Deregulated branched-chain amino acid synthesis in a Nicotiana plumbaginifolia cell line resistant to valine

A Nicotiana plumbaginifolia cell line able to grow in the presence of high doses of valine was isolated following -rays mutagenesis. The selected clone, named D5R5, showed a growth rate higher than that of wild-type. It was less sensitive also to an equimolar mixture of the three branched-chain amino acids, but did not display cross-resistance to isoleucine and leucine. The increased tolerance was due to neither a reduced valine uptake, nor a modification in the level or sensitivity to feed-back inhibition by valine of the first common enzyme (and the main regulative site) in isoleucine, leucine and valine synthesis, acetohydroxyacid synthase (AHAS). When wild-type cells were fed with valine or equimolar mixtures of the three aminoacids, a decrease in AHAS level was found. On the contrary, the level of extractable AHAS activity from D5R5 cells was significantly less affected by similar treatments, suggesting that some alteration in enzyme modulation mechanism(s) could account for valine resistance.Abbreviations AHAS acetohydroxyacid synthase - BCAA branched-chain amino acid - FAD flavin adenine dinucleotide - ILV equimolar mixture of isoleucine, leucine and valine - TPP thiamine pyrophosphate     

Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km(r)). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum DeltailvA DeltapanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km^r). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

Regulation of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) growth in McCoy cells by amino acid antagonism

Chlamydiae have amino acid requirements for growth in tissue culture as defined by those amino acids whose individual omission from the growth medium prevents chlamydial multiplication. We have tested the hypothesis that this inhibition of growth arises as a result of antagonism between particular amino acids such that inhibition occurs when the concentration of one amino acid is reduced in the presence of the antagonist amino acid at high concentration. Using the Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), in the presence of cycloheximide, the requirement for valine was abrogated by the simultaneous omission of isoleucine, that for phenylalanine by simultaneous omission of tryptophan and that for leucine by simultaneous omission of isoleucine plus valine. The antagonism shown between leucine and isoleucine plus valine appears to be unique among bacteria. In the absence of cycloheximide, GPIC had an additional need for tryptophan, tyrosine and isoleucine; these amino acid requirements were shown for both infected McCoy, HeLa and BHK cells. The results are consistent with a mechanism for regulation of parasite growth which depends on the balance of amino acid concentrations in the extracellular environment.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

The yeast ILV2 gene is under general amino acid control

The yeast ILV2 gene encodes acetolactate synthase, the first enzyme in the biosynthesis of isoleucine and valine. Its multiple regulation has precluded the clear demonstration of whether ILV2 is under general amino acid control. Nonderepressible gcn4 strains were used as recipients for transformation with a YCp plasmid carrying GCN4. Parental gcn4 cells and their isogenic GCN4 transformants were evaluated for ALS derepression following induced amino acid starvation. GCN4 cells showed 1.5- to 1.7-fold derepression but no derepression was observed in isogenic control gcn4 strains. A similar depression of ILV2 mRNA was also observed. Genetic evidence for general amino acid control was the gcn4 suppression of high level resistance to sulfometuron methyl by the SMRI-410 allele of ILV2.     

Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine

Addition of L-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 DeltailvA DeltapanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of L-isoleucine could relieve the valine effect on VAL1 whereas L-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.     

A general method for selection of alpha-acetolactate decarboxylase-deficient Lactococcus lactis mutants to improve diacetyl formation

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

Effect of Isoleucine, Valine, or Leucine Starvation on the Potential for Formation of the Branched-Chain Amino Acid Biosynthetic Enzymes

The derepression of the isoleucine and valine biosynthetic enzymes in Escherichia coli and Salmonella typhimurium was examined under conditions of restriction of isoleucine, valine, or leucine (the three amino acids needed for multivalent repression of these enzymes). A procedure was used that allowed the measurement of enzyme-forming potential that accumulated during the starvation period, but could not be expressed unless the missing amino acid was supplied. The threonine deaminase (the product of the ilvA gene)-forming potential that accumulated under such conditions was found to be unstable and decayed with a half-life of about 2.5 min (at 37 C). Evidence was obtained that indicates the threonine deaminase-forming potential that accumulates under conditions of isoleucine starvation is in the form of initiated (rifampin-resistant), but uncompleted (actinomycin D-sensitive), messenger ribonucleic acid chains. Furthermore, it appears that a large portion of the threonine deaminase- and dehydrase (the product of the ilvD gene)-forming potential, under such conditions, is in the form of initiated polypeptide chains. Based on these results and results obtained with SuA(-) strains, a model is presented that explains how the second gene (D) in the ilvADE operon can be partially transcribed and translated under conditions in which there are no completed messenger ribonucleic acids for the gene (A) transcribed before it.     

Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.     

Attenuation in the threonine operon: effects of amino acids present in the presumed leader peptide in addition to threonine and isoleucine

Summary We have studies in vivo the contribution of amino acids corresponding to codons of the leader sequence other than the so-called regulatory codons (for threonine and isoleucine) in the expression of the threonine operon. In the presence of threonine and isoleucine, addition of each amino acid encoded in the proximal part of the leader sequence resulted in a significant decrease of the expression of the operon, over and above that decrease observed in the sole presence of threonine and isoleucine. These effects were cumulative. No such effect was found with the amino acids encoded by the distal part of the leader sequence. These findings are discussed in the light of the current model of attenuation.