Analysis of ldh genes in Lactobacillus casei BL23: role on lactic acid production (original) (raw)
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Lactobacillus plantarum ldhL gene: overexpression and deletion
Journal of bacteriology, 1994
Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D i...
FEBS Letters, 1991
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(-)lactate was demonstrated,. The nucleotide sequence of the W-LDH gene (/&A) revealed the spontaneous inseetion of an E. coli insertion sequence IS2 upstream OF the gene cod&g region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LWH sequences but closely related to L. cosei W-hydroxyisocaproate dehydrogenase (W-HicDW). W-Lactate dehydtogenase; Ins&on sequence 1st; Lactobacillus delbrueckii subsp. bulgaricus; W-Hydroxyisocapeoate dehydrogenase Published by Elsevier Science Publishers B. V.
Pleiotropic effects of lactate dehydrogenase inactivation in Lactobacillus casei
Research in Microbiology, 2005
In lactic acid bacteria, conversion of pyruvic to lactic acid through the activity of lactate dehydrogenase (Ldh) constitutes the final step of the homofermentative pathway. Lactobacillus casei has two characterized genes encoding Ldh activities. The ldhL gene codes for an L-Ldh, which specifically catalyzes the formation of L-lactate, whereas the hicD gene codes for a D-hydroxyisocaproate dehydrogenase (HicDH), which catalyzes the conversion of pyruvate into D-lactate. In L. casei cells fermenting glucose, a mixture of L-/D-lactate with a 97:3% ratio was formed. Inactivation of hicD led to undetectable D-lactate levels after glucose fermentation, while L-lactate levels remained constant. Inactivation of ldhL did not abolish the production of L-lactate, but the lactate final concentration decreased about 25% compared to the wild type, suggesting the presence of at least a second L-Ldh. Moreover, part of the pyruvate flux was rerouted and half of the lactate produced was in the D-isomer form. ldhL inactivation in L. casei showed additional interesting effects. First, the glycolytic flux from pyruvate to lactate was redirected and other fermentation products, including acetate, acetoin, pyruvate, ethanol, diacetyl, mannitol and CO 2 , were produced. Second, a lack of carbon catabolite repression of lactose metabolism and N -acetyl-glucosaminidase activity was observed. This second effect could be partly avoided by growing the cells under aeration, since NADH oxidases could account for NAD + regeneration.
Characterisation of lactic dehydrogenase fromLactobacillus casei
Journal of Biosciences, 1979
Lactic dehydrogenase from Lactobacillus casei ATCC 7469 has been purified to homogeneity by a two-step affinity chromatography procedure which gave an yield of 35%. The enzyme specifically catalysed the conversion of pyruvate to lactate. The enzyme was maximally active at pH 4•6, which was shifted to 5•4 in the presence of fructose 1,6-biphosphate. The enzyme had a molecular weight of 70,800 with two identical subunits, unlike the lactic dehydrogenase from other sources. Histidine and primary amino groups appeared to be involved in catalysis.
Genetics of Lactic Acid Bacteria: Genetics of proteolysis in Lactococcus lactis
2003
Genetic and biochemical research over the past 15 years on milk protein degradation by Laetoeoeeus laetis has resulted in a detailed picture of how casein is broken down into its sub-fragments and used for cellular growth. Starting with the action of an extracellular but cell-wall-Iocated proteinase, milk protein is degraded into oligopeptides that are internalized by a specialized and essential oligopeptide permease (Opp) system. Without Opp, cells of Le. laetis cannot grow in milk. Notwithstanding this, the organism has two uptake systems for the internalization of di-and tripeptides. Once inside, a host of intracellular peptidases degrade the oligopeptides into smaller peptides and amino acids that are needed for cell growth. Both general aminopeptidases and more specialized enzymes are involved in this degradation process, as are a number of oligopeptidases. Most of the genes of the components making up the casein degradation pathway have been cloned and sequenced and most of these have been targeted by insertional mutagenesis to allow for an analysis of the function and importance of the components in the pathway. The casein degradation route has been engineered in various ways by the overexpression or stabilization of certain genes, by deleting genes, by mutating enzyme specificities or by expressing heterologous proteolytic enzymes in Le. laetis. In a number of cases, the modified strains have been used in (small-scale) cheese trials to assess the effects of the changes on the organoleptic quality of the fermentation product. More recently the regulation of the various genes in response to the growth phase of the cells and to medium composition has been studied. Even more recent is the discovery, through the determination of the nucleotide sequence of the chromosome of a widely studied laboratory strain of Le. laetis, of a number of additional proteinase genes not yet described in this organism. It is no exaggeration to claim that the caseinolytic pathway of Le. laetis is the
Applied Microbiology and Biotechnology, 2010
Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet™-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.
Journal of Bioscience and Bioengineering, 2017
The present study revealed the effect of nitrogen sources on lactic acid production and stimulation of D-and Llactate dehydrogenases (LDH) of parent Lactobacillus lactis NCIM 2368 and its mutant RM2-24 generated after UV mutagenesis. Both the parent and mutant strains were evaluated for D-lactic acid production in control and modified media. The modified media did not show remarkable effect on lactic acid production in case of parent whereas mutant exhibited significant enhancement in D-lactic acid production along with the appearance of L-lactic acid in the broth. Both LDH activities and specific activities were found to be higher in mutant than the parent strain. These results suggested that the diammonium hydrogen phosphate in modified media triggered the expression of LDH genes leading to enhanced lactic acid production. This observation has been proved by studying the expression levels of Dand L-LDH genes of parent and mutant in control and modified media using quantitative RT-PCR technique. In case of mutant, the transcriptional levels of D-LDH and L-LDH increased w17 fold and w1.38 fold respectively in modified medium compared to the values obtained with control medium. In case of parent, no significant change in transcriptional levels of D-and L-LDH was found when the cells were grown in either control medium or modified medium. This study suggested that the mutant, RM2-24 has L-LDH gene which is expressed in presence of (NH 4) 2 HPO 4 resulting in L-lactic acid production. Co-production of L-lactic acid in D-lactic acid fermentation may be detrimental in the PLA production.
1995
Recombinant plasmids containing the Pediococcus acidilactici L-(؉)-lactate dehydrogenase gene (ldhL) were isolated by complementing for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase-pyruvate formate lyase double mutant. The nucleotide sequence of the ldhL gene predicted a protein of 323 amino acids showing significant similarity with other bacterial L-(؉)-lactate dehydrogenases and especially with that of Lactobacillus plantarum. The ldhL transcription start points in P. acidilactici were defined by primer extension, and the promoter sequence was identified as TCAACT-(17 bp)-TATAAT. This sequence is closely related to the consensus sequence of vegetative promoters from gram-positive bacteria as well as from E. coli. Northern analysis of P. acidilactici RNA showed a 1.1-kb ldhL transcript whose abundance is growth rate regulated. These data, together with the presence of a putative rho-independent transcriptional terminator, suggest that ldhL is expressed as a monocistronic transcript in P. acidilactici.