高产双乙酰乳球菌的研究进展

双乙酰是乳制品中一种重要的风味物质。大多数乳酸茵（如乳球菌）都可以发酵葡萄糖和柠檬酸产生双乙酰。描述了在乳球菌中从柠檬酸代谢和糖酵解途径来生成双乙酰的代谢途径,以及利用基因工程和代谢工程技术来提高乳球菌的双乙酰生成量的策略。     

Transport and metabolism of citrate by Streptococcus mutans

Streptococcus mutans, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Two virulence determinants of S. mutans are its acidogenicity and aciduricity (the ability to produce acid and the ability to survive and grow at low pH, respectively). Citric acid is ubiquitous in nature; it is a component of fruit juices, bones, and teeth. In lactic acid bacteria citrate transport has been linked to increased survival in acidic conditions. We identified putative citrate transport and metabolism genes in S. mutans, which led us to investigate citrate transport and metabolism. Our goals in this study were to determine the mechanisms of citrate transport and metabolism in S. mutans and to examine whether citrate modulates S. mutans aciduricity. Radiolabeled citrate was used during citrate transport to identify citrate metal ion cofactors, and thin-layer chromatography was used to identify metabolic end products of citrate metabolism. S. mutans was grown in medium MM4 with different citrate concentrations and pH values, and the effects on the growth rate and cell survival were monitored. Intracellular citrate inhibited the growth of the bacteria, especially at low pH. The most effective cofactor for citrate uptake by S. mutans was Fe(3+). The metabolic end product of citrate metabolism was aspartate, and a citrate transporter mutant was more citrate tolerant than the parent.     

Energy transduction in lactic acid bacteria

Abstract: In the discovery of some general principles of energy transduction, lactic acid bacteria have played an important role. In this review, the energy transducing processes of lactic acid bacteria are discussed with the emphasis on the major developments of the past 5 years. This work not only includes the biochemistry of the enzymes and the bioenergetics of the processes, but also the genetics of the genes encoding the energy transducing proteins. The progress in the area of carbohydrate transport and metabolism is presented first. Sugar translocation involving ATP-driven transport, ion-linked cotransport, heterologous exchange and group translocation are discussed. The coupling of precursor uptake to product product excretion and the linkage of antiport mechanisms to the deiminase pathways of lactic acid bacteria is dealt with in the second section. The third topic relates to metabolic energy conservation by chemiosmotic processes. There is increasing evidence that precursor/product exchange in combination with precursor decarboxylation allows bacteria to generate additional metabolic energy. In the final section transport of nutrients and ions as well as mechanisms to excrete undesirable (toxic) compounds from the cells are discussed.     

Lactic acid bacteria, their metabolic products and interference with microbial growth

Abstract: Lactic acid bacteria produce a variety of metabolic products that are capable of interfering with the growth of other microbes. These bacterial end products have been applied to food systems to prevent the growth of certain undesirable bacteria. The following review will discuss the successful application of several of the metabolic products produced by lactic acid bacteria in food systems.     

Proteomic investigation of the adaptation of Lactococcus lactis to the mouse digestive tract

Lactic acid bacteria are used on an industrial scale for the manufacturing of dairy products. It is now intended to develop novel applications of lactic acid bacteria that could be used as living vehicles for the targeting of antigens or therapeutics to the digestive mucosa. The aim of this study was to analyze the adaptations of Lactococcus lactis, a model lactic acid bacteria to the digestive tract and to identify functions required for colonization of the intestine. For this purpose, we combined gnotobiology with proteomics: axenic mice were colonized with a dairy L. lactis strain and the bacterial proteome was examined by 2-DE. As compared to cultures in broth, the proteome profile of bacteria grown in the intestine indicates the activation of metabolic pathways involved in various carbon sources assimilation and suggests the adoption of a mixed acids fermentative metabolism. We identified the product of the ywcC gene as essential for the colonization of the digestive tract and demonstrated that the corresponding gene product (YwcC) possesses a phosphogluconolactonase activity, suggesting an important role of the pentose phosphate pathway for the development of L. lactis in the digestive environment.     

Citrate and Sugar Cofermentation in Leuconostoc oenos, a (sup13)C Nuclear Magnetic Resonance Study

(sup13)C nuclear magnetic resonance spectroscopy was used to investigate citrate-glucose cometabolism in nongrowing cell suspensions of the wine lactic acid bacterium Leuconostoc oenos. The use of isotopically enriched substrates allowed us to identify and quantify in the end products the carbon atoms derived from each of the substrates supplied; furthermore, it was possible to differentiate between products derived from the metabolism of endogenous carbon reserves and those derived from external substrates. Citrate-sugar cometabolism was also monitored in dilute cell suspensions for comparison with the nuclear magnetic resonance results. A clear metabolic shift of the end products from glucose metabolism was observed when citrate was provided along with glucose: ethanol was replaced by acetate, and 2,3-butanediol was produced. Reciprocally, the production of lactate and 2,3-butanediol from citrate was increased in the presence of glucose. When citrate was cometabolized with glucose, a 10-fold reduction in the intracellular concentration of glucose-6-phosphate was observed, a result in line with the observed citrate-induced stimulation of glucose consumption. The presence of citrate provided additional pathways for NADP(sup+) regeneration and allowed the diversion of sugar carbon to reactions in which ATP was synthesized. The increased growth rates and maximal biomass yields of L. oenos growing on citrate-glucose mixtures resulted from increased ATP synthesis both by substrate-level phosphorylation and by a chemiosmotic mechanism.     

Kinetics of lactate fermentation and citrate bioconversion by Lactococcus Iactis ssp. Iactis in batch culture

The growth kinetics of Lactococcus lactis ssp. lactis were studied in batch culture in conditions of non-limiting lactose and the presence of citric acid. The control of pH modified growth and citrate metabolism but did not change the yield of acid formation. At controlled pH the growth rate was unaffected by citrate metabolism. Lactose was transformed to L-lactate and assay of the metabolic by-products showed some heterofermentation at the end of the growth of cultures with low growth rates. This heterofermentation was interpreted as a slowing down of glycolysis with activation of both the pyruvate formate lyase (PFL) and the pyruvate dehydrogenase complex (PDHC). Under these conditions the presence of citric acid affected the activity of both the PDHC and the alcohol dehydrogenase (ADH). L-Lactate remains the major fermentation end-product and the sole inhibitor of fermentation, this inhibition was greater on growth than on lactic acid production.     

Bacteriocins of lactic acid bacteria

Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow, inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms. The following review will discuss biochemical and genetic aspects of bacteriocins that have been identified and characterized from lactic acid bacteria.     

A REVIEW : Arginine metabolism in wine lactic acid bacteria and its practical significance

This article begins with an introduction to malolactic fermentation in wine, followed by a review of the occurrence of arginine degradation in wine lactic acid bacteria and the pathway of arginine catabolism, the distribution of enzymes responsible, and the formation of products. The bioenergetics of wine lactic acid bacteria and arginine degradation are then reviewed. This is followed by a review of the possible mechanisms of arginine transport, and regulation of arginine metabolism and synthesis of the enzymes for arginine catabolism. Finally, the practical significance of arginine metabolism in wine lactic acid bacteria is reviewed with respect to taxonomic utility, biological significance and oenological implications.     

Substrate specificity of the citrate transporter CitP of Lactococcus lactis

The citrate transporter CitP of lactic acid bacteria catalyzes electrogenic precursor-product exchange of citrate versus L-lactate during citrate-glucose cometabolism. In the absence of sugar, L-lactate is replaced by the metabolic intermediates/end products pyruvate, α-acetolactate, and acetate. In this study, the binding and translocation properties of CitP were analyzed systematically for a wide variety of mono- and dicarboxylates of the form X-CR(2)-COO(-), where X represents OH (2-hydroxy acid), O (2-keto acid), or H (acid) and R groups differ in size, hydrophobicity, and composition. It follows that CitP is a very promiscuous carboxylate transporter. A carboxylate group is both essential and sufficient for recognition by the transporter. A C-2 atom is not essential, formate is a substrate, and C-2 may be part of a ring structure, as in benzoate. The R group may be as bulky as an indole ring structure. For all monocarboxylates of the form X-CHR-COO(-), the hydroxy (X = OH) analogs were the preferred substrates. The preference for keto (X = O) or acid (X = H) analogs was dependent on the bulkiness of the R group, such that the acid was preferred for small R groups and the 2-ketoacid was preferred for more bulky R groups. The C(4) to C(6) dicarboxylates succinate, glutarate, and adipate were also substrates of CitP. The broad substrate specificity is discussed in the context of a model of the binding site of CitP. Many of the substrates of CitP are intermediates or products of amino acid metabolism, suggesting that CitP may have a broader physiological function than its role in citrate fermentation alone.     

Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: a bioinformatics approach

AIMS: To identify potential pathways for citrate catabolism by Lactobacillus casei under conditions similar to ripening cheese. METHODS AND RESULTS: A putative citric acid cycle (PCAC) for Lact. casei was generated utilizing the genome sequence, and metabolic flux analyses. Although it was possible to construct a unique PCAC for Lact. casei, its full functionality was unknown. Therefore, the Lact. casei PCAC was evaluated utilizing end-product analyses of citric acid catabolism during growth in modified chemically defined media (mCDM), and Cheddar cheese extract (CCE). Results suggest that under energy source excess and limitation in mCDM this micro-organism produces mainly L-lactic acid and acetic acid, respectively. Both organic acids were produced in CCE. Additional end products include D-lactic acid, acetoin, formic acid, ethanol, and diacetyl. Production of succinic acid, malic acid, and butanendiol was not observed. CONCLUSIONS: Under conditions similar to those present in ripening cheese, citric acid is converted to acetic acid, L/D-lactic acid, acetoin, diacetyl, ethanol, and formic acid. The PCAC suggests that conversion of the citric acid-derived pyruvic acid into acetic acid, instead of lactic acid, may yield two ATPs per molecule of citric acid. Functionality of the PCAC reductive route was not observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This research describes a unique PCAC for Lact. casei. Additionally, it describes the citric acid catabolism end product by this nonstarter lactic acid bacteria during growth, and under conditions similar to those present in ripening cheese. It provides insights on pathways preferably utilized to derive energy in the presence of limiting carbohydrates by this micro-organism.     

Metabolic pathway engineering in lactic acid bacteria

Lactic acid bacteria (LAB) display a relatively simple carbon and energy metabolism where the sugar source is converted mainly to lactic acid. In Lactococcus lactis metabolic engineering has been very successful in the re-routing of lactococcal pyruvate metabolism to products other than lactic acid. Current metabolic engineering approaches tend to focus on more complex, biosynthetic pathways leading to end-products that generate a health benefit for the consumer (nutraceuticals). Several examples of research on these minor pathways in L. lactis have illustrated the potential of LAB as producers of these metabolites. Whole genome sequencing efforts and corresponding global technologies will have an impact on metabolic engineering in the future.     

Heterofermentative Carbohydrate Metabolism of Lactose-Impaired Mutants of Streptococcus lactis

Two mutants of Streptococcus lactis ATCC 11454 have been isolated which possess an impaired lactose-fermenting capacity; galactose utilization is also affected, but to a lesser extent. Although the Embden-Meyerhof-Parnas pathway is the major, if not the sole, pathway of carbohydrate metabolism in the three strains, the fermentation end products of the mutants are dramatically different from the typical homolactic pattern of the wild type. Under conditions of low oxygen tension and growth-limiting lactose concentrations, mutant strain T-1 produces largely formic acid, acetic acid (2:1), and ethanol rather than lactic acid. Aerated cultures produce acetic acid, CO(2) (1:1), acetyl-methylcarbinol, and diacetyl. When the mutants use galactose as an energy source, lactic acid is the major end product, but significant heterofermentative activity is observed. The aberrations responsible for the mutant phenotypes reside in the proteins which catalyze the transport and hydrolysis of galactosides. It is hypothesized that the impaired transport system of the mutants reduces the intracellular pool of glycolytic intermediates below that of the wild type. Since fructose-1, 6-diphosphate is an activator of lactic dehydrogenase in S. lactis, lactic acid production is reduced, and pathways leading to the formation of other products are expressed.     

Genetic organization and expression of citrate permease in lactic acid bacteria

Citrate is present in many natural substrates, such as milk, vegetables and fruits, and its metabolism by lactic acid bacteria (LAB) plays an important role in food fermentation. The industrial importance of LAB stems mainly from their ability to convert carbohydrates into lactic acid and, in some species, like Lactococcus lactis and Leuconostoc mesenteroides, to produce C4 flavor compounds (diacetyl, acetoin) through citrate metabolism. Three types of genetic organization and gene locations, involving citrate metabolism, have been found in LAB. Citrate uptake is mediated by a citrate permease, which leads to a membrane potential upon electrogenic exchange of divalent citrate and monovalent lactate. The internal citrate is cleaved into acetate and oxaloacetate by a citrate lyase, and oxaloacetate is decarboxylated into pyruvate by an oxaloacetate decarboxylase, yielding a pH gradient through the consumption of scalar protons.     

Metabolic engineering of lactic acid bacteria: overview of the approaches and results of pathway rerouting involved in food fermentations.

Lactic acid bacteria such as Lactococcus lactis are the microorganisms of choice for performing metabolic engineering in relation to food fermentation. These bacteria are used extensively in food fermentations, they have a simple and therefore controllable metabolism and the molecular genetics of these food bacteria is well-developed. There have been recent successes in metabolic engineering in these lactic acid bacteria, including examples of changes in both primary metabolism (diacetyl and alanine) and secondary metabolism (exopolysaccharides and flavour).     

Engineering metabolic highways in Lactococci and other lactic acid bacteria

Lactic acid bacteria (LAB) are widely used in industrial food fermentations and are receiving increased attention for use as cell factories for the production of food and pharmaceutical products. Glycolytic conversion of sugars into lactic acid is the main metabolic highway in these Gram-positive bacteria and Lactococcus lactis has become the model organism because of its small genome, genetic accessibility and simple metabolism. Here we discuss the metabolic engineering of L. lactis and the value of metabolic models compared with other LAB, with a particular focus on the food-grade production of metabolites involved in flavour, texture and health.     

Study of the Citrate Metabolism of Lactococcus lactis subsp. lactis Biovar Diacetylactis by Means of C Nuclear Magnetic Resonance

The metabolic fate of citrate and pyruvate in four strains of Lactococcus lactis subsp. lactis biovar diacetylactis has been studied by means of C nuclear magnetic resonance, using as a substrate either [3-C]pyruvic acid or custom-synthesized citric acid that is C labeled either at carbons 2 and 4 or at carbon 3. The fermentations were carried out batchwise in modified M17 broth. For the actual conversions of the C-labeled substrates, cells at the end of their logarithmic growth phase were used to minimize the conversion to lactic acid. A mass balance of the main citric acid metabolites was obtained; the four strains produced from 50 to 70% (on a molar basis) lactic acid from either citrate or pyruvate. The remaining 50 to 30% was converted mainly to either alpha-acetolactic acid (for one strain) or acetoin (for the other three strains). One of the strains produced an exceptionally high concentration of the diacetyl precursor alpha-acetolactic acid. Another strain (SDC6) also produced alpha-acetolactic acid, but this was decarboxylated to acetoin at a high rate. The C nuclear magnetic resonance method confirmed that the biosynthesis of alpha-acetolactic acid occurs via condensation of pyruvate and "active" acetaldehyde. Diacetyl was not found as a direct metabolite of citrate or pyruvate metabolism.     

End products of glucose and glutamine metabolism by L929 cells

Products of glucose and glutamine metabolism by L929 cells were detected and quantitated by gas chromatography and mass spectrometry of the oxime-trimethylsilyl derivatives. This method allowed detection and identification of all major carboxylic and amino acids produced in the system. Although lactic acid was expected to be the major product, alanine, citric, glutamic, aspartic, and pyruvic acids were also released into the culture medium at significant rates. Incorporation of labeled carbon from D-[U-13C]glucose showed that the alanine, lactic, and pyruvic acids were derived from glucose as was one-third of the citric acid carbon. The rate of glucose utilization for production of these end products was 29-fold greater than the rate of glucose oxidation to CO2, and calculated ATP production from alanine and pyruvate synthesis exceeded that from lactate synthesis by nearly 2-fold. Utilization of glutamine for synthesis of aspartic, glutamic, and citric acids also exceeded the rate of glutamine oxidation, thereby making end-product synthesis from glucose and glutamine the dominant cellular metabolic activity. In the absence of glucose, synthesis and intracellular levels of aspartic and glutamic acids increased, whereas synthesis and cell content of the other acids decreased markedly. This response is consistent with the metabolic pattern proposed by Moreadith and Lehninger (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221) in which much of the glutamine used by these cells is converted to aspartate in the absence of a pyruvate source and to aspartate or citrate in the presence of pyruvate.     

Barriers to application of genetically modified lactic acid bacteria

To increase the acceptability of food products containing genetically modified microorganisms it is necessary to provide in an early stage to the consumers that the product is safe and that the product provide a clear benefit to the consumer. To comply with the first requirement a systematic approach to analyze the probability that genetically modified lactic acid bacteria will transform other inhabitants of the gastro-intestinal (G/I) tract or that these lactic acid bacteria will pick up genetic information of these inhabitants has been proposed and worked out to some degree. From this analysis it is clear that reliable data are still missing to carry out complete risk assessment. However, on the basis of present knowledge, lactic acid bacteria containing conjugative plasmids should be avoided. Various studies show that consumers in developed countries will accept these products when they offer to them health or taste benefits or a better keepability. For the developing countries the biggest challenge for scientists is most likely to make indigenous fermented food products with strongly improved microbiological stability due to broad spectra bacteriocins produced by lactic acid bacteria. Moreover, these lactic acid bacteria may contribute to health.