2-Isoleucine-oxytocin and deamino-2-isoleucine-oxytocin: their synthesis and some of their pharmacologic activities.
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Leucine and isoleucine requirements of the kitten.
In separate experiments the isoleucine and leucine requirements of the kitten were determined on the basis of growth and nitrogen retention. The dietary concentrations of isoleucine tested were (g/kg diet) 1.4, 2.2, 3.0, 3.8, 4.6 and 9.0 with adequate (12.0 g/kg diet) leucine. The levels of leucine tested were (g/kg diet) 5.0, 7.5, 9.0, 10.5, 12.0 and 20.0 in diets containing adequate (9.0 g/kg diet) isoleucine. In both experiments six male and six female kittens received each dietary level of isoleucine or leucine for periods of 10 d in a balanced 6 X 6 Latin-square experimental design. Asymptotic curves were fitted to the response relationships and the minimal dietary requirements for maximal response were estimated from the values at 0.95 of the asymptote. On this basis, the requirements for maximal growth were 6.2 g isoleucine/kg and 7.8 g leucine/kg diet. The requirements for maximal N retention were higher; 8.4 g isoleucine and 10.6 g leucine/kg diet. The isoleucine requirements suggested by this method are probably overestimations and might be slightly above 4.6 g/kg diet. Plasma isoleucine and leucine concentrations were not useful in estimating the requirements. Plasma leucine increased rectilinearly with increasing dietary leucine while the response of plasma isoleucine to increasing dietary isoleucine was non-rectilinear. Neither response relationship exhibited a breakpoint at the level of requirement. Below the suggested minimal requirement for leucine there were significant increases in the concentrations of isoleucine and valine in the plasma. Dietary isoleucine below the level of requirement had no effect on plasma valine and leucine. Dietary leucine had no effect on the plasma concentrations of methionine, phenylalanine and threonine, suggesting that the effect of decreasing dietary leucine on plasma isoleucine and valine is a result of decreased oxidation rather than decreased protein anabolism. In a separate experiment six kittens, presented a diet containing 2.2 g isoleucine/kg, developed crusty exudates around their eyes within 27 d and six kittens, presented diets containing 3.8 g isoleucine/kg, showed this clinical sign but with less severity within 47 d. Cultures of conjunctival swabs taken from the most severely affected kittens showed the presence of staphylococcal species, suggesting that in isoleucine-deficient kittens there was impaired resistance to these dermal microbes.
Animals↗
Isoleucine, a blood glucose-lowering amino acid, increases glucose uptake in rat skeletal muscle in the absence of increases in AMP-activated protein kinase activity.
Leucine and isoleucine were shown to stimulate insulin-independent glucose uptake in skeletal muscle cells in vitro. In this study, we examined the effects of leucine and isoleucine on blood glucose in food-deprived rats and on glucose metabolism in skeletal muscle in vivo. Furthermore, we investigated the possible involvement of the energy sensor, 5'-AMP-activated protein kinase (AMPK), in the modulation of glucose uptake in skeletal muscle, which is independent of insulin, and also in leucine- or isoleucine-stimulated glucose uptake. Oral administration of isoleucine, but not leucine, significantly decreased the plasma glucose concentration. An i.v. bolus of 2-[1,2-3H]-deoxyglucose (2-[3H]DG) was administered to calculate glucose uptake. Glucose uptake in the skeletal muscle did not differ after leucine administration, but glucose uptake in the muscles of rats administered isoleucine was 73% greater than in controls, suggesting that isoleucine increases skeletal muscle glucose uptake in vivo. On the contrary, in the skeletal muscles, administration of leucine but not isoleucine significantly increased [U-14C]-glucose incorporation into glycogen compared with controls. AMPK alpha1 activity in skeletal muscle was not affected by leucine or isoleucine administration. However, isoleucine, but not leucine, significantly decreased AMPK alpha2 activity. The decrease in AMPK alpha2 activity was thought to be due to decreases in AMP content and the AMP:ATP ratio, which were related to the isoleucine administration. This is the first report of isoleucine stimulating glucose uptake in rat skeletal muscle in vivo, and these results indicate that there might be a relation between the reduction in blood glucose and the increase in skeletal muscle glucose uptake that occur with isoleucine administration in rats. The alterations in glucose metabolism caused by isoleucine may result in an improvement of the availability of ATP in the absence of increases in AMP-activated protein kinase activity in skeletal muscle.
AMP-Activated Protein Kinases↗
Increasing valine, isoleucine, and total branched-chain amino acids for lactating sows.
One hundred eighty-five (n = 24 to 27/group; average parity 1.3) sows (PIC, Line C-15) were used to evaluate effects of the interrelationship between isoleucine and valine on sow and litter performance. Diets were formulated to .90% total lysine with all amino acids other than isoleucine and valine at least 110% of their suggested requirement estimate relative to lysine using ratios derived from the National and Agricultural Research Councils. The control diet was formulated to .50% isoleucine and .72% valine. L-Valine and L-isoleucine replaced cornstarch to provide .72 or 1.07% dietary valine, and .50, .85, or 1.20% isoleucine. A seventh diet contained .50% isoleucine and 1.42% valine. Mean litter size after cross-fostering was 11.1 pigs, and average lactation length was 20.3 d. No valine x isoleucine interactions were observed (P > .10) for most response criteria. Number of pigs weaned (mean = 10.9), sow feed intake (mean = 6.13 kg), and lysine intake (mean = 55 g/d) were not affected by dietary isoleucine or valine. Litter weight and weight gain at weaning increased as dietary valine (P < .07), isoleucine (linear, P < .07), and total branched-chain amino acids (linear, P < .02) increased. Twelve sows per treatment (84 total) were milked manually on either d 17 or 18 of lactation. Increasing dietary valine increased milk DM and fat (linear, P < .01). Milk DM, CP, and fat increased (linear, P < .002) as dietary isoleucine increased. The casein fraction of milk protein increased (linear, P < .01) and whey and nonprotein N fractions decreased (linear, P < .06, P < .01, respectively) as dietary isoleucine increased. Based on these results, valine and isoleucine increased litter weights. The independent increases in litter weaning weights from adding valine and isoleucine suggest separate modes of action in lactating sows.
Amino Acids↗
Utilization of ileal digestible amino acids by growing pigs: isoleucine.
Two experiments were conducted to determine the utilization of ileal digestible isoleucine by growing pigs. In the first, the apparent ileal digestibility of amino acids in cottonseed meal, lupin-seed meal and soya-bean meal was determined in pigs fitted with 'T'-shaped cannulas. In the second, three isoleucine-deficient diets were formulated to 0.23 g ileal digestible isoleucine/MJ digestible energy (DE) with the three protein concentrates contributing the only source of isoleucine in sucrose-based diets. An additional three diets were formulated with supplements of isoleucine to confirm that isoleucine was limiting in the first three diets. The growth performance and retention of isoleucine by pigs given the six diets over the 20-45 kg growth phase were then determined. The apparent ileal digestibility of isoleucine in the three protein concentrates (proportion of total) was: cottonseed meal 0.68, lupin-seed meal 0.86, soya-bean meal 0.86. There were no significant differences (P > 0.05) in growth rates (g/d) and crude protein deposition rates (g/d) of the pigs given the three diets formulated to 0.23 g ileal digestible isoleucine/MJ DE: cottonseed meal 590, 84; lupin-seed meal 613, 87; soya-bean meal 594, 91 (SEM 13.0, 2.9) respectively. The response of pigs to the addition of isoleucine confirmed that isoleucine was limiting in these diets. The proportion of ileal digestible isoleucine retained by pigs given the cottonseed meal (0.65) was slightly lower than that retained by pigs given soya-bean meal (0.73; P < 0.05). These results indicate that values for the ileal digestibility of isoleucine in protein concentrates more closely reflect the proportion of isoleucine that can be utilized by the pig than occurs for other amino acids such as lysine, threonine and methionine.
Animal Feed↗
Mechanism and Regulation of Isoleucine Excretion in Corynebacterium glutamicum.
Whole cells of Corynebacterium glutamicum were loaded with high cytoplasmic l-isoleucine concentrations, and isoleucine excretion from these cells was studied in terms of mechanism and regulation. The transmembrane isoleucine flux could be differentiated into carrier-mediated uptake, carrier-mediated excretion, and diffusion. After discrimination from the other transmembrane solute movements, the outward-directed flux, which was due to the activity of the isoleucine excretion carrier, was characterized with respect to its energy dependence and its regulation at the level of expression. Isoleucine excretion was shown to function as a secondary transport process, driven by the membrane potential and coupled to the movement of protons, presumably with a stoichiometry of 2:1 (H(sup+)/isoleucine). Of a variety of putative transport substrates, only leucine was able to compete for isoleucine at the cis (cytosolic) side of the export carrier. Cytoplasmic isoleucine concentrations higher than 20 mM induce the activity of the isoleucine excretion system. This effect is specific for isoleucine and is inhibited by the presence of chloramphenicol. Apart from leucine, other amino acids and related amino acid analogs are not able to induce isoleucine excretion. The complex pattern of regulation of the isoleucine excretion system at the level of activity and expression is shown to be related to the pattern of regulation of the isoleucine uptake system in C. glutamicum in terms of physiological significance.
Journal Article↗
Leucine, isoleucine and valine interactions in turkey poults.
In four experiments, the interactions of leucine, isoleucine and valine in turkey poults were studied. The additon of 1.50% excess leucine to a 22% protein starter diet, marginal in isoleucine and valine, depressed growth. This growth depression was corrected by the addition of valine and isoleucine. The addition of the excess leucine caused a decrease in plasma valine and isileucine concentrations in experiments 3 and 4, and plasma valine concentration in experiment 1. The addition of valine caused a marked linear increase in plasma valine with little or no effect on plasma isoleucine. The addition of isoleucine to the diet caused an increase in plasma isoleucine. Plasma valine, however, was decreased by the addition of isoleucine to a high-leucine diet. It is concluded that interactions exist in turkey poults between leucine-valine, leucine-isoleucine and isoleucine-valine and that the growth reduction caused by added leucine can be partly alleviated by addition of valine or by valine plus isoleucine, but not be isoleucine alone.
Animal Feed↗
Studies on requirement and excess of isoleucine in laying hens.
Three production trials and one nitrogen balance trial were conducted with Lohmann Brown hens to determine the requirement for and effects of an excess of isoleucine in layers at different ages (24 to 32 and 46 to 54 wk of age). The trials were designed as dose-response studies where isoleucine-deficient basal rations with 11.4 MJ metabolizable energy per kilogram were supplemented with varying amounts of L-isoleucine. In the production trials, dietary isoleucine concentrations ranged from 0.37 to 1.05%. In the three production trials, maximum daily egg mass was achieved at dietary isoleucine concentrations of between 0.39 and 0.75% (25 to 32 wk of age, daily egg mass 53 g), 0.40 and 0.57% (24 to 32 wk of age, daily egg mass 57 g), and 0.40 and 0.81% (46 to 54 wk of age, daily egg mass 56 g). The corresponding ranges of daily isoleucine intakes were 412 to 770 mg, 436 to 624 mg, and 431 to 874 mg. In the nitrogen balance trial, maximum total nitrogen retention was achieved at dietary isoleucine concentrations of between 0.43 and 0.57%. Dietary isoleucine concentrations higher than 0.8% caused a reduction in hen BW. Dietary isoleucine concentrations higher than 1.0% additionally caused a reduction in the daily egg mass. The study thus shows that the margin between requirement and excess of isoleucine is narrow in laying hens.
Age Factors↗
Interactions of various supplies of isoleucine, valine, leucine and tryptophan on the performance of laying hens.
The present study was undertaken to investigate the interactions among the supplies of isoleucine, leucine, valine, and tryptophan in laying hens. A three-factor trial was conducted with laying hens in which the dietary concentrations of isoleucine (5.7, 8.0, and 11.5 g/kg), valine and leucine (6.3 and 7.2 g/kg and 10.1 and 11.5 g/kg, respectively), and tryptophan (1.5 and 2.4 g/kg) were varied. At the lowest concentration of valine + leucine, an increase in dietary isoleucine concentration led to a dose-dependent reduction in feed consumption, daily egg mass, and body weight gain and an increase of the isoleucine concentration in plasma. At a high dietary concentration of valine + leucine, excess dietary isoleucine concentration caused only a weak depression of performance parameters; the isoleucine concentration in plasma was independent of the dietary isoleucine concentration. Increasing the dietary tryptophan concentration did not influence the effect of an excessive dietary isoleucine concentration on performance parameters. Increasing the tryptophan concentration from 1.5 to 2.4 g/kg diet did, however, lead to a significant increase in feed consumption, irrespective of the supply of isoleucine, valine, and leucine. In conclusion, our study demonstrates that the supply of valine + leucine influenced the effects of excess dietary isoleucine in laying hens, whereas the supply with tryptophan did not.
Amino Acids↗
Isoleucine auxotrophy due to feedback hypersensitivity of biosynthetic threonine deaminase.
Isoleucine-deficient mutants of Salmonella typhimurium were isolated. Three groups of mutants can be discerned by their nutritional requirements and enzyme patterns. (i) Mutants which grow with isoleucine alone are devoid of biosynthetic threonine deaminase (TD). (ii) Mutants growing with isoleucine and valine are devoid of transaminase B. (iii) Mutants growing with either isoleucine or threonine have normal levels of TD. However, the sensitivity of this enzyme to feedback inhibition by isoleucine is greatly enhanced. The inhibitory effect of isoleucine can be counterbalanced by high concentrations of threonine. These results indicate that the production of isoleucine in the mutants is restricted to a low level not sufficient to support the growth of the cells. This hypothesis is confirmed by studies with revertants of an isoleucine-threonine mutant. In nine revertants, wild-type properties of TD have been restored. In four revertants, the hypersensitivity of TD is unchanged, but the strains produce a greatly enhanced quantity of threonine, which is excreted into the culture medium. It follows, that hypersensitivity of TD to inhibition by isoleucine is the cause of the nutritional requirement of isoleucine-threonine mutants.
Cell-Free System↗
NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase.
The 400-MHz 1H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS. The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche+ form about the C alpha-C beta bond and the trans form about the C beta-C gamma 1 bond. The IleRS-bound L-valine takes the gauche- form about the C alpha-C beta bond. Thus, the conformation of IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.
Amino Acyl-tRNA Synthetases↗
Modification of the effects of blood on amino acid metabolism by intravenous isoleucine.
The absence of isoleucine in the hemoglobin molecule has been suggested to contribute to increased urea production after a blood meal. To unravel the underlying mechanism, the effects of isoleucine infusion after blood ingestion in the healthy pig were studied. The isoleucine dose was chosen to induce an arterial isoleucine increase comparable to those observed for leucine or valine after blood ingestion. For the experiments, 10 female overnight-fasted pigs (20-25 kg) received 250 mL bovine erythrocytes intragastrically 1 week after catheter implantation for measuring hepatic, splanchnic, portal-drained viscera, and hindquarter fluxes of amino acids, urea, and ammonia. After the administration of erythrocytes, isoleucine or saline was administered i.v. for 6 hours. The data obtained show that the increase in arterial levels of urea and almost all amino acids was significantly greater in the control group (P less than 0.001) than in the isoleucine group. The net efflux of nearly all amino acids by the portal-drained viscera increased significantly less (P less than 0.001) in the isoleucine group. The liver uptake of amino acids increased after the blood meal, but the difference was not significant except for glutamine (P less than 0.001). Hindquarter amino acid net influx increased marginally. Splanchnic urea production increased more in the control group than in the isoleucine group (P less than 0.05). The data strongly suggest that i.v. administration of isoleucine enhanced the biological value of a blood meal, possibly by promoting amino acid retention in the portal drained viscera.
Amino Acids↗
Isoleucine, a potent plasma glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes.
To examine which branched-chain amino acids affect the plasma glucose levels, we investigated the effects of leucine, isoleucine, and valine (0.3 g/kg body weight p.o.) in normal rats using the oral glucose tolerance test (OGTT, 2 g/kg). A single oral administration of isoleucine significantly reduced plasma glucose levels 30 and 60 min after the glucose bolus, whereas administration of leucine and valine did not produce a significant decrease. Oral administration of valine significantly enhanced the plasma glucose level at 30 min after the glucose administration and leucine had a similar effect at 120 min. At each measurement timepoint, the insulin levels of the treated groups were lower than that of the control group. We then investigated the effects of leucine, isoleucine or valine at the same concentration (1 mM) on glucose metabolism in C(2)C(12) myotubes in the absence of insulin. Glucose consumption was elevated by 16.8% in the presence of 1 mM isoleucine compared with the control. Conversely, 1 mM leucine or valine caused no significant changes in glucose consumption in the C(2)C(12) myotubes. The 2-deoxyglucose uptake of C(2)C(12) myotubes significantly increased upon exposure to 1-10 mM isoleucine and 5-10 mM leucine. However, isoleucine caused no significant difference in glycogen synthesis in C(2)C(12) myotubes, although leucine and valine caused a significant increase in intracellular glycogen compared with the control. The isoleucine effect on glucose uptake was mediated by phosphatidylinositol 3-kinase (PI3K), but was independent of mammalian target of rapamycin (mTOR). These results suggest that isoleucine stimulates the insulin-independent glucose uptake in skeletal muscle cells, which may contribute to the plasma glucose-lowering effect of isoleucine in normal rats.
Administration, Oral↗
Effect of isoleucine on toxin production by Clostridium difficile in a defined medium.
Supplementation of a carbohydrate-free minimal medium with a high level (100 mM) of histidine, methionine, valine, isoleucine, proline and leucine, in particular isoleucine, markedly increased toxin production by Clostridium difficile VPI 10463. The effect of isoleucine was further examined. Increasing the concentration of isoleucine from 20 to 100 mM remarkably increased toxin production, while bacterial growth decreased gradually. Amino acid analysis of the culture revealed that, at 100 mM isoleucine, consumption of isoleucine was remarkably increased. During the incubation period when toxin titers increased markedly but bacterial growth was declining, isoleucine, leucine and cysteine were taken up preferentially and alanine and cystathionine, which were not found at 1 mM isoleucine, were produced in large quantities. These findings suggest that isoleucine may play an important role in toxin production by C. difficile and that alanine and cystathionine production may be co-regulated with the toxin production in the absence of fermentable carbohydrates.
Amino Acids↗
A member of the YER057c/yjgf/Uk114 family links isoleucine biosynthesis and intact mitochondria maintenance in Saccharomyces cerevisiae.
BACKGROUND: Two paralogs, YIL051c and YER057c, in the Saccharomyces cerevisiae genome are members of the YER057c/Yigf/Uk114 family, which is highly conserved among Eubacteria, Archaea and Eukarya. Although the molecular function of this protein family is not clear, previous studies suggest that it plays a role in the regulation of metabolic pathways and cell differentiation. RESULTS: Yil051cp is 70% identical in amino acid sequence to Yer057cp, and differs in that the former is longer by 16 amino acids containing, in part, the mitochondrial targeting signal at the N-terminus of the protein. An HA-tagged protein of Yil051cp is localized strictly in mitochondria, while that of Yer057cp is found in both cytoplasm and nucleus. Disruption of YIL051c (yil051cDelta) resulted in severe growth retardation in glucose medium due to isoleucine auxotroph, and no growth in glycerol medium due to the loss of mitochondria. An extract prepared from yil051cDelta cells showed no transaminase activity for isoleucine, while that for valine or leucine was intact. Haploid yil051cDelta cells newly isolated from the YIL051c/yil051cDelta hetero-diploids gradually lost mitochondrial DNA within 24 h in the absence of, but not in the presence of, an isoleucine. Mutants either requiring leucine (leu2-112) or isoleucine-valine (bat1Delta, bat2Delta) in a YIL051c background showed no changes in mitochondrial DNA maintenance in the absence of requirements. CONCLUSIONS: Based on these results, we named Yil051c as Ibm1 (Isoleucine Biosynthesis and Mitochondria maintenance1) and concluded that: (i) Ibm1p determines the specificity of isoleucine biosynthesis, probably at the transamination step, (ii) Ibm1p is required for the maintenance of mitochondrial DNA when isoleucine is deficient, and (iii) Isoleucine compensates for the lack of Ibm1p. Taken together, Ibm1p may act as a sensor for isoleucine deficiency as well as a regulator determining the specificity for branched amino acid transaminase.
Amino Acid Sequence↗
Isoleucine requirement of broiler chicks as affected by the concentrations of leucine and valine in practical diets.
1. Chicks were fed on an isoleucine-deficient diet, with 6 added concentrations of isoleucine to determine their isoleucine requirement and an additional 6 treatments were devised to determine their isoleucine requirement when dietary leucine and valine contents were increased. 2. The diet deficient in isoleucine contained 5.6 g/kg isoleucine with leucine and valine contents of 20.1 and 10.3 g/kg, respectively. Supplementation with leucine and valine increased these to 24.7 and 12.6 g/kg, respectively. 3. The isoleucine requirement was not affected by dietary leucine and valine contents in a diet with 13.4 MJ of ME per kg. Analysis of variance and Least Significant Difference of means indicated an isoleucine requirement of 7.2 g/kg. Non-linear regression of the same data indicated an isoleucine requirement of 8.44 g/kg, based on weight gain, or 8.19 g/kg based on food efficiency. 4. Reasons for the failure to find an imbalancing effect of branched chain amino acids in practical diets are discussed.
Analysis of Variance↗
Isoleucine imbalance using selected mixtures of imbalancing amino acids in diets of the broiler chick.
Three experiments were conducted to determine the isoleucine requirement of a broiler from the time of hatch to 16 d of age. Chicks in the experiments were fed an isoleucine-limiting diet composed of wheat and peanut meal as the primary protein sources; this diet was used to investigate various mixtures of amino acids as imbalancing agents for isoleucine. The isoleucine requirement for maximum weight gain and feed efficiency was determined on the basis of broken-line regression analysis to be 0.63 to 0.65% of the diet or 3.28 to 3.38% of dietary protein. A similar diet, marginally limiting in isoleucine, was used to investigate the response of chicks to the addition of various mixtures of amino acids to the diet. Chicks that received a 5% dietary addition of 11 amino acids consisting of equimolar concentrations of leucine, valine, histidine, methionine, phenylalanine, tryptophan, tyrosine, alanine, glycine, serine, and threonine had significantly lower weight gain and feed consumption and a higher feed conversion ratio than did chicks fed the basal diet. These adverse effects were only partly prevented by an isoleucine supplement. The large neutral amino acids, histidine, methionine, phenylalanine, tryptophan, and tyrosine, accounted for most of the effect of the mixture. No effect of a mixture of leucine and valine or a mixture of the small neutral amino acids (alanine, glycine, serine, and threonine) at the same concentrations as those in the mixture of the 11 amino acids was observed. Lysine and arginine were the only two indispensable amino acids not present in the mixture of 11 amino acids. A subsequent experiment demonstrated that these amino acids did not become co-limiting with isoleucine when the diet was imbalanced with the amino acid mixture. These results indicate that an isoleucine imbalance in chicks is readily precipitated by excessive dietary concentrations of large neutral amino acids in diets that are otherwise marginally adequate in isoleucine.
Aging↗
Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.
The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.
Biomass↗