Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of L-leucine and L-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in alpha-proteobacteria but not in other prokaryotes analyzed.     

Interactions between the branched-chain amino acids in the growth of Spirodela polyrhiza

The joint action of L-valine and L-isoleucine, L-leucine and L-isoleucine, and L-valine and L-leucine on the growth of Spirodela polyrhiza was established. The effect of one branched-chain amino acid on growth inhibition by another one was compared with the non-specific antagonisms which glycine and L-alanine exert on growth inhibition by singly supplied branched-chain amino acids. In this way specific and non-specific interactions could be distinguished. It appeared that: (1) L-isoleucine was a specific antagonist of L-valine; (2) L-leucine was a specific antagonist of L-isoleucine; (3) L-valine and L-leucine were synergistic growth inhibitors. Further, it was found that: (4) growth inhibition by L-leucine was specifically antagonized by simultaneously supplied L-valine and L-isoleucine; (5) an excess of L-isoleucine strongly inhibited the conversion of exogenous valine into leucine; (6) accumulation of valine was typical of isoleucine-induced growth inhibition. The results are consistent with the view that growth inhibition by L-valine and L-leucine is due to the blocking of acetohydroxy acid synthetase, the first common enzyme in the valine-isoleucine biosynthetic pathway. Growth inhibition by L-isoleucine, however, seems to result from inhibition of leucine synthesis at a step after 2-oxoisovaleric acid. Some aspects of the regulation of branched-chain amino acid biosynthesis in higher plants are discussed.     

代谢工程改造谷氨酸棒状杆菌合成及分泌途径生产L-缬氨酸

谷氨酸棒状杆菌是目前微生物发酵生产L-缬氨酸的主要工业菌株。文中首先在谷氨酸棒状杆菌VWB-1中敲除了alaT (丙氨酸氨基转移酶),获得突变菌株VWB-2,作为出发菌株。进而对L-缬氨酸合成途径关键酶——乙酰羟酸合酶 (ilvBN) 的调节亚基进行定点突变 (ilvBN1M13),解除L-缬氨酸对该酶的反馈抑制。然后辅助过量表达L-缬氨酸合成途径关键基因ilvBN1M13、乙酰羟酸异构酶 (ilvC)、二羟酸脱水酶 (ilvD)、支链氨基酸氨基转移酶 (ilvE),加强通往L-缬氨酸的碳代谢流,提高菌株的L-缬氨酸水平。最后,基于过量表达L-缬氨酸转运蛋白编码基因brnFE及其调控蛋白编码基因lrp1,提高细胞的L-缬氨酸转运能力。最终获得工程菌株VWB-2/pEC-XK99E-ilvBN1M13CE-lrp1-brnFE在5 L发酵罐中的L-缬氨酸产量达到461.4 mmol/L,糖酸转化率达到0.312 g/g葡萄糖。     

Effect of transport proteins on l-isoleucine production with the l-isoleucine-producing strain Corynebacterium glutamicum YILW

Previous studies have shown that the deletion of brnQ from the Corynebacterium glutamicum chromosome results in a significant reduction in L-isoleucine uptake rates, while overexpression of brnFE leads to enhanced L-isoleucine export rates. Given that net excretion rates would be an important factor for high titers of L-isoleucine accumulation, we have tested the notion that decreased L-isoleucine uptake combined with increased L-isoleucine excretion will further improve high-yield strains that are currently used for the industrial-scale production of L-isoleucine. To examine the effect of the two carriers on L-isoleucine accumulation in L-isoleucine producer C. glutamicum YILW, we constructed a brnQ deletion mutant (C. glutamicum YILW?brnQ) and two brnFE overexpressors (C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW?brnQpXMJ19brnFE). Compared to the original strain, the efflux rate of the brnQ mutant increased from 19.0 to 23.6?nmol?min(-1) mg (dry wt)(-1) and its L-isoleucine titer increased from 154.3?mM (20.2?g?l(-1)) to 170.3?mM (22.3?g?l(-1)). The efflux rates of C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW?brnQpXMJ19brnFE were 33.5 and 39.1?nmol?min(-1) mg (dry wt)(-1), and their L-isoleucine production titers were 197.2?mM (25.9?g?l(-1)) and 221.0?mM (29.0?g?l(-1)), respectively. Our results suggest that modifications of the transport system could provide a promising avenue for further increasing L-isoleucine yield in the L-isoleucine producer.     

Solubilization and reconstitution of sodium-dependent transport system for branched-chain amino acids from Pseudomonas aeruginosa

The sodium-dependent transport system for branched-chain amino acids of Pseudomonas aeruginosa was solubilized with n-octyl-beta-D-glucopyranoside and reconstituted into liposomes by a detergent-Sephadex G-50 gel filtration procedure. The reconstituted proteoliposomes exhibited Na+-dependent counterflow and Na+-gradient-driven transport of L-leucine, L-isoleucine, and L-valine. The leucine counterflow was specifically inhibited by only branched-chain amino acids and the uphill transport of two species of amino acids among the three was induced by counterflow of the other substrate. These results show that the transport system for branched-chain amino acids was reconstituted into liposomes from P. aeruginosa cells and strongly suggest that three branched-chain amino acids are transported by a common carrier system.     

Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli.

We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp.     

Functional differences in the catabolism of branched-chain L-amino acids in cultured normal and maple syrup urine disease fibroblasts

Possible functional differences in the catabolism of the four branched-chain L-amino acids in maple syrup urine disease were assessed using cultured human skin fibroblast stains. Transamination and oxidative decarboxylation were comparatively studied in 90-min incubations with 1 mmole/liter of 1-14C-labeled substrates. In normal cell strains (n = 5), apparent transamination rates (sum of branched-chain 2-oxo[14C]acid and 14CO2 release; means expressed in nmole/90 min/mg of cell protein) were in the order L-leucine (32) greater than L-valine (17) greater than or equal to L-isoleucine (14) greater than L-allo-isoleucine (8); 14CO2 production was in the order L-valine (9) greater than L-isoleucine (6) greater than or equal to L-leucine (5) greater than L-allo-isoleucine (2). In variant (n = 5) as well as classical (n = 2) MSUD cell lines, branched-chain 2-oxo-[14C]acid release rates were generally comparable to the control values. As compared to the 14CO2 release in controls (= 100%), branched-chain 2-oxo acid dehydrogenase activity in MSUD fibroblasts was individually reduced and varied considerably between strains (residual activity 2-38%). Within individual strains, only small differences in the residual decarboxylation activity were observed in incubations with L-valine, L-leucine, and L-isoleucine. It was remarkably high, however, when L-allo-isoleucine was applied as a substrate. With the exception of L-allo-isoleucine, apparent total transamination rates of branched-chain L-amino acids were therefore distinctly lower in MSUD cells than in normal cells.     

Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine, using artificial cells containing an immobilized multienzyme system and dextran-NAD+. 2. Yeast alcohol dehydrogenase for coenzyme recycling.

Semipermeable nylon-polyethylenimine artificial cells containing leucine dehydrogenase (EC 1.4.1.9), alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1.5), and dextran-NAD+ were prepared. Artificial cells could convert ammonia or urea into L-leucine, L-valine, and L-isoleucine. For batch conversion in 20.0 mM of ammonium acetate substrate solutions, in 2 h 0.2 ml of artificial cells could produce 4.48 mumol of L-leucine, 9.98 mumol of L-valine, or 5.96 mumol of L-isoleucine. The corresponding conversion ratios were 22.4, 49.9, and 29.8%. In 20.0 mM of urea substrate solutions, 13.71 mumol of L-leucine, 16.12 mumol of L-valine, or 13.44 mumol of L-isoleucine was produced and the conversion ratios were 68.6, 80.6, and 67.2%. The substrate specificity of leucine dehydrogenase for the reductive amination was determined. Of the three branched-chain amino acids produced, the production rates of L-valine were the highest. The apparent Km values were as follows: 0.32 mM for alpha-ketoisocaproate, 1.63 mM for alpha-ketoisovalerate, and 0.73 mM for Dl-alpha-keto-beta-methyl-n-valerate. The leucine dehydrogenase multienzyme system had a good storage stability. It retained 72.0% of the original activity with artificial cells were stored at 4 degrees C for 6 weeks. The optimum conversion pH and temperature were 8.5-9.0 and 35-40 degrees C. The effects of urea and ammonium salts on conversion rate were also studied. The relative activities in ammonium salts solutions were 45.1-75.9% of those in urea solutions.     

Reduction of large neutral amino acid levels in plasma and brain of hyperleucinemic rats

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly known. In the present study we investigated the effect of acute hyperleucinemia on plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected subcutaneously with 6 micromol L-leucine per gram body weight. Controls received saline in the same volumes. The animals were sacrificed 30--120 min after injection, blood was collected and their brain rapidly removed and homogenized. The amino acid concentrations were determined by HPLC using orthophtaldialdehyde for derivatization and fluorescence for detection. The results showed significant reductions of the large neutral amino acids (LNAA) L-phenylalanine, L-tyrosine, L-isoleucine, L-valine and L-methionine, as well as L-alanine, L-serine and L-histidine in plasma and of L-phenylalanine, L-isoleucine, L-valine and L-methionine in brain, as compared to controls. In vitro experiments using brain slices to study the influence of leucine on amino acid transport and protein synthesis were also carried out. L-Leucine strongly inhibited [14C]-L-phenylalanine transport into brain, as well as the incorporation of the [14C]-amino acid mixture, [14C]-L-phenylalanine and [14C]-L-lysine into the brain proteins. Although additional studies are necessary to evaluate the importance of these effects for MSUD, considering previous findings of reduced levels of LNAA in plasma and CSF of MSUD patients during crises, it may be speculated that a decrease of essential amino acids in brain may lead to reduction of protein and neurotransmiter synthesis in this disorder.     

Decrease in plasma tryptophan after tryptophan-free amino acid mixtures in man

Male healthy subjects, fasting 12 hours, ingested increasing amounts of a mixture containing a fixed proportion of seven essential amino acids (L-isoleucine 11.5%, L-leucine 18.0%, L-lysine 13.1%, L-methionine 18.0%, L-phenylalanine 18.0%, L-threonine 8.2%, L-valine 13.1%) and lacking tryptophan. The diets produced a rapid fall in plasma tryptophan which was proportional to the total amount of the amino acids ingested. Following the highest dose administered (36.6 g) plasma tryptophan fell to a minimum of about 35% the initial level and remained markedly reduced at 6 hours after treatment. The mechanism of this decrease and its potential clinical relevance are discussed.     

Nutritional requirements of Schistosoma japonicum eggs

Newly laid eggs of Schistosoma japonicum were cultured in a serum-free, chemically defined medium, RPMI 1640, which contained 20 amino acids, glutathione, 11 vitamins, and glucose in a balanced salt solution. The requirements for these components in the nutrition of the eggs was investigated by the deletion of single component from the medium. The following 14 amino acids were shown to be essential for the full development of the egg in the medium: L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. Choline chloride was the essential vitamin. The omission of nicotinamide from the medium affected maturation adversely. Glucose was also required by the eggs. Minimal concentration of glucose for maturation of the eggs was 0.02 mM, but concentrations ranging from 0.16 to 20.00 mM gave better results while the concentration of the other elements of the medium were kept constant.     

The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli.

Branched-chain keto acid dehydrogenase is a multienzyme complex which is required for the metabolism of the branched-chain amino acids in Pseudomonas putida. The structural genes encoding all four proteins of the bkd operon have been cloned, and their nucleotide sequences have been determined (G. Burns, K. T. Madhusudhan, K. Hatter, and J. R. Sokatch, p. 177-184 in S. Silver, A. M. Chakrabarty, B. Iglewski, and S. Kaplan [ed.], Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology, American Society for Microbiology, Washington D.C., 1990). An open reading frame which encoded a protein with 36.5% amino acid identity to the leucine-responsive regulatory protein (Lrp) of Escherichia coli was found immediately upstream of the bkd operon. Chromosomal mutations affecting this gene, named bkdR, resulted in a loss of ability to use branched-chain amino acids as carbon and energy sources and failure to produce branched-chain keto acid dehydrogenase. These mutations were complemented in trans by plasmids which contained intact bkdR. Mutations affecting bkdR did not have any effect on transport of branched-chain amino acids or transamination. Therefore, the bkdR gene product must affect expression of the bkd operon and regulation must be positive. Mutations affecting bkdR could also be complemented by plasmids containing lrp of E. coli. This is the first instance of a Lrp-like protein demonstrated to regulate expression of an operon outside of E. coli.     

Studies on the macroscopic protonation constants of some alpha-amino acids in ethanol-water mixtures

Both to demonstrate whether the predominant species are dipolar ion or the neutral form and to predict the change of dipolar form to neutral form ratio in ethanol-water mixtures, the macroscopic protonation constants of eight alpha-amino acid (glycine, L-alanine, L-valine, L-leucine, L-phenylalanine, L-serine, L-methionine, and L-isoleucine) were determined potentiometrically in 20-80% (v/v) ethanol-water mixtures at 25 degrees C with an ionic strength of 0.10 M. The calculation of the constants was carried out using a PKAS computer program. The effect of solvent composition on the protonation constants and the dipolar ionic to neutral form ratio of these acids in the mixed solvents are discussed. One can conclude that the dipolar form of amino acids, HA(+/-), dominates in ethanol-water mixtures.     

Synthesis, spectroscopic, mutagenic, and cytotoxicity studies of some mixed-ligand platinum(II) complexes of 2,2'-bipyridine and amino acids

Seven platinum(II) complexes of the type [Pt(bipy)(AA)]n+ (where n = 1 or 0 and AA is anion of L-valine, L-isoleucine, L-aspartic acid (dianion), L-glutamic acid (dianion), L-glutamine, L-proline, or S-methyl-L-cysteine) have been prepared and characterized. The modes of binding of amino acids in these complexes have been ascertained particularly by infrared and 1H NMR spectral studies. The L-glutamine complex shows a ID50 value (50% inhibitory dose) in the range of greater than 20 micrograms/ml to 100 micrograms/ml of the complex. However, the complexes of L-valine, L-isoleucine, L-aspartic acid, L-glutamic acid, L-proline, and S-methyl-L-cysteine show ID50 values greater than 100 micrograms/ml of the complex. The above complexes also show inferior growth inhibition of P-388 cells than platinum(II) complexes of 2,2'-bipyridine with L-alanine, L-leucine, L-methionine, and L-aspargine as reported earlier. The platinum(II) complexes of 2,2'-bipyridine with glycine (Gly), L-alanine (Ala), L-leucine (leu), L-valine (Val), L-methionine (Met), L-phenylalanine (Phe), L-serine (Ser), L-tyrosine (Tyr) and L-tryptophan (Trp) have been tested for mutagenesis using TA 100 and TA 98 strains. They show nonmutagenicity. This is in contrast to the cis-[Pt(NH3)2Cl2] showing a base pair substitution mutagenesis.     

Transport of branched-chain amino acids in membrane vesicles of Streptococcus cremoris.

The kinetics, specificity, and mechanism of branched-chain amino acid transport in Streptococcus cremoris were studied in a membrane system of S. cremoris in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (delta p)-generating system. Influx of L-leucine, L-isoleucine, and L-valine can occur via a common transport system which is highly selective for the L-isomers of branched chain amino acids and analogs. The pH dependency of the kinetic constants of delta p-driven L-leucine transport and exchange (counterflow) was determined. The maximal rate of delta p-driven transport of L-leucine (Vmax) increased with increasing internal pH, whereas the affinity constant increased with increasing external pH. The affinity constant for exchange (counterflow) varied in a similar fashion with pH, whereas Vmax was pH independent. Further analysis of the pH dependency of various modes of facilitated diffusion, i.e., efflux, exchange, influx, and counterflow, suggests that H+ and L-leucine binding and release to and from the carrier proceed by an ordered mechanism. A kinetic scheme of the translocation cycle of H+-L-leucine cotransport is suggested.     

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.