No abstract

We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus 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, stereospecific 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.

The UDP-MurNAc-pentapeptide is transferred to the outer face of the cell membrane by a lipid carrier and incorporated along with UDP-N-acetylglucosamine into the cell wall structure. The synthesis of other types of peptidoglycan precursors was demonstrated a few years ago in the context of several studies concerning vancomycin resistance. Vancomycin and other glycopeptide antibiotics can bind to the DAla-D-Ala terminus of pentapeptide-containing precursors by hydrogen bonding, thereby effectively blocking polymerization and preventing further cross-linking reactions (7, 41). Investigations of the molecular basis of vancomycin resistance started with strains of Enterococcus faecium and Enterococcus faecalis which showed inducible resistance to high levels of vancomycin and teicoplanin, another glycopeptide antibiotic. Examination of enzymes involved in cell wall synthesis in the resistant bacteria indicated that resistance to vancomycin was due to the synthesis of a novel type of peptidoglycan in which the terminal D-alanine residue was replaced by D-lactate, resulting in a drastic reduction of affinity for vancomycin (2,4,12,25,36). Two enzymes designated VanH and VanA are required for the synthesis of this alternative precursor (5). VanH is an ␣-ketoacid dehydrogenase that reduces pyruvate to D-lactate The synthesis of another type of peptidoglycan precursor has been described for Enterococcus gallinarum, which expresses inducible resistance to low levels of vancomycin but is susceptible to teicoplanin. In this case, the modified precursor terminates in D-serine instead of D-lactate (9). This feature results from the presence of another variant D-Ala-D-Ala ligase accepting D-serine (18).The genera Lactobacillus, Leuconostoc, and Pediococcus comprise strains and species constitutively resistant to vancomycin (15,20,33,39,46,49). Recently, peptidoglycan precursors from several of these lactic acid bacteria were analyzed. In Pediococcus pentosaceus and Lactobacillus casei (9, 26), the exclusive presence of a terminal D-lactate has been demonstrated. This presence could result from the action of a ligase which preferentially or exclusively catalyzes the synthesis of a D-Ala-D-Lac depsipeptide, as was suggested by Elisha and Courvalin (19). Analysis of Leuconostoc mesenteroides extracts identified a precursor that also terminates in D-lactate, but with an additional branched L-alanine In this paper, we report that the wild-type strain Lactobacillus plantarum NCIMB8826 is naturally resistant to high levels of vancomycin and teicoplanin and exclusively produces Dlactate-ending peptidoglycan precursors. We describe the construction of a strain defective for both D-and L-LDH, resulting in drastically reduced production of both isomers of lactate. We show that this alteration leads to the synthesis of a new type of precursor ending with D-alanine in addition to the usual muramyl depsipentapeptide observed in the wild-type strain,

The cell wall of lactic acid bacteria has the typical gram-positive structure made of a thick, multilayered peptidoglycan sacculus decorated with proteins, teichoic acids and polysaccharides, and surrounded in some species by an outer shell of proteins packed in a paracrystalline layer (S-layer). Specific biochemical or genetic data on the biosynthesis pathways of the cell wall constituents are scarce in lactic acid bacteria, but together with genomics information they indicate close similarities with those described in Escherichia coli and Bacillus subtilis, with one notable exception regarding the peptidoglycan precursor. In several species or strains of enterococci and lactobacilli, the terminal D-alanine residue of the muramyl pentapeptide is replaced by D-lactate or D-serine, which entails resistance to the glycopeptide antibiotic vancomycin. Diverse physiological functions may be assigned to the cell wall, which contribute to the technological and health-related attributes of lactic acid bacteria. For instance, phage receptor activity relates to the presence of specific substituents on teichoic acids and polysaccharides; resistance to stress (UV radiation, acidic pH) depends on genes involved in peptidoglycan and teichoic acid biosynthesis; autolysis is controlled by the degree of esterification of teichoic acids with D-alanine; mucosal immunostimulation may result from interactions between epithelial cells and peptidoglycan or teichoic acids.

No abstract

The Lactobacillus plantarum alr gene encoding alanine racemase was cloned by complementation of an Escherichia coli Alr ؊ DadX ؊ double mutant strain. Knockout of the alr gene abolished all measurable alanine racemase activity, and the mutant was shown to be strictly dependent on D-alanine for growth.Alanine racemases (EC 5.1.1.1) are unique prokaryotic enzymes that interconvert L-alanine and D-alanine (24). They are the sole identified biosynthetic pathway of D-alanine for bacterial cell wall synthesis (24). D-Ala is generally present as a dipeptide, D-alanyl-D-Ala, in the C-terminal position of the UDP-N-acetylmuramyl (MurNAc)-pentapeptide precursor (e.g., UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala) of the peptidoglycan (23). The penultimate D-Ala residue of this precursor is directly involved in the cross-linking of adjacent peptidoglycan strands in cell wall growth (16). Furthermore, the cell wall of gram-positive bacteria contains teichoic acids [poly(alditol phosphate) polymers] which could be of two types: wall teichoic acids, which are covalently linked to peptidoglycan, and lipoteichoic acids (LTA), which are anchored in the cytoplasmic membrane. These teichoic acids contain various substituents, such as D-Ala esters and glycosyl residues (7). Lactobacillus plantarum and the phylogenically close species L. casei contain LTA which are mainly (90%) or exclusively substituted with D-Ala esters, respectively (1, 9, 18). L. casei mutants deficient in LTA D-Ala ester substitutions are characterized by aberrant morphology and defective daughter cell separation (18). Thus, D-Ala is a central molecule in the biosynthesis of the two cell wall polymers peptidoglycan and teichoic acids.In Escherichia coli, two alanine racemases were identified. The alr-encoded alanine racemase (named biosynthetic racemase) is constitutively expressed (13,26,27), whereas the dadX-encoded enzyme (named catabolic racemase) is essential only for L-Ala catabolism, providing a substrate for a D-Alaspecific dehydrogenase encoded by the dadA gene. The dadX and dadA genes constitute an operon positively regulated by L-Ala and repressed by glucose (14, 27). The dadX gene product is the major source of alanine racemase activity (85% of total activity) and is probably a secondary source of D-Ala for cell wall biosynthesis (24). Only the Alr Ϫ DadX Ϫ double mutant is dependent on D-Ala for growth (27).In Bacillus subtilis, a single alanine racemase gene (dal) has been isolated (6). A Dal Ϫ mutant is dependent on D-Ala for growth only in rich medium and lyses in the absence of supplementation (2,6,8). Conversely, growth is not affected in minimal medium, except upon supplementation with L-Ala, which restores the D-Ala dependence for growth (2, 6). Ferrari et al. (6) suggested that a second, L-Ala-repressible racemase is present in B. subtilis.We have recently characterized the cell wall precursor of L. plantarum NCIMB8826: UDP-N-acetylmuramyl-L-Ala-DGlu-meso-diaminopimelic acid-D-Ala-D-lactate (5). The terminal depsip...

A straiti of Escherichiu coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LWH) was complemented with a genomic DNA iibc\lry from Laciobacilius delbrueckii subsp. Lirlgaricus. One positive clone showed LWH activity and production of W (-) The NADH-dependent lactate dehydrogenase (EDH) is a key enzyme in the fermentative metabolism of lactic acid bacteria, since it allows re-oxidation of the NAD needed for glycolysis through the coupled reduction of pyruvate to lactate. Two configurations are known for lactate: L(+) and D(-). Stereospecificity is achieved by distinct enzymes called L-LDH and D-EDH. Lactic acid bacteria produce either one or the two forms of lactate. For example, more than 90% of the lactate produced by Lactobacillus delbrueckii subsp.Little is known about the evolutionary relationship between L-and D-LDH. More than 25 years ago, the suggestion was made that these two enzymes had a common ancestry [2]. This claim was based upon limited peptide analysis and remained unchallenged, due to the lack of relevant information about D-LDH as opposed to L-LDH of which the primary and tertiary struclures are known in detail [3]. Two recent reports provide SOLO In this paper, we report for the first time the corr~pIete genomic sequence of a D-LDH gene cloned from L. bulgaricus. We show that this protein exhibits no similarity with the L-.LDH framework besides the co-enzyme binding site, but that it is closely related to L. casei D-HicDH. MATERIALS AND METHODSGeneral molecular biology and cultuee techniques were performed according to the instructions and recipes given by Sambrooket al. 671, 2. I. Bacterial strains and plasmids tictobacillus delbrucckii subsp. b&a&us LMG6901 (= NCIB 11778) was obtained from Wr W. Janssens (LMG Culture Collection, Lab. voor Microbiologic, Gent). E. coli FMJ 144 (pfl-Idh-CmR) was obtained from David P. Clark, Southern Illinois University, Carbondale [$I. The vectors used were pJWC9 [9] for complementation and pBluescript phagemids (Stratagene) for subcloning and sequencing.Abbreviations: L-LWH, L-lactate dehydrogenase (EC 1.1 .I .27); W-LDH, D-lactate dehydrogenase (EC 1 .l .1.28); W-HicDH, W-hydroxyisocaproate dehydrogenase. Growth and selection mediaL. bulgaricus was grown in 25.ml bottles at 37°C without shaking in MRS broth (Wifco 088 1). Forcomplementation, FMJ144 strain was grown anaerobically at 37OC on M9 minimal plates with glucose 0.4% and amino acids 0.2%. The selected clones were grown aerobically at 37°C in LB broth. Antibiotics were used at the following concentrations (in FgIml): ampicillin 100, chloramphenicol SO, erythromycin 250. .L. bulgaricus was grown to mid-log phase in 600 ml of MRS medium. Cells were pelleted and resuspended in 4 ml TEN buffer (TrisPublished by Elsevier Science Publishers B. V.

We have examined the metabolic consequences of knocking out the two ldh genes in Lactobacillus plantarum using 13 C nuclear magnetic resonance. Unlike its wild-type isogenic progenitor, which produced lactate as the major metabolite under all conditions tested, ldh null strain TF103 mainly produced acetoin. A variety of secondary end products were also found, including organic acids (acetate, succinate, pyruvate, and lactate), ethanol, 2,3-butanediol, and mannitol.