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Hyperproduction of aspartase of Escherichia coli K-12 by the use of a runaway plasmid vector
Journal
of Biorechnology,
6
31
(1987) 31-40
Elsevier JBT 00255
Hyperproduction of aspartase of Escherichia coli K-12 by the use of a runaway plasmid vector Noriyuki Research
Luboraroty
Nishimura, o/Applied
Saburo Komatsubara, Tomoyasu and Masahiko Kisumi Biochetnisrty,
Tanabe
Sei.vaku
Co. Ltd.
Taniguchi
Yodogawa-ku,
Osaka,
Japan
(Received 5 February 1987; accepted 12 March 1987)
Plasmid pYT125 bearing the aspartase gene (aspA) of Escherichia coli K-12 was constructed by the use of pSY343, runaway-replication plasmid vector. One of the transformants, strain MM294 (pYT125)S maintained pYT125 stably at 37’ C in a non-selective medium. This strain produced approx. 60-fold more aspartase than did the control strain after 30 cell generations. Polypeptide of the aspA product amounted to 25-30% of the total cellular protein. Plasmid DNA isolated from strain MM294 (pYT125)-S was very similar to that from unstable strain MM294 (pY’T125) in length and restriction sites. The copy number was 83 in strain MM294 @YT125)-S and was 380 in strain MM294 (pYT125). Vector pSY343 was also stabilized in cured strains, isolated from MM294 (pYT125)-S, accompanied by reduction of the copy number. Accordingly, stabilization of pYT125 was considered to be closely related to decrease of the copy number, owing to genetic alteration of MM294-S. Aspartase; Escherichia coli; Runaway-replication-plasmid;
Plasmid stability
Introduction Aspartase (EC 4.3.1.1) catalyzes the reversible conversion of fumaric acid and NH: to L-aspartic acid. L-aspartic acid has been industrially produced from fumaric acid and NH: with Escherichia cob cells (Kinoshita et al., 1958; Kisumi et al., 1960; Kitahara et al., 1960; Chibata et al., 1974; Nishida et al., 1979). Correspondence lo: N. Nishimura, Research Laboratory Ltd., Yodogawa-ku, Osaka 532, Japan.
0168-1656/87/$03.50
of Applied Biochemistry, Tanabe Seiyaku Co.
0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
32
Recently, we have isolated E. coli B mutants with high aspartase activity by mutational and transductional methods (Nishimura and Kisumi, 1984). The aspartase gene (as@) of E. coli K-12 was cloned with low copy vector and inserted into multicopy vector pBR322 (Komatsubara et al., 1986). This aspA recombinant plasmid elevated the aspartase formation in E. coli K-12. Since this recombinant plasmid was lost from cells at high frequencies, we stabilized it by insertion of the partition locus (par). Uhlin et al. (1979) have isolated plasmids that can be amplified to approx. 2000 copies per cell upon heat induction at 37’C, whereas the copy number is relatively low when cells are grown at 30°C. Replication of these plasmids is controlled at above 35 o C due to a temperature-sensitive mutation in repressor coded on plasmid DNA, but strictly controlled by repressor at a low temperature. Such plasmids, called runaway-replication plasmids or, simply, runaway plasmids, are useful as vectors for the overproduction of gene products. Several derivatives of these plasmids have been reported to be applied to overproduction of /3-lactamase (Uhlin et al., 1979), DNA replication proteins (Yasuda and Takagi, 1983), chloramphenicol acetyl transferase (Sninsky et al., 1981) and fi-galactosidase (Bittner and Vapnek, 1981). This paper deals with application of a runaway plasmid vector to aspartase hyperproduction. Materials and Methods Bacterial strains and plasmids.
E. co/i K-12 strains and plasmids used are listed in
Table 1. Media. L broth (Lennox, 1955) was routinely used as a rich medium. ASP medium contained 4% corn steep liquor, 2% Meast (Ebiosu Yakuhin Co. Ltd., Tokyo, Japan), 1.14% fumaric acid, 0.5% ammonium fumarate, 0.2% K,HPO,, and 0.05% MgSO, .7H,O (pH 7.0).
TABLE
1
BACTERIAL
STRAINS
AND
PLASMIDS
USED.
Strain
Characteristic
E. co/i K-12 MM294 MM294S
thi ksdR thi hsdR
Plasmid pSY 343 pYT482-1 pNKIO1 pYT125
Km’ pBR322-aspA Apr pBR322-aspA-par Ap’ pSY343-aspA Km’
a PL’ = stable
maintenance
a
Source
Backmann Derivative this paper
PLs
of plasmids;
or reference
Ap’ = resistance
et al. (1976) from MM294,
Y asuda and Takagi (1983) Komatsubara et al. (1986) Nishimura et al. (in press) This paper to ampicillin;
Km’
= resistance
to kanamycin.
33
DNA manipulation. Plasmid DNA was prepared from cells grown in L broth containing an antibiotic by the cleared lysate method (Maniatis et al., 1982a) and further purified by ethidium bromide-cesium chloride centrifugation (Maniatis et al., 1982b). Digestion and ligation were as described previously (Komatsubara et al., 1986). Transformation.
E. co/i cells were transformed
by the method of Cohen et al.
(1972). Agarose gel eiectrophoresis. Agarose gel electrophoresis was run with 0.7% agarose gel in the same buffer as described by Maniatis et al. (1982~). Estimation of plasmid stability. E. coli cells harboring plasmids were cultured on L broth agar slant containing an antibiotic. Cells were suspended at 5 X 10s ml-’ and cultured with shaking’for 12-16 h. After 10 cell generations of growth, culture was diluted 1 : 100 with fresh medium and further cultured. Dilution and incubation were repeated to obtain the cultures of 30 cell generations. Aliquots of the culture of 10, 20 and 30 cell generations were diluted and spread on L broth agar plates. After overnight incubation, 100 colonies arising from each culture were tested for antibiotic resistance. Determination of plasmid copy number. Cells were grown with shaking in ASP medium at 37 o C. Exponentially growing cells, 5 x log, were collected by centrifugation, washed twice with 10 mM Tris-hydrochloride, 1 mM EDTA (pH 8.0). To obtain DNA preparation, cells were treated with 200 ~1 of lysating buffer (50 mM Tris-hydrochloride, 50 mM EDTA, 15% (w/v) sucrose, 2 mg lysozyme ml-’ and 50 pg RNAase ml-‘) at 37’C for 30 min and with 10 ~1 of 20% sodium dodecyl sulfate at 37O C for 2 h. The copy number was determined by fluorescence densitometry as described by Projan et al. (1983) using the DNA sample prepared as described above.
Aspartase was determined by the method described by Nishida et al. (1979). The reaction mixtures containing 1 M ammonium fumarate (pH 8.5) 1 mM MgCl, and cell extract were incubated at 37” C. L-Aspartic acid formed was determined by bioassay with Leuconostoc mesenteroides P-60. Aspartase assay.
Other methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out by the method of Laemmli (1970). Protein concentration was determined by the method of Lowry et al. (1951). Materials. Restriction endonucleases and T4 DNA ligase were purchased from Takara Shuzo Co. Ltd., Kyoto, Japan. Other chemicals were purchased from Katayama Kagaku Co. Ltd., Osaka, Japan.
34
Results Construction of the aspA recombinant plasmid bearing runaway plasmid vector pSY343
pYT482-1 is composed of a 4.3 kb BamHI DNA fragment carrying the aspA gene and a 4 kb BamHI fragment of pBR322. Runaway plasmid pSY343 is ca. 9.5 kb in length and has the gene coding for kanamycin resistance (Km’) and a BamHI site that can be used as a cloning site without inactivating the Km’ gene (Yasuda and Takagi, 1983). To allow E. coli to overproduce more aspartase, we constructed the aspA recombinant plasmid bearing pSY343 as follows (Fig. 1). DNAs of pYT482-1 and pSY343 were digested with BamHI and subjected to ligation for insertion of the 4.3 kb fragment carrying the aspA gene into the BamHI site of pSY343. Five of 31 transformants of strain MM294, selected for Km’, were sensitive to Ap and had high aspartase activities. These five transformants harbored ‘plasmid DNAs which were cut into 4.3 kb and 9.5 kb fragments with BamHI. The above data indicated that we could obtain the expected recombinant plasmid (pSY343-aspA). The representative plasmid was denoted pYT125.
pBR322-aspA
(Ap’)
E pSY
343-
aSPA
B (Km’)
Fig. 1. Diagrammatic representation of the construction of pYT125(pSY343-uq4). Plasmid pYT125 was constructed by inserting a 4.3 kb BomHI fragment carrying the uspA gene into pSY343 at BamHI site. The abbreviations used for the restriction sites are: B, BumHI; E, EcoRI; H,HindlII.
35
Aspartase formation and stability of pYT125 in transformants of strain MM294
Cells of MM294, which was of wild-type for the aspA gene, were transformed with plasmid DNA of pYT125 and transformants were selected for Kmr. Maintenance stability of pYT125 was investigated by culturing these transformants at 37 o C in ASP medium for approx. 10 cell generations (Table 2). One of these 10 strains maintained pYT125 very stably and produced 60 times more aspartase than did the host strain. This strain (T-7) was re-named MM294 (pYT125)S. Furthermore, the stability of pYT125 was compared with those of vector pSY343 and another stable recombinant plasmid pNK.101 (pBR322-aspA-par), which was not a runaway plasmid, by culturing cells harboring these plasmids for approx. 30 cell generations (Fig. 2). Plasmid pYT125 was lost at only 0.3% per cell generation, whereas pSY343 was at a frequency as high as 3.2%, as judged from ratio of the number of Km’ colonies to that of total colonies. The stable plasmid pNKlO1 was lost at 0.4% per cell generation. Strain MM294 (pYT125)S produced 65 times more aspartase than did the control strain MM294(pSY343), even after 30 cell generations. Plasmid DNAs of 20 Km’ colonies from such a culture of MM294(pYTl25)-S were tested by agarose gel electrophoresis and found to be almost similar to pYT125 DNA in length and restriction sites. This result indicated that no deletion plasmids were produced. Therefore, we concluded that pYT125 was very stably maintained in strain MM294(pYT125)-S after many cell generations. Estimation of the intracellular amount of the aspA product
Cell extracts were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to identify the aspA product (Fig. 3). Plasmid pYT125 enhanced the TABLE 2 ASPARTASE strain B MM294 T-l 2 3 4 5 6 I 8 9 10
FORMATION
AND STABILITY
Sp act of aspartase b 10 310 350 600 230 560 230 640 420 100 400
OF pYT125 IN TRANSFORMANTS
OF MM294.
Plasmid stability ’ (4;) 17 21 88 7 69 8 100 32 0 27
a T-l to T-10 are pYT125 transformants of MM294. T-7 was re-named MM294(pYT125)-S studies. Cells were grown in ASP medium. b Specific activity is expressed as pmol of L-aspartic acid formed per min mg- ’ of protein. ’ Plasmid stability is expressed as percentage of Km’ cells.
for further
10
20 Generations
30
10 of
20
30
growth
Fig. 2. Maintenance stability of pYT125 in cells of E. co/i MM294(pYT125)-S and aspartase formation by MM294(pYT125)-S during 30 cell generations. Cells were grown in ASP medium at 37 ’ C. Plasmid: 0 = pYT125(pSY343-aspA) in MM294(pYT125)-S; 0 = pSY343 in MM294(pSY343); II = pNKlO1 (pBR322-arpA -par) in MM294(pNKlOl).
12345676
Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel analysis of total cellular protein. Cells were grown in ASP medium. Electrophoresis was carried out with a 12.5% sodium dodecyl sulfate-polyacrylamide gel. Extracts were applied to gel at 20 pg of protein per lane. Others were carried out as described by Laemmh (1970). Lane I= MM294-S at 30 o C; lane 2 = MM294-S at 37 o C; Lane 3 = MM294S(pSY343) at 30°C; lane 4 = MM294S(pSY343) at 37OC; lane 5 = MM294(pNKlOl) at 37°C; lane 6= MM294(pYT125)-S at 30 ’ C; lane 7 = MM294(pYT125)-S for 4 h after shifting the temperature from 30 o C to 37 ’ C at logarithmic growth phase; lane 8 = MM294(pYT125)-S at 37 o C.
37
production of polypeptide of molecular weight of approx. 50,000 to a large extent, whereas the vector pSY343 had no effect. When strain MM294(pYT125)-S was cultured, the aspA product was overproduced about two- to three-fold more abundantly at 37O C than at 30” C on densitometrical determination. This strain produced more polypeptide in cells cultured at 37” C during the entire growth period than in those cultured at 37 o C for 4 h after pre-incubation at 30’ C. These data indicated that the aspA product of pYT125 is polypeptide of molecular weight of approx. 50,000. When cultured at 37 OC, polypeptide of aspA product amounted to 25-30% of the total cellular protein by densitometrical determination. Analysis of plasmid DNA and host cell of strain MM294(pYT125)-S
The above results indicated that unexpectedly runaway plasmid pYT125 is stable in strain MM294(pYT125)-S. Therefore, we investigated whether stabilization of pYT125 was due to genetic alteration of plasmid DNA or that of host cell. First, plasmid DNAs were analyzed as follows. Cells of the wild-type strain MM294 were transformed with pYT125 DNA extracted from strain MM294(pYT125)-S. Of 10 transformants selected for Km’, 9 were unstable. Digestion of pYT125 DNAs isolated from these transformants with BamHI plus EcoRI and with BamHI plus Hind111 produced DNA fragments identical to those produced from pYT125 DNA isolated from strain MM294(pYT125). This indicated that the former pYT125 DNAs are very similar to the latter pYT125 DNA. Therefore, the stabilization of pYT125 in strain MM294(pYT125)-S was concluded not to be correlated with the structure of plasmid DNA. Subsequently, host cells were analyzed as follows. Ten cured strains were isolated from MM294(pYT125)-S. pYT125 DNA extracted from an unstable transformant was transferred into the above plasmid-free strains. 10 Km’ transformants were isolated from each of 10 cured strains. All of these Km’ strains maintained pYT125 stable. One of the plasmid-free strains deriving from MM294(pYT125)-S was denoted MM294-S. TABLE 3 COPY NUMBER
OF pYT125 IN MM294.
strain
Plasmid stability a (W)
Copy number b of plasmid per chromosome
MM294(pYT125) MM294(pYT125)-S MM294(pSY343) MM924S(pYT343)
59 99 59 91
3so*41.0 (4) 83k 2.7 (4) 52Ok72.0 (4) 103 f 16.0 (4)
a Plasmid stability is expressed as described in Table 2. b Copy number per chromosome was calculated by taking the molecular weight of the E. co/i chromosome to be 2.8 x 109, pSY343 as 9.5 x lo6 and pYT125 as 13.8 X 106. Values are the mean f standard deviation of independent trials and were calculated by considering values of plasmid stability. The number of trials is shown in parentheses. Cells were grown in ASP medium.
38
From the above results, stabilization genetic alteration of host cell. Plasmid copy number of pYTl25
of pYT125 was considered to be due to the
and pSY343 in E. cofi MM294 cells
Generally, high copy-number plasmids are unstable and low copy-number plasmids are stable. The stabilization of pYT125 is considered to be due to a reduction of copy number. Then, we investigated the copy number of pYT125 and vector pSY343 (Table 3). The copy number of pYT125 was 380 in the unstable strain and 83 in the stable strain. That of pYT343 was 520 in strain MM294 and 103 in strain MM294-S. The copy number of both plasmids was reduced in strain MM294-S. The above data indicated that stabilization of pYT125 and pSY343 is closely related to the decrease of the copy number of both plasmids.
Discussion To allow E. coli to overproduce more aspartase, we have constructed the aspA recombinant plasmid pYT125 bearing runaway plasmid vector, as described above. Strain MM294(pYT125)-S stably produced approx. 65-fold more aspartase than did the control strain. This aspartase-hyperproducing strain can be used for efficient production of aspartic acid from fumaric acid and ammonia. Since aspartase is produced in an amount of 25-30% of the total cellular protein, it could be purified to more than 90% only by ammonium sulfate fractionation of crude cell extracts (data not shown). Therefore, we will be able to obtain pure preparation of this enzyme readily in a large amount by the use of the above recombinant strain. Since the copy number of pYT125 was 83 in strain MM294-(pYT125)-S, the aspartase activity was nearly in proportion to the copy number of pYT125. When the ssb gene, related to DNA replication, was cloned on pSY343 and amplified, the level of overproduction has been reported to be approx. 60-fold higher over that in strain harboring the vector plasmid (Yasuda and Takagi, 1983). Thus, runaway plasmids are useful as cloning vectors for the extreme overproduction of products of the cloned genes. Gene amplification enables us to produce a single gene product in the maximal amount of 50% of total protein (Gelfand et al., 1978). The aspA product might amount to more than 50% of the total protein, if pYT125 exists stably at 200 copies in E. coli. However, since pYT125 was unstable in strain MM294 at more than 200 copies, strain MM294(pYT125) produced less aspartase than did strain MM294(pYT125)-S. When the culture of strain MM294(pYT125)-S was examined, the number of the viable cells was less than 10% of that of the total cells. Thus, the runaway replication is possibly lethal to the cells. pYT125 DNA was calculated to amount to approx. 30% of the total intracellular DNA in a stable strain. A drastic amplification of
39
plasmid DNA probably leads the cells to death by inhibiting the replication of chromosomal DNA. Aspartase activity of a stable strain was extremely high in a stable strain. Therefore, it may be one of the causes for lethality that the normal metabolism is remarkably disturbed by high activity of aspartase. Host strain MM294-S isolated from MM294(pYT125)-S stably maintained not only pYT125 but also vector pSY343. Furthermore, aspA-plasmid pYT482l(pBR322-aspA) bearing a multicopy vector was also stabilized in strain MM2946, and its copy number was decreased (data not shown). The above results also indicated that stabilization of these plasmids was closely related to a decrease of the copy number. Moreover, the decrease of copy number was considered to be due to the genetic alteration of strain MM294-S. Generally, the copy number of a plasmid is controlled not only by repressors encoded on plasmid DNA but also by chromosomal DNA-coded gene products of host cell. Therefore, the mutation on the latter gene of MM294-S might lead to decrease of the copy number of pYT125. Of 10 transformants, one was such a stable strain as MM294(pYT125)-S (Table 2 and Fig. 2). This frequency was unexpectedly high. Since the growth of unstable strain was slower in L broth than that of stable strain (data not shown), cells of stable transformants might be concentrated on selection plates. There are several genetic methods for stabilization of recombinant plasmids used for production purpose; device of selective pressure, control of copy number, regulation of gene expression, elimination of transposable elements, use of recombination-minus host and use of the partition locus (par). We have previously stabilized pBR322-aspA by insertion of the par locus. This method is the most simple and the most effective in stabilization of plasmid maintenance. We intended to stabilize runaway plasmid pYT125 by the use of the par locus. However, the copy number of pYT125 bearing the par locus was lowered to approx. 10 even at 37°C (data not shown). We have previously reported the isolation of aspartase-hyperproducing mutants of E. coli B (Nishimura and Kisumi, 1984). These mutants were resistant to catabolite repression for this enzyme. The introduction of the uspA plasmid described here into cells of such a strain is expected to allow E. co/i to produce aspartase in larger amounts. Acknowledgements We are grateful to I. Chibata, Senior Managing Director of Research and Development Headquaters of this company and to T. Tosa, General Manager of this laboratory, for encouragement. We also thank S. Yasuda for gift of pSY343 and F. Murakami for technical assistance. References Arai, K., Yasuda, S. and Kornberg, A. (1981) Mechanism of &rrrB protein action. I. Crystallization and properties of dnoB protein, an essential replication protein in Escherichia coli. J. Biol. Chem. 256, 52414252.
Backmann, K., Ptashne, M. and Gilbert, W. (1976) Construction of plasmids carrying the Cl gene of bacteriophage X. Proc. Natl. Acad. Sci. USA 73, 4174-4178. Bittner. M. and Vapnek, D. (1981) Versatile cloning vectors derived from the runaway-replication plasmid pKN402. Gene 15, 319-329. Chibata, I., Toss, T. and Sate. T. (1974) Immobilized aspartase-containing microbial cells: preparation and enzymatic properties. Appl. Microbial. 27, 878-885. Cohen, S.N., Chang, A.C.Y. and Hsu, CL. (1972) Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escl~ericl~ia co/i by R-factor DNA. Proc. Nat]. Acad. Sci. USA 69. 2110-2114. Gelfand, D.H., Shepard, H.M.. O’Farrell, P.H. and Polinsky, B. (1978) Isolation and characterization of a ColEl-derived plasmid copy-number mutant. Proc. Natl. Acad. Sci. USA 75, 5869-5873. Kinoshita, S., Nakayama, K. and Kitada, S. (1958) Production of aspartic acid from fumaric acid by microorganism. Hakko Kyokaishi 16, 517-520 (in Japanese). Kisumi, M., Ashikaga, Y. and Chibata, I. (1960) Studies on the fermentative preparation of L-aspartic acid from fumaric acid. Bull. Agric. Chem. Sot. Jpn 24, 296-305 (in Japanese). Kitahara, K., Fukui, S. and Misawa, M. (1960) Preparation of L-aspartic acid by bacterial aspartase. Nogeikagaku Kaishi 34, 44-48 (in Japanese). Komatsubara, S., Taniguchi, T. and Kisumi, M. (1986) Overproduction of aspartase of Esclren’clria co/i K-12 by molecular cloning. J. Biotechnol. 3, 281-291. Laemrnli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227.680-685. Lennox, E.S. (1955) Transduction of linked genetic characters of host by bacteriophage Pl. Virology 1. 190-206. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Mania& T., Fritsch, E.F. and Sambrook, J. (1982a) Molecular cloning, pp. 86-92. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982b) Molecular cloning, pp. 93-94. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982~) Molecular cloning, pp. 150-152. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Nishida, Y., Sato, T., Toss, T. and Chibata, I. (1979) Immobilization of Eschen’chia co/i cells having aspartase activity with carrageenan and locust bean gum. Enzyme Microb. Technol. 1, 95-99. Nishimura, N. and Kisumi, M. (1984) Aspartase-hyperproducing mutants of Escherichia co/i B. Appl. Environ. Microbial. 48, 1072-1075. Projan, S.J., Carleton, S. and Novick, R.R. (1983) Determination of plasmid copy number by fluorescence densitometry. Plasmid 9, 182-190. Sninsky, J.J., Uhlin, B.E., Gustafsson, P. and Cohen, S.N. (1981) Construction and characterization of a novel two-plasmid system for accomplishing temperature-regulated amplified expression of cloned adventitious genes in Escherichia coli. Gene 16, 275-286. Uhlin, B.E., Molin, S., Gustafsson, P. and Nordstrom, K. (1979) Plasmids with temperature-dependent copy number for amplification of cloned genes and their products. Gene 6, 91-106. Yasuda, S. and Takagi, T. (1983) Overproduction of Esclren’chio co/i replication protein by the use of runaway-replication plasmids. J. Bacterial. 154, 1153-1161.
of Biorechnology,
6
31
(1987) 31-40
Elsevier JBT 00255
Hyperproduction of aspartase of Escherichia coli K-12 by the use of a runaway plasmid vector Noriyuki Research
Luboraroty
Nishimura, o/Applied
Saburo Komatsubara, Tomoyasu and Masahiko Kisumi Biochetnisrty,
Tanabe
Sei.vaku
Co. Ltd.
Taniguchi
Yodogawa-ku,
Osaka,
Japan
(Received 5 February 1987; accepted 12 March 1987)
Plasmid pYT125 bearing the aspartase gene (aspA) of Escherichia coli K-12 was constructed by the use of pSY343, runaway-replication plasmid vector. One of the transformants, strain MM294 (pYT125)S maintained pYT125 stably at 37’ C in a non-selective medium. This strain produced approx. 60-fold more aspartase than did the control strain after 30 cell generations. Polypeptide of the aspA product amounted to 25-30% of the total cellular protein. Plasmid DNA isolated from strain MM294 (pYT125)-S was very similar to that from unstable strain MM294 (pY’T125) in length and restriction sites. The copy number was 83 in strain MM294 @YT125)-S and was 380 in strain MM294 (pYT125). Vector pSY343 was also stabilized in cured strains, isolated from MM294 (pYT125)-S, accompanied by reduction of the copy number. Accordingly, stabilization of pYT125 was considered to be closely related to decrease of the copy number, owing to genetic alteration of MM294-S. Aspartase; Escherichia coli; Runaway-replication-plasmid;
Plasmid stability
Introduction Aspartase (EC 4.3.1.1) catalyzes the reversible conversion of fumaric acid and NH: to L-aspartic acid. L-aspartic acid has been industrially produced from fumaric acid and NH: with Escherichia cob cells (Kinoshita et al., 1958; Kisumi et al., 1960; Kitahara et al., 1960; Chibata et al., 1974; Nishida et al., 1979). Correspondence lo: N. Nishimura, Research Laboratory Ltd., Yodogawa-ku, Osaka 532, Japan.
0168-1656/87/$03.50
of Applied Biochemistry, Tanabe Seiyaku Co.
0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
32
Recently, we have isolated E. coli B mutants with high aspartase activity by mutational and transductional methods (Nishimura and Kisumi, 1984). The aspartase gene (as@) of E. coli K-12 was cloned with low copy vector and inserted into multicopy vector pBR322 (Komatsubara et al., 1986). This aspA recombinant plasmid elevated the aspartase formation in E. coli K-12. Since this recombinant plasmid was lost from cells at high frequencies, we stabilized it by insertion of the partition locus (par). Uhlin et al. (1979) have isolated plasmids that can be amplified to approx. 2000 copies per cell upon heat induction at 37’C, whereas the copy number is relatively low when cells are grown at 30°C. Replication of these plasmids is controlled at above 35 o C due to a temperature-sensitive mutation in repressor coded on plasmid DNA, but strictly controlled by repressor at a low temperature. Such plasmids, called runaway-replication plasmids or, simply, runaway plasmids, are useful as vectors for the overproduction of gene products. Several derivatives of these plasmids have been reported to be applied to overproduction of /3-lactamase (Uhlin et al., 1979), DNA replication proteins (Yasuda and Takagi, 1983), chloramphenicol acetyl transferase (Sninsky et al., 1981) and fi-galactosidase (Bittner and Vapnek, 1981). This paper deals with application of a runaway plasmid vector to aspartase hyperproduction. Materials and Methods Bacterial strains and plasmids.
E. co/i K-12 strains and plasmids used are listed in
Table 1. Media. L broth (Lennox, 1955) was routinely used as a rich medium. ASP medium contained 4% corn steep liquor, 2% Meast (Ebiosu Yakuhin Co. Ltd., Tokyo, Japan), 1.14% fumaric acid, 0.5% ammonium fumarate, 0.2% K,HPO,, and 0.05% MgSO, .7H,O (pH 7.0).
TABLE
1
BACTERIAL
STRAINS
AND
PLASMIDS
USED.
Strain
Characteristic
E. co/i K-12 MM294 MM294S
thi ksdR thi hsdR
Plasmid pSY 343 pYT482-1 pNKIO1 pYT125
Km’ pBR322-aspA Apr pBR322-aspA-par Ap’ pSY343-aspA Km’
a PL’ = stable
maintenance
a
Source
Backmann Derivative this paper
PLs
of plasmids;
or reference
Ap’ = resistance
et al. (1976) from MM294,
Y asuda and Takagi (1983) Komatsubara et al. (1986) Nishimura et al. (in press) This paper to ampicillin;
Km’
= resistance
to kanamycin.
33
DNA manipulation. Plasmid DNA was prepared from cells grown in L broth containing an antibiotic by the cleared lysate method (Maniatis et al., 1982a) and further purified by ethidium bromide-cesium chloride centrifugation (Maniatis et al., 1982b). Digestion and ligation were as described previously (Komatsubara et al., 1986). Transformation.
E. co/i cells were transformed
by the method of Cohen et al.
(1972). Agarose gel eiectrophoresis. Agarose gel electrophoresis was run with 0.7% agarose gel in the same buffer as described by Maniatis et al. (1982~). Estimation of plasmid stability. E. coli cells harboring plasmids were cultured on L broth agar slant containing an antibiotic. Cells were suspended at 5 X 10s ml-’ and cultured with shaking’for 12-16 h. After 10 cell generations of growth, culture was diluted 1 : 100 with fresh medium and further cultured. Dilution and incubation were repeated to obtain the cultures of 30 cell generations. Aliquots of the culture of 10, 20 and 30 cell generations were diluted and spread on L broth agar plates. After overnight incubation, 100 colonies arising from each culture were tested for antibiotic resistance. Determination of plasmid copy number. Cells were grown with shaking in ASP medium at 37 o C. Exponentially growing cells, 5 x log, were collected by centrifugation, washed twice with 10 mM Tris-hydrochloride, 1 mM EDTA (pH 8.0). To obtain DNA preparation, cells were treated with 200 ~1 of lysating buffer (50 mM Tris-hydrochloride, 50 mM EDTA, 15% (w/v) sucrose, 2 mg lysozyme ml-’ and 50 pg RNAase ml-‘) at 37’C for 30 min and with 10 ~1 of 20% sodium dodecyl sulfate at 37O C for 2 h. The copy number was determined by fluorescence densitometry as described by Projan et al. (1983) using the DNA sample prepared as described above.
Aspartase was determined by the method described by Nishida et al. (1979). The reaction mixtures containing 1 M ammonium fumarate (pH 8.5) 1 mM MgCl, and cell extract were incubated at 37” C. L-Aspartic acid formed was determined by bioassay with Leuconostoc mesenteroides P-60. Aspartase assay.
Other methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out by the method of Laemmli (1970). Protein concentration was determined by the method of Lowry et al. (1951). Materials. Restriction endonucleases and T4 DNA ligase were purchased from Takara Shuzo Co. Ltd., Kyoto, Japan. Other chemicals were purchased from Katayama Kagaku Co. Ltd., Osaka, Japan.
34
Results Construction of the aspA recombinant plasmid bearing runaway plasmid vector pSY343
pYT482-1 is composed of a 4.3 kb BamHI DNA fragment carrying the aspA gene and a 4 kb BamHI fragment of pBR322. Runaway plasmid pSY343 is ca. 9.5 kb in length and has the gene coding for kanamycin resistance (Km’) and a BamHI site that can be used as a cloning site without inactivating the Km’ gene (Yasuda and Takagi, 1983). To allow E. coli to overproduce more aspartase, we constructed the aspA recombinant plasmid bearing pSY343 as follows (Fig. 1). DNAs of pYT482-1 and pSY343 were digested with BamHI and subjected to ligation for insertion of the 4.3 kb fragment carrying the aspA gene into the BamHI site of pSY343. Five of 31 transformants of strain MM294, selected for Km’, were sensitive to Ap and had high aspartase activities. These five transformants harbored ‘plasmid DNAs which were cut into 4.3 kb and 9.5 kb fragments with BamHI. The above data indicated that we could obtain the expected recombinant plasmid (pSY343-aspA). The representative plasmid was denoted pYT125.
pBR322-aspA
(Ap’)
E pSY
343-
aSPA
B (Km’)
Fig. 1. Diagrammatic representation of the construction of pYT125(pSY343-uq4). Plasmid pYT125 was constructed by inserting a 4.3 kb BomHI fragment carrying the uspA gene into pSY343 at BamHI site. The abbreviations used for the restriction sites are: B, BumHI; E, EcoRI; H,HindlII.
35
Aspartase formation and stability of pYT125 in transformants of strain MM294
Cells of MM294, which was of wild-type for the aspA gene, were transformed with plasmid DNA of pYT125 and transformants were selected for Kmr. Maintenance stability of pYT125 was investigated by culturing these transformants at 37 o C in ASP medium for approx. 10 cell generations (Table 2). One of these 10 strains maintained pYT125 very stably and produced 60 times more aspartase than did the host strain. This strain (T-7) was re-named MM294 (pYT125)S. Furthermore, the stability of pYT125 was compared with those of vector pSY343 and another stable recombinant plasmid pNK.101 (pBR322-aspA-par), which was not a runaway plasmid, by culturing cells harboring these plasmids for approx. 30 cell generations (Fig. 2). Plasmid pYT125 was lost at only 0.3% per cell generation, whereas pSY343 was at a frequency as high as 3.2%, as judged from ratio of the number of Km’ colonies to that of total colonies. The stable plasmid pNKlO1 was lost at 0.4% per cell generation. Strain MM294 (pYT125)S produced 65 times more aspartase than did the control strain MM294(pSY343), even after 30 cell generations. Plasmid DNAs of 20 Km’ colonies from such a culture of MM294(pYTl25)-S were tested by agarose gel electrophoresis and found to be almost similar to pYT125 DNA in length and restriction sites. This result indicated that no deletion plasmids were produced. Therefore, we concluded that pYT125 was very stably maintained in strain MM294(pYT125)-S after many cell generations. Estimation of the intracellular amount of the aspA product
Cell extracts were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to identify the aspA product (Fig. 3). Plasmid pYT125 enhanced the TABLE 2 ASPARTASE strain B MM294 T-l 2 3 4 5 6 I 8 9 10
FORMATION
AND STABILITY
Sp act of aspartase b 10 310 350 600 230 560 230 640 420 100 400
OF pYT125 IN TRANSFORMANTS
OF MM294.
Plasmid stability ’ (4;) 17 21 88 7 69 8 100 32 0 27
a T-l to T-10 are pYT125 transformants of MM294. T-7 was re-named MM294(pYT125)-S studies. Cells were grown in ASP medium. b Specific activity is expressed as pmol of L-aspartic acid formed per min mg- ’ of protein. ’ Plasmid stability is expressed as percentage of Km’ cells.
for further
10
20 Generations
30
10 of
20
30
growth
Fig. 2. Maintenance stability of pYT125 in cells of E. co/i MM294(pYT125)-S and aspartase formation by MM294(pYT125)-S during 30 cell generations. Cells were grown in ASP medium at 37 ’ C. Plasmid: 0 = pYT125(pSY343-aspA) in MM294(pYT125)-S; 0 = pSY343 in MM294(pSY343); II = pNKlO1 (pBR322-arpA -par) in MM294(pNKlOl).
12345676
Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel analysis of total cellular protein. Cells were grown in ASP medium. Electrophoresis was carried out with a 12.5% sodium dodecyl sulfate-polyacrylamide gel. Extracts were applied to gel at 20 pg of protein per lane. Others were carried out as described by Laemmh (1970). Lane I= MM294-S at 30 o C; lane 2 = MM294-S at 37 o C; Lane 3 = MM294S(pSY343) at 30°C; lane 4 = MM294S(pSY343) at 37OC; lane 5 = MM294(pNKlOl) at 37°C; lane 6= MM294(pYT125)-S at 30 ’ C; lane 7 = MM294(pYT125)-S for 4 h after shifting the temperature from 30 o C to 37 ’ C at logarithmic growth phase; lane 8 = MM294(pYT125)-S at 37 o C.
37
production of polypeptide of molecular weight of approx. 50,000 to a large extent, whereas the vector pSY343 had no effect. When strain MM294(pYT125)-S was cultured, the aspA product was overproduced about two- to three-fold more abundantly at 37O C than at 30” C on densitometrical determination. This strain produced more polypeptide in cells cultured at 37” C during the entire growth period than in those cultured at 37 o C for 4 h after pre-incubation at 30’ C. These data indicated that the aspA product of pYT125 is polypeptide of molecular weight of approx. 50,000. When cultured at 37 OC, polypeptide of aspA product amounted to 25-30% of the total cellular protein by densitometrical determination. Analysis of plasmid DNA and host cell of strain MM294(pYT125)-S
The above results indicated that unexpectedly runaway plasmid pYT125 is stable in strain MM294(pYT125)-S. Therefore, we investigated whether stabilization of pYT125 was due to genetic alteration of plasmid DNA or that of host cell. First, plasmid DNAs were analyzed as follows. Cells of the wild-type strain MM294 were transformed with pYT125 DNA extracted from strain MM294(pYT125)-S. Of 10 transformants selected for Km’, 9 were unstable. Digestion of pYT125 DNAs isolated from these transformants with BamHI plus EcoRI and with BamHI plus Hind111 produced DNA fragments identical to those produced from pYT125 DNA isolated from strain MM294(pYT125). This indicated that the former pYT125 DNAs are very similar to the latter pYT125 DNA. Therefore, the stabilization of pYT125 in strain MM294(pYT125)-S was concluded not to be correlated with the structure of plasmid DNA. Subsequently, host cells were analyzed as follows. Ten cured strains were isolated from MM294(pYT125)-S. pYT125 DNA extracted from an unstable transformant was transferred into the above plasmid-free strains. 10 Km’ transformants were isolated from each of 10 cured strains. All of these Km’ strains maintained pYT125 stable. One of the plasmid-free strains deriving from MM294(pYT125)-S was denoted MM294-S. TABLE 3 COPY NUMBER
OF pYT125 IN MM294.
strain
Plasmid stability a (W)
Copy number b of plasmid per chromosome
MM294(pYT125) MM294(pYT125)-S MM294(pSY343) MM924S(pYT343)
59 99 59 91
3so*41.0 (4) 83k 2.7 (4) 52Ok72.0 (4) 103 f 16.0 (4)
a Plasmid stability is expressed as described in Table 2. b Copy number per chromosome was calculated by taking the molecular weight of the E. co/i chromosome to be 2.8 x 109, pSY343 as 9.5 x lo6 and pYT125 as 13.8 X 106. Values are the mean f standard deviation of independent trials and were calculated by considering values of plasmid stability. The number of trials is shown in parentheses. Cells were grown in ASP medium.
38
From the above results, stabilization genetic alteration of host cell. Plasmid copy number of pYTl25
of pYT125 was considered to be due to the
and pSY343 in E. cofi MM294 cells
Generally, high copy-number plasmids are unstable and low copy-number plasmids are stable. The stabilization of pYT125 is considered to be due to a reduction of copy number. Then, we investigated the copy number of pYT125 and vector pSY343 (Table 3). The copy number of pYT125 was 380 in the unstable strain and 83 in the stable strain. That of pYT343 was 520 in strain MM294 and 103 in strain MM294-S. The copy number of both plasmids was reduced in strain MM294-S. The above data indicated that stabilization of pYT125 and pSY343 is closely related to the decrease of the copy number of both plasmids.
Discussion To allow E. coli to overproduce more aspartase, we have constructed the aspA recombinant plasmid pYT125 bearing runaway plasmid vector, as described above. Strain MM294(pYT125)-S stably produced approx. 65-fold more aspartase than did the control strain. This aspartase-hyperproducing strain can be used for efficient production of aspartic acid from fumaric acid and ammonia. Since aspartase is produced in an amount of 25-30% of the total cellular protein, it could be purified to more than 90% only by ammonium sulfate fractionation of crude cell extracts (data not shown). Therefore, we will be able to obtain pure preparation of this enzyme readily in a large amount by the use of the above recombinant strain. Since the copy number of pYT125 was 83 in strain MM294-(pYT125)-S, the aspartase activity was nearly in proportion to the copy number of pYT125. When the ssb gene, related to DNA replication, was cloned on pSY343 and amplified, the level of overproduction has been reported to be approx. 60-fold higher over that in strain harboring the vector plasmid (Yasuda and Takagi, 1983). Thus, runaway plasmids are useful as cloning vectors for the extreme overproduction of products of the cloned genes. Gene amplification enables us to produce a single gene product in the maximal amount of 50% of total protein (Gelfand et al., 1978). The aspA product might amount to more than 50% of the total protein, if pYT125 exists stably at 200 copies in E. coli. However, since pYT125 was unstable in strain MM294 at more than 200 copies, strain MM294(pYT125) produced less aspartase than did strain MM294(pYT125)-S. When the culture of strain MM294(pYT125)-S was examined, the number of the viable cells was less than 10% of that of the total cells. Thus, the runaway replication is possibly lethal to the cells. pYT125 DNA was calculated to amount to approx. 30% of the total intracellular DNA in a stable strain. A drastic amplification of
39
plasmid DNA probably leads the cells to death by inhibiting the replication of chromosomal DNA. Aspartase activity of a stable strain was extremely high in a stable strain. Therefore, it may be one of the causes for lethality that the normal metabolism is remarkably disturbed by high activity of aspartase. Host strain MM294-S isolated from MM294(pYT125)-S stably maintained not only pYT125 but also vector pSY343. Furthermore, aspA-plasmid pYT482l(pBR322-aspA) bearing a multicopy vector was also stabilized in strain MM2946, and its copy number was decreased (data not shown). The above results also indicated that stabilization of these plasmids was closely related to a decrease of the copy number. Moreover, the decrease of copy number was considered to be due to the genetic alteration of strain MM294-S. Generally, the copy number of a plasmid is controlled not only by repressors encoded on plasmid DNA but also by chromosomal DNA-coded gene products of host cell. Therefore, the mutation on the latter gene of MM294-S might lead to decrease of the copy number of pYT125. Of 10 transformants, one was such a stable strain as MM294(pYT125)-S (Table 2 and Fig. 2). This frequency was unexpectedly high. Since the growth of unstable strain was slower in L broth than that of stable strain (data not shown), cells of stable transformants might be concentrated on selection plates. There are several genetic methods for stabilization of recombinant plasmids used for production purpose; device of selective pressure, control of copy number, regulation of gene expression, elimination of transposable elements, use of recombination-minus host and use of the partition locus (par). We have previously stabilized pBR322-aspA by insertion of the par locus. This method is the most simple and the most effective in stabilization of plasmid maintenance. We intended to stabilize runaway plasmid pYT125 by the use of the par locus. However, the copy number of pYT125 bearing the par locus was lowered to approx. 10 even at 37°C (data not shown). We have previously reported the isolation of aspartase-hyperproducing mutants of E. coli B (Nishimura and Kisumi, 1984). These mutants were resistant to catabolite repression for this enzyme. The introduction of the uspA plasmid described here into cells of such a strain is expected to allow E. co/i to produce aspartase in larger amounts. Acknowledgements We are grateful to I. Chibata, Senior Managing Director of Research and Development Headquaters of this company and to T. Tosa, General Manager of this laboratory, for encouragement. We also thank S. Yasuda for gift of pSY343 and F. Murakami for technical assistance. References Arai, K., Yasuda, S. and Kornberg, A. (1981) Mechanism of &rrrB protein action. I. Crystallization and properties of dnoB protein, an essential replication protein in Escherichia coli. J. Biol. Chem. 256, 52414252.
Backmann, K., Ptashne, M. and Gilbert, W. (1976) Construction of plasmids carrying the Cl gene of bacteriophage X. Proc. Natl. Acad. Sci. USA 73, 4174-4178. Bittner. M. and Vapnek, D. (1981) Versatile cloning vectors derived from the runaway-replication plasmid pKN402. Gene 15, 319-329. Chibata, I., Toss, T. and Sate. T. (1974) Immobilized aspartase-containing microbial cells: preparation and enzymatic properties. Appl. Microbial. 27, 878-885. Cohen, S.N., Chang, A.C.Y. and Hsu, CL. (1972) Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escl~ericl~ia co/i by R-factor DNA. Proc. Nat]. Acad. Sci. USA 69. 2110-2114. Gelfand, D.H., Shepard, H.M.. O’Farrell, P.H. and Polinsky, B. (1978) Isolation and characterization of a ColEl-derived plasmid copy-number mutant. Proc. Natl. Acad. Sci. USA 75, 5869-5873. Kinoshita, S., Nakayama, K. and Kitada, S. (1958) Production of aspartic acid from fumaric acid by microorganism. Hakko Kyokaishi 16, 517-520 (in Japanese). Kisumi, M., Ashikaga, Y. and Chibata, I. (1960) Studies on the fermentative preparation of L-aspartic acid from fumaric acid. Bull. Agric. Chem. Sot. Jpn 24, 296-305 (in Japanese). Kitahara, K., Fukui, S. and Misawa, M. (1960) Preparation of L-aspartic acid by bacterial aspartase. Nogeikagaku Kaishi 34, 44-48 (in Japanese). Komatsubara, S., Taniguchi, T. and Kisumi, M. (1986) Overproduction of aspartase of Esclren’clria co/i K-12 by molecular cloning. J. Biotechnol. 3, 281-291. Laemrnli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227.680-685. Lennox, E.S. (1955) Transduction of linked genetic characters of host by bacteriophage Pl. Virology 1. 190-206. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Mania& T., Fritsch, E.F. and Sambrook, J. (1982a) Molecular cloning, pp. 86-92. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982b) Molecular cloning, pp. 93-94. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982~) Molecular cloning, pp. 150-152. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Nishida, Y., Sato, T., Toss, T. and Chibata, I. (1979) Immobilization of Eschen’chia co/i cells having aspartase activity with carrageenan and locust bean gum. Enzyme Microb. Technol. 1, 95-99. Nishimura, N. and Kisumi, M. (1984) Aspartase-hyperproducing mutants of Escherichia co/i B. Appl. Environ. Microbial. 48, 1072-1075. Projan, S.J., Carleton, S. and Novick, R.R. (1983) Determination of plasmid copy number by fluorescence densitometry. Plasmid 9, 182-190. Sninsky, J.J., Uhlin, B.E., Gustafsson, P. and Cohen, S.N. (1981) Construction and characterization of a novel two-plasmid system for accomplishing temperature-regulated amplified expression of cloned adventitious genes in Escherichia coli. Gene 16, 275-286. Uhlin, B.E., Molin, S., Gustafsson, P. and Nordstrom, K. (1979) Plasmids with temperature-dependent copy number for amplification of cloned genes and their products. Gene 6, 91-106. Yasuda, S. and Takagi, T. (1983) Overproduction of Esclren’chio co/i replication protein by the use of runaway-replication plasmids. J. Bacterial. 154, 1153-1161.