequency of abortive lysogeny was observed to be less than 0.01 per lysogenic cell. The system BK6::pMJR1750 has been observed to be highly unstable in the presence of up to 400 pg of ampicillin/mL in both batch and continuous cultures (Wood et al., 1990). We observed that the same plasmid was lost in less than 10 generations in supplemented M9CA at 200 and 800 pg of ampicillin/L. We then studied the stability of BK6::pTKWlOG that contains the parB locus. The inoculum was grown in the presence of 50 pg of kanamycin/ mL and was verified t o be 100% plasmid-bearing. Sequential batches were then grown in the absence of kanamycin in LB and supplemented MSCA. The plasmid pTKWlO6 was found to be 100% stable in both the complex and minimal media for the monitored period of 75 generations. Table I summarizes the results of our segregational stability studies. Measurement of &Galactosidase Production. The studies of @-galactosidaseproduction were carried out in LB. In lysogeny, both h systems produced about 1.0 unit of @-galactosidase/cellwhen grown at 30 "C with 0.1 mM IPTG in the culture medium. Figure 5 is a comparison of the time course of @-galactosidaseproduction in the two h systems. The abscissa in the plot is the time after IPTG addition in the expression phase. Production in Y1089hgtll reached a steady level of 8.0 (X10+? units/lysogenic cell after 60 min. On the other hand, JM109NM1070 continued to make increasing levels of @-galactosidase until a peak of 57.1 (XIO-s) units/lysogenic cell was reached at 120 min. Figure 6 is a detailed representation of expression in the system JM109-NM1070. There was partial lysis in the culture despite the mutation carried on NM1070 to prevent cell lysis in the lytic state. The cell lysis is indicated in Figure 6 by a decline in total culture intracellular @-galactosidase activity and a corresponding increase in total culture extracellular activity after 60 min of expression. The production of @-galactosidasein the plasmid system BK6::pTKWlOG was observed to be 10.1 (X10-s) units/ final induced cell a t 5.0 mM IPTG after 60 min of expression. Table 11 is a summary of @-galactosidase production levels in the expression systems studied.
Discussion Since the method of integration of the phage vector into the host chromosome is identical in each of our X lysogenic systems, the stability results of one are believed to be valid for the other in the same growth medium. We conclude that the product gene in a X vector is stable for at least 75 generations in lysogeny, in both complex and minimal media in batch culture. Other genetic approaches to enhancement of segrega-
0
x
Y1089-gill
0
JM109-NM1070
30
60 90 120 Time (minutes)
150
180
Figure 5. Total @-galactosidaseactivity vs time in Y1089Xgtll and JM109-NM1070. Time is measured in the expression phase after heat induction and IPTG addition. One unit of enzyme activity represents 1 @molof ONPG converted/min. Activity is expressed as units/lysogeniccell before heat induction of culture.
0
30
60 90 120 Time ( m i n u t e s )
150
180
Figure 6. Activity of @-galactosidasevs time in JM109NM1070. The units are the same as in Figure 5. Extracellular activity is that measured in the supernatant after spinning down cells in a centrifuge. Intracellular activity represents that in the cell homogenate.
Table 11. Production of &Galactosidase" system
JM109-NM1070 lysogen Y1089-Xgtll (abortive lysis) JM109-NM1070 (abortive lysis) BK6::pTKW 106
P-gal IPTG yield, 108 X expression concn, units/cell time, h mM 0.9 8.1 57.1
10.2
6.0 1.0
2.0 1.0
0.1 1.0 0.1
5.0
a The enzyme activity represents the peak level reached in the X expression system and the duration of expression to reach this level. The IPTG concentrations used were found to be optimal. The expression level in the plasmid system was not verified to be the peak level.
tional stability have been mainly restricted to insertion of stability loci on the plasmid. Nilsson and Skogman
28 1
Biotechnol. frog., 1990, Vol. 6, No. 4
(1986) observed a complete loss in 75 generations of plasmid-bearing cells carrying a parA or a p a r B locus in continuous culture. They constructed a plasmid carrying a ualS gene t h a t was stably retained under similar conditions for 150 generations. However, Wood et al. (1990) have reported complete stability of a parB+ plasmid in chemostat culture for a t least 75 generations. In other studies, the cos ends of X were shown to stabilize a plasmid for 330 generations (Edlin et al., 1984), and the presence of the cZ repressor gene on a plasmid resulted in its stable inheritance in a cI- lysogenic host strain for 132 generations in batch culture (Rosteck and Hershberger, 1983). A recent study has shown that the ssb gene present on the plasmid in a ssb- host strain prevents takeover of the reactor by plasmid-free cells (Porter et al., 1990). Our X vectors have a built-in stability mechanism due to their ability to integrate into the host chromosome, and they do not require either an external locus or a specific mutation in the host strain for stable inheritance. The 6-galactosidase activity in JM109-NM1070 shows about a 60-fold amplification in the abortive lytic state, whereas Y1089-Xgtll shows only an 8-fold higher activity over lysogeny (Table 11). Since the copy number of X DNA in the lytic state is about 100/cell, we should expect a high amplification of product activity in the abortive lytic state over lysogeny. The roughly 7-fold difference in the product activity of the two systems may be attributed to the packaging mutations carried on NM1070 that prevents packaging of X DNA in the lytic state. To investigate the possibility that the presence of the plasmid pMC9 in the host strain Y 1089 might negatively affect final product synthesis, we measured P-galactosidase production in JMlO9-Xgtll. The activity in JMlO9-Xgtll was found to be 6.3 (XIO-s) units/lysogenic cell, which is comparable to that obtained in Y1089-Xgtll. We conclude that the low product activity in the Xgtll systems is due to the absence of packaging mutations on the vector. The abortive lytic state of JM109-NM1070 exhibits partial cell lysis and releases some product into the extracellular medium. This may be attributed to two factors. First, the presence of a supE gene in JM109 may result in a low level of suppression of the amber mutations in NM1070, thereby leading to cell lysis. Second, the X gene R encodes an endolysin that, in a normal lytic state, breaks the cell wall along with the product of gene S. In the defective phage NM1070, the individual action of R may still weaken the cell wall and lysis may occur late in the expression phase or in the subsequent steps involved in harvesting of cells for product. The observed total product activity in JM109-NM1070 is close to that expected from a 100-fold amplification in product gene copy number. Analysis by SDS-polyacrylamide gel electrophoresis of cellular protein samples obtained after 120 min of expression from the JM109NM1070 system showed 6-galactosidase t o be approximately 20% of total intracellular protein. The extracellular samples were too dilute to show bands of protein on the gels. Plasmid expression systems commonly use the combination of either XPL or XPR with the temperaturesensitive CI repressor to achieve high cloned gene expression. A PL-CIplasmid system that is regulated by tryptophan instead of cultivation temperature produced 0-galactosidase as 21 % of total protein (Mieschendahl et al., 1986). Other systems with PL-CI~S have also shown high production levels depending on the nature of the product. Tryptophan synthetase A was produced as 40% of total protein in batch culture (Remaut et al., 1981) and 1015% in two-stage continuous culture (Siege1 and Ryu,
1985). Expression systems using PR-CI~S have produced 20% of total cell protein as chloramphenicol transacetylase (Wright et al., 1986) and 15% as active bovine growth hormone (George et al., 1985). The production level observed in our A expression system compares well with those of the plasmid systems above. It should be noted, however, that expression of the lacZ gene in our case was under the control of the much weaker lac promoter. The product yields from our X system may be further enhanced by using a stronger promoter for transcription of the cloned gene and by optimization of the X vector to minimize production of the nonessential head and tail proteins in the abortive lytic state. The high productivity in JM109-NM1070 may be also due to other aspects of the lytic state that are advantageous for overproduction of cloned gene product. The interactions between phage and host include association of phage proteins with host genes or proteins. If such interactions result in the cellular biosynthetic capacity being directed largely to the expression of the cloned gene product, then A-based vector systems have the potential for extremely high, controlled gene expression and excellent segregational stability. A defective phage system such as JM109-NM1070 offers an excellent vehicle for study of such interactions by monitoring the expression of a marker gene carried on the phage.
Conclusions The X expression systems studied here have produced two main findings. First, from the evaluation of segregational stability, the product gene carried on the X vector is 100% stable in batch culture for at least 75 generations in lysogeny. Second, the switch to an abortive lytic state, in which both packaging of X DNA and cell lysis are prevented, can be used to achieve a very high protein productivity. Although our study has been restricted to the E . coli protein P-galactosidase, this product gene may be easily replaced by another product gene. The use of specialized X vectors for expression shows strong promise for large-scale production of recombinant proteins.
Acknowledgment We thank Professor T. R. Klaenhammer for providing the strains Xgtll, Y1089, and Y1090 and for his technical assistance in this project. We also thank Professor Noreen Murray for donating the strain NM1070, Robert Kuhn for providing the strain BK6, and Tom Wood for providing the plasmid pTKWlO6.
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