APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1994, p. 126-132 Vol. 60, No. 10099-2240/94/$04.00+0Copyright X 1994, American Society for Microbiology

Stable Expression of hom-i-thrB in Corynebacterium glutamicumand Its Effect on the Carbon Flux to Threonine

and Related Amino AcidsDIETER J. REINSCHEID, WOLFGANG KRONEMEYER, LOTHAR EGGELING,

BERNHARD J. EIKMANNS,* AND HERMANN SAHMInstitut fur Biotechnologie 1 des Forschungszentrums Julich, D-52425 JJulich, Germany

Received 5 August 1993/Accepted 18 October 1993

The hom-l-thrB operon encodes homoserine dehydrogenase resistant to feedback inhibition by L-threonineand homoserine kinase. Stable expression of this operon has not yet been attained in different Corynebacteriumglutamicum strains. We studied the use of chromosomal integration and of a low-copy-number vector formoderate expression of the hom-l-thrB operon to enable an analysis of the physiological consequences of itsexpression in C. glutamicum. Strains carrying one, two, or three copies of hom-1-thrB were obtained. Theyshowed proportionally increased enzyme activity of feedback-resistant homoserine dehydrogenase and ofhomoserine kinase. This phenotype was stably maintained in all recombinants for more than 70 generations.In a lysine-producing C. glutamicum strain which does not produce any threonine, expression of one copy ofhom-l-thrB resulted in the secretion of 39 mM threonine. Additional copies resulted in a higher, although notproportional, accumulation of threonine (up to 69 mM). This indicates further limitations of threonineproduction. As the copy number ofhom-l-thrB increased, increasing amounts of homoserine (up to 23 mM) andisoleucine (up to 34 mM) were secreted. Determination ofthe cytosolic concentration of the respective amino acidsrevealed an increase of intracellular threonine from 9 to 100 mM and of intracellular homoserine from 4 to 74mM as the copy number of hom-l-thrB increased. These results suggest that threonine production with C.glutamicum is limited by the efflux system for this amino acid. Furthermore, the results show the successful useof moderate and stable hom-l-thrB expression for directing the carbon flux from aspartate to threonine.

L-Threonine biosynthesis starting with L-aspartate con-sists of five enzymatic steps (Fig. 1) and is part of thebranched pathway forming the aspartate family of aminoacids. In Corynebacterium glutamicum and its subspeciesBrevibacteriumflavum and Brevibacterium lactofermentum,L-threonine synthesis is controlled by the first enzyme,aspartate kinase, which is inhibited by lysine plus threonine(46), and by the third enzyme, homoserine dehydrogenase(HDH), which is inhibited by threonine (31). Moreover,homoserine kinase (HK) is weakly inhibited by threonine(31), and the synthesis of HDH and of HK is repressed bymethionine (14, 29).The C. glutamicum wild-type (WT) hom and thrB genes

coding for HDH and HK have been isolated and character-ized (14, 22, 37), and it was shown by Peoples et al. (37) thathom and thrB are expressed as an operon (hom-thrB). Fromtwo independently isolated mutants of C. glutamicum, bothpossessing an HDH insensitive to inhibition by threonine,the respective hom genes (originally designated hoMFBR andhoMdr and in the following referred to as hom-i and hom-2,respectively) were recently isolated and characterized on themolecular level (1, 13, 38).Whereas the WT hom gene or hom-thrB operon on plas-

mid was easily cloned and overexpressed in C. glutamicum(13, 14, 22), the cloning and expression of the mutant hom orhom-thrB alleles on plasmid turned out to be problematical.Nakamori et al. (35) and Morinaga et al. (34) reported thecloning of a mutant hom allele in B. lactofermentum; how-ever, the transformants obtained were quite heterogeneous,

* Corresponding author. Mailing address: Institut fur Biotechnol-ogie 1 des Forschungszentrums Julich, D-52425 Julich, Germany.Phone: 49-2461-613967. Fax: 49-2461-612710.

showed only weak overexpression, and were geneticallyunstable. The same complications were reported by Archeret al. (1) on attempts to transform C. glutamicum withplasmid carrying the hom-2-thrB operon. Also, the expres-sion of hom-i and of hom-l-thrB isolated by us was prob-lematic since, upon introduction into the original host, C.glutamicum DM 368-3, growth of the transformants wasimpaired and, in attempts to introduce hom-i or hom-i-thrBon plasmid in C. glutamicum WT or in a lysine-producingstrain, no stable transformants were obtained (13). Thus, upto now, it has not been possible to study the consequences ofthe overexpression of a hom allele coding for an HDHinsensitive to inhibition by L-threonine in a C. glutamicumWT background or in any given C. glutamicum strain, e.g.,in the L-lysine hyperproducer MH20-22B (41).

It seemed reasonable that the failure to clone the hom-i-thrB operon stably into C. glutamicum might be due to thehigh expression of the respective genes brought about by therelatively high copy numbers of the vectors used. To addressthis question, we tested for moderate and stable expressionof hom-i-thrB by using chromosomal integration techniquesand by the construction and use of a low-copy-numbervector. Employing these recombinant derivatives of C.glutamicum WT and the L-lysine hyperproducer C. glu-tamicum MH20-22B, we analyzed the physiological conse-quences of hom-i-thrB expression in C. glutamicum.

MATERUILS AND METHODS

Bacteria, plasmids, and culture conditions. C. glutamicumATCC 13032 (WT), the lysine producer C. glutamicumMH20-22B (41), Escherichia coli DH5 (17), and the mobiliz-ing donor E. coli S17-1 (47) were used. The plasmids

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STABLE EXPRESSION OF hom-1-thrB IN C. GLUTAMICUM 127

L-Aspartate

IysC Aspartatekinase

L-Aspartyl phosphateAspartate-

asd semialdehyde

srt dehydrogenaseL-Aspartate semialdehyde -.t L-Lysine

r homHomoserinedehydrogenase

L-Methionine4- Homoserine

(-- thrB

Homoserinephosphate

thrC

Homoserinekinase

Threoninesynthase

L-Isoleucine 4- L-Threonine

FIG. 1. Biosynthetic pathway of L-threonine and its regulation inC. glutamicum. -*, feedback inhibition of aspartate kinase byL-lysine plus L-threonine, and of HDH and HK by L-threonine; ,repression of the HDH and HK genes by L-methionine.

employed in this study were pEK-homFBR-thrB (13), themobilizable vector pSUP301 (47), pEKO (12), pJC1 (7), andpWKO. For construction of the latter plasmid, a 2.6-kbClaI-EcoRI fragment containing a corynebacterial origin ofreplication was isolated from the low-copy-number plasmidpNG2 (44) and ligated into the multiple cloning site ofplasmid pEM1 (42), which is a mobilizable E. coli vector. LBmedium (25) was used as complex medium for all organisms.The minimal media used for growth of C. glutamicum andfor analysis of amino acid excretion were described previ-ously (13). When appropriate, kanamycin (50 ,ug/ml) wasadded to the medium. The C. glutamicum cells were grownaerobically as 60-ml cultures in 500-ml baffled Erlenmeyerflasks at 30°C on a rotary shaker at 140 rpm. Growth wasfollowed by measuring the optical density at 600 nm.

Preparation of DNA, transformation, and conjugation.Chromosomal DNA from C. glutamicum was isolated asdescribed earlier (11). Plasmids from E. coli were isolated asdescribed by Birnboim (3), and those from C. glutamicumwere isolated by the same method with prior incubation (1 h,37°C) of the cells with lysozyme (15 mg/ml). E. coli wastransformed by the CaCl2 method (5), and C. glutamicumcells were transformed via electroporation as described byLiebl et al. (26). Conjugation between E. coli S17-1 and C.glutamicum was performed as describeld by Schafer et al.(40), and transconjugants were selected on LB agar platescontaining kanamycin (25 ,ug/ml) and nalidixic acid (50Fxg/ml).DNA manipulations. All restriction enzymes, T4 DNA

ligase, Klenow polymerase, and calf intestine phosphatasewere obtained from Boehringer (Mannheim, Germany) andused as specified by the manufacturer.For DNA hybridization, about 10 ,ug of Sall-restricted

genomic DNA of the C. glutamicum strains was size-fractionated on 0.8% agarose gels and transferred onto anylon membrane Nytran 13 (Schleicher und Schull, Dassel,Germany) by vacuum-supported diffusion using VacuGene(Pharmacia LKB, Freiburg, Germany). A 1.48-kb KpnI

fragment from plasmid pEK-homFBR-thrB was digoxigenin-dUTP-labeled and used as a probe. Labeling, hybridization,washing, detection, and size determination were performedwith a nonradioactive DNA labeling and detection kit (Boehr-inger).Enzyme assays. For the determination of enzyme activi-

ties, cells were grown to the late exponential phase, washedtwice in 20 ml of 100 mM potassium phosphate buffer, pH7.6, and resuspended in 2 ml of the same buffer containing30% glycerol. Cells were disrupted by sonication (13), andafter centrifugation (30 min at 13,000 x g) the supematantwas used for the assays. The protein concentration wasdetermined by the Biuret method (15), with bovine serumalbumin as the standard. HDH was assayed essentially asdescribed previously (13) at 30°C in 1 ml of 100 mMpotassium phosphate buffer, pH 7.0, containing 0.25 mMNADPH and 5 mM aspartate semialdehyde. HK was as-sayed at 30°C in 1 ml of 100 mM HEPES (N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 7.8, con-taining 250 mM KCl, 3.3 mM ATP, 0.25 mM NADH, 5 mMphosphoenolpyruvate, 10 mM MgCl2, 10 U of pyruvatekinase, 25 U of lactate dehydrogenase, and 5 mM L-homo-serine (13). One unit of enzyme activity corresponds to theoxidation of 1 ,mol of NADPH (HDH) or NADH (HK) permin.Amino acid analysis. For the analysis of amino acid accu-

mulation in the culture fluid, aliquots of 1 ml were withdrawnand the cells were removed by centrifugation (5 min at13,000 x g). For cytosolic amino acid determinations, the C.glutamicum cells were grown to the exponential growthphase and then separated from the medium and inactivatedby the silicone oil centrifugation method (23). The actualintracellular amino acid concentrations were calculated ac-cording to the method of Schrumpf et al. (42). Amino acidswere analyzed as ortho-phthaldialdehyde derivatives byreversed-phase chromatography, as described previously(42).

RESULTS

Chromosomal integration and expression of hom-l-thrB inC. glutamicum. In order to integrate the hom-l-thrB operonas a stable copy into the genome of C. glutamicum, we usedinterspecies conjugal transfer of a mobilizable E. coli vectorwhich is nonreplicative in C. glutamicum. For the construc-tion of this vector, the hom-l-thrB operon from C. glu-tamicum DM 368-3 was isolated as a 3.7-kb SalI fragmentfrom plasmid pEK-homFBR-thrB and ligated into vectorpSUP301. The resulting plasmid pSUP-homl was trans-ferred from the mobilizing donor strain, E. coli S17-1, to theC. glutamicum WT and to the L-lysine-producing strain C.glutamicum MH20-22B. About 100 kanamycin-resistanttransconjugants of each strain were obtained. The kanamy-cin-resistant phenotype indicated cointegrate formation viahomologous recombination between the plasmid-borne hom-1-thrB fragment and the respective hom-thrB region on theC. glutamicum chromosome. Six transconjugants of eachstrain were tested and confirmed plasmid free by analyzingthe total DNA. The C. glutamicum WT transconjugantswere designated WT-DR1 to -DR6, and the C glutamicumMH20-22B transconjugants were designated MH20-22B-DR1 to -DR6.To assess the expression of hom-l-thrB in the 12 C.

glutamicum transconjugants, the specific enzyme activitiesof HDH and HK were determined (Table 1). Compared withthe parental strains, all recombinant strains, except the

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TABLE 1. Specific activity of HDH in the absence or in thepresence of 25 mM L-threonine and specific activity of HK

Sp act (U/mg of protein)

C glutamicum strain(s) HDH- ~~~HK- Threonine + Threonine

WT 0.87 0.02 0.035WT-DR1 (DR2-DR6)a 1.83 0.86 0.081WT(pWK-homl) 3.60 3.01 0.156

MH20-22B 0.88 0.02 0.030MH20-22B-DR1 (DR2, DR4-DR6)b 1.86 1.04 0.054MH20-22B-DR3 2.67 2.13 0.100MH20-22B-DR17 3.23 2.73 0.132MH20-22B(pWK-homl) 3.50 2.61 0.146

a As the specific HDH activities of C. glutamicum WT-DR1 to WT-DR6were almost identical (in the absence of [-] threonine, 1.6 to 1.9 U/mg ofprotein; in the presence of [+] threonine, 0.8 to 0.9 U/mg of protein), onlythose of one representative strain, WT-DR1, are given.

b As the specific HDH activities of C glutamicum M20-22B-DR1, -DR2,and -DR4 to -DR6 were almost identical (- threonine, 1.6 to 1.9 U/mg ofprotein; + threonine, 0.8 to 1.0 U/mg of protein), only those of onerepresentative strain, MH20-22B-DR1, are given.

strain MH20-22B-DR3, possessed the expected twofoldHDH and HK activities due to the presence of both theoriginal genomic copy of hom-thrB and the copy of hom-l-thrB introduced by vector integration. In the presence ofL-threonine (5 mM, 10 mM, or 25 mM) the HDH activity ofall recombinant strains (again, except MH20-22B-DR3) wasapproximately 50% (Table 1). This indicates full activity ofthe HDH encoded by the introduced hom-i gene and inhi-bition of the HDH encoded by the WT hom allele. Thedeviating strain MH20-22B-DR3 showed approximatelythreefold the specific HDH and HK activities of MH20-22B,and more than 70% of the HDH activity was retained in thepresence of 25 mM L-threonine (Table 1). These resultssuggested that in strain MH20-22B-DR3 the hom-l-thrBoperon was integrated twice, and this prompted us to test afurther 25 transconjugants of C. glutamicum MH20-22B forHDH activity. By this screening, we obtained strain MH20-22B-DR17, which, compared with MH20-22B, exhibitedfourfold specific HDH and HK activities, with about 80%residual HDH activity in the presence of 25 mM L-threonine(Table 1). These data suggest that with strain MH20-22B-DR17 we obtained a transconjugant expressing as many asthree copies of the hom-l-thrB operon.To relate the evidence for one, two, or three copies of

hom-l-thrB to the chromosomal organization in the respec-tive C. glutamicum strains, we analyzed genomic DNAs ofthese strains by Southern hybridization. Sall-restrictedDNA from C. glutamicum WT and WT-DR1 and from C.glutamicum MH20-22B, MH20-22B-DR1, MH20-22B-DR3,and MH20-22B-DR17 was hybridized to a digoxigenin-la-beled probe derived from the cloned hom-l-thrB operon.This probe hybridized to chromosomal DNA fragments of3.7 kb in the cases of the WT (Fig. 2A, lane 1) andMH20-22B (Fig. 2B, lane 1), corresponding to the chromo-somal organization reported for the C. glutamicum hom-thrBlocus (14). In the cases of strains WT-DR1 (Fig. 2A, lane 2)and MH20-22B-DR1 (Fig. 2B, lane 2), the probe hybridizedto fragments of about 13 kb. A size of 12.8 kb was expectedupon single integration of plasmid pSUP-homl (which is 9.1kb and contains no Sall site) into the chromosomal 3.7-kbSall fragment. For strain MH20-22B-DR3, hybridization

A B1 2 1 2 3 4

.- 23.1 kb

4- 9.4 kb

6.6 kb

4.3 kb

2.4 kb

FIG. 2. Southern blot analysis of genomic DNA from C. glu-tamicum WT (A, lane 1) and WT-DR1 (A, lane 2) and from C.glutamicum MH20-22B, MH20-22B-DR1, MH20-22B-DR3, andMH20-22B-DR17 (B, lanes 1 to 4, respectively). The chromosomalDNAs were restricted with SalI and probed with a digoxigenin-labeled hom-specific probe. Size standards are given on the right.

resulted in a signal at about 22 kb (Fig. 2B, lane 3) and forstrain MH20-22B-DR17, a signal occurred at about 30 kb(Fig. 2B, lane 4). These results were expected on twofold(two times 9.1 plus 3.7-kb) and threefold (three times 9.1 plus3.7-kb) integration of plasmid pSUP-homl in the respectivestrains. Thus, the results obtained by analysis of the chro-mosomal organization are consistent with the data obtainedby analyzing HDH and HK enzyme activities in the recom-binant C. glutamicum strains.

Expression of the hom-l-thrB operon via a low-copy-num-ber vector. In parallel with the approach via chromosomalintegration, a second approach was made to attain moderateexpression of the hom-l-thrB operon in C. glutamicum. Forthis purpose, a new C. glutamicum-E. coli shuttle vectorwith a low copy number in C. glutamicum was constructed(see Materials and Methods). This 6-kb vector, designatedpWKO and depicted in Fig. 3, has single restriction sites forBamHI, XbaI, Sall, PstI, SphI, and EcoRI replicates in C.glutamicum and E. coli, confers kanamycin resistance toboth hosts, and is mobilizable. The copy number ofpWKO inC. glutamicum was estimated relative to those of the well-known C. glutamicum-E. coli vectors pEKO and pJC1. Aftertransformation of the C. glutamicumr WT with either pWKO,pEKO, or pJC1, plasmid DNA from the same number of cells(1-ml culture of the same optical density) was isolated,restricted, and compared by agarose gel electrophoresis.The amount of pWKO proved to be approximately 1/10 thatof pEKO or pJC1. In addition, identical aliquots of the sameplasmid DNA preparations were used to transform E. coliDH5. This resulted in 40 E. coli transformants obtained withpWKO, compared with 360 and 440 transformants obtainedwith pEKO and pJC1, respectively, thus confirming the10-fold lower copy number of pWKO in C. glutamicum.By ligating the 3.7-kb Sall fragment containing hom-l-

thrB into Sall-restricted pWKO, we obtained plasmid pWK-homl. This vector was introduced into the C. glutamicumWT and MH20-22B, resulting in strains WT(pWK-homl)and MH20-22B(pWK-homl), and the HDH and HK activi-

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STABLE EXPRESSION OF hom-l-thrB IN C. GLUTAMICUM 129

BamHI

pEMI-Fragment

pNG2-Fragment

FIG. 3. Restriction map of the C. glutamicum-E. coli shuttlevector pWKO with a low copy number in C. glutamicum. Abbrevi-ations: KmR, kanamycin resistance determinant; oriV, E. coli originof vegetative replication; oriC, corynebacterial origin of replication;oriT, origin of transfer replication.

ties of the transformants were determined (Table 1). Bothrecombinant strains exhibited about fourfold higher specificactivities of both enzymes than the respective host strains,and 75 to 80% of the HDH activity was resistant to feedbackinhibition by L-threonine (Table 1). These results show thatthe plasmid-bome hom-l-thrB operon is functionally ex-pressed in both C. glutamicum strains and indicate thatpWK-homl has a copy number of three to four.Growth and stability of the recombinant- C. glutamicum

strains. Since, so far, the hom-l-thrB operon has only beenexpressed in its original host with severely reduced growthrates as a consequence (13), it was important to study thegrowth and stability of the C. glutamicum strains carryinghom-l-thrB on their chromosomes or on plasmid pWK-homl. For this purpose, the transconjugants WT-DR1,MH20-22B-DR1, MH20-22B-DR3, and MH20-22B-DR17and the transformants WT(pWK-homl) and MH20-22B(pWK-homl) were inoculated into minimal medium plusglucose and growth was followed for 30 h. Compared with

the parental strains, all recombinants showed the samedoubling times of approximately 130 min (WT derivatives)and 200 min (MH20-22B derivatives). Stability, the mostimportant characteristic of the recombinant C. glutamicumstrains, was assessed by transferring them sequentially ontocomplex medium without kanamycin for more than 70 gen-erations. Fifty single colonies from each strain were thentested for kanamycin resistance, and all were found to beresistant to the antibiotic. Furthermore, six colonies of eachstrain were also tested for HDH activity. They all exhibitedthe same specific enzyme activity and the same degree ofinhibition by L-threonine as the respective strains fromwhich they derived (data not shown). These results show thesuccessful use of chromosomal integration and of plasmidpWKO for stable expression of hom-i in C. glutamicum.Amino acid formation by recombinant C. guttamicum

strains. To study the physiological consequences of hom-l-thrB expression with respect to the carbon flux to threonineand related amino acids, the amino acid profiles of therecombinant C. glutamicum strains were determined andcompared with those of their parents. The organisms weregrown on minimal medium plus 10% glucose, and afterconsumption of the substrate, the culture fluid was analyzedfor amino acids (Table 2). C. glutamicum WT accumulated15 mM glycine, but hom-l-thrB expression from the inte-grated copy or from pWK-homl had no significant effect oneither glycine accumulation or the formation of any otheramino acid. This is probably due to the tight control of thecarbon flux by the aspartate kinase, which (in the WT strain)is inhibited by L-threonine plus L-lysine (Fig. 1). In contrast,the lysine producer C. glutamicum MH20-22B possesses anaspartate kinase which is resistant to feedback inhibition bythreonine plus lysine (41). In this strain, expression of onecopy of hom-l-thrB resulted in substantial threonine forma-tion and, according to the higher gene dose, increasing copynumbers of hom-l-thrB led to increased threonine concen-trations of up to 69 mM (Table 2). Surprisingly, with increas-ing HDH activity the recombinant MH20-22B strains alsoaccumulated up to 23 mM homoserine (Table 2), the directproduct of the HDH reaction. In addition, up to 34 mMisoleucine, which derives from threonine (Fig. 1), and up to35 mM glycine were formed (Table 2). Simultaneously, thelysine concentration in the culture fluid decreased drasticallywith increasing copy numbers of hom-J-thrB, indicating thatthreonine, homoserine, isoleucine, and glycine formationoccurred at the expense of lysine.The accumulation of homoserine, isoleucine, and glycine

in the culture fluid of the recombinant C. glutamicumMH20-22B derivatives was unexpected and prompted us to

TABLE 2. Amino acid excretion by C. glutamicum WT, MH20-22B, and their derivatives

C. glutamicum strain Amino acid concn (mM) ± SD in culture fluidb(copy no.)' Threonine Homoserine Isoleucine Glycine Lysine

WT(0)

130 REINSCHEID ET AL.

TABLE 3. Cytosolic amino acid concentrations in C. glutamicum MH20-22B and its derivatives

C. glutamicum strain Cytosolic amino acid concn (mM) ± SD'(copy no.)' Threonine Homoserine Isoleucine Glycine Lysine

MH20-22B(0) 9 + 1 4 ± 1 4 ± 2 3 ± 1 26 ± 2MH20-22B-DR1(1) 32 ± 5 31 ± 3 8 ± 2 14 ± 1 25 ± 2MH20-22B-DR3 (2) 49 ± 4 45 ± 5 4 ± 3 15 ± 3 19 ± 2MH20-22B-DR17 (3) 100 ± 14 74 ± 12 7 ± 2 13 ± 2 20 ± 2

a The copy numbers are for hom-l-thrB operons.b Values ± standard deviations were obtained from three independent cultivations by two determinations per experiment.

also determine the amino acid concentrations in the interiorof the cells. The four strains compared were again grown onminimal medium plus glucose, and the cytosolic amino acidprofiles were determined by the silicone oil centrifugationmethod. In the series investigated, the lysine and the iso-leucine concentrations were hardly influenced and theglycine concentration was moderately increased by thepresence of hom-1-thrB in the cell (Table 3). However, adrastic effect of hom-l-thrB expression was found for thethreonine and homoserine concentrations. As a function ofthe presence and copy number of hom-l-thrB, up to 100 mMthreonine accumulated in the cytosol of the recombinantMH20-22B derivatives, whereas only 9 mM was present inthe parental strain (Table 3). Similarly, the intracellularhomoserine concentration in the recombinants was up to 74mM, which is 20 times that of the parent (Table 3). Theseresults show a drastic influence of hom-l-thrB expression onmetabolite formation within the C. glutamicum cells.

DISCUSSION

Overexpression of genes coding for enzymes of biosyn-thetic pathways provides a powerful tool for influencing thecarbon flux in microorganisms. In C. glutamicum, many WTgenes encoding enzymes involved in amino acid biosynthesishave been overexpressed from plasmids without difficulty,e.g., those of lysine, threonine, and isoleucine biosynthesis,and thus, studies on carbon flux alterations have becomepossible (6-8, 13, 27, 28, 49). However, the plasmid-borneexpression of some mutant genes involved considerableproblems, e.g., in the case of the IYSCFBR gene coding for afeedback-resistant aspartate kinase (8) or in the case of homalleles coding for feedback-resistant HDHs (see the intro-duction). It should be noted that the transformation of E. coliand of B. flavum with plasmids carrying E. coli genes codingfor deregulated aspartate kinase and/or HDH also resulted ininstability of the transformants (32, 36). As reported here,we have been successful in expressing the hom-l-thrBoperon in a stable fashion in the C. glutamicum WT and in alysine hyperproducer by introducing only a limited numberof gene copies (up to three) into the cells. This was achievedby (i) chromosomal integration of a nonreplicative plasmidcontaining hom-l-thrB and (ii) the use of a newly con-structed low-copy-number shuttle vector. The chromosomalintegration technique, only recently established for C. glu-tamicum (24, 39, 43), resulted mainly in strains carrying onecopy of the vector and thus of hom-J-thrB. However, asevidenced by Southern hybridization and enzymatic analy-sis, we also found C. glutamicum clones carrying two orthree copies of the vector integrated at the same site. Anamplification of integrated vectors or of resistance genes hasbeen described previously for several gram-positive bacteriaincluding C. glutamicum (20, 24, 33, 50). Because of the

higher copy number of the respective resistance gene, suchclones show enhanced antibiotic resistance and, in fact, itwas reported for B. subtilis that it is possible to screendirectly for gene amplification by the use of increasingantibiotic concentrations as selective pressure (20, 50).Mechanistically, the amplification might be brought about byhomologous recombination of DNA sequences bracketed bydirect sequence repeats (51). Another explanation for the C.glutamicum derivatives with two or three copies of thevector is that the donor plasmid population contained dimersand trimers, as well as monomers, and that these multimericplasmids were integrated. In addition to the graduated ex-pression of hom-l-thrB via chromosomal integration, weobtained stable and moderate overexpression of this operonby using the low-copy-number shuttle vector pWKO. Thisvector might be useful not only for moderate overexpressionof a given gene but also, in particular, for the isolation of C.glutamicum genes which are detrimental when expressedtoo highly in this host, e.g., genes encoding transport ormembrane proteins. Thus, compared with high-level expres-sion with conventional plasmids, the use of chromosomalintegration techniques and of the low-copy-number vectoropens new horizons for studying and directing the metaboliteflux in C. glutamicum.Most C. glutamicum mutants possessing an HDH insen-

sitive to feedback inhibition by threonine were obtained onlyby repeated rounds of mutagenesis and subsequent strongselection pressure with the threonine analog a-amino-1-hydroxyvaleric acid (1, 21, 46). This and the failure to clonehom-2-thrB in C. glutamicum led to the hypothesis thatmutants with deregulated HDH require further mutationswithin linked biochemical pathways to counteract the meta-bolic effect of deregulation (1). Since in our experimentshom-l-thrB was introduced and expressed in the C. glu-tamicum WT without any obvious negative effect on growthor metabolism, the hypothesis of additional mutations as aprerequisite for expression of hom-l-thrB can be ruled out.On the basis of studies with threonine producers obtained byrandom mutagenesis, it has also been suggested that aderegulated HDH alone leads to threonine production incoryneform bacteria (45). However, since expression ofhom-l-thrB in the C. glutamicum WT did not result in anexternal accumulation of threonine, it is obvious that aderegulated HDH is not the only prerequisite for threonineproduction. On the basis of our studies, we think that theminimal requirement for this purpose is the deregulation ofboth HDH and aspartate kinase.The expression of only one copy of the hom-l-thrB

operon in the strain with deregulated aspartate kinase led tomassive threonine accumulation in the medium. This showsthat the carbon flux to threonine is tightly controlled byHDH and/or HK. However, the stepwise increase of hom-1-thrB expression did not correlate with a linear increase of

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STABLE EXPRESSION OF hom-l-thrB IN C. GLUTAMICUM 131

the amount of threonine excreted. This suggests an as yetunknown limitation of threonine production by C. glu-tamicum. Further information was obtained by determiningthe intracellular amino acid concentrations which, in con-trast to external amino acid accumulation, reflect the steady-state situation within the recombinant cells. It is striking thatwith increasing expression of hom-l-thrB the intracellularthreonine concentration rose to values which were signifi-cantly higher than the external threonine concentration inthe culture fluid. The high intracellular threonine accumula-tion in the recombinant strains indicates that the efflux ofthreonine from the cells into the medium is limiting. It is notyet known whether active threonine export systems exist inbacteria and it is conceivable that simple diffusion and/or carrier-mediated export are responsible for the efflux.Knowledge of bacterial amino acid export systems is limited,and active export systems for lysine, isoleucine, and gluta-mate have been only recently described for C. glutamicum(4, 9, 16, 18).

In parallel with the increase of intracellular threonine, theintracellular homoserine concentration in the recombinantC. glutamicum MH20-22B strains also rose drastically withincrease in the copy number of hom-1-thrB. The highintracellular homoserine accumulation is most probablybrought about by the inhibition of HK at higher threonineconcentrations. HK was reported to be inhibited half-maxi-mally at about 25 mM threonine (31). This concentration isregarded as unphysiological under normal conditions, but itwas exceeded in all C. glutamicum strains expressing one ormore copies of the hom-l-thrB operon.Another consequence of hom-l-thrB expression in C.

glutamicum MH20-22B was a significant accumulation ofisoleucine and glycine in the culture medium. Isoleucine issynthesized from threonine in a sequence of five reactions.Threonine dehydratase and acetohydroxy acid synthase, thefirst two enzymes involved, are feedback-inhibited by iso-leucine (10, 30). Obviously, this regulation did not preventan increased carbon flux towards isoleucine in our recombi-nant strains. This can be explained by the fact that theintracellular isoleucine concentration in the recombinantstrains remained as low as that in the parental strain and thusboth enzymes were probably not inhibited in their activity.The low intracellular isoleucine concentration and the exter-nal accumulation of isoleucine indicate that this amino acidis rapidly excreted by the efflux carrier system previouslydescribed by Ebbighausen et al. (9). The increased glycineformation by the recombinant C. glutamicum MH20-22Bstrains is probably due to the degradation of accumulatedthreonine via threonine dehydrogenase and 2-amino-3-ox-obutyrate-CoA ligase. Both enzymes have been shown to bepresent as threonine-inducible enzymes in corynebacteria(2).Apart from the analysis of our recombinant C. glutamicum

strains, it is tempting to speculate why the homologouscloning and expression of hom-l-thrB on vectors like pEKOor pJC1 causes problems. Although we do not rule out otherpossibilities, in our opinion the most likely reason is that ahigh activity of feedback-resistant HDH leads to abnormallyhigh and toxic levels of pathway intermediates or derivativesthereof. Moderate expression has already led to high internalconcentrations of threonine and homoserine, and from thestepwise upmodulation of hom-l-thrB expression, it can beextrapolated that the intracellular concentrations of bothamino acids would be tremendous if the hom-l-thrB genedose were further increased. High intracellular threoninecould lead to an accumulation of 2-oxobutyrate, which is the

product of the threonine dehydratase reaction and has beenshown to be toxic (48). Furthermore, assuming that thereactions from homoserine to threonine are in equilibrium, itis likely that the intracellular concentration of homoserinephosphate is also highly elevated and excessive accumula-tion of organophosphates has been discussed previously asinhibitory to the growth of bacteria (19).

ACKNOWLEDGMENTS

We are grateful to H. Cichorius and J. Engelmann for theirexcellent technical assistance, S. Peters for preparing the photos,and M. Heinz and J. Carter-Sigglow for critical reading of themanuscript.

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