APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1990, p. 129~1302
`0099-2240/90/051296-07$02.00/0
`Copyright © 1990, American Society for Microbiology
`
`Vol. 56, No.5
`
`Improved Expression of Human Interleukin-2 in High-Cell-Density
`Fermentor Cultures of Escherichia coli K-12 by a
`Phosphotransacetylase Mutant
`KEITH A. BAUER, ARIE BEN-BASSAT, MIKE DAWSON, VIRGINIA T. DE LA PUENTE,
`AND JUSTIN 0. NEWA Y*
`Department of Fermentation Research and Development, Cetus Corporation, 1400 Fifty-third Street,
`Emeryville, California 94608
`
`Received 13 November 1989/Accepted 12 February 1990
`
`A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium
`during growth to high cell density in fermentor cultures and was shown to be defective in ito; phosphotrans(cid:173)
`acetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2
`(IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent
`during expression under control of the temperature-sensitive PL promoter. In flask cultures at lower cell
`density, the mutant again produced less acetate than the parent, although both showed a much lower level of
`acetate accumulation than that seen in fermentors at high cell density. Both showed a higher specific expression
`level of IL-2 in flask cultures, and there was a greater ditference between the mutant and its parent in the final
`extent of specific IL-2 accumulation in fermentor cultures compared with flask cultures. Thus, the concentra(cid:173)
`tion of acetate in the medium, which was much higher in fermentor cultures (2:300 mM after 5 h of induction)
`than in flask cultures (:53 mM) of the parent organism, was a significant factor in limiting expression of the
`heterologous protein product, IL-2. The acetate kinase-phosphotransacetylase pathway was therefore a major
`source of acetate formation in these cultures. Blocking this pathway improved accumulation of IL-2 and did not
`slow growth.
`
`Organic acids accumulate in the culture medium during
`aerobic growth of Escherichia coli on glucose (17). The most
`abundant organic acid is often acetic, and its concentration
`can build up to levels that are inhibitory to growth (1, 13).
`In a previous study (10), we showed that intracellular
`accumulation of interleukin-2 (IL-2), under control of the
`temperature-sensitive bacteriophage lambda PL promoter,
`was inversely correlated with cell density and acetate accu(cid:173)
`mulation in fermentor cultures. These observations provided
`circumstantial evidence that acetate was at least partially
`responsible for the cessation of product accumulation during
`expression of heterologous genes in E. coli and suggested
`that higher levels of IL-2 accumulation could be expected if
`acetate formation could be blocked.
`Two enzymatic pathways for acetate formation in E. coli
`have been identified. Acetate can be derived directly from
`pyruvate by pyruvate oxidase, but the activity of this en(cid:173)
`zyme in E. coli is thought to be too low to account for the
`amount of acetate produced (4). Acetate can also be derived
`from acetylcoenzyme A (CoA) by the acetate kinase-phos(cid:173)
`photransacetylase (ACK-PTA) pathway (3). In E. coli B,
`PTA is activated by pyruvate and inhibited by NADH (14).
`The genes coding for PTA and ACK have been mapped and
`form an operon in E. coli and Salmonella typhimurium which
`is induced as much as twofold under anaerobic conditions
`(8). Mutants defective in both activities can be isolated by
`selection for fluoroacetate resistance (3).
`In the work presented here, we examined the role of the
`ACK-PTA pathway in the formation of acetate during intra(cid:173)
`cellular accumulation of IL-2. We isolated 52 fluoroacetate(cid:173)
`resistant mutants from E. coli MM294-1 and partially char-
`
`* Corresponding author.
`
`acterized them. Some of these mutants had reduced acetate(cid:173)
`forming ability, as low as 10 to 20% of parental levels in flask
`cultures, with near-normal aerobic growth rates on glucose.
`We describe here the characterization of one of these
`mutants and its performance as a host strain for PL promot(cid:173)
`er-driven accumulation of IL-2.
`
`MATERIALS AND METHODS
`Strains. All strains used in this study were derived from E.
`coli K-12 and are listed in Table 1. Phage stocks used for
`testing cl857 function were the gift of D. Gelfand. HW21 was
`the gift of H. C. Wong. The poxB strains were the gift of
`J. E. Cronan and the pta-ack deletion strains were the gift of
`G. F.-L. Ames.
`Media. N8-2 medium consisted of NH4Cl (10 mM),
`KH2P04 (21.9 mM), Na2HP04 (28.1 mM), K2S04 (9 mM),
`MgS04 (0.2 mM), MnS04 (3 JJ.M), ZnS04 (3 JJ.M), and
`CuS04 (0.1 JJ.M) in deionized water. The medium was
`sterilized by autoclaving, after which the following sterile
`additions were made: glucose (2 g/liter); thiamine hydrochlo(cid:173)
`ride (10 mg/liter); FeS04 (10 JJ.M).
`Minimal agar plates were of similar composition to defined
`flask medium (see below), except that the trace metals
`solution was 1 mllliter, MgS04 was at 1 mM, and agar was
`added to 1.5% (wt/vol). Alternate carbon sources (galactose
`or sodium pyruvate) were also used at 5 g/liter. Ampicillin
`(50 mg/liter) and tetracycline (15 mg/liter) were used as
`indicated. Sodium monofluoroacetate (Tull Chemical Co.,
`Inc., Oxford, Ala.) was added to 25 mM as indicated.
`R2-4 agar plates were standard rich-medium plates de(cid:173)
`scribed elsewhere (10).
`Isolation plates were composed ofN~HP04 (5.68 g/liter),
`KH2P04 (3.54 g/liter), trisodium citrate · 2H20 (0.44 g/liter),
`a solution containing ZnS04 (30 mM), MnS04 (30 mM), and
`
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`VoL. 56, 1990
`
`PTA- MUTANT OF E. COLI SHOWING IMPROVED IL-2 EXPRESSION
`
`1297
`
`TABLE 1. Strains used
`
`Strain
`
`Relevant characteristics
`
`Source
`
`DG116
`
`cl857+, derived from
`MM294-1
`Thi+ derivative of 00116
`HW21
`HW21(pFC54.t) TS 11 copy number (16), PL(cid:173)
`IL-2 (15) in HW21
`Thi+ derivative of MD050
`KB100
`KB100(pFC54.t) TS copy number (16), PL-
`IL-2 (15) in KB100
`Pta-
`MD050
`Pta-, cl857+ (lysogen)
`MD050L
`MD050L(pFC54.t) TS copy number (16), PL-
`IL-2 (15) in MD050L
`Tn5 inserted into poxB
`(PoxB-)
`endAJ hsdR17 supE thi,
`derived from MM294 (11)
`Gal- strain for P1
`transduction
`pta
`ack
`ll(ack, pta, hisP-hisQ)
`Ack+ Pta+
`TnlO inserted near poxB
`(PoxB+)
`
`MH6
`
`MM294-1
`
`MM294-1
`galE::TnlO
`TA3514
`TA3515
`TA3516
`TA3521
`YYC201
`
`CMcca
`
`CMCC
`This work
`
`This work
`This work
`
`This work
`This work
`This work
`
`J. E. Cronan
`
`CMCC
`
`D. Gelfand
`
`G. F.-L. Ames
`G. F.-L. Ames
`G. F.-L. Ames
`G. F.-L. Ames
`J. E. Cronan
`
`trifuged at 28,000 x g for 60 min at 5°C and stored at - 20°C
`prior to use.
`(ii) PTA activity. PTA activity was measured by the
`method of Brown et al. (3). In this assay, the conversion of
`acetylphosphate to acetyl-CoA is coupled to NADH forma(cid:173)
`tion through malate dehydrogenase and citrate synthase.
`The reaction mixture contained the following components in
`1 ml: Tris hydrochloride (pH 8.0 at room temperature), 100
`JJ.mol; MgCl2 , 5 JJ.mol; NAD+, 0.5 JJ.mol; CoA, 0.5 JJ.mol;
`malate, 5 JJ.mol; acetylphosphate, 10 JJ.mol; malate dehydro(cid:173)
`genase, 27.5 U; and citrate synthase, 6.75 U. One unit of
`PTA activity was defined as the amount of enzyme required
`to allow the formation of 1 JJ.mol of NADH per min.
`(iii) ACK activity. ACK activity was measured by the
`method of Fox and Roseman (6). In this assay, ADP forma(cid:173)
`tion from A TP and acetate is coupled to NADH consump(cid:173)
`tion through pyruvate kinase and lactate dehydrogenase.
`The reaction mixture contained the following components in
`1 ml: PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)](cid:173)
`KOH (pH 7.0 at room temperature; Sigma Chemical Co.), 50
`JJ.mol; glycerol, 100 mg; MgCl2 , 5 JJ.mol; dithiothreitol, 1
`JJ.mol; phosphoenolpyruvate, 7 JJ.mol; potassium acetate, 300
`JJ.mol; ATP, 30 JJ.mol; NADH, 1 IJ.mol; pyruvate kinase, 50
`U; and lactate dehydrogenase, 10 U. One unit of ACK
`activity was defined as the amount of enzyme required to
`allow the oxidation of 1 JJ.mol of NADH per min.
`For both enzyme assays, the reactions were started by
`addition of extract and followed by measuring the change in
`A340 over time at room temperature, using a Hewlett(cid:173)
`Packard HP8452A spectrophotometer equipped with en(cid:173)
`zyme kinetics software. PTA activity was shown to be
`completely dependent on the presence of both primary
`substrates, acetylphosphate and CoA. ACK activity was
`dependent on the presence of A TP and was greatly reduced
`but not eliminated when acetate was omitted. This was
`probably due to the presence of other A TPases in the crude
`extracts. Boiling the extracts eliminated all activity in both
`assays. Both assays were used in the linear range of enzyme
`activity.
`Genetic techniques. (i) Selection of low-acetate-producing
`strains. The ACK-PTA pathway is bi-directional and allows
`entry of acetate into central metabolism via acetyl-CoA.
`Mutants with low acetate-producing ability were therefore
`selected by isolating strains resistant to the tricarboxylic
`acid cycle inhibitor fluoroacetate (3, 9). E. coli MM294-1 was
`grown in nutrient broth to a cell density of approximately 2.0
`OD680 (approximately 3 x 1lf' cells per ml). Cells were then
`plated at approximately 2 x 107 , 2 x 108 , and 2 x 109 cells
`per plate on isolation plates containing either lactate plus
`fluoroacetate or pyruvate plus fluoroacetate and incubated at
`37°C for 3 days. A total of 52 individual colonies were picked
`from these plates, streaked for isolation onto the same
`selective medium, and incubated at 37°C. Because there was
`still perceptible growth of carry-over wild-type cells on some
`plates, the 52 isolates were again picked from single colo(cid:173)
`nies, streaked for a second time onto fresh plates containing
`the same selective medium, and incubated at 37°C. Single
`colonies were again picked after 48 h, suspended in 1 ml of
`saline, and streaked for a third time onto fresh selective
`medium. The same isolates were also streaked onto medium
`containing acetate as sole carbon source to test for differ(cid:173)
`ences in ability to grow on acetate.
`Strains were evaluated for acetate production and growth
`rate by inoculating 125-ml flasks containing 25 ml of pre(cid:173)
`warmed N8-2 medium at an initial cell density of0.05 OD680•
`Cultures were incubated at 37°C on a New Brunswick rotary
`
`a CMCC, Cetus Master Culture CoUection.
`b TS, Temperature sensitive.
`
`CuS04 (30 mM) at 0.2 ml/liter; (NH4hS04 (2.0 g/liter),
`MgS04 • 7H20 (102 mg/liter), and agar (1.5%, wt/vol). The
`liquid was sterilized by autoclaving, and the following sterile
`additions were made: FeS04 (200 mM) in 2.5 mM H2S04 ,
`0.36 mVliter, CaCl2 (1.0 M), 0.1 mVliter, thiamine hydrochlo(cid:173)
`ride (1.0%), 2.0 mVliter. Pyruvate, lactate, acetate, or glu(cid:173)
`cose was added at 0.5% (wt/vol) as carbon source. Sodium
`monofluoroacetate was added at a concentration of 25 mM
`along with lactate or pyruvate to select for putative mutants
`with reduced ability to produce acetate.
`Defined flask medium consisted of trisodium citrate (4
`mM), (NHJ2S04 (10 mM), KH2P04 (50 mM), and trace
`metals solution (2) (4 mVliter). The pH of the medium was
`adjusted to 6.8 with NaOH, and the solution was autoclaved.
`Glucose (5 g/liter), thiamine hydrochloride (20 mg/liter), and
`MgS04 (3 mM) were added to the cooled medium from
`sterile concentrated stock solutions.
`For fermentor medium, the medium of Bauer and Shiloach
`(2) with some modifications was used as described previ(cid:173)
`ously (10), except that 20 mg of thiamine hydrochloride per
`liter was added after autoclaving. The 14-liter Chemap
`fermentors as well as inoculation and operating conditions
`used in this study were as described previously (10).
`Assays. Assays for acetate, cell growth, and IL-2 in
`isolated refractile bodies, as well as the various conversion
`factors used, were as described previously (10).
`(i) Preparation of ceU extracts. Cell extracts for assays of
`PTA and ACK activities were prepared as follows: 100 ml of
`a defined flask medium culture in exponential growth was
`centrifuged at 3,000 x g for 10 min at 5°C and then washed
`twice in 30 ml of 10 mM sodium phosphate buffer, pH 7.5 (at
`room temperature), which included 10 mM MgCl2 and 1 mM
`EDTA. The cells were suspended in the same buffer to a final
`total protein concentration of 2 to 2.4 mg/ml (based on
`optical density at 680 nm [OD680] converted to protein
`content) and sonicated on ice to completion (approximately
`5 min, microscopic observation). Sonic extracts were cen-
`
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`1298
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`BAUER ET AL.
`
`APPL. ENVIRON. MICROBIOL.
`
`shaker at 200 rpm, and density readings were taken approx(cid:173)
`imately every 0.5 h. At approximately 1.2 OD680, small
`culture samples were taken for acetate analysis. Many of the
`52 isolat«:s showed greatly reduced acetate. accumulation,
`some as httle as 10% of parental levels. Stram MD050 were
`selected for further experimentation because it consistently
`gave the lowest levels of acetate accumulation and had a
`growth rate essentially indistinguishable from that of its
`parent (approximately 0.6 h- 1).
`(ii) Pl transduction. Genetic elements coding for markers
`such as cl857, Thi+, and poxB::Tn5 were transferred by Pl
`transduction, essentially as described by Miller (12). To
`select for cl857, recipient strains were first made Gal-, using
`a lysate grown on MM294:ga/E::Tnl0. This intermediate
`was then transduced back to Gal+ by using a lysate grown on
`DG116 (a strain containing cl857 in the chromosome). Col(cid:173)
`onies were screened for cotransduction of the tightly linked
`cl857 marker by testing for lambda resistance at 30 and 41 oc.
`To select for the Thi+ phenotype, strains were plated on
`minimal glucose agar plates without thiamine. The resulting
`colonies were restreaked for purity and then grown in
`glucose-containing defined flask medium lacking thiamine to
`confirm that their growth rate in this medium was identical to
`that of the parent strain in thiamine-containing medium.
`(iii) poxB-containing strains. To create a Pta- poxB double
`mutant, the poxB::Tn5 mutation from MH6 was transduced
`into KB100. To confirm that the poxB mutation had been
`transferred, a lysate was grown on the putative double
`mutant and used to transduce YYC201 to kanamycin resis(cid:173)
`tance. The resulting transductants were shown to be poxB on
`pyruvate tetrazolium plates, as described by Chang and
`Cronan (5).
`
`RESULTS
`Characterization of MDOSO. (i) PTA activity. Selection for
`fluoroacetate resistance results in mutations in at least two
`genes, pta and ack (3, 9). MD050 was a presumptive Pta(cid:173)
`mutant based on its reduced acetate secretion levels and
`slow growth rate on acetate-containing plates. To confirm
`this, PTA and ACK activities were compared
`in
`MD050L(pFC54.t) and HW21(pFC54.t) grown at 30°C in
`defined me~ium. The two host strains were isogenic, except
`for the les10n recovered by fluoroacetate selection; they
`were both derived from the same MM294-1 parent. The only
`other difference between them was that HW21 was Thi+
`allowing it to grow without the addition of thiamine to th~
`medium. This change made no discernible difference to
`either acetate formation or IL-2 expression in HW21 when
`compared with its parent MM294-1 (data not shown). All
`defined media used here included thiamine.
`Three sets of extracts were prepared on separate days
`from shake flask cultures grown at 30°C (i.e., repressed IL-2
`expression). The results (Table 2) clearly showed that
`MD050L had greatly reduced PTA activity. No PTA activity
`was detected in the mutant in the first set of assays (detec(cid:173)
`tion limit, <1% of wild type), and small amounts (1.5% or
`less of wild type) were detected in the subsequent two tests.
`The low PTA activity in the second extract of MD050L
`(Table 2, set 2) was dependent on the presence of
`acetylphosphate in the assay mixture, but was still present
`when CoA was omitted. Furthermore, doubling the amount
`of Co A in the reaction mix inhibited the residual PTA
`activity in MD050L extracts. In contrast, the PTA activity in
`HW21 extracts had the expected dependence on both CoA
`and acetylphosphate. A comparison of ACK and PTA levels
`
`TABLE 2. Specific enzyme activities of PTA and ACK
`
`Strain
`
`Set 1
`HW21(pFC54. t)
`MD050L(pFC54. t)
`
`Set 2
`HW21(pFC54.t)
`MD050L(pFC54. t)
`
`Set 3
`HW21(pFC54.t)
`MD050L(pFC54. t)
`
`Activity (U/mg of protein)
`
`PTA
`
`1.2
`<0.01
`
`0.48
`0.007
`
`1.5
`0.002
`
`ACK
`
`0.45
`1.2
`
`0.26
`0.40
`
`0.14
`0.19
`
`in samples sonicated for different lengths of time (data not
`shown) showed that release of ACK activity was maximal
`under the sonication conditions used to generate the assay
`results shown in Table 2. Further sonication resulted in a
`small increase in the PTA activity seen in HW21 extracts,
`but the minute amount of activity seen in MD050L extracts
`was lost altogether. The observed enzyme activity differ(cid:173)
`ences were therefore not due to differences in the extent of
`cell breakage between the two strains, and the small amount
`of apparent PTA activity in MD050L was probably due to an
`activity other than normal PTA.
`ACK activity in the mutant strain was higher than that in
`the PTA-competent strain by 2.7-fold in the first assay,
`1.5-fold in the second, and 1.3-fold in the third assay.
`Enhanced ACK activity in pta mutants has not been re(cid:173)
`ported in the literature; in fact, in one report, mutations in
`pta were said to have no effect on ACK activity (3).
`(ii) Acetate accumulation. To determine the extent of
`acetate accumulation in flask cultures, HW21 (Pta+) and
`KB100 (Pta-) were grown for 15 generations in glucose(cid:173)
`containing defined medium at 30°C to achieve balanced
`growth and to eliminate carry-over of undefined nutrients
`from the inoculum. Growth and acetate accumulation rates
`were measured over an OD680 range of 0.02 to 0.3 in which
`growth was exponential and the supply of nutrients (includ(cid:173)
`ing oxygen) was not limiting. Data from these experiments
`are shown in Table 3. The Pta- mutant and nonmutant
`strains both had very similar specific growth rates of 0.59 to
`0.60 h- 1 • The specific acetate production rate of KB100 in
`
`TABLE 3. Growth rate and acetate production
`
`Expt and straina
`
`Expt 1 (flask
`cultures)
`HW21
`HW21(pFC54.t)
`KB100
`
`Expt 2 (flask
`cultures)
`TA3521
`TA3514 (pta)
`T A3515 (ack)
`T A3516 [~(ack-
`pta-hisQ-hisP)]
`
`Specific growth
`rate (h- 1)
`
`300C
`
`37"C
`
`Specific acetate
`production
`(mmollg [dry wt}
`per h)
`
`0.60
`0.50
`0.59
`
`0.69
`0.55
`0.49
`0.56
`
`4.62
`3.55
`0.59
`
`6.07
`1.38
`2.74
`1.40
`
`a Medium and conditions were as described in the text.
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`

`
`VoL. 56, 1990
`
`PTA- MUTANT OF E. COLI SHOWING IMPROVED IL-2 EXPRESSION
`
`1299
`
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`Culture Age (Hours)
`FIG. 1. Mock inductions in fermentor cultures: growth (closed
`symbols) and acetate production (open symbols) during mock in(cid:173)
`ductions of the Pta- mutant MD050 (triangles) and a Pta nonmutant
`strain, HW21 (circles). In each case, the temperature was shifted
`from 30 to 37•c when the 00680 reached 20.
`
`these flask cultures was approximately sevenfold lower than
`that of HW21.
`To explore acetate accumulation by the Pta- mutant in
`fermentor cultures, mock inductions of strains MD050 and
`HW21 were performed by shifting the temperature from 30
`to 37•c at an 00680 of 20. These two strains did not contain
`plasmid and thus did not express IL-2. The results are shown
`in Fig. 1. The Pta- mutant and nonmutant strains had similar
`growth kinetics at both temperatures, while specific acetate
`production in the mutant was lower by a factor of approxi(cid:173)
`mately twofold. Specific acetate production rates were lower
`in fermentors than in flasks for both strains, with the
`difference being more pronounced in HW21 than in MD050.
`This may have been due to simple inhibition of the reversible
`ACK-PT A pathway, caused by the higher levels of acetate in
`the medium of fermentor cultures of HW21. There was little
`change in specific acetate production rates in the parent
`strain before and after mock induction. This was in contrast
`to the increase of 16-fold in specific acetate productivity that
`occurs when PTA-competent cells contain a plasmid allow(cid:173)
`ing IL-2 expression (10).
`Acetate accumulation in flasks was also measured in an
`isogenic set of E. coli K-12 strains which included a different
`pta mutant (T A3514) and a pta-ack double deletion mutant
`(T A3516). These strains were grown in defined medium plus
`histidine (100 1-Lg/ml to satisfy an auxotrophic requirement) at
`37•c for 20 h prior to analyzing the supernatant for acetate.
`Both the deletion strain and our Pta- strain (MD050) were
`found to produce lower amounts of acetate compared with
`their respective nonmutant parents (Table 3).
`Finally, we studied the effect of a mutation in pyruvate
`oxidase (poxB) on acetate accumulation in our Pta- mutant.
`A Tn5 insertion in the pyruvate oxidase gene was transferred
`into KB100 by P1 transduction. Acetate accumulation and
`
`growth rate in this double mutant were measured in glucose(cid:173)
`containing defined medium at 37•c and were found to be
`indistinguishable from those in its parent.
`EIJ'ect of the Pta- mutation on IL-2 production. The effects
`of the Pta- mutation on acetate and IL-2 accumulation were
`examined in flask and fermentor cultures, after lysogenizing
`KB100 (Table 1) to carry the cl857 gene in the chromosome,
`and transforming both it and HW21 with a plasmid (pFC54.t)
`which allowed intracellular accumulation of IL-2 under
`control of the temperature-sensitive PL promoter.
`(i) Shake Basks. Cultures, 100 ml, of KB100(pFC54.t) and
`HW21(pFC54.t) (Table 1), growing exponentially in glucose(cid:173)
`containing defined medium, were added to 400-ml portions
`of prewarmed (38°C) fresh medium in Fembach flasks and
`shaken at 3s•c for up to 5 h. Samples for high-pressure liquid
`chromatography analysis of IL-2 were taken at 3 and 5 h of
`induction. Growth kinetics were similar in the two strains,
`with the mutant growing slightly more in the later hours of
`induction, while specific acetate accumulation was calcu(cid:173)
`lated to be approximately sevenfold higher in the parent
`strain than in the mutant. IL-2 expression (as a percentage of
`total intracellular protein) after 3 and 5 h was 16 and 19%,
`respectively, for the HW21 strain and 18 and 21%, respec(cid:173)
`tively, for the KB100 strain. Therefore, the Pta- mutation
`resulting in lower acetate accumulation in KB100 did not
`negatively affect either growth or IL-2 accumulation and
`may have enhanced them slightly.
`(ii) Fermentor cultures. M0050L(pFC54.t) was grown in a
`fermentor as described above and induced by shifting the
`temperature from 30 to 37•c at cell densities of approxi(cid:173)
`mately 20 and 40 00680 in two separate runs. For compari(cid:173)
`son, HW21(pFC54.t) was grown similarly, with induction of
`IL-2 expression by temperature shift at 20 and 40 00680 • The
`kinetics of growth, acetate production, and IL-2 expression
`in these experiments are presented in Fig. 2. While both
`strains had a similar 2.5-fold increase in 00680 in the first 3
`h of induction in flasks, the Pta+ strain stopped growing after
`a cell density increase of only 1.6-fold in the 20-00680
`fermentor induction and 1.5-fold in the 40-00680 fermentor
`induction. In contrast, the Pta- mutant grew substantially
`more during induction at both cell densities. Specific acetate
`production during the 5-h induction period was seven- to
`ninefold less for the mutant than for the wild type at the
`different cell densities tested.
`Specific IL-2 accumulation in the transformed Pta- mu(cid:173)
`tant was 23% lower in fermentor cultures than observed in
`flask cultures (17 versus 21% in flasks), while in the wild type
`it was 36% lower than that seen in flask cultures (14 versus
`19% in flasks). The data for specific IL-2 accumulation in the
`PTA-competent strain at the induction cell densities tested in
`these studies were consistent with our previous observations
`(10) in which IL-2 expression was less affected by the cell
`density at induction in the 20- to 40-00680 range. Figure 20
`shows that the Pta- mutant accumulated more total IL-2 in
`the fermentor as a result of the increase in specific IL-2
`accumulation combined with continued growth during induc(cid:173)
`tion.
`Effect of Pta- mutation on ethanol, lactate, and pyruvate
`accumulation. In fermentor cultures of the PTA-competent
`strain HW21(pFC54.t) grown in glucose-containing defined
`medium, o-lactate and pyruvate were not produced in large
`amounts. During the 5-h induction period, the concentration
`of o-lactate increased from approximately 1 to approxi(cid:173)
`mately 4 mM, and that of pyruvate increased from approx(cid:173)
`imately 0.1 to approximately 7 mM. There was a higher level
`of accumulation of these acids during growth and induction
`
`BEQ 1029
`Page 4
`
`

`
`1300
`
`BAUER ET AL.
`
`APPL. ENVIRON. MICROBIOL.
`
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`Q) u c 8 150.0
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`Time After Induction (h)
`Time After Induction (h)
`FIG. 2. Growth (A), acetate production (B), specific IL-2 production (C), and total IL-2 production (D) in fermentor cultures: Pta- mutant
`MD050L(pFC54.t) (filled symbols) and a Pta nonmutant strain, HW21(pFC54.t) (open symbols) during induction at OD680s of 20 (triangles)
`and 40 (circles). Both strains carried a plasmid which allowed expression of IL-2 when the temperature was shifted from 30 to 37°C at the time
`of induction. The expression level of IL-2 is reported as the weight percentage of total intracellular protein.
`
`when MD050L(pFC54.t) was used. In these fermentor cul(cid:173)
`tures, o-lactate accumulated to 9 mM by the start of induc(cid:173)
`tion and subsequently rose to 15 mM at 3 h of induction
`before falling to 5 mM by the end of the 5-h period. Pyruvate
`accumulation increased from 1.5 to 8.4 mM during the same
`period. Ethanol was not produced in amounts greater that 1
`mM in either culture before or during induction. Clearly, the
`lowered amount of acetate produced in the Pta- mutant was
`not completely compensated for by the slight increases in
`D-lactate and pyruvate production, particularly when the
`differences in cell density are taken into account.
`
`DISCUSSION
`
`The data presented here provide evidence that MD050 is a
`Pta- mutant. At least two groups (3, 9) have reported that
`the method used to select MD050 yields two classes of
`mutants, pta and ack. Both classes of mutants are reported
`to have near-normal aerobic growth rates on glucose, but
`have a greatly reduced growth rate on acetate, particularly
`the pta mutants. Brown et al. (3) showed that pta mutants
`have greatly reduced acetate production, while ack mutants
`accumulate near-normal levels of acetate. This could be
`
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`Page 5
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`

`
`VoL. 56, 1990
`
`PTA- MUTANT OF E. COU SHOWING IMPROVED IL-2 EXPRESSION
`
`1301
`
`because acetylphosphate, which is expected to be accumu(cid:173)
`lated by ack mutants, is unstable at physiological pH and
`temperature and may decompose to acetate. The lack of
`growth of MD050 on acetate-containing plates and its re(cid:173)
`duced acetate accumulation implied that it was a Pta(cid:173)
`mutant rather than an Ack- mutant. The absence of PTA
`activity in MD050 extracts provided further proof. When
`transformed for IL-2 production under control of the tem(cid:173)
`perature-sensitive PL promoter, the Pta- mutant continued
`to grow during induction to a greater extent than its parent.
`It produced less acetate and greater amounts ofiL-2 in terms
`of both specific and total IL-2 accumulation. These proper(cid:173)
`ties made MD050 superior to its parent, MM294-1, as a host
`organism for expression of IL-2 and possibly other heterol(cid:173)
`ogous or homologous protein products.
`We believe that MD050 was nearly devoid of PTA activ(cid:173)
`ity, despite the fact that it still produced some acetate. Two
`lines of evidence support this view: first, only trace amounts
`of PTA activity could be detected in crude extracts; and
`second, strains carrying a known deletion of PTA continued
`to produced acetate. However, the precise location and
`nature of the genetic lesion in MD050 have not been deter(cid:173)
`mined by mapping or other more rigorous means, and we
`cannot completely rule out the possibility that some of the
`remaining acetate production in MD050 was due to residual
`PTA activity. Other possible sources of acetate production
`include the enzyme pyruvate oxidase, which catalyzes the
`oxidative decarboxylation of pyruvate, and spontaneous
`hydrolysis of acetyl-CoA. Our results indicate that pyruvate
`oxidase was not responsible for the remaining acetate pro(cid:173)
`duced by MD050.
`The 27% improvement in specific IL-2 accumulation
`achieved in this study by lowering acetate accumulation,
`using the Pta- mutant, was slightly better than the 17%
`improvement seen in our previous study, which used me(cid:173)
`dium perfusion to keep acetate concentrations low (10). If
`this difference is significant, then it may result from the fact
`that, in the perfusion cultures, although its concentration
`was kept below 100 mM, acetate may still have been
`somewhat inhibitory to maximal IL-2 accumulation. It could
`also be explained if the blockage in the Pta- mutant used in
`the present study led to improved utilization of acetyl-CoA
`through the tricarboxylic acid cycle and oxidative phosphor(cid:173)
`ylation. This may have allowed the formation of more
`intermediates and ATP for IL-2 synthesis than would be
`expected from waste acetate formation. Comparisons of the
`flux rates through the tricarboxylic acid cycle and through
`oxidative phosphorylation before and after induction of IL-2
`synthesis will provide additional information in this regard.
`The reason E. coli makes acetate during aerobic growth on
`glucose is a matter for speculation. It is clear that the bulk of
`the acetate accumulating in the medium during IL-2 expres(cid:173)
`sion came directly from acetyl-CoA via the PT A-ACK
`pathway (i.e., it appeared not to be made directly from
`pyruvate). Presuming that pyruvate formate lyase was not
`active under these conditions, this probably means that
`pyruvate dehydrogenase is sufficiently active to supply both
`the tricarboxylic acid cycle (for oxidative phosphorylation
`and amino acid biosynthetic needs) as well as acetate secre(cid:173)
`tion. Our data show that reducing the acetate formation rate
`by as much as sevenfold by mutation of pta had no measur(cid:173)
`able effects on growth rate. We did, however, observe that
`greater amounts of o-lactate and pyruvate were produced,
`but not in sufficient amounts to make up the difference in
`acetate accumulation. Perhaps the production of these acids
`reflects an imbalance between the cell's glycolytic capacity
`
`and its respiratory capacity (7). The excess glycolytic capac(cid:173)
`ity is shunted into acetate or, failing that, into o-lactate and
`pyruvate. Conditions that result in increased organic acid
`production may be those in which there is reduced respira(cid:173)
`tory flux, exacerbating the imbalance between glycolysis and
`respiration. High-level IL-2 expression may be one such
`condition. It may inhibit respiratory flux directly, by inter(cid:173)
`fering with some component of the respiratory machinery, or
`indirectly, perhaps by excessive shunting of carbon through
`the glyoxylate bypass to provide sufficient intermediates for
`amino acid biosynthesis, but at the expense of e