Bbtechnol. R o ~1993, . 9, 31-39
51
Effects of Plasmid Copy Number and Runaway Plasmid Replication on Overproduction and Excretion of @-Lactamasefrom Escherichia coli A. Paul Togna,?Michael L. Shuler,' and David B. Wilson$ School of Chemical Engineering and Section of Biochemistry, Molecular and Cell Biology, Cornel1 University, Ithaca, New York 14853
Runaway plasmid replication can be used to increase target gene dosage and thereby overproduce proteins within the bacterium Escherichia coli. However, the presence of excessive plasmid DNA often alters normal cell functions. High copy number plasmids with strong promoters place a severe metabolic burden on the cell, causing a decreased specific growth rate and changes in cell physiology. Induction of j3-lactamase synthesis from the tac promoter on plasmid pKN causes runaway plasmid replication and excretion of j3-lactamase. Runaway plasmid relication results from readthrough of tac promoter transcript9 into the replication region of the plasmid. Both high plasmid copy numbers and a strong promoter (tac) are necessary t o achieve the level of overproduction necessary for excretion of j3-lactamase, but high-level target protein synthesis is detrimental to the cell. A derivative of pKN which is more easily regulated was constructed by adding the lac1 gene t o the plasmid.
Introduction In order to maintain plasmids within bacterial cells, plasmid DNA replication must occur one or more times during the bacterial division cycle. The replication mechanisms of many plasmids, including ColE1, R1, and pSC101, have been elucidated (1, 2). The plasmid replication region contains information that plays a role in maintaining a stable and characteristic plasmid copy number within a cell. Plasmids have been isolated containing mutations within their replication regions that cause an increase in plasmid copy number (3-6).In some cases, these mutations are conditional and only cause an increase in copy number when the temperature is raised above 40 "C (5,6).These plasmids can be used as inducible cloning vectors to produce large quantities of target protein (7-9). Remaut et al. (8)were able to produce high levels of T4 DNA ligase within Escherichia coli from a temperature-dependent runaway replication mutant of plasmid R1 containing the T4 DNA ligase gene under the control of the XPL promoter. During the 3-h induction period a t 42 "C, T4 DNA ligase protein synthesisamounted to roughly 40% of net protein synthesis (8). Runaway plasmid replication and the associated highlevel plasmid-encoded protein production place a severe metabolic burden on the cell, resulting in a decreased growth rate and, in some cases, cell death (5,8,10).This is clearly not attractive for long-term, continuous target protein production. The problems of plasmid instability and decreased productivity at high plasmid copy numbers have prompted detailed investigations into host-plasmid interactions (11-14). The plasmid pKN used in this investigation is a derivative of pBR322 which contains a ColEl-type replicon (15).The replication mechanism of ColEl-type plasmids has been extensively studied (I), and the interactions within E. coli regulating the ColEl replicon have been mathematically modeled (16-18). Mutations within RNA I or RNA I1 that alter the t
Present Address: Envirogen, Inc., Lawrenceville, NJ 08648. Section of Biochemistry, Molecular and Cell Biology. 8756-7938/93/3009-003 1$04.00/0
secondary structure of these transcripts can cause an increase in plasmid copy number (4,6).The copy number can also be increased by inserting strong promoters, such as the lacUV5 (19,20) or XPL (21) promoters, upstream from the native RNA I1 promoter. Transcription readthrough into the replication region causes high levels of RNA I1 to be synthesized and results in an increased plasmid copy number. Larsen et al. (22)used a similar approach to induce runaway replication of an R1 derivative. The XPR promoter was inserted upstream from the repA gene product, the rate-limiting plasmid initiator protein of R1. Stueber and Bujard (23)demonstrated that, when the strong coliphage T5 promoter was inserted downstream from the origin of replication in pBR322, transcription readthrough in the direction of RNA I caused a decreasein copy number. This decreasecan be attributed to higher production of Rom protein and increased numbers of RNA I transcripts. Panayotatos (24)constructed a derivative of ColEl with the lacUV5 promoter and the gene for human interferon a2 inserted upstream from the RNA I1 promoter. With this construction, the same overproduced transcript can serve as a template for translation of human interferon a2 and as a primer precursor for initiation of plasmid replication (24). Within E. coli RB791, this plasmid is maintained a t 1-5 copies per cell when lactose is not included in the growth medium. But when cells are induced with lactose, the copy number increases to approximately 600 copies per cell, and human interferon a2 accumulates to 10-20% of the total cell protein. More recently, Chew and Tacon (25)constructed a plasmid with the pBR322 origin of replication region immediately downstream of an anthranilate synthase-human epidermal growth factor fusion gene (trpE-hEGF), both under common trp promoter control such that the primer of replication is cotranscribed with the trpE-hEGF gene. Upon induction, the copy number increases up to 6-fold, and the TrpE-hEGF fusion protein accumulates to approximately 12% of the total cell protein (25). We have previously developed an inducibleE. coli hoetplasmid expression system for j3-lactamase [RB791-
0 1993 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1993, Vol. 9, No. 1
32
Table 11. Description of Plasmids
Table I. Escherichia coli Strains ~
strain RB791 W3110 JM109
DH5a KL16-21-23
description F,A-, lacIqL8
F,x-
recA, A(lac, pro), endAl, gyrA96, thi-1, hsdR17, supE44, relAl, F'(traA36, prom+, lacIq, zAM15) supE44, AlacU169(~801acZAM15), hsdR17, recAl, endAl, gyrA96, thi-1, relAl A-, A(ptsM-kdgR)82,ptsF3, relAl, spoT1, thi-1, Hfr
ref@) 29-31 29 32
33 34,35
(ptacll)]in which selective excretion of the target protein from the periplasm is observed without cell lysis (26,273. It has been shown by Georgiou et al. (27) that excretion of p-lactamase from RB79l(ptacll) cells is closely linked to high-level expression of the target protein and is not accompanied by release of cytoplasmic proteins. The pKN plasmid used in this investigation is a derivative of the ptacll plasmid used by Georgiou et al. (28). In this article, it is shown that the copy number of pKN increases dramatically after induction of cells containing this plasmid and that runaway replication of pKN is caused by transcription readthrough into the replication region of the plasmid. More importantly, it is shown that both a high plasmid copy number and a strong 8-lactamase promoter (tac) are required to achieve the level of overproduction necessary for excretion of plasmid-encoded 6-lactamase from E. coli cells.
Materials and Methods Bacterial Strains and Plasmids. The E.coli strains used in this investigation are listed in Table I and the plasmids in Table 11. All constructed plasmids are derivatives of pKN (28),which contains the tac promoter inserted upstream of the 8-lactamase gene. j3-Lactamase production from pKN can be induced by adding isopropyl 6-D-thiogalactopyranoside(IPTG) to the medium. Plasmid pKN also confers host resistance to the antibiotic neomycin. Plasmid pDW17, isolated by Georgiou et al. (27), is a down-promoter tac mutant of plasmid ptacll. Plasmid ptacll is the parent plasmid of pKN (28) and does not contain a gene for neomycin resistance. All enzymes and buffers were purchased from New England Biolabs, Inc. (Beverly, MA), Boehringer Mannheim Biochemicals (Indianapolis, IN), or Amersham Corp. (Arlington Heights, IL) and were used according to the manufacturers' instructions. Large quantities of plasmid DNA for ligation were prepared by growing cells in 1L of LB medium at 37 OC and amplifying the plasmid by adding 170 pg/mL of chloramphenicol (Sigma Chemical Co., St. Louis, MO). Plasmid was isolated using the alkaline lysis method and purified by CsCl ethidium bromide density gradient centrifugation. DNA fragments used for ligation were isolated from agarose gels by electroelution. Plasmid DNA was isolated for restriction analysis using the alkaline lysis miniprep procedure. All strains except for KL1621-23[pPC130] were transformed by the standard calcium chloride method. KL16-21-23[pPC130] was transformed by electroporation (39). Detailed descriptions of the recombinant DNA techniques can be found in Maniatis et al. (40) and Sambrook et al. (33). Medium and Growth Conditions. Cells were grown at 20 "C in Tanaka medium (41), pH 7.2, supplemented with 0.2 % casein amino acids, acid hydrolysate (Sigma), 0.2% glucose (Sigma or Mallinckrodt, Inc., Paris, KY), and 50 pg/mL of neomycin sulfate (Sigma) for cells
Dlasmid PKN pDW17 pKK62B-7 pKNter pYM058 pKNI pPC130
descrbtion Neo' (APH(3')-Zo,tacAmp' tacdownAmpr source of rrnBTlT2 Neo' (APH(S')-ZZ),tacAmpr,rrnBTIT2 source of lac1 Neo' (APH(3')-ZZ),tacAmp', lac1 derivative of pMMB22 with Gentr inserted between PstI sites, lacIq
refW 28 27 36 a
37 a
38
This work.
containing pKN-related plasmids or 50 pg/mL of ampicillin (Sigma) for cells containing pDW17. At temperatures greater than 20 OC, overproduction of 8-lactamase causes inclusion body formation (28). B-Lactamase synthesis was induced by adding IPTG (Sigma or Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) to the medium. Assays and Cell Counting Techniques. Assays for 8-lactamase activity were performed in 50 mM phosphate buffer at pH 7.0 using 0.5 mg/mL penicillin G (Sigma) as substrate and monitoring the rate of decreasein absorbance at 240 nm (42) with a Beckman ACTA MVI Spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). A unit of activity was defined as 1 pmol of penicillin G degraded per minute at 25 OC using an extinction coefficient of 0.57 A240 unit per pmol/mL of penicillin G. The specificactivity of p-lactamase under these conditions is 3550 units/mg (27). Total 8-lactamaseactivity (internal and external)was determined by rupturing cells in a Carver Model C laboratory press (Fred S. Carver Inc., Menomonee Falls, WI) at 20 000 psi and separating the soluble lysate from the cell debris by centrifugation at 13600g for 5 min in a Fisher Model 235C microcentrifuge (Fisher Scientific, Pittsburgh, PA). The culture supernatant was assayed directly for external 8-lactamase after removal of cells by centrifugation at 13600g for 5 min. Total soluble protein of cell extracts was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA) with bovine serum albumin as the standard. For viable cell counts, each dilution was plated on three nonselective plates and on three selective plates. Plates were incubated at 37 OC for 16-24 h. Only those plates which contained between 30 and 300 colonies were counted. Nonselective plates contained LB medium (10 g/L Bactotryptone, 5 g/L Bacto-yeast extract, and 10 g/L NaC1) with 1.5% Bacto-agar (Bacto-products from Difco Laboratories, Detroit, MI). Selective plates were supplemented with 50 pg/mL of neomycin sulfate (Sigma) when plating cells containing pKN-related plasmids or 50 pg/ mL of ampicillin (Sigma) when plating cells containing pDW17. A Coulter Counter Model ZB (Coulter Electronics, Inc., Hialeah, FL) was used to determine total cell counts Cells were diluted in Isoton I1 (Coulter Diagnostics, Hialeah, FL) before they were counted. Plasmid Isolationfor Copy Number Determination. Plasmid DNA for copy number determination was isolated essentially by the alkaline lysis method of Birnboim and Doly (40). Purified plasmid DNA was cut with EcoRI and loaded onto either a 0.6% or 0.8% agarose gel containing 0.5 pg/mL of ethidium bromide (Sigma). Known weight standards of HindIII/EcoRI A-DNA (International Biotechnologies, Inc., New Haven, CT) were also loaded. Gels were run at 30 V in TBE buffer containing 0.5 pg/mL of ethidium bromide overnight and photographed on a UV transilluminator with type 55 Polaroid film (positive/negative) through a yellow filter. The intensities of bands on the negative were analyzed
Bbtechnol. Prog., 1993, Vol. 9, No. 1
EcoRl0.00
XmnI 3.40r
Figure 1. Partial restriction map of pKN.
with either a Bio-Rad 620 video densitometer (Bio-Rad Laboratories, Richmond, CA) or a Quick Scan flur-vis densitometer (Helena Laboratories, Beaumont, TX). Plasmid copy numbers (plasmids/cell) were determined by dividing the number of plasmid molecules per milliliter of culture by the total number of plasmid-containing cells per milliliter of culture, determined by subtracting the plasmid-free cell number from the total number of cells (Coulter counted cells). It was assumed that nonviable cells contained plasmid and that plasmid recovery was complete. Comparison of Induced and Uninduced RB791(pKN). Cells stored at -70 "C were transferred to either a New Brunswick Model F-2000 MultiGen fermenter (working volume = 1.5 L) or a New Brunswick Model C30 BioFlow fermenter (working volume = 350 mL) (New Brunswick Scientific Co., Inc., Edison, NJ). Temperature was maintained at 20 f 1 "C. The pH was maintained at 7.2 f 0.1 by the controlled addition of 0.25 N HC1 or 2 N NaOH (Chemtrix Type 45A pH controller, Whatman Labsales, Inc., Hillsboro, OR). For the induced cultures, 1X 10" M IPTG was added when the cell density reached -5 X lo8 cells/mL. Periodically, 1.5 mL of culture was harvested for sampling and centrifuged at 13600g for 5 min. Cell pellets were saved frozen at -70 "C for plasmid copy number determinations. Comparison of Different Plasmid Constructions. Cells stored at -70 "C were transferred to either Bellco 50-mL (containing 10 mL of medium) or 250-mL (containing 50 mL of medium) triple baffled shake flasks (Bellco Glass Inc., Vineland, NJ). The shake flasks were agitated at 3W350 rpm in a New Brunswick Model G24 environmental incubator shaker (New Brunswick Scientific Co.) at 20 f 1OC. Cultures were induced with IPTG at different cell densities. At the end of growth, appropriate sampleswere frozen at -20 "C for the determination of total and external j3-lactamase, and cells were plated and counted with the Coulter Counter. Aliquots (1.5 mL) of each culture were centrifuged at 13600g for 5 min, and cell pellets were stored at -70 O C for copy number determinations. Plasmid Constructions. Construction of pKNter. To determine whether readthrough of j3-lactamase transcripts into the replication region of pKN was responsible for the observed runaway replication, the strong rrnBTrT2 transcription terminators were inserted between the end of the j3-lactamase gene and the replication region. These terminators are effective in preventing readthrough of m-RNA transcripts originating from the tac promoter (43). Figure 1shows that two DraI sites are conveniently located within pKN between the end of the j3-lactamase gene and the replication region. These sites are located 110 and
-
-40 bp away from the RNA I1 promoter and the 8-lactamase gene, respectively (15, 44,45). Therefore, insertion of DNA fragments into these sites does not interfere with transcription of either RNA I1or @-lactamase m-RNA. The rrnBTlT2 terminators were obtained from plasmid pKK62b-7 (361,purchased from Pharmacia. The T1T2 terminators are contained within a 0.5-kbp EcoRI fragmentof this plasmid. This fragmentwas electroeluted from a 1%agarose gel, and the ends were filled in with Klenow polymerase. pKN plasmid DNA was partially digested with DraI, and the 5.7-kbp fragments were electroeluted from a 1% agarose gel. The ends were dephosphorylated by treatment with alkaline phosphatase, and the pKN fragments and the rrnBT1Tz fragment were ligated. The ligation mixturewas used to transformKL1621-23[pPC130]. Selection was made on LB plates containing 100 rg/mL ampicillin (Sigma). In this way, only plasmids containing rrnBTlT2 inserts into the DraI site( 8 ) located after the j3-lactamase gene were selected. Plasmid DNA was isolated from a number of colonies for restriction enzyme analysis. An XmnI site located at one end of the rrnBTlT2 fragment (46)was used to determine the orientation of the insert. Two plasmids, designated pKNter and pKNter-, were isolated for further study with the rrnBT1Tz fragment inserted in the same orientation as is found within the rrnB operon and in the opposite orientation, respectively. In both of these plasmids, the two DraI sites after the j3-lactamase gene were missing. Construction of pKNI. The lac1 gene was added to pKN to create the pKNI plasmid. This plasmid was constructed with the expectation of obtaining a controllable copy number derivative of pKN. The lac1 gene was obtained from plasmid pYM058, kindly provided by Dr. Masayori K. Inouye (37). The lac1gene is contained within a 2.4-kbp EcoRI fragment. This fragment was isolated from a 1%agarose gel by electroelution, and the ends were filled in with Klenow polymerase. pKN plasmid DNA was digested with HindIII, and the ends were filled in with Klenow polymerase and dephosphorylatedby treatment with alkaline phosphatase. The DNA fragments were ligated, and the ligation mixture was used to transform DH5a. Selection was made on LB plates containing 50 pg/mL neomycin sulfate (Sigma). Since DH5a is not a lacIq strain, only plasmids with the lac1 insert were selected. Previous studies by Georgiou et al. (27)have demonstrated that hosts without the lacIq mutation cannot be transformed with pKN. Plasmid DNA was isolated from a number of colonies for restriction enzyme analysis. An HpaI site located asymmetrically within the lac1 gene fragment (37) was used to determine the orientation of the insert (HpaIIBglII digestion). Plasmid pKNI, which contains the lac1 gene oriented so that it is transcribed in the opposite direction as the j3-lactamase gene, was chosen for further study.
Results Response of RB791(pKN) to IPTG Induction. Figure 2 is an agarose gel of plasmid taken from uninduced and induced RB79l(pKN) cells in batch culture. It can be seen from Figure 2 that plasmid DNA within induced cells increases dramatically after IPTG addition. Runaway replication of plasmid pKN within another lacIq strain of E. coli, JM109, was also observed, but the rate of plasmid increase after induction was approximatelyone-half that for RB79l(pKN) (data not shown). Figure 3 shows the total and external j3-lactamase specific activities in shake flask cultures of RB79l(pKN) as a function of the cell number at induction as measured
Bbtechrwl. Rug., 1993, Vd. 9, No. 1
34
Induced
Uninduced
A B C D
A B D
10,
i
+
9 l
0
h
1
+
PKN 106
1
1b 7
- . . ..... 1b8
- . . .....
. . . ..
1b9
CellslmL at Induction
Figure 4. Final total,viable, and viable plasmid-containing cell numbers in shake flask cultures of RB79l(pKN). Cells were induced with lo-’ M IPTG. 0 = Coulter Counter. A = selective plates (antibiotic resistance). + = nonselective plates.
RNA
Figure 2. Agarose gel electrophoresis of plasmid taken from uninduced and induced RB791 (pKN). Equal volumes were loaded. The two cultures were grown in parallel. One culture was induced a t -17 h (-5 X loHcells/mL) with M IPTG. Samples were removed a t A = -17 h, B = -23 h, C = -30 h, and D = -42 h. *0°
m 600
O0 0
2 a
400
1 I
0 0
0
440
I
CellslmL at Induction
Figure 3. Total and external & l a c b a s e specific activity in shake flask cultures of RB791(pKN). Cells were induced with M IPTG. 0 = total 8-lactamase. A = external 6-lactamase. Numbers refer to final plasmid copy numbers.
with a Coulter Counter. Also shown in Figure 3 are the final plasmid copy numbers. Figure 4 shows the final cell counts. Three types of cell counts were measured: (1)the absolute cell count, measured with a Coulter Counter; (2) the viable nonselective plate count; and (3) the selective plate count, yielding an estimate of the number of viable plasmid-bearing cells. From Figures 3 and 4, it can be seen that RB79l(pKN) cells producing high levels of 8-lactamase lose viability and that by the end of growth plasmid-freeRB791 cells dominate batch cultures induced at cell densities between 1 X lo8 and 5 X lo8 cells/mL. RB79l(pKN) cells are apparently capable of degrading most of the neomycin in the medium by the time the cell density has reached 1 X 108 cells/mL. Plasmid instability only partially accounts for the low final levels of 8-lactamase observed when shake flask cultures are induced a t cell densities lower than 1 X lo8 cells/mL. Cultures induced a t inoculation (- 1x 107ce11s/ mL) produce low levels of 8-lactamase even though plasmid instability is not observed (see Figures 3 and 4). Induction
of shake flask cultures a t low cell densities select13 for a subpopulation of variant cells capable of forming colonies M IPTG on selective plates supplemented with 3 x (seeFigure 5). Unaltered RB79l(pKN) cellsare incapable of forming visible colonieson plates containing IPTG due to high-level 8-lactamase production and decreased viability (47). The variant cells produce approximately 75 % less 8-lactamase than unaltered cellsand have been shown through [&met/~yZ-~~C J thiogalactopyranoside([I4C]TMG) uptake experimentsto be ZQC permease mutants of RB791(pKN) (48). Induction of RB79l(pKN) lac permease M IPTG does not cause immediate mutants with runaway plasmid replication. The mutants therefore have a selective growth advantage over unaltered cells and quickly displace the unaltered cell population after induction during continuous culture (48). As seen in Figure 5, because of the low viability of the unaltered cell population after induction, the lac permease mutantstake over batch cultures if IPTG is added early. Runaway Plasmid Replication and Excretion. The copy number of pKN increases dramatically after induction of E. coli cells containing this plasmid. Within E. coli strain RB791, pKN increases to a level of -6600 plasmids per cell from a basal level of 10-40 plasmids per cell before induction (see Figures 2 and 3). To determine whether transcription read through into the replication region of pKN is responsible for this observed increase in plasmid copy number, the plasmid pKNter was constructed (see the plasmid constructions section). This plasmid contains the strong r r n B T T ~2 transcription terminators inserted between the 8-lactamase gene and the replication region of pKN (see Figure 1). Figure 6 shows the final 8-lactamase specific activity and plasmid copy number of RB79l(pKNter) cultures as a function of cell number a t induction as measured with a Coulter Counter. Figure 7 shows the final cell counts. Cells were induced with lo4 M IPTG, a level of IPTG sufficient to cause runaway replication of plasmid pKN. The copy number of plasmid pKNter increases approximately 6-fold after addition of IPTG, compared to the 25-fold increase in copy number of plasmid pKN. The transcription terminators not only prevent severerunaway plasmid replication but also, as a consequence, prevent severe cell death and plasmid instability (compare Figure 7 with Figure 4). Interestingly, the terminators appear to function just as well, if not better, in the opposite orientation (data not shown). I t has been postulated that therrnBT1terminator can function bidirectionally, since the G/C-rich stem structure, characteristic of all rho-independent transcription terminators, can be extended with A/U pairing (49). An rho-independent transcription terminator with ex-
Biotechnol. hog... 1993, Vol. 9, No. 1
35
0
f 0
O
0
I
d
0
RB791 (pKNter) 102
108
20
0
40
60
10
80
Hours After Inoculation
Cells/mL at Induction
Figure 5. Selection of variant RB79l(pKN)subpopulation (lac
permease mutants) in batch culture. Cells were induced at inoculation with lo4 M IF'TG. 0 = Coulter Counter. h = selective plates (antibiotic resistance). 0 = selective plates (antibiotic resistance) supplemented with 3 X 10"' M IPTG. ann
RB791 (pKNter)
12
4001
,"'
50
'
l
O
d
-
6 00
._ E
40
Y
0
c
E
°
I
500
c
1
300
138791 (pDW17) 106
0
1
CellsimL at Induction
Figure 7. Final total,viable, and viable plasmid-containing cell numbers in shake flask cultures of RB79l(pKNter)and RB791(pDW17). Cells were induced with 10"' M IPTG. 0 = Coulter Counter. A = selective plates (antibiotic resistance). + = nonelective plates.
CellsimL at Induction
-
.f
e
c
-m L
F
y
-
600
RB791(pDW17) 500: F o o 2 1 0 2 2 0 0
300 -
0
400
200
140 0
90
-
O
8
.-C
60
I
3
1000
0
A
.A
.be...
.
. .*.ol
a
040
- .....~1 0
Figure 6. Final plasmid copy number and j3-lactamasespecific activity in shake flask cultures of RB79l(pKNter)and RB791(pDW17). Cells were induced with lo4 M IPTG. 0 = total j3-lactamase. A = external 8-lactamase. Numbers refer to final
plasmid copy numbers.
tended A/U pairing has been shown by Postle and Good (50) to function in either direction, both in vivo and in vitro. RB791(pKN) cells induced in shake flasks a t -2 X lo8 cells/mL excrete about 5040% of the 8-lactamase they produce (see Figure 3). In contrast, RB79l(pKNter) cells induced a t -2 X lo8 cells/mL only excrete about 5% of their j3-lactamase (see Figure 6). The total level of j3-lactamase produced in both cases is approximately 400 unita/mg of total protein, but when the presence of plasmid-free cells is taken into account, RB79l(pKN) cells induced at 2 X lo8cells/mL produce about 1.5-fold more 8-lactamase than RB79l(pKNter) cells induced at the same cell density, assuming that nonviable cells contain plasmid. RB79UpKNter) cells will excrete a maximum of about 20% of the 8-lactamase they produce in batch
-
culture if induced a t inoculation (-2 X lo7 cells/mL), which is still 3-fold lower than the 50430% excretion of 8-lactamase from induced RB79l(pKN) cells (see Figure 3). An increase in the number of tac promoters after induction, from a basal level of 10-40 promoters (plasmids) per cell before induction, is required to attain the level of 8-lactamase production necessary for excretion. The maximumdifferential rate (Aunita/A(mgof total protein) of 8-lactamase production from RB79l(pKN) cells (-600 plasmids per cell) in batch culture is -800 Aunits/A(mg of total protein), approximately 22% of total protein production, while the maximum differential rate of 8-lactamase production from RB79l(pKNter) cells (-60 plasmids per cell) in batch culture is -400 Aunits/A(mg of total protein), approximately 11%of total protein production. The plasmid pDW17 is a down-promoter mutant of plasmid ptacll (27). This plasmid contains a mutation within the -35 region of the tac promoter that results in a 4-fold decrease in production of 8-lactamase activity at 37 "C (27). Figure 6 shows the final 8-lactamase specific activity and plasmid copy number in batch cultures of RB791(pDW17) at 20 OC as a function of the cell density at the time of induction as measured with a Coulter Counter. Induced cellscontaining plasmid pDW17 behave almost identically to induced cells containing plasmid pKNter. The plasmid copy number of pDW17 increases approximately 6-fold after induction to a level of -250 plasmids per cell from a basal level of -40 plasmids per cell before induction, but the lower rate of transcription initiation from the tacdoWn promoter compared to the tac promoter prevents severe runaway plasmid replication. As a consequence, severe cell death and plasmid instability are also prevented, as shown in Figure 7. Note that the rate of 8-lactamaseproduction from RB791(pDW17)cells,
Blotechnol. Prog., 1993, Vol. 9, No. 1
36
I I
A
n
O I . , 0.0
0.5
.
I
1.0
I
,
1.5
I
I
.
2.0
I
2.5
0 .E
I
E 10'1
10
.
3.0
IPTG (moles/L) x 10,000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
IPTG (moles/L) x 10,000
Figure 8. Total 0-lactamase specific activity in shake flask cultures of RB79l(pKN) and RB79l(pKNI). Cultures were induced at cell densities between 5 X 108 and 1 X logcells/mL. 0 = RB79l(pKN). A = RB79l(pKNI).
Figure 9. Final plasmid content within shake flask cultures of RB79l(pKN)and RB79l(pKNI). Cultures were induced at cell densities between 5 X 10s and 1 X lo@cells/mL. 0 = RB791(pKN). A = RB79UpKNI).
even with 250 plasmids per cell, is insufficient to cause excretion of 8-lactamase from this strain (see Figure 6). Georgiou et al. (27)observed this same phenomenon at 37 "C. Therefore, both a strong promoter (tac) and a large number of plasmid molecules per cell are necessary for leakiness. Copy Number Control. As shown above, an increase in the copy number of pKN after induction is required for excretion of &lactamase from E. coli RB79l(pKN) cells. However, if the plasmid copy number becomes too high, excreting cells are quickly outgrown by plasmid-free cells or low-producing, nonexcreting lac permease mutants (see Figures 4 and 5 and ref 46). Unfortunately, the plasmid copy number of pKN cannot be easily controlled by adjusting the amount of IPTG added to the culture medium. IPTG controls the rate of transcription initiation at the tac promoter, and therefore the rate of plasmid replication after induction, by binding the lac repressor and preventing the lac repressor from binding to the tac promoter. It is possible to regulate the degree of IPTG binding to the lac repressor by regulating the intracellular concentration of IPTG, but once the plasmid copy number increases beyond some threshold level, the lac repressor is titrated out and the plasmid copy number increases in an uncontrolled manner. Induction of pKN is therefore analogous to an on/off switch. The copy number of pKN can only be controlled if the intracellular concentration of the lac repressor increases along with plasmid copy number. The plasmid pKNI was developed as a controllable copy number derivative of pKN. This plasmid contains the lac repressor gene (lad) inserted at the Hind111 site of pKN (see the plasmid constructions section). The behavior of plasmids pKN and pKNI within strain RB791 are compared in Figures 8 and 9. Figure 8 shows the final j3-lactamase specific activity in batch culture as a function of the amount of added IPTG. Figure 9 shows the final plasmid content as a function of added IPTG. Cultures were induced a t cell densities between 7 X lo8 and 1 X lo9 cells/mL, conditions leading to high 8-lactamase production and low plasmid instability within induced batch cultures of RB79l(pKN). These figures show that pKNI is a more controllable plasmid than pKN, but the extent of control is limited. Figure 9 shows that runaway replication of plasmid pKN occurs when cells are induced with IPTG concentrations greater than 3 X 10-6 M, while the copy number of pKNI can be regulated within the range 3 X to 7 X M IPTG. In the experiments described above, the cells were able to grow only approximately 2-3 doubling5 in the induced state before glucose was depleted. There is likely to be
some lag between the time when the plasmid copy number increases and the lac repressor is expressed from the plasmid. Uhlin et al. (IO) have mathematically shown that during runaway plasmid replication, the amplification of the plasmid is not accompanied by proportional amplification of the cloned gene product, even without transcriptional and translational limitations. The lac repressor will be proportional to the plasmid copy number only during balanced growth. Therefore, the full negative feedback effects of the pKNI plasmid are likely to be seen only in continuous culture. In fact, it is possible that the results shown in Figures 8 and 9 merely reflect differences in initial lac repressor levels. Figure 10 shows the final plasmid copy number and 8-lactamase specific activity in batch cultures of RB791(pKNI) as a function of both the cell number a t the time of induction and the level of added IPTG. When RB791(pKNI) cells are partially induced with 5 X M IPTG, the plasmid copy number of pKNI appears to be regulated at -350 plasmids per cell, and the cells excrete about 50 % of the 8-lactamase they produce (see Figure 10).When cells are fully induced with higher levels of IPTG (1X 10-4 or 2 X M IPTG), they behave essentially like fully induced cells containing the parent plasmid pKN (see Figure 3). The final pKNI copy numbers within cultures producing the highest levels of 8-lactamase are -600 plasmids per cell, and lac permease mutants of RB791(pKNI) take over cultures induced at inoculation, similar to the behavior of RB791 cells containing plasmid pKN (data not shown).
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Discussion It has been shown by Georgiou et al. (27)that excretion of @-lactamasefrom E. coli RB791 cells containing pKN or related plasmids is not accompanied by release of cytoplasmic proteins and is therefore not caused by cell lysis. This was confirmed in these investigations by comparing the release of 8-lactamase activity with the release of &galactosidase (a cytoplasmic protein) activity from cells induced at -108 cells/mL in batch culture. External 8-lactamase activity amounted to roughly 40% of the total 8-lactamase activity, while external 8-galactosidase activity accounted for only 5% of the total &galactosidase activity (data not shown). The copy number of pKN increases dramatically after induction (see Figure 2), and the simultaneous copy number increase and induction of target protein synthesis result in high-level production and excretion of the target protein, p-lactamase (seeFigure 3). By inserting the strong rmBT1T2 transcription terminators directly after the
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1000
360
e
c
P
600
c
.
5
400
e
200
:
01 o 7
CellsimL 108 at Induction 109
10'0
Figure 10. Final plasmid copy number and j3-lactamasespecific activity in shake flask cultures of RB79l(pKNI) induced with 5 X 10-5 M IPTG. 0 = total j3-lactamase. A = e x t e n d j3-lactamase. Numbers refer to final plasmid copy numbers. 8-lactamase gene, it was shown that readthrough of tac promoter transcripts into the replication region of pKN is responsible for the observed runaway replication after induction (see Figure 6). Transcripts originating from the tac promoter are apparently able to serve as both template for 8-lactamase translation and preprimer for plasmid replication. This is the simplest explanation for the observed behavior. Uhlin and Nordstrom (5)observed that when 8-lactamase, transcribed from its native promoter, was expressed with E. coli from a runaway replication mutant of plasmid R1, some of the 8-lactamase produced by the cells was released into the extracellular medium. Abrahmsh et al. (51) observed excretion of plasmid-encoded Staphylococcal protein A from E. coli when the gene for this protein was transcribed from a plasmid in the same direction as the plasmid preprimer. When the orientation of this gene was reversed, the expression level was reduced 20-fold and excretion was greatly reduced. However, the observed directional dependence in that case may not have been due to transcription readthrough since insertion of transcription terminators directly following the Staphylococcal protein A gene did not influence the orientation effect (511. More recently, Ryan and Parulekar (52,531 observed excretion of 8-lactamase from E. coli JM103 cells harboring the high copy number plasmid pUC8. Ryan and Parulekar observed that, within continuous culture, as the average plasmid copy number increased (with decreasing dilution rate), the percentage of 8-lactamaseexcreted also increased (52). Georgiou et al. (27)have developed a preliminary model to explain why overproduction of 8-lactamase from ptacll leads to excretion of periplasmic proteins from E. coli. They found that even though high-level 8-lactamase synthesis inhibits processing of its own precursor, it does not inhibit processing of the outer membrane proteins OmpA and OmpC. Instead, overproduction of 8-lactamase inhibits the synthesis of these two outer membrane proteins. On the basis of these and other results, Georgiou et al. (27) hypothesize that high-level synthesis of 8-lactamase interferes with the synthesis of cell envelope proteins, causing changes in outer membrane structure which ultimately affect outer membrane permeability. According to their model, active cell growth is necessary for excretion. The results reported in this article are consistent with this model. The increase in plasmid copy number after induction places a severe metabolic burden on the cell as plasmids compete with the host cell chromosome for the protein synthetic machinery of the cell. This competition interferes with the synthesis of normal levels of outer membrane proteins. Full induction
of -60 tac promoters per cell is not sufficient to cause disruption of outer membrane structure (see Figure 6), while partial induction of -350 tac promoters is sufficient (seeFigure 10). Induced RB'lOl(pKNter1 cells containing -60 plasmids (-60 tac promoters) excrete only about 5-20% of the 8-lactamase they produce (see Figure 5), while induced RB79l(pKN) cells containing -600 plasmids (-600 tac promoters) excrete about 5 0 4 0 % of their 8-lactamase (see Figure 3). The 8-lactamase synthesis rate from cells containing -60 copies of pKNter is -400 Aunits/A(mg of total protein) (-11% of total protein production), while the synthesis rate from cells containing -600 copiesof pKN is 800 Aunits/ A(mg of total protein) (-22% of total protein production). The threshold level of j3-lactamase production for excretion therefore appears to be between 400 and 800 Aunits/A(mg of total protein) or 11 and 22% of total protein production. In glucoseminimal medium without added amino acids,fuUy induced RB79l(pKN) cells excrete 90-100% of the produced j3-lactamase (46). The model proposed by Ryan and Parulekar (53) for excretion of 8-lactamase from E. coli JM103(pUCS) cells is similar to the model proposed above for excretion of 8-lactamasefrom induced E. coli RB79l(pKN) cells. Ryan and Parulekar found that the presence of the high copy number plasmid pUC8 within JM103 cells affected the integrity of the outer cell membrane, causing leakage of 8-lactamase into the medium. The authors concluded that the increased permeability of the outer membrane was caused by the metabolic burden imposed on the host cell by plasmid-related activities. However, in their study, Ryan and Parulekar observed that nongrowing cells appeared to have less intact membranes than growing cells. They concluded that cell growth was not necessary for excretion of 8-lactamase and that growing cells were probably better able to repair their leaky outer membranes than nongrowing cells. The 8-lactamase within shake flask cultures of RB791(pKNter) and RB791(pDW17) reaches levels greater than 400 units per mg of total protein (11% of total protein) with minimal cell death (see Figures 6 and 71, and most of the j3-lactamase produced is not excreted. It may therefore be concluded that p-lactamase itself is not toxic to the cell and that the decrease in viability observed when RB79l(pKN) cells are induced with IPTG is due to the high rate of 8-lactamase production from these cells. The possibility of controlling the plasmid copy number at levels which will allow for excretion without cell death led to the development of plasmid pKN1. Plasmid pKNI, an 8.1-kbp controllable copy number derivative of pKN, was constructed by adding the lac1 gene to pKN at the Hind111 site. Cells containing the plasmid pKNI can be partially induced in batch culture with IPTG in the range 3X to 7 X 10" M (see Figures 8-10). The construction of plasmid pKNI also allows high-level 8-lactamase production and excretion to be studied in E. coli strains that do not contain lacIq. Within W3110, the parent strain of RB791, the plasmid pKNI was stably maintained, and WBllO(pKN1) cells produced over 1000 units of P-lactamase per mg of total protein in batch culture. It is possible that runaway replication alone, and not high-level 8-lactamase synthesis, may have been responsible for leakage of 8-lactamase in this investigation. This is difficult to determine from these experiments alone since the target protein, 8-lactamase,was always produced from the strong tac promoter in all experiments which resulted in excretion. There is evidence, however, that high-level protein synthesis, in general, causes periplasmic leakiness.
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Georgiou (47) has found that induction of the cytoplasmic C l phage X protein from the tac promoter causes leakage of j3-lactamase produced from ita native promoter and an overall reduction of outer membrane proteins. However, there is evidence to the contrary. Uhlin and Nordstrtim (5)observed that when j3-lactamase, transcribed from its native promoter, was expressed within E. coli from a runaway replication mutant of plasmid R1,some of the j3-lactamase produced by the cells was released into the extracellular medium.
Acknowledgment This work was supported, in part, by NSF Grant EET8513612.
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(47) Georgiou, G. Inducible overproduction and excretion of a periplasmic protein (8-lactamase) in Escherichia coli. Ph.D. Thesis, Cornell University, Ithaca, NY, 1987. (48) Fu, J.; Togna, A. P.; Shuler, M. L.; Wilson, D. B. Escherichia coli host cell modifications in continuous culture affecting heterologous protein overproduction: a population dynamics study. Biotechnol. B o g . 1992,8, 340-346. (49) Liebke, H.; Hatfull, G. The sequence of the distal end of the E . coli ribosomal RNA rrnE operon indicates conserved features are shared by rrn operons. Nucleic Acids Res. 1985, 13,5515-5525. (50) Postle, K.; Good, R. F. A bidirectional rho-independent transcription terminator between the E . coli tonB gene and an opposing gene. Cell 1985,41, 577-585. (51) Abrahmsbn, L.; Moks, T.; Nilsson, B.; U h l h , M. Secretion of heterologous gene products to the culture medium of Escherichia coli. Nucleic Acids Res. 1986, 18, 7487-7500. (52) Ryan, W.; Parulekar, S. J. Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboringa high copy number plasmid. Biotechnol. Bioeng. 1991,37, 415-429. (53) Ryan, W.; Parulekar, S. J. Recombinant protein excretion in Escherichia coli JM103(pUC8): Effects of plasmid content, ethylenediaminetetraacetate, and phenethyl alcohol on cell membrane permeability. Biotechnol. Bioeng. 1991,37,430444. Accepted October 12, 1992.
