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The world market for amino acids amounts to about US$3 billion.More than half of theL-amino acids (Ala, Asn, Gln, His, Ile, Leu,Met, Pro, Ser, Tyr, and Val) are produced in the range of 1,000tons/year with an average price of $50/kg1. With the sole exception ofmethionine, the L-form of amino acids is required for most foodindustry and pharmaceutical applications1. L-alanine is currentlyused as a food sweetener13 and for pharmaceutical applications inwhich it is incorporated together wi th several otherL-amino acids instandard infusions for parenteral administration in clinical pre- andpostoperative nutr it ion therapy1.

A number of microorganisms (i.e.,Corynebacterium gelat inosum,Arthrobacter oxydans, Brevibacterium lactofermentum, Clostr idi umsp. P2, and Pyrococcus fur iosus) are able to produce alanine fromsugar fermentation1,47. However, the maximal sugar conversion rateis 5060%, and a mixture of both isomers is generally obtained dueto the presence of an alanine racemase, which is required forD-ala-nine production for cell wall biosynthesis1. An industrial process hasbeen developed for the exclusive production ofL-alanine through L-aspartate decarboxylation1,8. This process uses immobilized cells orsuspensions ofPseudomonas dacunhaeas biocatalysts with a yield

>90%1,8. A metabolic engineering approach for L-alanine produc-tion was recently attempted inEscherichia coliby the coexpression offormate dehydrogenase and alanine dehydrogenase9. Alanine (80%L-isoform) was produced directly from pyruvate, but the yielddecreased upon increasing pyruvate concentration and, moreover,the relativeD-alanine isomer concentration increased with incuba-tion t ime due to alanine racemase activit y9. Metabolic engineering toproduce alanine from glucose has been achieved in Zymomonasmobilisby expressing an alanine dehydrogenase, but the end prod-ucts were a mixture of ethanol and alanine (presumably both iso-mers) with a glucose conversion rate into alanine of 85%. However,the maximal glucose consumption was low (30%), and the final ala-nine concentration did not exceed 80 mM10,11.

Lactococcus lacti sbelongs to the lactic acid bacteria (LAB) group

and is widely used as a starter for the production of fermented dairyproducts. The extensive knowledge of this species at the genetic andphysiological levels has permit ted the rerout ing of various catabolicpathways in bioreactors or fermented food products12. The mainadvantage of LAB for metabolic engineering stems from the nearlycomplete uncoupling of the basic catabolic and cell biosyntheticpathways. Lactococcus lacti sconverts excess sugars primarily intoL-lactate through glycolysis (homolactate fermentation)12. As withother LABs,L. lacti scontains neither a functional electron transportchain nor a cit ric acid cycle. ATP is generated mainly from glycolysisby two substrate-level phosphorylation reactions coupled to thereduction of NAD+ into NADH. The NAD+ cofactor is regeneratedthrough the action ofL-lactate dehydrogenase (L-LDH), which con-verts pyruvate intoL-lactate (Fig. 1).

This work reports homoalanine fermentation in an L. lacti sL-LDH-deficient strain overexpressing L-alanine dehydrogenase(L-AlaDH). Exclusive production ofL-alanine was achieved by dis-rupt ion of the endogenous alanine racemase gene.

Results and discussion

Overexpression of the Bacill us sphaeri cusalanine dehydrogenasegene (alaD). AlaDH and LDH both use pyruvate as substrate andNADH as cofactor (Fig. 1). Furthermore, the KM values of theB.sphaericusL-AlaDH13 for pyruvate and NADH are very close to thoseof theL. lacti sL-LDH14 (Fig. 1). We expressedL-AlaDH from a nisin-inducible promoter because of the ability to fine-tune its activity byvarying the levels of inducer and because expression at levels of up to50% of total cell protein have been reported with this system15,16.

The B. sphaeri cus alaDgene17 was fused to thenisA promoter(pNZ2650) and introduced into L. lacti sstrain NZ3900 (ref. 15),which expressed thenisRKregulatory genes stably integrated at thepepN locus. As competition between L-AlaDH and L-LDH wasexpected in the wild-type strain, we created an isogenicL-LDH-defi-cient strain, NZ3950 by homologous recombination. NZ3950 dis-

Conversion ofLactococcus lactisfromhomolactic to homoalanine fermentation

through metabolic engineeringPascal Hols1,2*, Michiel Kleerebezem1, Andr N. Schanck3, Thierry Ferain2, Jeroen Hugenholtz1,

Jean Delcour2, and Willem M. de Vos1

1Microbial Ingredients Secti on, NIZO Food Research, P.O. Box 20, 6710 BA Ede, The Netherlands. 2Laboratoire de Gntique Molculai re, Pl ace Croi x du Sud 5,and3Laboratoi re de Chimie Physique et de Cristal lographie, Place Loui s Pasteur 1, Un iversitcatholi que de Louvai n, 1348 Louvain-La-Neuve, Belgium.

*Correspondi ng author (e-mail : [email protected]).

Received 6 October 1998; accepted 29 March 1999

We report the engineering ofLactococcus lactis to produce the amino acid L-alanine. The primary end

product of sugar metabolism in wild-typeL. lactis is lactate (homolactic fermentation). The terminal enzy-

matic reaction (pyruvate + NADHL-lactate + NAD+) is performed byL-lactate dehydrogenase (L-LDH). We

rerouted the carbon flux toward alanine by expressing theBacillus sphaericus alanine dehydrogenase (L-

AlaDH; pyruvate + NADH + NH4+L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient

strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stere-

ospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase,

opening the door to the industrial production of this stereoisomer in food products or bioreactors.

Keywords: alanine, alanine racemase, alanine dehydrogenase,Lactococcus lactis

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played no L-LDH activit y and produced only t race amounts ofL-lac-tate (data not shown), as previously observed for a similar strain18.L-AlaDH was expressed in NZ3950 cells, and its specific activity was

determined as a function of increasing concentrations of nisin in themedium (01 ng/ml). A linear relationship between nisin concentra-tion and L-AlaDH-specific activity was observed (Fig. 2A), rangingfrom 0.05 U/mg protein to a maximum of 28 U/mg protein at 0.75ng/ml nisin (550-fold induction). I ncreasing the nisin concentrationto 1 ng/ml resulted in both decreased growth andL-AlaDH activi ty(data not shown). Since purified L-AlaDH f rom B. sphaericushad aspecific activity of 157 U/mg protein13, the expressed L-AlaDH wasestimated to represent approximately 20% of cell proteins. Analysisof cell extracts by SDSPAGE revealed a protein band with the pre-dicted molecular mass (39.5 kDa; Fig. 2B). At maximal inductioncondit ions, theL-AlaDH protein amounted to about 3040% of totalsoluble protein. Similar results were obtained with wild-typeNZ3900 cells expressingL-AlaDH (data not shown). The discrepan-

cy in quantification compared with the 20% evaluation based onspecific activiti es could be explained if only a fraction of the overex-pressed protein is active or, alternatively, if some inhibitory com-pounds affect the enzymatic assay performed on crude extracts.

Use ofL. lacti sresting cells as biocatalyst for alanine production.In order to assay alanine production,L-AlaDH was induced in bothwild-type and LDH-deficient cells, which were then washed andresuspended at a concentration of approximaetly 3.3 g dry weight/Lin potassium sodium phosphate buffer (100 mM, pH 7.0) supple-mented wi th glucose and ammonium acetate. An evaluation of vari -ous production parameters was performed with small-scale (4 ml)cell suspensions. Both strains produced alanine at a rate that was

Figure 1. Rerouting carbon flux toward L-alanine by overproductionof alanine dehydrogenase (L-AlaDH) in an L-lactate dehydrogenase(L-LDH) and alanine racemase (ALR)-deficient strain. The alternativepathways of pyruvate catabolism in the L-LDHdeficient strain18 takeplace through -acetolactate synthase (-ALS), pyruvate dehydro-genase complex (PDHC), and pyruvate formate lyase (PFL). The KMvalues for pyruvate and NADH of L-LDH and L-AlaDH are indicatedbetween brackets.

Figure 2. Nisin-dependent overproduction of alanine dehydrogenase.(A) Effect of nisin concentration on L-AlaDH specific activity. (B)Coomasie brilliant blue-stained gel after SDSPAGE of cell extractsfrom cultures induced with increasing concentrations of nisin (ng/ml)as indicated above each lane. Molecular mass markers (inkilodaltons) are indicated on the left, and the L-AlaDH band isindicated by arrow.

A

B

Figure 3. Alanine production as a function of nisin concentration andammonium supply. (A) Effect of nisin concentration on alanineproduction by cell suspensions of the wild-type NZ3900 strain (blackbars) and the L-LDHnegative NZ3950 strain (hatched bars). (B) Effectof ammonium acetate supply on alanine production. (Experimentalconditions were as in (A) except that the cells were induced at a fixednisin concentration of 0.5 ng/ml). Symbols as in (A).

BA

Figure 4. End products formation by cell suspensions of the wild-typeNZ3900 strain (black bars) and the L-LDHdeficient NZ3950 strain(hatched bars), each harboring pNZ2650. Cells were induced at afixed nisin concentration of 0.5 ng/ml. Production of acetate, 2,3-butanediol, and pyruvate was not observed.


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dependent on the nisin concentration (Fig. 3A) and the ammoniumsupply (Fig. 3B). Alanine production was always higher and requiredlower nisin concentrations and ammonium amounts in theL-LDH-deficient strain relative to the wild-type strain (Fig. 3). This wasprobably due to the absence of competition betweenL-AlaDH and L-LDH in theL-LDH mutant. Carbon balances (corrected for CO2pro-duction) were calculated from the concentration of the different end

products for each strain and were found to vary between 95 and110%. At maximal L-AlaDH induction, the end products of glucosefermentation in the wild- type strain (Fig. 4) were a mixture ofL-lac-tate (65% [percentage of total amount of carbon converted]) andalanine (35%). In theL-LDH mutant (Fig. 4), the end products weremainly a mixture of alanine (50%) and acetoin (35%) together withsmall amounts of ethanol (8%), formate (2%), and lactate (1.5%).These data show that L-AlaDH is able to compete withL-LDH in the

wild-type strain and even more efficiently with the alternative path-ways of pyruvate catabolism responsible for a mixed fermentationprofile in theL-LDH-deficient strain (Fig. 1). Residual L-lactate pro-duction in the L-LDH mutant suggests the presence of alternativepathways for lactate production as reported with similar mutantsobtained in LAB18,19. The influence of increasing the ini tial pH in themedium from 5 to 8 was also examined. Alanine production gradu-

ally increased up to a maximum at pH 8 (internal pH of 8.5, ref. 20)(data not shown), in agreement with the reported pH optimum(8.59) for amination11.

As the pH played a critical role in alanine production, a numberof fermentations were conducted using larger cell suspensions (400ml) maintained at a fixed pH of 7.5. Figure 5 illustrates the timecourse of the most important products (glucose, alanine, and lac-tate) for fermentation performed with ini tial concentrations of glu-

cose and (NH4)2SO4 of 100 mM each. Ammoniumacetate was replaced by (NH4)2SO4 because consump-tion of acetate by theL-LDH mutant complicated thecalculation of the carbon balances (data not shown).The wild-type strain (Fig. 5A) produced a mixture oflactate (104 mM), alanine (68 mM), and acetate (8mM) with 35% rerouting toward alanine. Thus, con-

trolling the pH in this case does not improve thererouting, probably because L-LDH can still competewith L-AlaDH at an alkaline pH. Conversely, the L-LDH mutant (Fig. 5B) under similar conditionsshowed an improved rerouting toward alanine (140mM, 75%) in comparison with uncontrolled pH con-ditions. The following additional end products weredetected in low amounts: pyruvate, lactate, acetate, andformate, altogether accounting for

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LDH-negative strain was observed within the first 4 h. This wasreduced to 4% by increasing the initial amount of ammonium sul-fate (150 mM) during the fermentation. The higher lysis level (12%)observed at low ammonium sulfate concentration is probably causedby ammonium limitation resulting in less efficient fermentation.Preliminary data obtained with alginate-immobilized cells are veryencouraging in terms of cell stability and alanine production effi-

ciency, which is maintained at the initial level for at least 24 h, indi-cating that the level of cell lysis must be very low (data not shown).Should autolysis become a problem for long-term cell stability inprocess development, the major autolysin ofL. lacti scould be inacti -vated, which should result in strongly reduced autolytic behaviorwithout negatively affecting growth24.

Exclusive production ofL-alanine isomer in an alanine racemaseknockout mutant. L-alanine is an important isomer for applicationsin the food, feed, and pharmaceutical industries1. The production ofeach isomer was determined from samples obtained from small- orlarge-scale fermentations using enzymatic assays. A mixture of bothisomers was observed in all cases with the D-isomer amounting to1015% of the total, independent of fermentation time (data notshown). This proportion ofD-alanine is rather low in comparison tothe racemic mixture generally observed with strains that naturally

produce alanine1,2,4. D-alanine is essential for peptidoglycan biosyn-thesis and alanine racemase is responsible for its conversion fromL-alanine25. The racemization observed with the constructed recom-binant strains is the result of an enzymatic conversion: incubationwith D-chloroalanine (50 g/ml), a specific inhibitor of alanine race-mase26, led to a signif icant reduction in the proport ion of theD-iso-mer (99%). In contrast toE. colior B. subti li s, in whichtwo alanine racemases were identified27,28, L. lacti sapparently con-tains a single racemase.

In conclusion, the use of a straightforward metabolic engineeringstrategy consisting of the controlled overproduction ofL-AlaDH anddisruption ofldhandalrgenes led to a complete conversion ofL. lac-ti sfrom the naturally occurring homo-L-lactate fermentation into ahomo-L-alanine fermentation. To our knowledge, this is the firstexample of a complete metabolic rerouting toward a metaboli te thatis not a normal end product in the host. Nearly complete rerouting

toward an endogenous end product (ethanol) has previously beenreported inE. coli30.

These results confirm the suitability ofL. lacti s, which displays asimple and efficient fermentation metabolism both aerobically andanaerobically, as a host for metabolic engineering12. The use of restingcells of L. lacti sas biocatalysts for the conversion of cheap sugarsources (i.e., sucrose, whey, and starch) intoL-alanine opens interest-ing perspectives for the development of a new process for the produc-tion of this amino acid as a fine chemical. Such a process could usegel- immobilized steady-state cells (as was already implemented forL-lactate production)31. Another interesting perspective could be thedevelopment of alanine-producing dairy starters for in situ produc-tion of the natural sweetener alanine in dairy products such as cheese,buttermilk, or yogurt .

Experimental protocolBacterial strains and media. E. coliMC1061 and TG1 were grown in LuriaBroth medium with aeration at 37C32,33. L. lactisNZ3900 (ref. 15) and itsderivatives were grown at 30C in M17 medium (Merck, Darmstadt, Germany)supplemented with 0.5% (wt/vol) glucose (GM17). D-alanine was added at afinal concentration of 2.25 mM to ensure growth of the alanine racemasemutants. Chloramphenicol, erythromycin, and tetracycline were used at 20g/ml, 250 g/ml , 12.5 g/ml, respectively, for E. coli , and at 10 g/ml (5 g/ml

when used in combination), 5 g/ml, and 1 g/ml, respectively, for L. lacti s.DNA techniques and transformation. E. coliMC1061 or TG1 strains wereused as intermediate hosts for cloning. Lactococcal plasmid and chromoso-mal DNA were isolated as described previously34,35. DNA sequencing was per-formed with an Applied Biosystems model 377 DNA sequencer and a dye-labeled terminator sequencing kit (Applied Biosystems, Foster City, CA).PCR reactions were performed with the Expand High Fidelity PCR system(Boehringer, Mannheim, Germany) in a DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT) with the following regimen: denaturation at 95C for 1min, annealing at 40C for 1 min, and extension at 72C for 1 min for a totalof 25 cycles. All other DNA manipulations were performed using establishedprocedures33. L. lactiswas electroporated as reported before36.

Construction of plasmids and mutant strains. The promoterless B.sphaeri cus alaDgene was isolated as a 1.22 kb SmaI-HindIII fragment fromplasmid pBM20alaD(a gift from G.A. Sprenger, Institut fr Biotechnologie,Jlich, Germany) and cloned intoHpaI-HindIII digested pGIT032 (ref. 37),

to generate pGIT503. This intermediate plasmid positioned the strong L.plantarum ldhL terminator37 downstream of the alaDopen reading frame(ORF). ThealaDORF-ldhL terminator module was then cloned as a 1.64 kbBamHI-XbaI fragment into simi larly digested pNZ8020 (ref. 15). The result-ing pNZ2650 expresses thealaDgene from thenisA promoter.

To create thealrgene knockout, partial sequence (5 end region) of thealrgene from L. lacti sIL1403 was kindly provided by P. Renault (INRA, Jouy enJosas, France). A 0.692 kb PCR product (corresponding to a 5 and 3 trun-cated alrgene) was amplified from NZ3900 chromosomal DNA usingprimers derived from the IL1403 DNA sequence (LLALR1: 5-CGAGGATC-CGGCTCGGTTGAGGTTTCTAAAGCGG-3 containing a BamHI site[underlined] and LLALR2: 5-CGCGAGCTCACTTGTTTCATAAGGCAC-CGTAACC-3 containing an SstI site [underlined]). The DNA fragment wasidentified by sequencing and displayed 94% identity to the IL1403 DNAsequence (data not shown). The fragment was then restricted with BamHIand SstI and cloned into the corresponding sites of the suicide plasmidpJDC9 (ref. 38) to generate pGIP011.

The L-LDHdeficient derivative of NZ3900 (NZ3950) was obtained bysingle crossover chromosomal integration of plasmid pNZ2007 (5 and 3truncated ldh, tetM) and validated as described previously18. The Alr- strainswere constructed by transformation of pGIP011 DNA into strains NZ3900and NZ3950. Transformants corresponding to single crossover chromosomalintegration of pGIP011 were selected on GM17 containing D-alanine anderythromycin. The result ing Alr-deficient strain PH3900 and itsL-LDHdefi-cient variant PH3950 were identi fied by PCR using primers located 5 and 3to the fragment used for homologous recombination (data not shown). TheAlrdeficient phenotype was validated on the basis of absolute dependenceupon D-alanine for growth.

Nisin induction and fermentation. An overnight culture ofL. lactiscon-taining pNZ2650 was diluted (1:20) in GM17 supplemented with the appro-priate antibiotics and grown unti l an OD600nmof 0.5. The cells were inducedwith different concentrations of nisin A (referred to as nisin) and incubateduntil an OD600nm of 2.0, at which point the cells were either harvested for

extraction or used as cell suspensions. Small-scale cell suspensions made useof a 20 ml culture that had reached an OD600nmof 2.0. Cells were harvested bycentrifugation (12,000g, 10 min, 20C), washed with an equal volume of 100mM K-Na PO4 (pH 7.0) and resuspended in 4 ml of the same buffer supple-mented with 50 mM glucose and different concentrations of ammoniumacetate. The cell suspensions were subsequently incubated under shaking for1 h at 30C followed by centr ifugation and supernatant recovery for analysisof end products. Large-scale cell suspensions (400 ml) were obtained usingthe same procedure as described above, but nisin concentration was fixed at0.75 ng/ml, the initial culture volume was increased to 2 L and ammoniumacetate was replaced by ammonium sulfate (100 or 150 mM). The cell sus-pension was transferred to a 1-liter fermentor (Applikon DependableInstruments, Schiedam, The Netherlands). The pH was kept at 7.5 by theaddition of 1 M NaOH, the temperature was maintained at 30C, and thestirrer speed was set at 120 r.p.m.


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Cell extracts, enzyme assays, and protein analysis. Preparation of cellextracts and protein assays were performed as described37. L-LDH activitywas determined by the method of Hillier and Jago14. L-AlaDH activity wasmeasured according to the procedure of Ohashima and Soda13. Specific activ-ities were expressed as units (U, mol of NADH/min) per mg protein.SDSPAGE was performed according to the protocol of Laemmli 39. Samples(10 g of total proteins) were heated for 1 min at 100C and loaded on a 10%polyacrylamide gel. The gels were stained with Coomasie bril liant blue. The

prestained molecular weight markers were obtained from Bio-RadLaboratories (Richmond, CA). The protein fractions were quantified as apercentage of the total intracellular proteins as reported before15.

Analysis of fermentation products. Glucose, lactate, acetate, formate,ethanol, pyruvate, acetoin, and 2,3-butanediol were analyzed by HPLC asdescribed before40. Total alanine (L- and D-isomers) analysis was performedon a 4151 Alpha Plus amino acid analyzer (Pharmacia Biotech, Uppsala,Sweden) after appropriate dilut ions of the samples. Enzymatic determinationofL-alanine was perfomed by coupling the glutamatepyruvate transaminase(pig heart, 10 mg/ml; Boehringer) reaction to the L-LDH (rabbit muscle, 5mg/ml; Boehringer) reaction according to the enzyme manufacturersinstructions. The enzymatic measurement ofD-alanine concentration madeuse of the same solutions as for L-alanine quanti fication, but the init ial mix-ture contained 550 l of triethanolamine HCl (750 mM)EDTA (7.5 mM),950 l of H2O, 50 l of sample at appropriate dilution, and 30 l ofD-aminoacid oxidase (hog kidney, 5 mg/ml, Boehringer) . The mixture was incubated

for 1 h at 37C under strong shaking, 50 l of NADHNa2 2H2O (6mM)NaHCO3 (120 mM) were added and a first measurement at 340 nmwas performed. The reaction was initiated with 10 l of L-LDH (rabbit mus-cle, 5 mg/ml; Boehringer). The next steps were identical to L-alanine assays.13C NMR experiments were conducted as fol lows: 0.5 ml of D2O was added toa 2 ml sample in a 10 mm tube. 13C spectra were recorded at 298 K using anAM 500 Bruker instrument operating at 125.7 MHz. Fourier t ransform para-meters were: spectral width, 26 kHz; data points, 32 K; pulse angle, 90(23 s); recycle time, 9 s. After 5,000 transients were accumulated, a li nebroadening of 1.5 Hz was applied to the free induction decay before Fouriertransformation. Chemical shifts were accurate at 0.05 p.p.m.

Nucleotide sequence accession number. The nucleotide sequence of the 5and 3 truncated alrgene from L. lacti sNZ3900 (MG1363 derivative) hasbeen submi tted to the EMBL Nucleotide Sequence Database under accessionnumber Y18148.

AcknowledgmentsWe thank G.A. Sprenger for provi ding plasmid pBM20alaD; P. Renaul t for

sequence informati on on the alr gene from L. lactis IL1403 preceding publica-

ti on; and R. Holl eman, H. Kosters, and M . Star renburg for their techni cal help

in HPLC analyses and fermentat ion. This research was carr ied out in the frame-

work of the Communi ty Research Programme BIOTECH ( contr act no. BIO4-

CT96-0498). P.H. holds a fellowship of the EC BIOTECH programme (contract

no. BIO4-CT96-5093).

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