Fermentative Production of L-Alanyl-L-Glutamine by a Metabolically Engineered Escherichia coli Strain Expressing L-Amino Acid {alpha}-Ligase

In spite of its clinical and nutritional importance, L-alanyl-L-glutamine(Ala-Gln) has not been widely used due to the absence of anefficient manufacturing method. Here, we present a novel methodfor the fermentative production of Ala-Gln using an Escherichiacoli strain expressing L-amino acid ${alpha}$ -ligase (Lal), which catalyzesthe formation of dipeptides by combining two amino acids inan ATP-dependent manner. Two metabolic manipulations were necessaryfor the production of Ala-Gln: reduction of dipeptide-degradingactivity by combinatorial disruption of the dpp and pep genesand enhancement of the supply of substrate amino acids by deregulationof glutamine biosynthesis and overexpression of heterologousL-alanine dehydrogenase (Ald). Since expression of Lal was foundto hamper cell growth, it was controlled using a stationary-phase-specificpromoter. The final strain constructed was designated JKYPQ3(pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (laland ald expressed under the control of the uspA promoter) orpPE177 (lal and ald expressed under the control of the rpoHpromoter). Either strain produced more than 100 mM Ala-Gln extracellularly,in fed-batch cultivation on glucose-ammonium salt medium, withoutadded alanine and glutamine. Because of the characteristicsof Lal, no longer peptides (such as tripeptides) or dipeptidescontaining D-amino acids were formed.

INTRODUCTION

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INTRODUCTION

Glutamine (Gln) is the most abundant free-form amino acid inhuman cells. Gln is not an essential amino acid for humans,but it is considered to be a conditionally essential amino acidbecause of its central role in nitrogen metabolism (7, 40).In spite of such nutritional importance, Gln has hardly beenused as a component of parenteral nutrition due to its low solubilityand instability in solution. One approach to overcome the physicochemicallimitations of Gln is to supply the amino acid as a dipeptideby conjugation with other amino acids. Alanyl-glutamine (Ala-Gln)is the most suitable Gln-containing dipeptide. Since there havebeen a number of reports on the safety and effectiveness ofthis dipeptide (2, 7), the clinical and nutritional importanceof Ala-Gln has already been established.

Several methods for producing Ala-Gln have been described; chemicalor enzymatic condensation of protected alanine (Ala) and Gln(3, 22, 24, 43) and a chemical synthetic process via D-2-chloropropionyl-glutamine(32). These methods, however, have not been satisfactory inregard to cost-effectiveness and quality. Most methods needprotection steps and sometimes deprotection steps, loweringproduction efficiency (8). The protecting reactions also triggerracemization of the substrate amino acid(s), which results inthe formation of an undesired by-product, D-alanyl-glutamine.These chemical and enzymatic methods have also been suggestedto form other by-products, such as different dipeptides (forexample, alanyl-glutamic acid) or longer oligopeptides (forexample, tripeptides) (32, 43).

Recently, we identified a new enzyme named L-amino acid ${alpha}$ -ligase(Lal) in Bacillus subtilis (38). This enzyme catalyzes dipeptidesynthesis from unprotected amino acids in an ATP-dependent manner.The substrate specificity was so wide that it could synthesize44 different dipeptides, including Ala-Gln and alanyl-alanine(Ala-Ala), but not glutaminyl-alanine and alanyl-glutamic acid.Lal never formed tripeptides or longer peptides and did notreact with D-amino acids. These characteristics prompted usto explore the possibility of producing Ala-Gln using an Escherichiacoli strain expressing Lal directly from glucose and ammonia,identical to amino acid fermentation.

However, the dipeptide-degrading activity of the cells, whichis apparently necessary for protein turnover and homeostasis(20), had to be taken into consideration; E. coli possessesmore than 40 peptide bond-breaking activities (20). Four peptidases,encoded by pepN, pepA, pepB, and pepD, have been reported todegrade a broad spectrum of dipeptides (16, 20). Unfortunately,it is unclear to what extent these peptidases contribute tothe total dipeptide degradation in the cell, although disruptionof the peptidases made the strain unable to grow on certaindipeptides (21). Another consideration when using E. coli expressingLal is the low affinity of Lal for Gln. The K_m of Lal for Glnhas been reported to be 107 mM (38), whereas the intracellularconcentration of Gln in E. coli under normal conditions hasbeen reported to be around 1.2 mM (29), implying that the enzymeworks poorly in vivo.

In the present report, we show how to overcome the above-mentionedcomplications and demonstrate a novel fermentative process forthe production of Ala-Gln.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Microorganisms, plasmids, and media.
The E. coli strains and plasmids used in this study are listedin Table 1. Strain NM522 was used for plasmid construction.For test tube cultivation, 0.8 ml of an overnight culture inLB medium (10 g/liter Bacto tryptone, 5 g/liter yeast extract,and 5 g/liter NaCl) was transferred into a test tube with 8ml of TT medium [10 g/liter glucose, 16 g/liter K₂HPO₄, 14 g/literKH₂PO₄, 2 g/liter (NH₄)₂SO₄, 5 g/liter Casamino acid, 1 g/litercitric acid, 2 g/liter MgSO₄·7H₂O, 50 mg FeSO₄·7H₂O,10 mg/liter MnSO₄·7H₂O, and 10 mg/liter thiamine HCl]and vigorously shaken (220 rpm) for 24 h at 30°C. To maintainthe plasmid(s), 100 mg/liter of kanamycin and/or 25 mg/literof chloramphenicol was added and 0.2 g/liter of proline (Pro)was provided for the proline auxotroph strain. For the amino-acidfeeding experiment, 2.5 g/liter each of Ala and Gln was addedinitially. For cultivation in a jar fermentor, 100 ml of LBmedium with kanamycin cultured in a 1-liter Erlenmeyer flaskat 30°C for 16 h was transferred into a 5-liter jar fermentor(Mitsuwa Rikagaku Kogyo, Japan) containing 1.9 liters of J medium(10 g/liter glucose, 6 g/liter Na₂HPO₄, 3 g/liter KH₂PO₄, 5g/liter NaCl, 1 g/liter NH₄Cl, 5 g/liter yeast extract, 10 mg/literMnSO₄·7H₂O, 2 g/liter MgSO₄·7H₂O, 200 mg/literFeSO₄·7H₂O, and 10 mg/liter thiamine HCl) with kanamycinand Pro (0.3 g/liter) and cultivated at 30°C with aeration(2 liter/min) and agitation (600 rpm). When the initial glucosewas depleted, continuous feeding of a glucose solution (600g/liter) was begun. One liter of the glucose solution was suppliedat a controlled rate to keep the glucose concentration of theculture medium at 30 ± 10 g/liter. During cultivation,the pH was kept at 7.0 with 28% (vol/vol) NH₄OH. At each timepoint indicated (see Fig. 4), a sample (5 ml) was taken anda portion (1 ml) was used to measure the optical density at660 nm. The rest of the sample was centrifuged immediately toget a supernatant, which was used for the assay of glucose,amino acids, and dipeptides concentrations. The resting-cellreaction for measuring the Ala-Gln degradation rate was carriedout as follows. A test tube with LB medium from the overnightculture was centrifuged, and the cells were washed twice withice-cold 50 mM KH₂PO₄ (pH 7.2). The cells were suspended inthe same buffer at an optical density of 4 (660 nm). Two millilitersof cell suspension was mixed with 0.1 ml of Ala-Gln solution(100 g/liter); part of the sample (0.1 ml) was taken as timepoint zero, and the rest was incubated at 30°C with vigorousshaking. Samples (0.1 ml) were taken every 3 h and centrifuged.The Ala-Gln concentrations of the supernatants were assayedas described in "Analytical procedures" below. As the degradationrates were almost constant during the first 9 h, the averagevalue during this period was taken as the degradation rate.

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TABLE 1. Strains and plasmids used in this study

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FIG. 4. Time course of dipeptide fermentation. JKYPQ2/pPE86 (a), JKYPQ3/pPE167 (b), or JKYPQ3/pPE177 (c) was cultivated with J medium in a fed-batch manner. Cell growth (x) and the amounts of Ala ( ${diamond}$ ), Gln ( ${square}$ ), Ala-Gln (•), and Ala-Ala ( ${blacktriangleup}$ ) that accumulated in the cultivation medium were measured. Each strain was cultivated at least three times, and representative results are shown.

DNA manipulation.
DNA manipulations were basically performed according to themethod of Sambrook et al. (31). The primers used in this studyare listed in Table 2. The pTK31 plasmid, a derivative of pTrS30(23) containing the replication origin of pBR322, a kanamycinresistance gene from pUC4K (Pharmacia Biotech), and the tryptophanpromoter from E. coli K-12 was used as an expression vector(see the supplemental material). The lal gene was amplifiedfrom pQE60ywfE (38) by PCR to be flanked by an XhoI site anda BamHI site. The trp promoter region was amplified from pTrS30by PCR to be flanked by an EcoRI site and an XhoI site. The1.4-kb lal gene PCR product was digested with NcoI and BamHI,and the 0.4-kb trp promoter PCR product was digested with EcoRIand XhoI and inserted between the EcoRI and BamHI sites of pTrS30.The resulting plasmid, designed to express lal under the controlof the trp promoter, was named pPE56. The L-alanine dehydrogenase(ald) gene was amplified from the B. subtilis (ATCC 15245) genomeby PCR to introduce BamHI sites at both termini. The 1.2-kbald gene PCR product was digested with BamHI and inserted betweenthe BamHI sites of pPE56 in the same direction as lal to yieldpPE86. The ribosome-binding site (RBS) of pQE-60 (QIAGEN, Germany)was amplified by PCR to be flanked by a ClaI site and a SphIsite. The amplified 0.3-kb RBS region was digested with ClaIand SphI and inserted between the ClaI and SphI sites of pTK31to yield pTKQE31. The uspA promoter region was amplified fromthe E. coli W3110 genome by PCR to be flanked by an EcoRI siteand a ClaI site. The rpoH promoter region was amplified fromthe E. coli W3110 genome by PCR to be flanked by an EcoRI siteand a ClaI site. The 0.4-kb PCR product of the uspA promoterregion was digested with EcoRI and ClaI and inserted betweenthe EcoRI and ClaI sites of pTKQE31 to yield pUATKQE31. The0.4-kb rpoH promoter region amplified by PCR was similarly insertedinto pTKQE31 to yield pRHTKQE31. The uspA promoter and RBS regionwere cut from pUAKQE31 with EcoRI and NcoI and inserted betweenthe EcoRI and NcoI sites of pPE86. In the resulting plasmid,pPE167, the lal and ald genes were downstream of the uspA promoter-RBSregion. A similar construct, but with the rpoH promoter andthe RBS region of pRHTKQE31 instead of the uspA-RBS region,was named pPE177.

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TABLE 2. Primers

Gene disruptions were performed based on the phage lambda Redrecombinase system (5). pKD46, pKD3, pKD13, and pCP20 were obtainedfrom the E. coli Genetic Stock Center. The primer sets usedfor disruption are listed in Table 2. The 1.0-kb FLP recombinationtarget-flanked kanamycin or chloramphenicol resistance genewas introduced into the strain carrying the Red helper plasmid(pKD46) by electroporation. Following PCR to check for correctintegration, the kanamycin or chloramphenicol resistance genewas eliminated by introducing a FLP recombinase expression plasmid(pCP20).

Analytical procedures.
Bacterial growth was expressed as the optical density at 660nm after dilution with distilled water. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) of proteins was carried out accordingto the method of Laemmli (15). The glucose concentration wasmeasured using Detaminar GL-E (Kyowa Medex, Japan). Dipeptidesand amino acids were derivatized using 9-fluorenylmethoxy carbonylchloroformate and measured by high-performance liquid chromatographyas described previously (38), except that the gradient programwas slightly modified as follows: 0 to 2 min, solvent A-solventB at 75:25; 2 to 26 min, a linear increase in solvent B to A-Bat 55:45; 26 to 28 min, a linear increase to A-B at 5:95; 28to 33 min, held at A-B at 5:95; 33 to 35 min, a linear decreasein solvent B to A-B at 75:25. Intracellular amino acids or dipeptideswere extracted with ice-cold ethanol as previously described(29). For determination of the dry weight of cells, 5-ml ofculture was filtered through a 0.45-µm-pore-size filter(diameter, 47 mm), the filter was washed with 10 ml of waterand dried at 105°C to a constant weight, and the mean bacterialdry weight was calculated to be 0.33 mg ml^–1 A₆₆₀ unit^–1for the TT medium culture. The intracellular volume of the cellwas calculated based on 1.6 µl mg dry cell weight^–1(29). Data are expressed as the mean values of at least threeindependent experiments, except for one figure that shows representativedata (see Fig. 4).

RESULTS

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RESULTS

Lal expression in a strain with the wild-type background.
We first examined whether the expression of Lal in a wild-typeE. coli strain evoked Ala-Gln dipeptide accumulation. An E.coli strain expressing Lal under the control of the trp promoterwas constructed (JM101/pPE56). Expression of the enzyme wasconfirmed by SDS-PAGE analysis of cell extracts (Fig. 1). Whenthe strain was cultivated in TT medium in a test tube, no dipeptide(either Ala-Gln or other dipeptides) was detected in the cultivationfluid. Inside the cells, only Ala-Ala was detected (Table 3).Since the JM101 host strain alone never accumulated dipeptides(Table 3), the occurrence of Ala-Ala in the JM101/pPE56 cellssuggested the expression of active Lal in the strain. This viewwas supported by the fact that JM101 expressing His-tagged Lalalso accumulated Ala-Ala intracellularly, and furthermore, bythe fact that His-tagged Lal purified from cells using a nickelcolumn confirmed activity (data not shown).

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FIG. 1. Confirmation of Lal and Ald expression by SDS-PAGE analysis. Shown are crude cell extracts of JM101/pTK31 (lane 1), JM101/pPE56 (lane 2), and JM101/pPE86 (lane 3). Lane M, marker. The arrows show the expected band positions of Lal (52.3 kDa) and Ald (39.7 kDa).

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TABLE 3. Impact of enhancement of amino acid biosynthesis on the accumulation of amino acids and dipeptides

The observation that Ala-Gln was not detected inside or outsidethe cells, despite the presence of an active Lal, prompted usto investigate what obstructed the formation of Ala-Gln.

Influence of peptide-degrading activities.
To examine whether inactivation of one or a few dipeptidasesand aminopeptidases encoded by the pepD, pepN, pepA, and pepBgenes exerted a positive effect on accumulation of the Ala-Glndipeptide, single, double, or triple disruptants of pepD, pepN,pepA, and pepB were constructed. Each strain was incubated withAla-Gln, and the extracellular concentration of Ala-Gln wasmeasured periodically to assess the Ala-Gln degradation rate.

As shown in Fig. 2a, single disruption of each gene slightlyreduced the rate of degradation of Ala-Gln, except for the pepNdisruptant (JKYP4, bar 2), which showed degrading activity identicalto that of the parental strain (JM101, bar 1). The degradationrate of the ${Delta}$ pepD ${Delta}$ pepN strains was almost the same as that ofthe ${Delta}$ pepD strain (JKYP3, bar 3), supporting the hypothesis thatPepN hardly contributes to Ala-Gln degradation. On the otherhand, a combination of ${Delta}$ pepD, ${Delta}$ pepN, and ${Delta}$ pepB (JKYP7, bar 8) reducedthe degradation rate. It should be noted that the growth rateof the double or triple disruptant (JKYP6 or JKYP7) was slightlylower than that of the wild-type strain, JM101 (data not shown).

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FIG. 2. Influence of disruption of the dpp or pep gene on the Ala-Gln degradation rate (a) and extracellular dipeptide accumulation (b). (a) Each strain was incubated with Ala-Gln, and the rate of Ala-Gln degradation was measured. (b) The same strain containing pPE56 was cultivated in a test tube with TT medium supplemented with Ala and Gln for 24 h. The amounts of Ala-Ala (empty bars) and Ala-Gln (filled bars) that accumulated in the medium (extracellularly) were assayed. The relevant genotype of the strain shown in panel a, which was used as the host strain in panel b, is indicated at the bottom. The open triangles show the genes disrupted. The strains employed were JM101 (column 1), JKYP4 (column 2), JKYP1 (column 3), JKYP3 (column 4), JKYP2 (column 5), JKYP5 (column 6), JKYP6 (column 7), JKYP7 (column 8), JKYP8 (column 9), JKYP9 (column 10), JKYP10 (column 11), and JKYP11 (column 12). While each value is the mean of at least three experiments, standard deviations are not shown, since each measurement was within 10% of the mean value.

It has been demonstrated that E. coli imports dipeptides viaa transport system coded for by the dpp operon (1). The influenceof inactivation of this system was examined (Fig. 2a). Disruptionof dpp alone (JKYP5, bar 6) showed an effect similar to disruptionof pepD. However, the combination of ${Delta}$ dpp and ${Delta}$ pepD exerted asynergistic effect rather than an additive effect (JKYP8, bar9). The degradation rate was further reduced and was six timeslower than the wild-type level when pepA disruption was introducedinto the ${Delta}$ pepD ${Delta}$ pepN ${Delta}$ dpp strain (JKYP10, bar 11).

Since a reduction in degradation activity toward externallyadded Ala-Gln was confirmed, the effect of the above-describedgenetic manipulation on internally synthesized Ala-Gln was investigated.The Lal-expressing plasmid pPE56 was introduced into each mutantstrain and cultivated in a test tube in the presence of Alaand Gln. After 24 h of cultivation, the amounts of dipeptidesaccumulated extracellularly were assayed (Fig. 2b). Althoughnegligible amounts were detected in strains with the wild-typebackground, when each single mutation was conferred, small butsignificant amounts of dipeptides (Ala-Gln and Ala-Ala) appeared.More extensive disruption of peptidases was associated withgreater accumulation of dipeptides. Simultaneous disruptionof the dpp and pep genes boosted this accumulation. These resultsalmost correlated with those of the experiments in which Ala-Glnwas added to E. coli strains (Fig. 2a). The differences werethe effect of ${Delta}$ pepN and the degree of synergy between the disruptionsof the dpp and pep genes. While the reason for these differencesis not clear at present, the reduction in dipeptide-degradingactivity by combinatorial inactivation of the dpp and pep geneswas effective at increasing the production of Ala-Gln.

Effectiveness of enhancement of the amino acid supply.
One possibility for Lal to efficiently synthesize dipeptidesin vivo is to elevate the intracellular concentration of thesubstrate amino acid(s) to levels high enough for Lal activity.This can be achieved by increasing the metabolic flux towardsubstrate amino acids. In E. coli, Gln biosynthesis is knownto be negatively regulated by GlnE and GlnB (18, 28); therefore,we performed an experiment in which regulation of Gln biosynthesiswas attenuated by constructing a strain from JM101 by disruptingthe glnE and glnB genes. The glnE-glnB-deficient strain, JKYQ1,was transformed with pPE56, a Lal expression plasmid, and cultivatedin the absence of Ala and Gln. As shown in Table 3, the glnE-glnBdeficiency resulted in an increase in intracellular Gln andexcretion of the amino acid, confirming that the genetic manipulationderegulated Gln biosynthesis. On this background, expressionof Lal resulted in the intracellular appearance of Ala-Gln,although the amount of the dipepide detected in cells was smalland variable (Table 3).

Whether simultaneous elevation of Ala and Gln levels boostedAla-Gln production was further assessed. To enhance Ala synthesis,L-alanine dehydrogenase from B. subtilis (34) was used. A plasmidcoexpressing the gene (ald) coding for L-alanine dehydrogenaseand Lal was constructed (pPE86). Expression of Ald and Lal wasconfirmed by SDS-PAGE analysis (Fig. 1). The intracellular concentrationsof Ala and Ala-Ala were increased by the additional expressionof Ald (compare JM101/pPE56 and JM101/pPE86 or JKYQ1/pPE56 andJKYQ1/pPR86 in Table 3), confirming the contribution of Aldto Ala synthesis. Corresponding to the elevation in intracellularconcentrations of Ala and Gln, the amount of Ala-Gln that accumulatedin cells increased severalfold (Table 3). It should be notedthat Ala-Gln was detected even outside JKYQ1/pPE86 cells. Theseresults suggest that a simultaneous increase in the intracellularlevels of both substrate amino acids made it possible for cellswith Lal activity to synthesize the dipeptide faster than therate of degradation of the dipeptide.

Construction of an Ala-Gln-producing strain.
As described above, the reduction in dipeptide-degrading activitiescaused by a combinatorial disruption of the dpp and pep genesand elevation of intracellular concentrations of the substrateamino acids was found to have a large impact on Ala-Gln production.Thus, these two strategies were integrated by constructing astrain with higher productivity of Ala-Gln.

The starting strain was the pepD-, pepN-, and dpp-deficientmutant JKYP9. This strain did not accumulate Gln in the cultivationmedium, but as in strain JM101, disruption of glnE and glnB(JKYPQ1) conferred Gln overproductivity (Fig. 3b, bar 4). Onthis genetic background (pepD pepN dpp glnE glnB), expressionof Lal alone (JKYPQ1/pPE56) triggered extracellular accumulationof a significant amount of Ala-Gln (Fig. 3a, bar 5). Coexpressionof Ald and Lal almost doubled the Ala-Gln titer (JKYPQ1/pPE86,bar 6). The fact that the amount of accumulated dipeptide inJKYPQ1/pPE86 was 50-fold higher than in JKYQ1/pPE86 clearlyindicates the importance of limiting dipeptide-degrading activity.

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FIG. 3. Influences of metabolic manipulations on the production of Ala-Gln, amino acids, and cell growth. Each strain was cultivated in a test tube with TT medium for 24 h. The amounts of Ala-Gln (a), Ala (b, empty bars), and Gln (b, filled bars) that accumulated in the medium (extracellularly) and of cell growth (c) were measured. Ala-Ala was not detected in the medium. The relevant genotype of the strain is indicated at the bottom. +, overexpression of Lal or Ald; +*, Lal expression under the control of the uspA promoter; ${triangleup}$ , disruption of the gene(s). The strains employed were JM101 (column 1), JKYQ1 (column 2), JKYQ1/pPE86 (column 3), JKYPQ1 (column 4), JKYPQ1/pPE56 (column 5), JKYPQ1/pPE86 (column 6), JKYPQ2/pPE86 (column 7), JKYPQ2/pPE167 (column 8), and JKYPQ3/pPE167 (column 9). While each value is the mean of at least three experiments, standard deviations are not shown, since each measurement was within 10% of the mean value.

The pepA gene was further deleted from JKYPQ1. The putA genewas also disrupted to reduce the amount of proline, which wasrequired by the pepD-disrupted strain in our experiments. Bythe additional disruption of pepA (JKYPQ2/pPE86, bar 7) theAla-Gln titer was raised, but cell growth was greatly impaired(Fig. 3c). The growth rate remained low even when the strainwas cultivated in a jar fermentor under sufficient aerationand pH-controlled conditions (Fig. 4a). The Ala-Gln titer was7.4 mM after 47 h of cultivation.

Since the expression of Lal apparently exerted a negative effecton cell growth (Fig. 3c), separating the production period fromthe growth period could improve both growth and productivity.For this purpose, Lal was expressed under the control of therpoH or uspA promoter, since expression of these genes has beenreported to be elevated in the stationary phase (9, 25). Thismodification of Lal expression yielded a small increase in growth,an increase in the amount of Gln produced, and a slight decreasein the Ala-Gln titer (Fig. 3, column 8), suggesting an alleviatedgrowth-inhibitory effect of Lal.

Thus, we constructed a host strain containing all of the usefulgenotypes described above by introducing a pepB disruption intothe KYPQ2 strain. The resultant strain was designated KYPQ3(pepA pepB pepD pepN dpp glnE glnB putA). To confirm phase transitionduring fermentation, the final strains (JKYPQ3/pPE167 and JKYPQ3/pPE177)were cultured in a jar fermentor in a fed-batch manner (Fig.4b and c). In both cases, the growth rates were higher thanthat of JKYPQ2/pPE86, and when cells entered the stationaryphase as a result of consumption of proline, which was required,Ala-Gln rapidly accumulated in the medium. After 47 h of cultivation,the extracellular concentrations of Ala-Gln and Ala-Ala reached100.3 mM (24.7 g/liter) and 26.2 mM (4.2 g/liter), respectively,in JKYPQ3/pPE167 and 100.1 mM (24.6 g/liter) and 26.8 mM (4.3g/liter), respectively, in JKYPQ3/pPE177. Molar yields of Ala-Glnagainst consumed glucose were 11.5% and 11.6% for JKYPQ3/pPE167and JKYPQ3/pPE177, respectively. No other dipeptides, includingD-amino-acid-containing dipeptides or longer oligopeptides,were detected in the cultivation medium (Fig. 5). These resultsclearly demonstrate that separating the production phase fromthe growth phase is effective at increasing the production ofAla-Gln.

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FIG. 5. High-performance liquid chromatography analysis of the fermentation medium. (a) Authentic samples of Ala, Gln, glutamic acid (Glu), Ala-Gln, and Ala-Ala. (b) The supernatant from 47-h cultivation of JKYQ3/pPE167. The peaks indicated by asterisks were derived from 9-fluorenylmethoxy carbonyl reagent.

DISCUSSION

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DISCUSSION

In this report, we demonstrate that the dipeptide Ala-Gln canbe produced through a fermentative process with Lal in a geneticallyengineered E. coli strain. This fermentative process never formslonger oligopeptides or dipeptides containing D-amino acid(s)and does not need any protection or deprotection steps or additionof substrate amino acids. The only dipeptide by-product, Ala-Ala,can be easily separated from Ala-Gln by chromatography or crystallization(unpublished observation). Therefore, Ala-Gln fermentation seemsto be very advantageous in regard to quality and cost-effectivenesscompared with other Ala-Gln-manufacturing methods.

There have been many reports on dipeptides as microbial metabolites(11, 12, 14, 17, 27, 30, 39), but all of them except one (14)concern dipeptides containing an unusual amino acid(s), suchas bacilycin (30) or alahopcin (11, 12). In contrast to thenumber of reports on the occurrence of dipeptides in nature,their biosynthesis has rarely been studied. Only bacilycin synthesisin B. subtilis has been studied to some extent; it relies onthe bac operon, which involves bacD (a synonym for lal) (36),and expression of the bac operon is regulated by guanosine 3'diphosphate 5' diphosphate (13). It was also reported that Salmonellaenterica serovar Typhimurium lacking peptidases N, A, B, andD accumulated a heterogeneous mixture of small peptides (42).

While Lal activity is indispensable for Ala-Gln fermentation,two metabolic manipulations (reduction of dipeptide degradationand elevation of intracellular levels of the substrate aminoacids) were shown to be essential. Since it is impossible toinactivate all dipeptide-degrading activities, the combinationof the two metabolic manipulations is apparently necessary forAla-Gln production. It is worth noting that the necessity forincreasing the amino acid supply had an unexpected benefit,since it prevented the formation of other dipeptides despitethe wide substrate specificity of Lal.

Peptidases had a cumulative effect on the Ala-Gln breakdown(Fig. 3), as expected from their overlapping substrate specificities.Despite a long history of peptidase research, the contributionof each peptidase to the overall peptide-degrading activityof the cell is still largely unknown. It was reported that PepA,-B, -D, and -N have cysteinylglycine-hydrolyzing activity inE. coli (37). PepA, -B, -D, and -N have been shown to participatein protein breakdown in S. enterica serovar Typhimurium (41).On the other hand, Chandu and Nandi reported that PepN was exclusivelyresponsible for the breakdown of several chromogenic peptidasesubstrates (4), implying that its contribution to hydrolysiswould differ for each peptide.

Ala-Gln and Ala-Ala accumulated inside JKYQ/pPE86 cells, whereasonly Ala-Gln was detected extracellularly (Table 3). This unbalanceddistribution could be attributed to preferences in the dipeptideuptake system. DppA, the periplasmic substrate binding proteinof the Dpp system, is known to prefer Ala-Ala over Ala-Gln (35).Other peptide uptake systems, Opp (6) and/or Tpp (26), mightalso be involved. In addition to these possibilities, completelyunknown processes that release dipeptides into extracellularspaces could also be involved.

One unexpected finding of the current study was the growth-inhibitoryeffect of Lal. This effect seemed to be more severe when Lalexpression was combined with pep disruption. It was previouslyreported that a pepA, -B, -D, and -N-deficient mutant grew moreslowly than the wild-type strain (10). Certain small peptides,such as leucine-containing peptides, have been reported to inhibitE. coli growth (19). Taken together, these data indicate thatdipeptides accumulating in cells due to peptidase deficiencymay inhibit cell growth. However, at present, we cannot specifythe dipeptides that cause this growth inhibition, since exogenouslyadded Ala-Gln or Ala-Ala had no effect on growth (unpublishedobservation) and we could not find any other peptide accumulatingin the cell.

Considering the wide substrate specificity of Lal and a relatedenzyme, D-alanine-D-alanine ligase (33), other dipeptides couldbe produced in a manner similar to that presented here. To explorethese possibilities, more precise studies of the intracellulardegradation and synthesis rates and the transport systems ofeach dipeptide are needed.

ACKNOWLEDGMENTS

We thank Aya Kubota-Hada, Reiko Kohda, and Mayumi Fukano fortheir technical assistance. We also thank Akio Ozaki and YoshiyukiYonetani for helpful discussions.

FOOTNOTES

* Corresponding author. Mailing address: Technical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 1-1 Kyowa-cho, Hofu-shi, 747-8522 Yamaguchi, Japan. Phone: 81-835-22-2518. Fax: 81-835-22-2466. E-mail: shashimoto{at}kyowa.co.jp

Published ahead of print on 24 August 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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