Regulation Of Lysine Biosynthesis Research Articles

The AMP-Activated Protein Kinase (AMPK) Positively Regulates Lysine Biosynthesis Induced by Citric Acid in Flammulina filiformis.

Flammulina filiformis, the most produced edible mushroom species in China, is rich in lysine. Further enhancing its lysine biosynthesis is vital for improving its quality in industrialized cultivation. Citric acid induction significantly increases both the biomass and growth rate of F. filiformis hyphae, as well as the lysine content. The genes encoding enzymes in the lysine biosynthesis pathway were detected under the optimal induction, revealing that the expression levels of hcs, hac, and hah were 2.67, 1.97, and 1.90 times greater, respectively, relative to the control, whereas no significant difference was seen for hdh, aat, sr, and shd, and the expression of aar decreased. Furthermore, the transcriptional levels of Ampk, GCN2, GCN4, and TOR were found significantly upregulated, with the most upregulated, Ampk, reaching a level 42.68 times greater than that of the control, while the phosphorylation of AMPK rose by nearly 54%. In AMPK-silencing strains under the optimal induction, however, the phosphorylation increment dropped to about 16% and the lysine content remained at the same level as in the WT. Thus, AMPK is presented as the critical intermediary in citric acid's regulation of lysine biosynthesis in F. filiformis.

Involvement of the arginine repressor in lysine biosynthesis of Thermus thermophilus

Lysine biosynthesis of Thermus thermophilus proceeds in a similar way to arginine biosynthesis, and some lysine biosynthetic enzymes from T. thermophilus so far investigated have the potential to function in arginine biosynthesis. These observations suggest that arginine might regulate the expression of genes for lysine biosynthesis. To test this hypothesis, the argR gene encoding the regulator of arginine biosynthesis was cloned from T. thermophilus and its function in lysine biosynthesis was analysed. The addition of arginine to the culture medium inhibited the growth of an arginase gene knockout mutant of T. thermophilus, which presumably accumulates arginine inside the cells. Arginine-dependent growth inhibition was not alleviated by the addition of ornithine, which is a biosynthetic intermediate of arginine and serves as a peptidoglycan component of the cell wall in T. thermophilus. However, the growth inhibition was cancelled either by the simultaneous addition of lysine and ornithine or by a knockout of the argR gene, suggesting the involvement of argR in regulation of lysine biosynthesis in T. thermophilus. Electrophoretic mobility shift assay and DNase I footprinting revealed that the ArgR protein specifically binds to the promoter region of the major lysine biosynthetic gene cluster. Furthermore, an alpha-galactosidase reporter assay for this promoter indicated that arginine repressed the promoter in an argR-dependent manner. These results indicate that lysine biosynthesis is regulated by arginine in T. thermophilus.

Regulation of aspartokinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase in Lactobacillus plantarum

The use of a lysine-overproducing strain of Lactobacillus plantarum in food or feed fermentations may lead to the production of lysine-rich products. The availability of functional genes and information on the regulation of lysine biosynthesis are required to develop a lysine-overproducing strain. The genome sequence of L. plantarum revealed putative lysine biosynthetic genes, some of which may produce isozymes. This study examined the functionality of the genes and the regulation of the first four enzymes of lysine biosynthesis, together with homoserine dehydrogenase, in L. plantarum. The genes were expressed in Escherichia coli, and the regulation of the enzymes was studied in cell extracts of both recombinant E. coli and L. plantarum. Among seven lysine biosynthetic genes studied (aspartokinase genes, thrA1 and thrA2; aspartate semialdehyde dehydrogenase genes, asd1 and asd2; dihydrodipicolinate synthase genes, dapA1 and dapA2; and the dihydrodipicolinate reductase gene, dapB) plus two homoserine dehydrogenase genes (hom1 and hom2), the products of six genes, i.e. thrA2, asd2, dapA1, dapB, hom1 and hom2, showed obvious enzyme activities in vitro. The product of one of the homoserine dehydrogenase genes, hom1, exhibited both homoserine dehydrogenase and aspartokinase activities. However, the aspartokinase activity was mainly due to ThrA2 and was inhibited by L-lysine and repressed by L-threonine, and the homoserine dehydrogenase activity was mainly due to Hom2 and was inhibited by L-threonine. The aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase were not regulated by the end-products of the pathway.

Regulation of lysine biosynthesis and transport genes in bacteria: yet another RNA riboswitch?

Comparative analysis of genes, operons and regulatory elements was applied to the lysine biosynthetic pathway in available bacterial genomes. We report identification of a lysine-specific RNA element, named the LYS element, in the regulatory regions of bacterial genes involved in biosynthesis and transport of lysine. Similarly to the previously described RNA regulatory elements for three vitamins (riboflavin, thiamin and cobalamin), purine and methionine regulons, this regulatory RNA structure is highly conserved on the sequence and structural levels. The LYS element includes regions of lysine-constitutive mutations previously identified in Escherichia coli and Bacillus subtilis. A possible mechanism of the lysine-specific riboswitch is similar to the previously defined mechanisms for the other metabolite-specific riboswitches and involves either transcriptional or translational attenuation in various groups of bacteria. Identification of LYS elements in Gram-negative gamma-proteobacteria, Gram-positive bacteria from the Bacillus/Clostridium group, and Thermotogales resulted in description of the previously uncharacterized lysine regulon in these bacterial species. Positional analysis of LYS elements led to identification of a number of new candidate lysine transporters, namely LysW, YvsH and LysXY. Finally, the most likely candidates for genes of lysine biosynthesis missing in Gram- positive bacteria were identified using the genome context analysis.

Degradation of lysine in rice seeds: Effect of calcium, ionic strength, S‐adenosylmethionine and S‐2‐aminoethyl‐ l‐cysteine on the lysine 2‐oxoglutarate reductase‐saccharopine dehydrogenase bifunctional enzyme

Lysine biosynthesis has been extensively studied and the regulatory enzymes characterized in some of the most important crop plants, however, much less is known about the lysine degradation pathway. Lysine 2‐oxoglutarate reductase (LOR) and saccharopine dehydrogenase (SDH) have recently been partially purified and characterized from plants, and have been shown to exist as a single bifunctional polypeptide. We have further characterized these enzymes from rice endosperm in relation to Ca2+ and ionic strength modulation. Optimum pH values of 7.0 and 8.0 were obtained for LOR and SDH, respectively. The LOR domain of the polypeptide was modulated by Ca2+ and ionic strength, whereas the SDH domain was not. It would appear that the modulation by Ca2+ and ionic strength of LOR is a common feature among plant LOR enzymes. S‐adenosylmethionine (SAM) did not produce any significant effect on either enzyme activity, indicating that it only plays a role in the regulation of lysine biosynthesis. The effect of S‐2‐aminoethyl‐ l‐cysteine (AEC) as both a substrate and an inhibitor of LOR activity was also tested. AEC was shown to partially substitute for lysine as a substrate for LOR, but was also able to inhibit LOR activity, possibly competing with lysine at the active site. The higher Km for AEC compared to lysine may reflect a lower binding affinity for AEC.

Regulation of aspartokinase in Lactobacillus plantarum.

As a rational approach to the genetic development of a stable lysine overproducing strain of Lactobacillus plantarum for the fermentation of 'ogi', a Nigerian fermented cereal porridge, regulation of lysine biosynthesis in this species was investigated. Spontaneous lysine overproducing mutants of Lact. plantarum were obtained and their aspartokinase activities compared with those of wild-type strains under different conditions. Results showed that aspartokinase activity of Lact. plantarum cell extracts was not inhibited by either lysine, threonine, methionine or combinations of lysine and threonine. Instead, methionine enhanced aspartokinase activity in vitro. Results indicated that lysine biosynthesis in Lact. plantarum could be regulated by lysine via the control of aspartokinase production in a way different to that described for other bacteria.

Regulation of aspartokinase in Lactobacillus plantarum

As a rational approach to the genetic development of a stable lysine overproducing strain of Lactobacillus plantarum for the fermentation of ‘ogi’, a Nigerian fermented cereal porridge, regulation of lysine biosynthesis in this species was investigated. Spontaneous lysine overproducing mutants of Lact. plantarum were obtained and their aspartokinase activities compared with those of wild‐type strains under different conditions. Results showed that aspartokinase activity of Lact. plantarum cell extracts was not inhibited by either lysine, threonine, methionine or combinations of lysine and threonine. Instead, methionine enhanced aspartokinase activity in vitro. Results indicated that lysine biosynthesis in Lact. plantarum could be regulated by lysine via the control of aspartokinase production in a way different to that described for other bacteria.

Regulation of alpha-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from alpha-aminoadipate into penicillin biosynthesis.

The activity and regulation of alpha-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for alpha-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of alpha-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest alpha-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5-1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM alpha-aminoadipate, highest penicillin production was observed in those strains whose alpha-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of alpha-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of alpha-aminoadipate, and hence penicillin.

Regulation of lysine and dipicolinic acid biosynthesis in Bacillus brevis ATCC 10068: significance of derepression of the enzymes during the change from vegetative growth to sporulation.

Lysine biosynthetic pathway enzymes of Bacillus brevis ATCC 1068 were studied as a function of stage of development (growth and sporulation). The synthesis of aspartic-2-semialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHDPA-reductase and diaminopimelate decarboxylase (DAP-decarboxylase) was found not to be co-regulated, since lysine was not a co-repressor for these enzymes. Unlike the aspartokinase isoenzymes, the other enzymes of the lysine pathway were not derepressed in thiosine-resistant, lysine-excreting mutants. Thus, the aspartokinase isoenzymes were the key enzymes during growth and regulation of lysine biosynthesis through restriction of L-ASA synthesis via feedback control by lysine on the aspartokinases was therefore suggested. In contrast to other Bacillus species, the levels of the lysine biosynthetic pathway enzymes of strain ATCC 10068 were not derepressed during the change from vegetative growth to sporulation. Two control mechanisms, enabling the observed preferential channelling of carbon for the synthesis of spore-specific diaminopimelic acid (DAP) and dipicolinic acid (DPA) were a) loss of DAP-decarboxylase, b) inhibition of DHDPA-reductase by DPA. Increase in the level of the DAP pool during sporulation, as a consequence of the loss of DAP-decarboxylase, and its relevance to the non-enzymatic formation of DPA has been discussed.

Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris (Spegazzini and Comes)

Two S-(2-aminoethyl)L-cysteine (AEC) resistant lines were isolated by screening mutagenized protoplasts from diploid N. sylvestris plants. Both lines accumulated free lysine at levels 10 to 20-fold higher than in controls. Lysine overproduction and AEC-resistance were also expressed in plants regenerated from the variant cultures. A feedback insensitive form of dihydrodipicolinate synthase (DHPS), the pathway specific control enzyme for lysine synthesis, was detected in callus cultures and leaf extracts from the resistant lines. Aspartate kinase (AK), the other key enzyme in the regulation of lysine biosynthesis, was unaltered in the mutants. Crosses with wild type plants indicated that the mutation conferring insensitivity to feedback in DHPS, with as result overproduction of lysine and resistance to AEC, was inherited as a single dominant nuclear gene.

Regulation of lysine biosynthesis in Brevibacterium flavum

AbstractL‐lysine synthesis pathway enzyme activities: β‐aspartate kinase (EC.2.7.2.4), diaminopimelate decarboxylase (EC.4.1.1.20) for two L‐lysine producing strains Brevibacterium flavum 22LD and RC‐115 were studied. It has been found that β‐aspartate kinase and diaminopimelate decarboxylase in the Br. flavum RC‐115 are less sensitive to feed‐back inhibition by lysine and threonine. It is supposed that desensitized β‐aspartate kinase in the Br. flavum RC‐115 can be determined by genetical changes of the regulatory properties of the β‐aspartate kinase.Auxotrophity in the locus of homoserine dehydrogenase was tested and no homoserine dehydrogenase (EC.1.1.1.3) activity was found in either strain.The combination of these both types of mutation supplemented by the lack of catabolic repression in the RC‐115 strain makes it an active lysine producer in the medium with high carbohydrates content.

The relA locus and the regulation of lysine biosynthesis in Escherichia coli.

The allelic state of relA influences the phenotype of Escherichia coli strains carrying the lysA22 mutation:lysA22 relA strains are Lys- where lysA22 relA+ strains grow (slowly) in the absence of lysine. This physiological effect has been related to an effect of the expression of the relA locus on the regulation of lysine biosynthesis. The fully derepressed levels of some lysine enzymes (aspartokinase III, aspartic semialdehyde dehydrogenase, didhydrodipicolinate reductase) are observed under lysine limitation only in rel+ strains. And the induction of DAP-decarboxylase by DAP is much higher in rel+ than in rel- strains when an amino acid limitation of growth is also realised. These results are in agreement with the hypothesis of Stephens et al. (1975) on a possible role of the stringent regulation as a general signal for amino acid deficiency.

Biosynthesis of L-lysine and L-threonine in Brevibacterium. I. Pathway and regulation of lysine biosynthesis in Brevibacterium lactofermentum.

Biosynthesis of L-lysine and L-threonine in Brevibacterium. I. Pathway and regulation of lysine biosynthesis in Brevibacterium lactofermentum.

Regulation of lysine biosynthesis by leucine in Brevibacterium lactofermentum.

The effect of L-leucine on L-lysine production was investigated in Brevibacterium lactofermentum.L-Lysine production was inhibited by L-leucine specifically, and this inhibition was recovered completely by L-valine. The present study indicated that the inhibition of L-lysine production by the addition of excess L-leucine was accompanied with the repression of dihydrodipicolinate (DDP) synthetase. DDP synthetase was the first specific enzyme in lysine biosynthesis.On the other hand, α-isopropylmalate (IPM) synthetase, which was the key enzyme and the first specific enzyme in leucine biosynthesis was inhibited by L-leucine, while L-lysine caused the complete reversion from its inhibition.From the above results, the possible physiological meanings of this coregulation mechanism of lysine and branched-chain amino acids biosynthesis in B. lactofermentum was discussed.

Effect of mutations affecting lysyl-tRNALys on the regulation of lysine biosynthesis in Escherichia coli

When studying mutants affecting lysyl-tRNA synthetase or tRNAlys (hisT, hisW), a lack of correlation is clearly observed between the amount of lysyl-tRNA and the level of derepression of several lysine biosynthetic enzymes. This exlcudes the possible role of lysyl-tRNA as the specific corepressor of the lysine regulon. However, the level of derepression of DAP-decarboxylase, the last enzyme of the lysine pathway, is very low in the hisT mutant; this indicates that tRNAlys is a secondary effector involved in the regulation of the synthesis of this enzyme.

Role of lysyl-tRNA in the regulation of lysine biosynthesis in Escherichia coli K12

A mutant of lysyl-tRNA synthetase has been isolated in Escherichia coli K12. With this strain the Kmapp for lysine is 25 fold higher than with the parental strain. The percentage of charged tRNAlys in vivo is only 7 per cent (as against 65 per cent with HFR H). Under these conditions no derepression of synthesis is observed for three lysine biosynthetic enzymes (AK III, ASA-dehydrogenase, DAP-decarboxylase) ; a partial derepression is obtained in the case of the dhdp-reductase. Thus lysyl-tRNA does not act as the only corepressor molecule in the lysine regulon.

Overproduction of lysine by mutant strains of Escherichia coli with defective lysine transport systems.

Mutants selected on the basis of their resistance to S-(beta-aminoethyl) cysteine and overproduction of lysine were found to be defective in the lysine transport system. The overproduction of lysine was not due to mutation affecting either of the two regulatory enzymes aspartokinase and dihydrodipicolinic acid synthetase. Uptake of labeled lysine by the lysine-specific transport system was reduced to a negligible level, while uptake by the lysine, ornithine, arginine system was also affected. A hypothesis regarding the nature of these mutations and their effects on the regulation of lysine biosynthesis is discussed.

Consequences of lysine oversynthesis in Pseudomonas mutants insensitive to feedback inhibition. Lysine excretion or endogenous induction of a lysine-catabolic pathway.

Regulation of lysine biosynthesis was studied in Pseudomonas putida and Pseudomonas acidovorans. In both species, aspartokinase was subject to feedback inhibition by lysine and threonine and dihydrodipicolinate synthetase was inhibited by lysine. P. acidovorans could not degrade lysine whereas P. putida degraded lysine through an inducible lysine oxygenase. P. putida mutants with an aspartokinase insensitive to lysine inhibition were obtained. They did not excrete lysine but their lysine oxygenase was endogenously induced. However mutants obtained from a mutant lacking lysine oxygenase still did not excrete lysine. P. acidovorans mutants with a dihydrodipicolinate synthetase desensitized towards lysine inhibition were obtained. From them double mutants with both dihydrodipicolinate synthetase and aspartokinase desensitized were also obtained. All of these mutants excreted lysine.

Enzymatic and Physiological Properties of the Yeast Glutamate-α-Ketoadipate Transaminase

In a study of differences between the two glutamate-α-ketoadipate transaminases of Saccharomyces, it was observed that glutamate-α-ketoadipate transaminase I exhibited lower substrate specificity, functioning with aspartate in addition to glutamate as amino donor but not with alanine. Glutamate-α-ketoadipate transaminase II functioned with glutamate but not with aspartate or alanine. Glutamate-α-ketoadipate transaminase II readily catalyzed the reverse reaction (toward α-ketoadipate), whereas glutamate-α-ketoadipate transaminase I exhibited only weak activity in the reverse direction. A kinetic study of the forward reaction revealed that α-ketoglutarate, a product, behaved as a strong noncompetitive inhibitor of glutamate-α-ketoadipate transaminase I but not of glutamate-α-ketoadipate transaminase II. On the other hand, glutamate-α-ketoadipate transaminase II formation was derepressed by growth on α-aminoadipate, and to a lesser extent by lysine, and was repressed by glucose, while glutamate-α-ketoadipate transaminase I was scarcely affected by the growth conditions. These mechanisms may play a role in the regulation of lysine biosynthesis in yeast.