Appl Microbial Biotechnol (1989) 31:163-167
`
`Glucose as a substrate in recombinant
`strain fermentation technology
`
`By-product formation, degradation
`and intracellular accumulation of recombinant protein
`
`Applied
`.
`•nd Mterohw/ogy
`Biotechnology
`© Springer-Verlag 1989
`
`Ursula Rinas 1·*, Heinrich-Andreas Kracke-Helm 2
`
`, and Karl Schiigerl2
`
`1 Institut fiir Biophysik und Physikalische Biochemie, Universitat Regensburg, Universitatsstrasse 31, D-8400 Regensburg,
`Federal Republic of Germany
`2 Institut fiir Technische Chemie, Universitat Hannover, Callinstrasse 3, D-3000 Hannover, Federal Republic of Germany
`
`to complex
`Summary. Glucose supplements
`growth media of Escherichia coli affect the pro(cid:173)
`duction of a recombinant model protein under the
`control of a temperature-sensitive expression sys(cid:173)
`tem. The bacterial "Crabtree effect", which occurs
`in the presence of glucose under aerobic condi(cid:173)
`tions, not only represses the formation of citric
`acid cycle enzymes, but also represses the forma(cid:173)
`tion of the plasmid-encoded product even though
`the synthesis of this protein is under the control
`of the temperature-inducible lambda PR-promot(cid:173)
`er/cl857-repressor expression system. When the
`recombinant E. coli is grown at a moderate tem(cid:173)
`perature (35° C) with protein hydrolysate and glu(cid:173)
`cose as substrates, a biphasic growth and produc(cid:173)
`tion pattern is observed. In the first phase, the
`cells grow with a high specific growth rate, uti(cid:173)
`lizing glucose and forming glutamate as a by(cid:173)
`product. The intracellular level of recombinant
`protein is very low in this phase. Later, glutamate
`is consumed, indicating an active citric acid cycle.
`The degradation of glutamate is accompanied by
`the intracellular accumulation of high amounts of
`recombinant protein.
`
`Introduction
`
`Genetic engineering methods are already in in(cid:173)
`dustrial use to produce many types of proteins us(cid:173)
`ing fast-growing microorganisms such as the bac(cid:173)
`terium Escherichia coli. For industrial use, it is im(cid:173)
`portant to have a recombinant bacterial strain that
`provides high productivity and sufficient genetic
`
`*Present address: Department of Chemical Engineering, Cali(cid:173)
`fornia Institute of Technology, Pasadena, CA 91125, USA
`Offprint requests to: K. Schiigerl
`
`stability. For this purpose it is often convenient to
`choose expression systems in which cell growth
`and formation of recombinant protein appear in
`different cultivation phases. In order to separate
`growth and production phases, chemically induci(cid:173)
`ble promoters, such as the lac, tac, or trp promot(cid:173)
`er, and temperature-inducible expression systems,
`such as the lambda promoter/lambda cl857-re(cid:173)
`pressor system, are commonly used.
`For the economical use of recombinant micro(cid:173)
`organisms that form intracellular products, it is
`important to utilize a fermentation process that
`results in a high intracellular level of product and
`a high cell concentration in the fermentor. An
`inexpensive and convenient substrate to obtain a
`high cell mass is a simple carbohydrate, such as
`glucose. Glucose feeding leads to a bacterial
`"Crabtree effect" under aerobic conditions, re(cid:173)
`sulting in the formation of acetate and other me(cid:173)
`tabolic by-products (Anderson and von Meyen(cid:173)
`burg 1980). To prevent accumulation of by-prod(cid:173)
`ucts, which reduce the growth yield, fed-batch
`strategies have been developed for non-recombi(cid:173)
`nant E. coli (Gleiser and Bauer 1981; Mori et al.
`1979). In cultivations of recombinant E. coli it was
`also observed that accumulation of acetate in the
`culture medium is concomitant with low levels of
`recombinant protein production (Brown et al.
`1985; Meyer et al. 1984). Reducing the growth
`rate by decreasing the dilution rate in continuous
`culture or the substrate feed in fed-batch culture
`results in decreased acetate formation and in in(cid:173)
`creased production of recombinant protein
`(Brown et al. 1985; Meyer et al. 1984; Zabriskie
`and Arcuri 1986). When complex (instead of de(cid:173)
`fined) media are supplemented with glucose as an
`additional carbon source, a stronger reduction in
`growth rate is necessary to prevent the formation
`of acetate (Meyer et al. 1984).
`
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`164
`
`U. Rinas et a!.: Glucose as a substrate in recombinant strain fermentation technology
`
`The development of a control-scheme process
`for use in recombinant strain fermentation tech(cid:173)
`nology requires knowledge about the connection
`between substrate utilization and product forma(cid:173)
`tion. Therefore, the objective of this work was to
`correlate substrate consumption, metabolic by(cid:173)
`product formation, and by-product degradation
`with the intracellular accumulation of a recombi(cid:173)
`nant model protein in E. coli grown in batch cul(cid:173)
`ture on complex medium supplemented with glu(cid:173)
`cose.
`We investigated an E. coli host in which the
`temperature-sensitive lambda cl857 -repressor is
`formed by a chromosomally integrated, defective
`lambda lysogen. The formation of plasmid en(cid:173)
`coded product, a cro-lacZ fusion protein with /3-
`galactosidase activity, is under the control of the
`lambda Pwpromoter (Zabeau and Stanley 1982).
`We observed not only that the temperature de(cid:173)
`pendent interaction between promoter and re(cid:173)
`pressor is responsible for the induction of product
`formation, but also that substrate supply and sub(cid:173)
`strate-induced repression and derepression affect
`the extent of product formation.
`
`Materials and methods
`
`Bacterial strain and plasmid. Escherichia coli K12 MF
`(F- trp;m /ac;m SmR) with the chromosomal lambda lysogen
`(lambda Nam7 Nam53 cl857~HI) was used as a host (Caste(cid:173)
`lazzi eta!. I972). The plasmid utilized was pCL47~Y-T. This
`plasmid, carrying a cro-lacZ fusion under control of the
`lambda PR-promoter, is described by Zabeau and Stanley
`(I982). Host and plasmid were kindly provided by K. Stanley
`(EMBL, Heidelberg, FRG).
`
`Medium and culture conditions. LB medium, containing IO g/1
`casein peptone (Difco, Detroit, Mich.), 5 g/1 yeast extract (Sig(cid:173)
`ma, St. Louis, Mo), and 9 g/1 NaCI, was supplemented with
`IO g/1 glucose and IOO mg/1 ampicillin. The cultivation was
`carried out in a 10-1 stirred-tank reactor (Chemap, Volkets(cid:173)
`will). The aeration rate was 0.5 vvm at 500 rpm. The pH was
`kept constant at pH 7 with 4 M NaOH and 2M H2S04. An
`overnight culture (LB medium supplemented with IOO mg/1
`ampicillin) prepared from a 2-day-old, single colony was used
`as inoculum (I% v/v).
`
`Electrophoresis of proteins. Sodium dodecyl sulphate (SDS)(cid:173)
`polyacrylamde gel electrophoresis was performed in 7.5% poly(cid:173)
`acrylamide slab gels according to the method of Laemmli
`(I970). For sample preparation cells corresponding to I mg
`dry weight (DW) were recovered by centrifugation (7000 g,
`5 min). Harvested cells were washed in 0.9% NaCI solution
`and centrifuged at 7000 g for 5 min. The remaining cell pellet
`was resuspended in IOO ~1 sample buffer. Samples were then
`boiled for IO min. Insoluble material was removed by centrifu(cid:173)
`gation. Supernatant (IO ~1) was loaded onto the 7.5% SDS-poly(cid:173)
`acrylamide gel in the presence of O.I o/o SDS. The composition
`of the sample buffer was as follows: 4% SDS, IO mM dithio(cid:173)
`threitol, 20% glycerol, I25 mM TRIS/hydrochioride (pH 6.8).
`
`The gels were stained with Coomassie brilliant blue R 250.
`To estimate the amount of cro-p-galactosidase, the stained gels
`were scanned with a Camaq TLC scanner (Muttenz, CH).
`
`P-Ga/actosidase activity. Samples (I ml) removed from batch
`culture were centrifuged at 7000 g for 5 min, and the cells re(cid:173)
`suspended in 0.9% NaCI solution. The cells were disrupted
`with an ultrasonic disintegrator (Labsonic I5IO, IOO watts, B.
`Braun, Melsungen, FRG) for 5 min on ice. Cell debris was re(cid:173)
`moved by centrifugation at 7000 g for 5 min and fi-galactosi(cid:173)
`dase activity was assayed by measuring the rate of hydrolysis
`of o-nitrophenol-P-n-galactopyranoside (ONPG) according to
`the method of Miller (I982). A unit of activity was defined as
`I ~mol o-nitrophenolliberated per minute. Specific activity of
`the cro-p-galactosidase was reported as total units of enzyme
`per gram of total DW of the recombinant cells.
`
`Other methods. The growth curve was determined by measur(cid:173)
`ing the turbidity of the culture medium at 600 nm against a
`blank of sterile culture medium and by obtaining the cells
`DW. Drying was performed at 105° C to constant weight.
`Glucose concentration in the medium was analysed by
`HPLC using refractive index detection. A Shodex Ionpak S801
`(8*500) column (Macherey Nagel, Duren, FRG) was em(cid:173)
`ployed at 75° C and 2 mM H2S04 was used as the eluent at a
`flow rate of 1.0 mllmin.
`The concentration of amino acids were determined by
`HPLC with the o-phthaldialdehyde/fi-mercaptoethanol preco(cid:173)
`lumn derivatization method (Qureshi et a!. I984).
`The dissolved-oxygen probe was of the polarographic
`type (Ingold, Urdort). The medium saturated with air or ni(cid:173)
`trogen was defined as IOO% or Oo/o dissolved oxygen, respec(cid:173)
`tively.
`
`Results and discussion
`
`The usual derepression condition of the lambda
`promoter/ cl857 repressor system is a temperature
`shift from 30° C to 42° C. Our observation is that
`"inclusion body" formation is prevented, and that
`high amounts of soluble and biologically-active
`product are found when the lambda cl857 repres(cid:173)
`sor is only partially inactivated, and thus the
`lambda PR-promoter is not fully induced. A culti(cid:173)
`vation temperature of 35° C allows the formation
`of soluble and biologically-active cro-lacZ fusion
`protein (manuscript in preparation).
`When LB medium is supplemented with glu(cid:173)
`cose, the concentration of dissolved oxygen is
`crucial for the attainable intracellular product
`concentration. The following figures document
`the results of an aerobic batch fermentation at
`35° C in LB medium supplemented with 1 o/o glu(cid:173)
`cose. Figure 1a shows the courses of cell growth,
`intracellular accumulation of product, and con(cid:173)
`centration of dissolved oxygen. The growth and
`production phases were separate. Specific prod(cid:173)
`uct formation occurred in two steps. The first,
`smaller step occurred when the growth rate de(cid:173)
`creased, the concentration of dissolved oxygen
`
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`

`
`U. Rinas et al.: Glucose as a substrate in recombinant strain fermentation technology
`
`165
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`Fig. 1 a, b. Growth, intracellular concentration of biologically active cro-p-galactosidase, and by-product formation and degrada(cid:173)
`tion of recombinant cells during aerobic growth on LB medium supplemented with 1 o/o glucose. The lines without symbols represent
`the concentration of dissolved oxygen in the culture fluid (% saturation). a. Cell growth is given as the natural logarithm of the
`recombinant cells dry weigth (ln DW) -
`r:J -. Specific product concentration is shown as total units of jJ-galactosidase activity per
`total dry weight (U/g DW) -0-. Roman numerals correspond to the protein gel electrophoresis of total cell extracts in Fig. 3. b.
`The concentration of glucose (g/1) - r:J- and glutamate (mg/1) -0 - in the culture fluid of the recombinant cells
`
`reached a minimum, and amino acids, such as ser(cid:173)
`ine and aspartic acid had already been consumed
`(Fig. 2a, b). The second step occurred when the
`concentration of dissolved oxygen reached a max(cid:173)
`imum and the consumption of glutamic acid be(cid:173)
`gan (Fig. 1 a, b).
`Glutamic acid seems to be the key compound
`in this process. On the one hand, it is formed as a
`by-product of incomplete glucose oxidation,
`while on the other hand, it is used as a carbon
`source when substrates such as serine, aspartic
`acid, glucose, and threonine are already con(cid:173)
`sumed (Figs. 1a, 2a, b). The beginning of glutamic
`acid consumption did not immediately follow the
`disappearance of glucose from the culture me(cid:173)
`dium. Instead there was a delay of about 2 h (Fig.
`1 b), and the organisms did not start to degrade
`glutamic acid before the amino acid threonine
`was consumed (Fig. 2a).
`The aerobic formation of glutamic acid during
`growth on glucose (in the presence of ammonium)
`
`indicates that the formation of 2-ketoglutarate de(cid:173)
`hydrogenase, the key enzyme of the citric acid cy(cid:173)
`cle, was repressed (Yamada et al. 1972; Gray et al.
`1966; Amarasingham and Bernhard 1965). As
`long as the organisms can generate sufficient
`amounts of ATP by substrate phosphorylation,
`and the citric acid cycle is not necessary for the
`synthesis of cell metabolites (since they are pres(cid:173)
`ent as components of the complex medium), the
`organisms do not need an active Krebs cycle and
`the formation of these enzymes is repressed (Gray
`et al. 1966; Amarashingham and Bernhard 1965).
`When the cell metabolism changed, and the
`Krebs cycle was activated (indicated by the onset
`of glutamic acid degradation), formation of re(cid:173)
`combinant protein increased significantly (Fig.
`la, b). The formation and degradation of acetate,
`another by-product of incomplete glucose oxida(cid:173)
`tion, are correlated with the accumulation of the
`cro-p-galactosidase fusion protein in a similar
`manner (Kracke-Helm, unpublished data). Ace-
`
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`
`166
`
`U. Rinas et a!.: Glucose as a substrate in recombinant strain fermentation technology
`
`p0 2 Maximum
`
`!
`
`o Aspartate
`x Asparagine
`o Threonine
`<> Alanine
`
`p0 2 Maximum
`
`j
`
`o Glycine
`x Tryptophan
`o Serine
`
`40
`
`2a
`
`a
`
`b
`
`10
`
`30
`20
`Time [hi
`Fig. 2a, b. The concentration of amino acids in the culture fluid during aerobic growth of the recombinant cells on LB medium
`supplemented with 1% glucose. Arrows indicate the cultivation times when the concentration of dissolved oxygen reached mini(cid:173)
`mum and maximum values. a. Aspartic acid, asparagine, threonine, and alanine. b. Glycine, tryptophan, and serine
`
`40
`
`50
`
`tate, like glutamate, can only be degraded via the
`Krebs cycle.
`When cells grow on substrates which can only
`be utilized via the citric acid cycle and respiration,
`it is evident that the oxygen supply to the cells be(cid:173)
`comes the critical variable in the fermentation
`process. Oxygen-limiting conditions result in an
`
`accumulation of metabolic by-products which
`cannot be degraded, and, consequently, in a low
`intracellular concentration of recombinant pro(cid:173)
`tein. When oxygen was not the limiting substrate,
`cells accumulated the product in quantities up to
`25% of the total cell protein (Fig. 3), even though
`the lambda Pwpromoter was not fully induced.
`
`[Dalton)
`
`116000-
`
`57000-
`
`40000-
`
`25000-
`
`17 800-
`
`·(~-Gal)
`
`I
`
`ll m
`
`Fig. 3. Protein gel electrophoresis of total cell extracts.
`Samples were prepared as described in Materials and
`methods. After 50 h cultivation the intracellular level of
`cro-p-galactosidase reached 25% of total cell protein (deter(cid:173)
`mined by scanning). The Roman numerals refer to the sam(cid:173)
`ple times shown in Fig. 1a
`
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`
`U. Rinas et al.: Glucose as a substrate in recombinant strain fermentation technology
`
`167
`
`The intracellular accumulation of the recom(cid:173)
`binant cro-p-galactosidase
`in
`the
`stationary
`growth phase is in part explained by an increase
`in plasmid copy number with decreasing growth
`rate, although a simple correlation between plas(cid:173)
`mid copy number and product expression does
`not exist (Rinas 1987). Changes in the transcrip(cid:173)
`tion level of the product and/ or repressor gene
`and/ or changes of the translation level of product
`and/or repressor mRNA may be other possible
`reasons to explain the observed effect.
`In conclusion utilization of glucose as a sub(cid:173)
`strate in recombinant DNA fermentations in or(cid:173)
`der to achieve high amounts of cell mass and high
`intracellular product concentrations requires a
`fermentation strategy that facilitates the degrada(cid:173)
`tion of metabolic by-products that are formed by
`incomplete glucose oxidation. Oxygen-limiting
`conditions should be avoided, and, when controll(cid:173)
`ing the glucose feed rate in fed-batch culture, the
`measurements of dissolved oxygen, glucose, and
`especially of by-product concentration should be
`used.
`
`References
`
`Amarasingham CR, Bernhard DD (1965) Regulation of 2-ke(cid:173)
`toglutarate dehydrogenase formation in Escherichia coli. J
`Bioi Chern 240:3664-3668
`Anderson KB, Meyenburg K von (1980) Are growth rates of
`Escherichia coli in batch cultures limited by respiration? J
`Bacteriol144:114-123
`Brown SW, Meyer H-P, Fiechter A (1985) Continuous produc(cid:173)
`tion of human leukocyte interferon with Escherichia coli
`and continuous cell lysis in a two stage chemostat. Appl
`Mirobiol Biotechnol 23:5-9
`
`Castellazzi M, Brachet P, Eisen H (1972) Isolation and charac(cid:173)
`terization of deletion in bacteriophage lambda residing as
`prophage in E. coli K12. Mol Gen Genet 117:211-218
`Gleiser IE, Bauer S (1981) Growth of E. coli W to high cell
`concentrations by oxygen level linked control of carbon
`source concentration. Biotechnol Bioeng 23: 1015-1021
`Gray CT, Wimpenny IWT, Mossmann MR (1966) Regulation
`of metabolism in facultative bacteria. II. Effects of aero(cid:173)
`biosis, anaerobiosis and nutrition on the formation of
`Krebs cycle enzymes in Escherichia coli. Biochim Biophys
`Acta 117:33-41
`Laemmli UK (1970) Cleavage of structural proteins during the
`assembly of the head of bacteriophage T4. Nature
`227:680-685
`Meyer H-P, Leist C, Fiechter A (1984) Acetate formation in
`continuous culture of Escherichia coli K12 D1 on defined
`and complex media. J Biotechnol 1:355-358
`Miller JH (1982) Experiments in molecular genetics. Cold
`Spring Harbour Laboratory, New York
`Mori H, Yano T, Kobayashi T, Shimizu S (1979) High density
`cultivation of biomass in fed-batch system with DO-stat. J
`Chern Eng Jpn 12:313-319
`Qureshi AG, Fohlin L, Bergstrom J (1984) Application of
`high-performance liquid chromatography to the determi(cid:173)
`nation of free amino acids in physiological fluids. J Chro(cid:173)
`matogr 297:91-100
`Rinas U (1987) Produktbildung und Stabilitlit eines rekombi(cid:173)
`nanten,
`temperatursensitiven Escherichia coli-Stammes.
`PhD Thesis University of Hannover, FRG
`Yamada K, Kinoshita S, Tsunoda T, Aiba K (1972) The micro(cid:173)
`bial production of amino acids. Wiley, New York
`Zabeau M, Stanley K (1982) Enhanced expression of cro-P(cid:173)
`galactosidase fusion proteins under the control of the Pw
`promoter of bacteriophage lambda. EMBO J 1:1217-1224
`Zabriskie DW, Arcuri EJ (1986) Factors influencing produc(cid:173)
`tivity of fermentations employing recombinant microor(cid:173)
`ganisms. Enzyme Microb Techno! 8:706-717
`
`Received 18 August 1988/ Accepted 28 February 1989
`
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