scholarly journals Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2102 ◽  
Author(s):  
Li-Yan Feng ◽  
Jian-Zhong Xu ◽  
Wei-Guo Zhang
Keyword(s):  
Corynebacterium Glutamicum ◽  
Branched Chain Amino Acids ◽  
Gene Products ◽  
Branched Chain Amino Acid ◽  
Synthesis Activity ◽  
Branched Chain ◽  
Aromatic Aminotransferase ◽  
First Time ◽  
Branched Chain Aminotransferase

The production of branched-chain amino acids (BCAAs) is still challenging, therefore we rationally engineered Corynebacterium glutamicum FA-1 to increase the l-leucine production by optimizing the aminotransferases. Based on this, we investigated the effects of the native aminotransferases, i.e., branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) and aspartate aminotransferase (AspB; encoded by aspB) on l-leucine production in C. glutamicum. The strain FA-1△ilvE still exhibited significant growth without leucine addition, while FA-1△ilvE△aspB couldn’t, which indicated that AspB also contributes to L-leucine synthesis in vivo and the yield of leucine reached 20.81 ± 0.02 g/L. It is the first time that AspB has been characterized for l-leucine synthesis activity. Subsequently, the aromatic aminotransferase TyrB and the putative aspartate aminotransferases, the aspC, yhdR, ywfG gene products, were cloned, expressed and characterized for leucine synthesis activity in FA-1△ilvE△aspB. Only TyrB was able to synthesize l-leucine and the l-leucine production was 18.55 ± 0.42 g/L. The two putative branched-chain aminotransferase genes, ybgE and CaIlvE, were also cloned and expressed. Both genes products function efficiently in BCAAs biosynthesis. This is the first report of a rational modification of aminotransferase activity that improves the l-leucine production through optimizing the aminotransferases.

scholarly journals Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence of Corynebacterium glutamicum

2005 ◽  
Vol 187 (22) ◽  
pp. 7639-7646 ◽  
Author(s):  
Jan Marienhagen ◽  
Nicole Kennerknecht ◽  
Hermann Sahm ◽  
Lothar Eggeling
Keyword(s):  
Corynebacterium Glutamicum ◽  
Branched Chain Amino Acids ◽  
The Other ◽  
Deletion Mutants ◽  
Cluster Assembly ◽  
Sulfur Transfer ◽  
Amino Donor ◽  
Putative Aminotransferase ◽  
Branched Chain

ABSTRACT Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with l-glutamate, l-aspartate, and l-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) l-glutamate > 2-aminobutyrate > l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes l-alanine to aminate 2-oxo-isovalerate, the l-valine precursor, and 2-oxo-butyrate. A second AT active with the l-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for l-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.

scholarly journals Arteriovenous differences for amino acids across control and acid-secreting rat stomach in vivo

1983 ◽  
Vol 210 (2) ◽  
pp. 451-455 ◽  
Author(s):  
N G Anderson ◽  
P J Hanson
Keyword(s):  
Amino Acids ◽  
Ammonia Concentration ◽  
Branched Chain Amino Acids ◽  
Stomach Wall ◽  
Rat Stomach ◽  
Branched Chain Amino Acid ◽  
Acid Secreting ◽  
Branched Chain ◽  
Stimulation Of

1. A method is described for measuring arteriovenous differences across the rat stomach in vivo. 2. Notable results were the uptake of branched-chain amino acids, the uptake of arginine, which was approximately balanced by an output of ornithine, and the output of alanine. 3. The fractional extraction of glutamine from the blood by the stomach wall of pentagastrin-stimulated rats was 4.7%. 4. The arteriovenous differences for ammonia depended upon the blood ammonia concentration. 5. Arteriovenous differences were not affected by the stimulation of acid secretion with pentagastrin. 6. It is concluded that the high activity of branched-chain-amino-acid aminotransferase (EC 2.6.1.42) in the gastric mucosa is associated with metabolism of these amino acids, but that the stomach wall is a less avid user of glutamine than is the small intestine.

scholarly journals Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2978-2987 ◽  
Author(s):  
Disha Awasthy ◽  
Sheshagiri Gaonkar ◽  
R. K. Shandil ◽  
Reena Yadav ◽  
Sowmya Bharath ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Mutant Strain ◽  
Branched Chain Amino Acids ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Kinetics Of

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C6 or C2 carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the ΔilvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the ΔilvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

scholarly journals Impaired branched chain amino acid metabolism alters feeding behavior and increases orexigenic neuropeptide expression in the hypothalamus

2011 ◽  
Vol 212 (1) ◽  
pp. 85-94 ◽  
Author(s):  
Megan N Purpera ◽  
Li Shen ◽  
Marzieh Taghavi ◽  
Heike Münzberg ◽  
Roy J Martin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Food Intake ◽  
Feeding Behavior ◽  
Control Diet ◽  
Peripheral Tissues ◽  
Protein Status ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Branched Chain Aminotransferase ◽  
Deficient Mice

Elevation of dietary or brain leucine appears to suppress food intake via a mechanism involving mechanistic target of rapamycin, AMPK, and/or branched chain amino acid (BCAA) metabolism. Mice bearing a deletion of mitochondrial branched chain aminotransferase (BCATm), which is expressed in peripheral tissues (muscle) and brain glia, exhibit marked increases in circulating BCAAs. Here, we test whether this increase alters feeding behavior and brain neuropeptide expression. Circulating and brain levels of BCAAs were increased two- to four-fold in BCATm-deficient mice (KO). KO mice weighed less than controls (25.9 vs 20.4 g,P<0.01), but absolute food intake was relatively unchanged. In contrast to wild-type mice, KO mice preferred a low-BCAA diet to a control diet (P<0.05) but exhibited no change in preference for low- vs high-protein (HP) diets. KO mice also exhibited low leptin levels and increased hypothalamicNpyandAgrpmRNA. Normalization of circulating leptin levels had no effect on either food preference or the increasedNpyandAgrpmRNA expression. If BCAAs act as signals of protein status, one would expect reduced food intake, avoidance of dietary protein, and reduction in neuropeptide expression in BCATm-KO mice. Instead, these mice exhibit an increased expression of orexigenic neuropeptides and an avoidance of BCAAs but not HP. These data thus suggest that either BCAAs do not act as physiological signals of protein status or the loss of BCAA metabolism within brain glia impairs the detection of protein balance.

Branched chain amino Acids as in vitro and in vivo Anti-Oxidation Compounds

2021 ◽  
pp. 3899-3904
Author(s):  
Moath Alqaraleh ◽  
Violet Kasabri ◽  
Ibrahim Al-Majali ◽  
Nihad Al-Othman ◽  
Nihad Al-Othman ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Amino Acids ◽  
Branched Chain Amino Acids ◽  
Raw 264.7 ◽  
Raw 264.7 Cell ◽  
Branched Chain ◽  
Raw 264.7 Cell Line

Background and aims: Branched chain amino acids (BCAAs) can be tightly connected to metabolism syndrome (MetS) which can be counted as a metabolic indicator in the case of insulin resistance (IR). The aim of this study was to assess the potential role of these acids under oxidative stress. Material and Methods: the in vitro antioxidant activity of BCAAs was assessed using free radical 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging assays. For further check, a qRT-PCR technique was madefor detection the extent of alterations in gene expression of antioxidative enzymes (catalase and glutathione peroxidase (Gpx)) in lipopolysaccharides (LPS(-induced macrophages RAW 264.7 cell line. Additionally, BCAAs antioxidant activity was evaluated based on plasma H2O2 levels and xanthine oxidase (XO) activity in prooxidative LPS-treated mice. Results: Different concentrations of BCAAs affected on DPPH radical scavenging activity but to lesser extent than the ascorbic acid. Besides, BCAAs obviously upregulated the gene expression levels of catalases and Gpx in LPS-modulated macrophage RAW 264.7 cell line. In vivo BCAAs significantly minimized the level of plasma H2O2 as well as the activity of XO activity under oxidative stress. Conclusion: our current findings suggest that BCAAs supplementation may potentially serve as a therapeutic target for treatment of oxidative stress occurs with atherosclerosis, IR-diabetes, MetS and tumorigenesis.

scholarly journals Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

2014 ◽  
Vol 83 (3) ◽  
pp. 1019-1029 ◽  
Author(s):  
Julienne C. Kaiser ◽  
Sameha Omer ◽  
Jessica R. Sheldon ◽  
Ian Welch ◽  
David E. Heinrichs
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Defined Medium ◽  
Infection Model ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The branched-chain amino acids (BCAAs; Ile, Leu, and Val) not only are important nutrients for the growth ofStaphylococcus aureusbut also are corepressors for CodY, which regulates virulence gene expression, implicating BCAAs as an important link between the metabolic state of the cell and virulence. BCAAs are either synthesized intracellularly or acquired from the environment.S. aureusencodes three putative BCAA transporters, designated BrnQ1, BrnQ2, and BrnQ3; their functions have not yet been formally tested. In this study, we mutated all threebrnQparalogs so as to characterize their substrate specificities and their roles in growthin vitroandin vivo. We demonstrated that in the community-associated, methicillin-resistantS. aureus(CA-MRSA) strain USA300, BrnQ1 is involved in uptake of all three BCAAs, BrnQ2 transports Ile, and BrnQ3 does not have a significant role in BCAA transport under the conditions tested. Of the three, only BrnQ1 is essential for USA300 to grow in a chemically defined medium that is limited for Leu or Val. Interestingly, we observed that abrnQ2mutant grew better than USA300 in media limited for Leu and Val, owing to the fact that this mutation leads to overexpression ofbrnQ1. In a murine infection model, thebrnQ1mutant was attenuated, but in contrast,brnQ2mutants had significantly increased virulence compared to that of USA300, a phenotype we suggest is at least partially linked to enhancedin vivoscavenging of Leu and Val through BrnQ1. These data uncover a hitherto-undiscovered connection between nutrient acquisition and virulence in CA-MRSA.

Abstract P388: Extra-cardiac BCAA Catabolism Lowers Blood Pressure And Protects From Heart Failure

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Danielle Murashige ◽  
Cholsoon Jang ◽  
Michael Neinast ◽  
Michael Levin ◽  
Jae Woo Jung ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Blood Pressure ◽  
Mendelian Randomization ◽  
Multiple Models ◽  
Direct Effects ◽  
Heavy Isotope ◽  
Branched Chain Amino Acid ◽  
Branched Chain

Pharmacologic activation of branched chain amino acid (BCAA) catabolism is protective in numerous models of heart failure (HF). How this protection occurs has remained unclear, although a causative block in cardiac BCAA oxidation has been proposed. We use here in vivo heavy isotope infusion studies to show that cardiac preference for BCAA oxidation increases, rather than decreases, in multiple models of HF. We use various genetic models to show that cardiac-specific activation of BCAA oxidation does not protect from HF, even though systemic activation of BCAA oxidation does. Lowering plasma and cardiac BCAAs by genetic means is also not sufficient to confer protection comparable to that conferred by pharmacologic activation of BCAA oxidation, suggesting alternative mechanisms of protection. Surprisingly, telemetry and invasive hemodynamic studies showed that pharmacological activation of BCAA catabolism lowers blood pressure, a well-established cardioprotective mechanism. The effects on blood pressure occurred independently of nitric oxide (NO), and reflected a vascular resistance to adrenergic constriction. Finally, mendelian randomization studies revealed that elevations in plasma BCAAs portend higher blood pressure in large human cohorts. Together, these data indicate that activation of BCAA oxidation lowers blood pressure and protects from heart failure independently of any direct effects on the heart itself.

Production and utilization of amino acids by ovine placenta in vivo

1998 ◽  
Vol 274 (1) ◽  
pp. E13-E22 ◽  
Author(s):  
Misoo Chung ◽  
Cecilia Teng ◽  
Michelle Timmerman ◽  
Giacomo Meschia ◽  
Frederick C. Battaglia
Keyword(s):  
Amino Acids ◽  
Branched Chain Amino Acids ◽  
Plasma Amino Acids ◽  
Physiological Conditions ◽  
Ovine Placenta ◽  
Branched Chain ◽  
Glutamine Production

Uterine and umbilical uptakes of plasma amino acids were measured simultaneously in eighteen singleton pregnant ewes at 130 ± 1 days gestation for the purpose of establishing which amino acids are produced or used by the uteroplacenta under normal physiological conditions and at what rates. The branched-chain amino acids (BCAA) had uterine uptakes significantly greater than umbilical uptakes. Net uteroplacental BCAA utilization was 8.0 ± 2.5 μmol ⋅ kg fetus−1 ⋅ min−1( P < 0.005) and represented 42% of the total BCAA utilization by fetus plus uteroplacenta. There was placental uptake of fetal glutamate (4.2 ± 0.3 μmol ⋅ kg fetus−1 ⋅ min−1, P < 0.001) and no uterine uptake of maternal glutamate. Umbilical uptake of glutamine was ∼61% greater than uterine uptake, thus demonstrating net uteroplacental glutamine production of 2.2 ± 0.9 μmol ⋅ kg fetus−1 ⋅ min−1( P < 0.021). In conjunction with other evidence, these data indicate rapid placental metabolism of glutamate, which is in part supplied by the fetus and in part produced locally via BCAA transamination. Most of the glutamate is oxidized, and some is used to synthesize glutamine, which is delivered to the fetus. There was net uteroplacental utilization of maternal serine and umbilical uptake of glycine produced by the placenta. Maternal serine utilization and glycine umbilical uptake were virtually equal (3.14 ± 0.50 vs. 3.10 ± 0.46 μmol ⋅ kg fetus−1 ⋅ min−1). This evidence supports the conclusion that the ovine placenta converts large quantities of maternal serine into fetal glycine.

Effect of carnitine on branched-chain amino acid oxidation by liver and skeletal muscle.

1978 ◽  
Vol 234 (5) ◽  
pp. E494 ◽  
Author(s):  
H S Paul ◽  
S A Adibi
Keyword(s):  
Amino Acids ◽  
Liver Mitochondria ◽  
Branched Chain Amino Acids ◽  
Diabetic Rats ◽  
Acid Oxidation ◽  
Acyltransferase Activity ◽  
Remarkable Effect ◽  
Carnitine Acyltransferase ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The effect of L-carnitine (0.5-2.0 mM) on the rates of alpha-decarboxylation of 1-14C-labeled branched-chain amino acids by gastrocnemius muscle and liver homogenates of fed rats was investigated. Carnitine increased the rate of alpha-decarboxylation of leucine (125%) and valine (28%) by muscle, but it was without effect on the oxidation of these amino acids by liver. Carnitine increased the rate of alpha-decarboxylation of alpha-ketoisocaproate by both tissues. This effect was more pronounced in muscle (130% increase) than in liver (41% increase). The activity of carnitine acyltransferase, with isovaleryl-CoA as a substrate, was 18 times higher in muscle mitochondria than in liver mitochondria. Both starvation and diabetes increased the rate of alpha-decarboxylation of leucine by muscle without having a remarkable effect on the concentration of carnitine or the activity of carnitine acyltransferase. We conclude that: a) carnitine stimulates decarboxylation of branched-chain amino acids by increasing the conversion of their ketoanalogues into carnitine esters, b) a greater carnitine acyltransferase activity in muscle than in liver may be responsible for the greater carnitine effect in muscle, c) carnitine does not appear responsible for the enhancement of leucine oxidation by muscle of starved and diabetic rats.

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