Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts

When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.     

Effect of carbon monoxide on fermentation of fiber, starch, and amino acids by mixed rumen microorganisms in vitro.

When 1 atm (101.3 kPa) of carbon monoxide was added to mixed rumen bacterial incubations containing timothy hay, methane production was inhibited by 88% without an increase in hydrogen. The molar ratio of propionate to acetate increased from 0.83 to 1.53, extracellular ammonia declined from 5.2 to 2.4 mM, and hemicellulose and cellulose digestions were inhibited by 40 and 27%, respectively. Even low levels of carbon monoxide (less than 0.1 atm [10.13 kPa]) significantly changed the products of fermentation. With starch, methane production was once again inhibited, but the magnitude of starch fermentation was unaffected. Decrease in acetate was accompanied by an equal molar increase in lactate. Ammonia production from the amino acid source, Trypticase, declined 20% as carbon monoxide was increased to 1.0 atm, and 93% of this decrease was explained by a selective inhibition of branched-chain amino acid fermentation.     

Effect of carbon monoxide on fermentation of fiber, starch, and amino acids by mixed rumen microorganisms in vitro

When 1 atm (101.3 kPa) of carbon monoxide was added to mixed rumen bacterial incubations containing timothy hay, methane production was inhibited by 88% without an increase in hydrogen. The molar ratio of propionate to acetate increased from 0.83 to 1.53, extracellular ammonia declined from 5.2 to 2.4 mM, and hemicellulose and cellulose digestions were inhibited by 40 and 27%, respectively. Even low levels of carbon monoxide (less than 0.1 atm [10.13 kPa]) significantly changed the products of fermentation. With starch, methane production was once again inhibited, but the magnitude of starch fermentation was unaffected. Decrease in acetate was accompanied by an equal molar increase in lactate. Ammonia production from the amino acid source, Trypticase, declined 20% as carbon monoxide was increased to 1.0 atm, and 93% of this decrease was explained by a selective inhibition of branched-chain amino acid fermentation.     

Branched-chain Amino Acid Metabolon: INTERACTION OF GLUTAMATE DEHYDROGENASE WITH THE MITOCHONDRIAL BRANCHED-CHAIN AMINOTRANSFERASE (BCATm)*

The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain α-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain α-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5′-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5′-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5′-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.     

Regulation of NAD(H) pool and NADH/NAD(+) ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111

Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD(+) regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD(+) ratio from 0.64 to 0.13, in particular, the concentration of NAD(+) increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD(+) for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD(+) ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD(+) ratio was very important for succinic acid production.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.     

Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli.

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.     

A method for branched-chain amino acid aminotransferase activity in microgram and nanogram tissue samples

A method for branched-chain amino acid aminotransferase is described which is based on running the reaction in the reverse of the usual direction with glutamate and alpha-ketoisocaproate as substrates. The alpha-ketoglutarate generated is reduced with glutamate dehydrogenase and NADH. For sensitivity in the nanomole range, the NAD+ generated is measured directly by converting to the highly fluorescent strong alkali product. For smaller samples, down to the 0.2- to 2-pmol range, the NAD+ is amplified by enzymatic cycling.     

The stimulus-secretion coupling of amino acid-induced insulin release. XI. Kinetics of deamination and transamination reactions

L-Leucine and its nonmetabolized analogue, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH) activate glutamate dehydrogenase in pancreatic islets, whether the reaction velocity is measured in the direction of glutamate synthesis or glutamate deamination. The rate of glutamate oxidative deamination is increased by ADP and inhibited by 2-ketoglutarate, NH4+ and GTP. The islet homogenate catalyzes the transamination between L-glutamate and either 2-ketoisocaproate or pyruvate, and between 2-ketoglutarate and L-leucine, L-aspartate, L-alanine, L-isoleucine, L-valine, L-norvaline or L-norleucine, but not b (+/-) BCH. The glutamate-aspartate transaminase is preferentially located in mitochondria relative to other transaminases. The parallel effects of L-leucine and BCH on glutamate dehydrogenase and their vastly different abilities to act as transamination partners may account for both analogies and discrepancies in the metabolic and functional responses of the islets to these two branched-chain amino acids.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

Ethanol and oleate inhibition of alpha-ketoisovalerate and 3-hydroxyisobutyrate metabolism by isolated hepatocytes.

Ethanol inhibited glucose synthesis from alpha-ketoisovalerate by isolated rat hepatocytes without significant inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex. Accumulation of 3-hydroxyisobutyrate, an intermediate in the catabolism of alpha-ketoisovalerate, was increased by ethanol, indicating inhibition of flux at the level of 3-hydroxyisobutyrate dehydrogenase. 3-Hydroxybutyrate caused the same effects as ethanol, suggesting inhibition was a consequence of an increase in the mitochondrial NADH/NAD+ ratio. Flux through the 3-hydroxyisobutyrate dehydrogenase was more sensitive to regulation by the mitochondrial NADH/NAD+ ratio than flux through the branched-chain alpha-ketoacid dehydrogenase. Oleate also inhibited glucose synthesis from alpha-ketoisovalerate, but marked inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex was caused by this substrate.     

Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18.

NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.     

胞内氧化还原水平对嗜热厌氧乙醇菌 发酵代谢的影响

辅酶NADH/NAD+在细胞内氧化还原反应中起着重要的作用,是细胞生长和能量代谢必不可少的辅因子。调节微生物胞内NADH/NAD+的比率是定向改变微生物代谢,高效获得目标代谢产物的有效手段。嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus)是高温厌氧菌中乙醇产量较高的代表性菌株,本文利用不同氧化还原态的碳源改变T.ethanolicus的胞内NADH/NAD+含量和比例,进而研究了其对细胞生长、代谢产物分布的影响。以不同比例的葡萄糖/甘露醇作为混合碳源发酵,胞内氧化还原水平、细胞的生长特性、代谢产物都发生了不同程度的差异,以葡萄糖作为唯一碳源进行培养时,T.ethanolicus生长良好,乙醇产量为0.79g/L,但胞内NADH/NAD+比值和乙醇/乙酸的比值都比较低,分别为0.47和4.82;随着葡萄糖在混合碳源中比例的下降,NADH/NAD+比值增高,发酵产物中乙醇/乙酸比值也呈现上升的趋势。而以甘露醇作为唯一碳源时,发酵产物中乙醇浓度为0.389g/L,NADH/NAD+比值和乙醇/乙酸的比值分别为1.04和16.0。     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.     

Deamination of amino acids by Bacillus pasteurii

Resting cells of Bacillus pasteurii as employed in the treatment of distillery waste showed deamination of amino acids. The deamination of l-glutamic acid and dl-aspartic acid was found to be oxidative while that of dl-serine, dl-threonine and l-asparagine was non-oxidative. dl-Alanine and glycine were not deaminated when present individually but when incubated together showed oxidative deamination. NAD stimulated the oxidation of l-glutamic acid and α,α′-dipyridyl completely inhibited it.     

Stimulation of transport into Escherichia coli membrane vesicles by internally generated reduced nictotinamide adenine dinucleotide

Membrane vesicles containing internal alcohol dehydrogenase and nicotinamide adenine dinucleotide (NAD) were prepared from Escherichia coli ML308-225. Upon the addition of ethanol, these vesicles respired and transported several amino acids. Transport rates driven by internally generated reduced NAD (NADH) were about 60 to 80% of that stimulated by d-lactate. This transport was inhibited by cyanide and anaerobiosis. Ferricyanide, a nonpermeable electron acceptor, inhibited transport stimulated by external NADH, but not that stimulated by internally generated NADH.     

Why Does Clostridium acetireducens Not Use Interspecies Hydrogen Transfer for Growth on Leucine?

Clostridium acetireducens is the first reported anaerobic bacterium that is dependent on acetate as an electron acceptor for growth on branched-chain amino acids and alanine. The fermentation pathway of leucine and its deamination product α-ketoisocaproate were studied in this organism. Addition of Methanobacterium formicicum to pure cultures of C. acetireducens stimulated the degradation of α-ketoisocaproate but not the degradation of leucine, indicating that the electrons produced during the oxidative deamination of leucine were not transferred to hydrogen. This conclusion is supported by the observed low NAD(P)H ferredoxin reductase activity. Not only acetate but also crotonate proved to be an appropriate electron sink for the regeneration of NAD(P)⁺ in this bacterium. Interestingly, C. acetireducens was shown to form polyhydroxybutyrate during growth on leucine plus acetate. Received: 2 December 1996 / Accepted: 4 March 1997     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Catabolism of leucine to branched-chain fatty acids in Staphylococcus xylosus

AIMS: Staphylococcus xylosus is an important starter culture in the production of flavours from the branched-chain amino acids leucine, valine and isoleucine in fermented meat products. The sensorially most important flavour compounds are the branched-chain aldehydes and acids derived from the corresponding amino acids and this paper intends to perspectivate these flavour compounds in the context of leucine metabolism. METHODS AND RESULTS: GC and GC/MS analysis combined with stable isotope labelling was used to study leucine catabolism. This amino acid together with valine and isoleucine was used as precursors for the production of branched-chain fatty acids for cell membrane biosynthesis during growth. A 83.3% of the cellular fatty acids were branched. The dominating fatty acid was anteiso-C(15:0) that constituted 55% of the fatty acids. A pyridoxal 5'-phosphate and alpha-ketoacid dependent reaction catalysed the deamination of leucine, valine and isoleucine into their corresponding alpha-ketoacids. As alpha-amino group acceptor alpha-keto-beta-methylvaleric acid and alpha-ketoisovaleric acid was much more efficient than alpha-ketoglutarate. The sensorially and metabolic key intermediate on the pathway to the branched-chain fatty acids, 3-methylbutanoic acid was produced from leucine at the onset of the stationary growth phase and then, when the growth medium became scarce in leucine, from the oxidation of glucose via pyruvate. CONCLUSIONS: This paper demonstrates that the sensorially important branched-chain aldehydes and acids are important intermediates on the metabolic route leading to branched-chain fatty acids for cell membrane biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic information obtained is extremely important in connection with a future biotechnological design of starter cultures for production of fermented meat.