A General Method for Selection of α-Acetolactate Decarboxylase-Deficient Lactococcus lactis Mutants To Improve Diacetyl Formation

1999 ◽  
Vol 65 (3) ◽  
pp. 1202-1206 ◽  
Author(s):  
Mirjana Curic ◽  
Birgitte Stuer-Lauridsen ◽  
Pierre Renault ◽  
Dan Nilsson
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Genetically Modified Organisms ◽  
Defined Medium ◽  
Isoleucine Leucine ◽  
Genes Encoding ◽  
Resistant Mutants ◽  
Branched Chain ◽  
Selection Of

ABSTRACT The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the α-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp.lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains.

scholarly journals Amino Acid Metabolism of Thermoanaerobacter Strain AK90: The Role of Electron-Scavenging Systems in End Product Formation

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Sean Michael Scully ◽  
Johann Orlygsson
Keyword(s):  
Amino Acids ◽  
Fatty Acids ◽  
Amino Acid ◽  
Electron Acceptor ◽  
Detailed Examination ◽  
Product Formation ◽  
Isoleucine Leucine ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The catabolism of the 20 amino acids by Thermoanaerobacter strain AK90 (KR007667) was investigated under three different conditions: as single amino acids without an electron-scavenging system, in the presence of thiosulfate, and in coculture with a hydrogenotrophic methanogen. The strain degraded only serine without an alternative electron acceptor but degraded 11 amino acids (alanine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, and valine) under both of the electron-scavenging systems investigated. Acetate was the dominant end product from alanine, cysteine, lysine, serine, and threonine under electron-scavenging conditions. The branched-chain amino acids, isoleucine, leucine, and valine, were degraded to their corresponding fatty acids under methanogenic conditions and to a mixture of their corresponding fatty acids and alcohols in the presence of thiosulfate. The partial pressure of hydrogen seems to be of importance for the branched-chain alcohol formation. This was suggested by low but detectable hydrogen concentrations at the end of cultivation on the branched-chain amino acid in the presence of thiosulfate but not when cocultured with the methanogen. A more detailed examination of the role of thiosulfate as an electron acceptor was performed with Thermoanaerobacter ethanolicus (DSM 2246) and Thermoanaerobacter brockii (DSM 1457).

scholarly journals Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

2012 ◽  
Vol 79 (4) ◽  
pp. 1095-1101 ◽  
Author(s):  
Agata M. Pudlik ◽  
Juke S. Lolkema
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Expression System ◽  
Ph Profile ◽  
Content Type ◽  
Citrate Transporter ◽  
Keto Acids ◽  
Branched Chain ◽  
Mode Of Transport

ABSTRACTTransamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells ofLactococcus lactisIL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence ofl-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.

scholarly journals Plasma Branched-Chain Amino Acids and Incident Cardiovascular Disease in the PREDIMED Trial

2016 ◽  
Vol 62 (4) ◽  
pp. 582-592 ◽  
Author(s):  
Miguel Ruiz-Canela ◽  
Estefania Toledo ◽  
Clary B Clish ◽  
Adela Hruby ◽  
Liming Liang ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Amino Acids ◽  
Cardiovascular Death ◽  
Branched Chain Amino Acids ◽  
Control Group ◽  
Cvd Risk ◽  
Hazard Ratios ◽  
Increased Risk ◽  
Branched Chain

Abstract BACKGROUND The role of branched-chain amino acids (BCAAs) in cardiovascular disease (CVD) remains poorly understood. We hypothesized that baseline BCAA concentrations predict future risk of CVD and that a Mediterranean diet (MedDiet) intervention may counteract this effect. METHODS We developed a case-cohort study within the Prevención con Dieta Mediterránea (PREDIMED), with 226 incident CVD cases and 744 noncases. We used LC-MS/MS to measure plasma BCAAs (leucine, isoleucine, and valine), both at baseline and after 1 year of follow-up. The primary outcome was a composite of incident stroke, myocardial infarction, or cardiovascular death. RESULTS After adjustment for potential confounders, baseline leucine and isoleucine concentrations were associated with higher CVD risk: the hazard ratios (HRs) for the highest vs lowest quartile were 1.70 (95% CI, 1.05–2.76) and 2.09 (1.27–3.44), respectively. Stronger associations were found for stroke. For both CVD and stroke, we found higher HRs across successive quartiles of BCAAs in the control group than in the MedDiet groups. With stroke as the outcome, a significant interaction (P = 0.009) between baseline BCAA score and intervention with MedDiet was observed. No significant effect of the intervention on 1-year changes in BCAAs or any association between 1-year changes in BCAAs and CVD were observed. CONCLUSIONS Higher concentrations of baseline BCAAs were associated with increased risk of CVD, especially stroke, in a high cardiovascular risk population. A Mediterranean-style diet had a negligible effect on 1-year changes in BCAAs, but it may counteract the harmful effects of BCAAs on stroke.

Role of insulin and branched-chain amino acids in regulating protein metabolism during fasting

1990 ◽  
Vol 258 (6) ◽  
pp. E907-E917 ◽  
Author(s):  
M. Frexes-Steed ◽  
M. L. Warner ◽  
N. Bulus ◽  
P. Flakoll ◽  
N. N. Abumrad
Keyword(s):  
Amino Acids ◽  
Protein Metabolism ◽  
Insulin Dose ◽  
Group Iii ◽  
Group I ◽  
Tissue Sensitivity ◽  
Branched Chain ◽  
Independent Effects ◽  
Dose Dependent

This study examines the independent effects of insulin and amino acids on protein metabolism after a 12-h and 4-day fast in healthy volunteers. Leucine (Leu) kinetics were examined during sequential insulin infusions of 0 (group I) or 0.0125 (groups II and III), 1.2, and 10 mU.kg-1.min-1. Plasma Leu was maintained at 12-h fasted levels in groups I and II and at 84-h fasted levels in group III. Four-day fast (vs. 1 day, P less than 0.01) was associated with a 79% drop in plasma insulin and elevations in plasma Leu (122%), Leu rates of appearance (Ra) (21%), and Leu oxidation (56%), and no change in nonoxidative rates of disappearance (Rd). Insulin resulted in a dose-dependent suppression of endogenous Leu Ra with group III = I greater than II. Leu oxidation rose 1.7-fold in group III at the highest insulin dose but remained stable in the two other groups. In conclusion, 4-day fasting is associated with enhanced proteolysis and Leu oxidation with no change in nonoxidative Rd (protein synthesis). Elevated branched-chain (and other) amino acids were required to restore tissue sensitivity and specificity to the effects of insulin on protein metabolism after 4 days of fasting.

scholarly journals Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 652
Author(s):  
Tiffany Bergot ◽  
Eric Lippert ◽  
Nathalie Douet-Guilbert ◽  
Séverine Commet ◽  
Laurent Corcos ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Cancer ◽  
Myeloid Cell ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Small Nuclear Ribonucleoprotein ◽  
Genes Encoding ◽  
Protein Isoform ◽  
Nuclear Ribonucleoprotein

Deregulation of pre-mRNA splicing is observed in many cancers and hematological malignancies. Genes encoding splicing factors are frequently mutated in myelodysplastic syndromes, in which SF3B1 mutations are the most frequent. SF3B1 is an essential component of the U2 small nuclear ribonucleoprotein particle that interacts with branch point sequences close to the 3’ splice site during pre-mRNA splicing. SF3B1 mutations mostly lead to substitutions at restricted sites in the highly conserved HEAT domain, causing a modification of its function. We found that SF3B1 was aberrantly spliced in various neoplasms carrying an SF3B1 mutation, by exploring publicly available RNA sequencing raw data. We aimed to characterize this novel SF3B1 transcript, which is expected to encode a protein with an insertion of eight amino acids in the H3 repeat of the HEAT domain. We investigated the splicing proficiency of this SF3B1 protein isoform, in association with the most frequent mutation (K700E), through functional complementation assays in two myeloid cell lines stably expressing distinct SF3B1 variants. The yeast Schizosaccharomyces pombe was also used as an alternative model. Insertion of these eight amino acids in wild-type or mutant SF3B1 (K700E) abolished SF3B1 essential function, highlighting the crucial role of the H3 repeat in the splicing function of SF3B1.

scholarly journals From mitochondria to healthy aging: The role of branched-chain amino acids treatment: MATeR a randomized study

2020 ◽  
Vol 39 (7) ◽  
pp. 2080-2091 ◽  
Author(s):  
Ilaria Buondonno ◽  
Francesca Sassi ◽  
Giulia Carignano ◽  
Francesca Dutto ◽  
Cinzia Ferreri ◽  
...  
Keyword(s):  
Amino Acids ◽  
Healthy Aging ◽  
Randomized Study ◽  
Branched Chain Amino Acids ◽  
Branched Chain

scholarly journals CodY-Regulated Aminotransferases AraT and BcaT Play a Major Role in the Growth of Lactococcus lactis in Milk by Regulating the Intracellular Pool of Amino Acids

2003 ◽  
Vol 69 (6) ◽  
pp. 3061-3068 ◽  
Author(s):  
Emilie Chambellon ◽  
Mireille Yvon
Keyword(s):  
Amino Acids ◽  
Growth Inhibition ◽  
Lactococcus Lactis ◽  
Double Mutant ◽  
Wild Type Strain ◽  
Aromatic Amino Acids ◽  
Nitrogen Sources ◽  
Nutritional Factors ◽  
Keto Acids ◽  
Branched Chain

ABSTRACT Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.

Amino acid transportation, sensing and signal transduction in the mammary gland: key molecular signalling pathways in the regulation of milk synthesis

2020 ◽  
Vol 33 (2) ◽  
pp. 287-297
Author(s):  
Zhihui Wu ◽  
Jinghui Heng ◽  
Min Tian ◽  
Hanqing Song ◽  
Fang Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammary Gland ◽  
Milk Protein ◽  
Building Blocks ◽  
Branched Chain Amino Acids ◽  
Signalling Pathways ◽  
Recent Advances ◽  
Branched Chain ◽  
Molecular Signalling Pathways

AbstractThe mammary gland, a unique exocrine organ, is responsible for milk synthesis in mammals. Neonatal growth and health are predominantly determined by quality and quantity of milk production. Amino acids are crucial maternal nutrients that are the building blocks for milk protein and are potential energy sources for neonates. Recent advances made regarding the mammary gland further demonstrate that some functional amino acids also regulate milk protein and fat synthesis through distinct intracellular and extracellular pathways. In the present study, we discuss recent advances in the role of amino acids (especially branched-chain amino acids, methionine, arginine and lysine) in the regulation of milk synthesis. The present review also addresses the crucial questions of how amino acids are transported, sensed and transduced in the mammary gland.

scholarly journals Inactivation of an Iron Transporter in Lactococcus lactis Results in Resistance to Tellurite and Oxidative Stress

2007 ◽  
Vol 73 (19) ◽  
pp. 6144-6149 ◽  
Author(s):  
Mark S. Turner ◽  
Yu Pei Tan ◽  
Philip M. Giffard
Keyword(s):  
Oxidative Stress ◽  
Lactococcus Lactis ◽  
Oxygen Toxicity ◽  
Phosphate Transport ◽  
Lag Phase ◽  
Uptake System ◽  
Wild Type ◽  
Oxidative Defense ◽  
Genes Encoding ◽  
Resistant Mutants

ABSTRACT In Lactococcus lactis, the interactions between oxidative defense, metal metabolism, and respiratory metabolism are not fully understood. To provide an insight into these processes, we isolated and characterized mutants of L. lactis resistant to the oxidizing agent tellurite (TeO3 2−), which generates superoxide radicals intracellularly. A collection of tellurite-resistant mutants was obtained using random transposon mutagenesis of L. lactis. These contained insertions in genes encoding a proton-coupled Mn2+/Fe2+ transport homolog (mntH), the high-affinity phosphate transport system (pstABCDEF), a putative osmoprotectant uptake system (choQ), and a homolog of the oxidative defense regulator spx (trmA). The tellurite-resistant mutants all had better survival than the wild type following aerated growth. The mntH mutant was found to be impaired in Fe2+ uptake, suggesting that MntH is a Fe2+ transporter in L. lactis. This mutant is capable of carrying out respiration but does not generate as high a final pH and does not exhibit the long lag phase in the presence of hemin and oxygen that is characteristic of wild-type L. lactis. This study suggests that tellurite-resistant mutants also have increased resistance to oxidative stress and that intracellular Fe2+ can heighten tellurite and oxygen toxicity.

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