Isoleucine Limitation Research Articles

Efficient hydroxyproline production from glucose in minimal media by Corynebacterium glutamicum.

The efficient coupling of biotransformation steps to an existing fermentation pathway is an interesting strategy to expand the product portfolio of Corynebacterium glutamicum as whole-cell biocatalyst. This is especially challenging if the biotransformation step comprises a direct link to central metabolism, as in the case of α-ketoglutarate-dependent oxygenase catalysis. Aiming at trans-4-hydroxy-L-proline (Hyp) production from glucose in a minimal medium, the proline-4-hydroxylase gene from Dactylosporangium sp. strain RH1 was introduced into a proline-producing, isoleucine-bradytroph C. glutamicum strain. The production of proline was found to be induced by isoleucine limitation. Proline and Hyp production were found to depend differently on isoleucine limitation. Severe isoleucine limitation was shown to result in proline accumulation and low hydroxylation rates both in batch and continuous cultivation set-ups. The investigation of different steady states with various glucose/isoleucine molar ratios revealed that optimal conditions for Hyp production are met around a molar ratio of 46:1, where isoleucine limitation is sufficient to trigger proline production but the hydroxylation rate is high enough to convert the majority of formed proline to Hyp. A high cell-density fed-batch set-up was designed, capable of producing 7.1 g L(-1) of Hyp from glucose in 23 h with 98.5% conversion of proline to Hyp. Reaction engineering, specifically the fine-tuning of the glucose/isoleucine concentration ratio, enabled control of the fermentation profile and thus the accumulation of the desired product Hyp from glucose in minimal and defined media.

Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC)

Cell growth limitation is known to be an important condition that enhances L: -valine synthesis in Corynebacterium glutamicum recombinant strains with L: -isoleucine auxotrophy. To identify whether it is the limited availability of L: -isoleucine itself or the L: -isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of L: -valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel (-) ) C. glutamicum DeltailvA DeltapanB Deltarel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and L: -valine synthesis in rel (+) and rel (-) strains under conditions of L: -isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in L: -valine synthesis were observed in both rel (+) and rel (-) cells exposed to L: -isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and L: -valine synthesis in cells during the stationary growth phase.

Metabolic network analysis during fed-batch cultivation of Corynebacteriumglutamicum for pantothenic acid production: first quantitative data and analysis of by-product formation

A first generation genetically modified strain of Corynebacterium glutamicum has been assessed for its potential to synthesise and accumulate the vitamin pantothenic acid in the medium using fed-batch cultivation technology, with biomass concentration controlled by isoleucine limitation. Kinetic analysis of specific rates throughout the process has been used to model carbon flux through both central metabolism and the specific pathways involved in product formation. Flux towards pantothenic acid is potentially high but much of this flux is dissipated as by-products within associated pathways, notably linked to amino acid synthesis. The major limitation of vitamin production in this strain is linked to the tenfold higher flux of keto-isovalerate towards valine rather than pantothenic acid. Attempts to modify this ratio by imposing nitrogen limitation provoked carbon overflow as unidentified non-nitrogenous compounds. The observed accumulation of glycine suggests that the flux towards pantothenate production may by limited by the rate of the pathway intermediate (5,10-methylene-tetrahydrofolate) regeneration.

Global Expression Profiling and Physiological Characterization ofCorynebacterium glutamicumGrown in the Presence ofl-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.

Branched-chain amino acid biosynthesis in Salmonella typhimurium: a quantitative analysis.

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.

A novel RNA product of thetyrToperon ofEscherichia Coli

The tyrT operon of E. coli and several other tRNA operons of E. coli show striking structural features: they contain repeated sequence units including a 19bp motif resembling the 3' end of the corresponding mature tRNA. A novel RNA, encoded by the repeated sequence of the tyrT operon, was identified. The RNA, characterized by primer extension and Nuclease-S1 analysis, contained 171 nucleotides and terminated with the 19bp motif of the CCA-end of tRNA(1Tyr). The RNA, designated as rtT RNA, is probably released from the primary transcript of tyrT during tRNA processing, it includes the coding capacity for the arginine rich peptide Tpr. Predictions of secondary folding resulted a rather stable RNA structure with a free energy of -44.3kcal/mol. A weak ribosome binding site was found, preceding the second possible AUG initiator codon for Tpr. The comparison of rtT RNA with putative transcripts from the repeated sequences associated with related tRNA genes showed common features with respect to primary structure, arrangement and secondary folding. In E. coli cultures the lag-phase during growth, caused by transient glycine or by isoleucine limitation, was found to be overcome or markedly shortened in the presence of rtT RNA. These and previously reported results suggest a modulatory effect of rtT RNA on stringent response.

Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

We have proposed that guanosine tetraphosphate produced in Escherichia coli cells subjected to an isoleucine limitation inhibits pBR322 DNA replication [1]. In E. coli relA which cannot synthesize guanosine tetraphosphate (ppGpp) upon amino acid limitation pBR322 DNA is amplified after arginine starvation. The yield of plasmid DNA amplified either by chloramphenicol (Cm) or by arginine limitation is compared. The plasmid yield per cell is equal in amino acid-starved cells and in cells treated with Cm. To increase the plasmid content per ml of cell suspension the growth medium was supplemented with increasing amounts of nutrients. Plasmid DNA can be isolated in large quantities by this procedure. This simple method can be used for the enrichment of pBR325 DNA which cannot be amplified by Cm treatment. Our results indicate that E. coli relA strains might be suitable hosts for the amplification of pBR322 and related plasmids in E. coli.

Evidence for cAMP-mediated control of isoleucyl-tRNA synthetase formation in Escherichia coli K-12.

The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl-tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP.

3H-Thymidininkorporation nach Isoleucinlimitation in “stringent” und “relaxed” kontrollierten Stämmen vonEscherichia coli

Stringent and relaxed strains of E. coli subjected to isoleucine starvation were examined by follow-wing the incorporation of 3H-thymidine into chromosomal DNA. After valine treatment to trigger an isoleucine deprivation (p)ppGpp is synthesized in the stringent strain only. Remarkable differences in the morphology of the amino acid starved cells of the stringent and relaxed strains can be observed. Upon isoleucine limitation 3H-thymidine incorporation into DNA is reduced in both strains, but this inhibition is remarkably delayed in the relaxed strain. Our result show that the reduction of chromosomal DNA synthesis during amino acid limitation occurs also without ppGpp, but in the presence of ppGpp this process is accelerated.

3H‐Thymidininkorporation nach Isoleucinlimitation in “stringent” und “relaxed” kontrollierten Stämmen von Escherichia coli

Stringent and relaxed strains of E. coli subjected to isoleucine starvation were examined by follow-wing the incorporation of 3H-thymidine into chromosomal DNA. After valine treatment to trigger an isoleucine deprivation (p)ppGpp is synthesized in the stringent strain only. Remarkable differences in the morphology of the amino acid starved cells of the stringent and relaxed strains can be observed. Upon isoleucine limitation 3H-thymidine incorporation into DNA is reduced in both strains, but this inhibition is remarkably delayed in the relaxed strain. Our result show that the reduction of chromosomal DNA synthesis during amino acid limitation occurs also without ppGpp, but in the presence of ppGpp this process is accelerated.

Cell cycle regulation of ribonucleoside diphosphate reductase activity in permeable mouse L cells and in extracts.

Ribonucleoside diphosphate reductase (EC1.17.4.1) was previously characterized in exponentially growing mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (Kucera and Paulus, 1982b). This characterization has now been extended to cells in specific phases of the cell cycle and in transition between cell cycle phases, with activity studied both in situ (permeabilized cells) and in cell extracts. Cells at various stages in the cell cycle were obtained by unit-gravity sedimentation employing a commercially available reorienting chamber device, by G1 arrest induced by isoleucine limitation, and by metaphase arrest induced by Colcemid. G1 cells from both cycling and noncycling populations had negligible levels of ribonucleotide reductase activity as measured by CDP reduction both in situ and in extracts. When G1 arrested cells were allowed to progress to S phase, ribonucleotide reductase activity increased in parallel with [3H]thymidine incorporation into DNA. Ribonucleotide reductase activity in extracts increased at a somewhat greater rate than in situ activity. S phase ribonucleotide reductase activity measured in situ resembled the previously characterized activity in exponentially growing cells with respect to an absolute dependence on ATP or its analogs as positive allosteric effector, sensitivity to the negative allosteric effector dATP, and low susceptibility to stimulation by NADPH, dithiothreitol, and FeCl3. Disruption of permeabilized cells caused reductase activity to become highly dependent on the presence of both dithiothreitol and FeCl3. As synchronized cultures progressed from S into G2/M phase, no significant change in ribonucleotide reductase activity was seen. On the other hand, when cells that had been arrested in metaphase by Colcemid were allowed to resume cell cycle traversal by removing the drug, in situ ribonucleotide reductase activity decreased by 75% within 2.5 h. This decrease seemed to be a late mitotic event, since it was not correlated with the percentage of cells entering G1 phase. The cause of a subsequent slight increase of in situ ribonucleotide reductase activity is not clear. Parallel measurements of ribonucleotide reductase activity in cell extracts indicated also an initial decline accompanied by increasing dependence on added dithiols and FeCl3, followed by complete activity loss. Our results suggest a cell cycle pattern of ribonucleotide reductase activity that involves negligible levels in G1 phase, a progressive increase of activity upon entry into S phase paralleling overall DNA synthesis, continued retention of significant ribonucleotide reductase activity well into the metaphase period of mitosis, and a very rapid decline in activity during the later phases of mitosis. The periods of increase and decrease of ribonucleotide reductase activity were accompanied by modulation of the properties of the enzyme as indicated by differential changes in enzyme activity measured in situ and in extracts.

Cysteine starvation, isoleucyl-tRNAIle, and the regulation of the ilvGEDA operon of Escherichia coli.

The involvement of undermodified tRNA in the regulation of the ilvGEDA operon has been investigated using Escherichia coli C6, a relA-, Cys-, Met- mutant. This strain accumulates thionucleotide-deficient or methyl-deficient tRNA when starved for cysteine or methionine, respectively. The levels of threonine deaminase, the ilvA gene product, and transaminase B, the ilvE gene product, were both lower in cysteine-starved cells, as compared with either growing or methionine-starved cultures. When cysteine was added to cysteine-starved cells, growth ensued promptly and both enzyme activities returned to control levels. Treatment of recovering cultures with valine limited growth by isoleucine limitation, but did not cause a derepression of the ilvGEDA operon. Valine treatment of nonstarved or methionine-starved cells led to the expected increase in threonine deaminase and transaminase B activities. Cysteine-starved cells slowly regained the ability to derepress the operon after 3 h of recovery in complete medium. In contrast, the induction of the lac operon was normal in cysteine-starved cultures, even in the presence of valine. The loss of derepressibility of the ilvGEDA operon was correlated with the presence of a kinetically and chromatographically altered tRNAIle in cysteine-starved cells. No changes in tRNAIle were observed after methionine starvation. Using the periodate method, we found that the charging of tRNAIle increased from the normal level of 60 to 80% or greater after starvation for cysteine. Under conditions where the ilvGEDA operon was fully derepressed in nonstarved cells, the charging of tRNAIle fell to 27%. Unexpectedly, nearly identical results were obtained with cysteine-starved cells after an identical derepression test. These results suggest that factors other than the aminoacylation state of tRNAIle may be important in the regulation of this operon. In particular, modifications to tRNA which involve cysteine may be necessary for controlling the expression of the ilvGEDA operon in E. coli.

Regulation of Escherichia coli aspartate transcarbamylase synthesis by guanosine tetraphosphate and pyrimidine ribonucleoside triphosphates

The effects of guanosine tetraphosphate (ppGpp) and pyrimidine ribonucleoside triphosphates on Escherichia coli aspartate transcarbamylase (ATCase) synthesis were examined. To determine the effect of ppGpp, a stringent (relA+) and relaxed (relA) isogenic pair of E. coli K-12 strains was starved for isoleucine, and the residual rate of synthesis of this enzyme was measured. It was necessary to starve the strains for uracil before the isoleucine limitation to maintain similar, low levels of UTP, the putative pyrimidine effector of ATCase synthesis. The isoleucine starvation of the stringent strain caused an immediate 10-fold increase in the intracellular concentration of ppGpp, which was coincident with the cessation of the synthesis of the enzyme. The elevated level of ppGpp then decayed until it reached an intracellular concentration similar to that found in unstarved cells. Enzyme synthesis resumed at this time. In the relaxed strain, the intracellular concentration of ppGpp did not increase upon isoleucine starvation and synthesis of the enzyme was not repressed. These experiments strongly indicated that ppGpp acts as a negative effector of ATCase synthesis. The repression of ATCase synthesis by ppGpp was demonstrated directly by using a Salmonella typhimurium (relA) in vitro coupled transcription-translation system with a lambda specialized transducing phage carrying the E. coli K-12 operon encoding the subunits of this enzyme (pyrBI) as a source of DNA. This in vitro system was also used to measure the effects of UTP and CTP on ATCase synthesis. Increasing the concentration of UTP in the in vitro reaction mixture resulted in strong repression of this synthesis, whereas increasing the CTP concentration did not affect synthesis significantly. Possible mechanisms for the regulation of pyr gene expression, including attenuation control, are discussed.

Genetic organization of the Salmonella typhimurium ilv gene cluster

A number of Salmonella typhimurium ilv::Tn10 insertion strains were used to analyze the Salmonella ilv gene cluster. Tn10 generated ilv deletion mutants were employed in mapping experiments to conclusively define the gene order as ilvG-E-D-A-C. Examination of ilv enzyme levels confirms that the direction of transcription of ilvGEDA is from ilvG to ilvA. The major control locus, designated ilvO, is located before ilvG forming an ilvOGEDA transcriptional unit that is multivalently repressed by isoleucine, valine and leucine. Two internal promoters, one before ilvE and anonother before ilvD, are identified and are shown to provide repressed levels of the ilvE, D and A gene products. Possible regulation of transcription from these promoters in response to isoleucine limitation is discussed in terms of attenuation.

Regulation of isoleucine and valine biosynthesis in Salmonella typhimurium: The effect of hisU on repression control

The control of isoleucine and valine biosynthesis was examined in a hisU mutant of Salmonella typhimurium. It was found that the levels of expression of the ilvEDA operon and the ilvC gene were significantly reduced relative to an isogenic normal strain when grown in unsupplemented medium. In contrast, this hisU mutant exhibited only a slight reduction in total acetohydroxy acid synthase activity relative to that of the wild type. The hisU and hisU + strains were examined to determine their derepressibility upon either leucine, valine or isoleucine limitation. Only during leucine limitation did the hisU strain exhibit impaired derepressibility relative to the hisU + strain. In addition, repression control of threonine deaminase (the ilvA product of the ilvEDA operon) in this hisU mutant was refractory to exogenous supplementation with either leucine or valine. This response is in distinct contrast to that of the normal strain, in which the single addition of leucine or valine results in a significant reduction in the level of threonine deaminase.

Isolation and properties of a plasmid which expresses the E. coli Su+7 amber suppressor tRNA gene

The gene of the amber suppressor tRNA derived from tRNATry, Su+7, has been inserted into a col E1-derived vehicle by selecting for its expression. Despite selection for a suppressor phenotype, and the plasmid's stable presence at ca. 180 copies cell during balanced growth, the level mature tRNA maintained by the gene is less than that of the normal haploid tRNATry locus in the bacterial chromosome. Transfer RNA genes, both the plasmid Su+7 gene and chromosomal tRNA's are expressed during inhibition of protein synthesis. During, e.g. chloramphenicol inhibition, Su-7 and Su+7 tRNA can be elevated similarly in the plasmid-containing cell; Su+7 reaches levels of molecules/cell which ordinarily characterize a major tRNA. The recombinant plasmid, but not the cloning vehicle alone, has a more general effect on tRNA levels; accumulation of tRNA from three chromosomal tRNA loci including tRNATry, continues during extensive isoleucine limitation. The plasmid therefore contains a locus which probably alters the relaxed-stringent circuit, whose effect is disseminated to at least 3 widely separated loci.

Synthesis and activities of branched-chain aminoacyl-tRNA synthetases in threonine deaminase mutants of Escherichia coli

Valyl-, isoleucyl-, and leucyl-tRNA synthetase activities were examined in an Escherichia coli K-12 strain that possessed a deletion of three genes of the ilv gene cluster, ilvD, A, and C, and in a strain with the same deletion that also carried the lambdadilvCB bacteriophage. It was observed that the branched-chain tRNA synthetase activities of both strains were considerably less than those of the normal strain during growth in unrestricted medium. Furthermore, during an isoleucine limitation, there was a further reduction in isoleucyl-tRNA synthetase activity and an absence of the isoleucine-mediated derepression of valyl-tRNA synthetase formation in both of these mutants, as compared with the normal strain. In addition, it was observed that these branched-chain synthetase activities were reduced in steady-state cultures of several ilvA point mutants. However, upon the introduction of the ilv operon to these ilvA mutants by use of lambda bacteriophage, there was a specific increase in the branched-chain synthetase activities to levels comparable to those of the normal strain. These results support our previous findings that the stability and repression control of synthesis of these synthetases require some product(s) missing in the ilvDAC deletion strain and strongly suggest this component is some form of the ilvA gene product, threonine deaminase.

Threonyl-transfer ribonucleic acid synthetase and the regulation of the threonine operon in Escherichia coli

Two threonine-requiring mutants with derepressed expression of the threonine operon were isolated from an Escherichia coli K-12 strain containing two copies of the thr operon. One of them carries a leaky mutation in ilvA (the structural gene for threonine deaminase), which creates an isoleucine limitation and therefore derepression of the thr operon. In the second mutant, the enzymes of the thr operon were not repressed by threonine plus isoleucine; the threonyl-transfer ribonucleic acid(tRNA) synthetase from this mutant shows an apparent Km for threonine 200-fold higher than that of the parental strain. The gene, called thrS, coding for threonyl-tRNA synthetase was located around 30 min on the E. coli map. The regulatory properties of this mutant imply the involvement of charged threonyl-tRNA or threonyl-tRNA synthetase in the regulation of the thr operon.

Thialysine-resistant mutant of Salmonella typhimurium with a lesion in the thrA gene.

A mutant of Salmonella typhimurium was selected for its spontaneous resistance to the lysine analog, thialysine (S-2-aminoethyl cysteine). This strain, JB585, exhibits a number of pleiotropic properties including a partial growth requirement for threonine, resistance to thiaisoleucine and azaleucine, excretion of lysine and valine, and inhibition of growth by methionine. Genetic studies show that these properties are caused by a single mutation in the thrA gene which encodes the threonine-controlled aspartokinase-homoserine dehydrogenase activities. Enzyme assays demonstrated that the aspartokinase activity is unstable and the threonine-controlled homoserine dehydrogenase activity absent in extracts prepared from the mutant. These results explain the growth inhibition by methionine because the remaining homoserine dehydrogenase isoenzyme would be repressed by methionine, causing a limitation for threonine. The partial growth requirement for threonine during growth in glucose minimal medium may also, by producing an isoleucine limitation, cause derepression of the isoleucine-valine enzymes and provide an explanation for both the valine excretion, and azaleucine and thiaisoleucine resistance. The overproduction of lysine may confer the thialysine resistance.

Arrest of C1300 neuroblastoma cells by limiting serum or isoleucine: Implications for growth control in malignant cells

The arrest of C1300 neuroblastoma cells by limiting serum or isoleucine in the growth medium is described. The resumption of DNA synthesis after the return of the cells to complete medium indicates that they stop in the early G 1 (or G 0) phase of the cell cycle with both arrest procedures. However, the isoleucine limitation procedure also arrests about half of the cells in the G 2 phase of the cell cycle. This result is used to modify a recent model for growth control of transformed cells.