A Mutant of Arabidopsis thaliana lpar;L.) Heynh. with Modified Control of Aspartate Kinase by Threonine

Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grow on 2 m M lysine has been used to isolate mutants that may contain an aspartate kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an aspartate kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of aspartate kinase, were crossed to assess the effects of the simultaneous presence of these different aspartate kinase forms. A double mutant (RLT40 × RL4) was isolated and characterized by two feedback-desensitized isozymes of aspartate kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double aspartate kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1and ak2) were found to be unlinked.     

Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes

The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B.␣lactofermentum ATCC 13869. The hom- and thrB- disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine. Received: 23 January 1996 / Received last revision: 28 July 1996 / Accepted: 5 August 1996     

Lysine-requiring mutants ofAspergillus ochraceus

Seven mutants ofAspergillus ochraceus unable to produce lysine have been selected by treating conidia of the wild type with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), at pH 6.4. Complementation analysis revealed that MNNG had caused a mutation at a single locus. Growth studies indicated the growth requirement for lysine in the mutants. Lysine-requiring mutants were further characterized by measurement of colony extension rate at various lysine concentrations.     

Isolation of regulatory mutants of Pseudomonas acidovorans by use of amino acid analogs

Mutants resistant to various combinations of threonine, lysine and/or their analogs were obtained and characterized in Pseudomonas acidovorans. In particular, mutants resistant to aminoethylcysteine had a dihydrodipicolinate synthetase insensitive to lysine inhibition whereas mutants resistant to threonine plus a low concentration of aminoethylcysteine had a feedback-insensitive aspartokinase.     

L-lysine production at 65°C by auxotrophic-regulatory mutants ofBacillus stearothermophilus

Summary The amino acid L-lysine was produced from auxotrophic-regulatory mutants ofBacillus stearothermophilus at a temperature of 60–65°C. One of the mutants (AEC 12 A5, S-(2-aminoethyl)-cysteine^r, homoserine^–), produced L-lysine at the concentration of 7.5 g/l in shaken flasks in minimal medium containing 5% glucose. Culture conditions for optimizing L-lysine production were not investigated. The aspartokinase activity of the wild strainB. stearothermophilus Zu 183 was inhibited by lysine alone and by threonine plus lysine. AEC resistant mutants showed an aspartokinase activity genetically desensitized to the feedback inhibition. Optimal temperature and pH of aspartokinase were 45°C and 9.5, respectively. The data provide significant evidence that mutants of the speciesB. stearothermophilus have a potential value for amino acid production.     

Genetic and amino-acid analysis of two maize threonine-overproducing,lysine-insensitive aspartate kinase mutants

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37–54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.     

Lysine Overproduction Mutations in the YeastSaccharomyces cerevisiae Are Introduced into Industrial Yeast Strains

Yeast mutants resistant to a toxic lysine analog, thialysine were obtained by a method described in the literature [1]. A strain excreting the maximum amount of lysine (0.45 g/l) was selected from these mutants. The intracellular content of lysine was also increased by 30%. The genetic nature of lysine overproduction was studied in this strain. An increase in the amount of excreted lysine was shown to be determined by at least two genes, one of which carries a mutation of thialysine resistance manifesting the pleiotropic effect of lysine overproduction (Th1 ^R) and the other is involved in the regulation of lysine production (PRL). Linkage groups of these genes were determined: the first gene was mapped to the IV chromosome and the second, to the XV chromosome. Both genetic characters were introduced into industrial baker's yeast strains via a series of backcrosses. The stabilization of the genome in the newly derived strains was confirmed by electrokaryotyping.     

Protein lysine methylation contributes to modulating the response of sensitive and tolerant Arabidopsis species to cadmium stress

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analysed the importance of protein lysine methylation for plants to cope with cadmium. We analysed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs are regulated by cadmium. Also, we showed that 9 out of 23 A. thaliana mutants disrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, and the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress.     

Improved L-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation

The amino acid L-lysine is produced on a large scale using mutants of Corynebacterium glutamicum. However, as yet recombinant DNA techniques have not succeed in improving strains selected for decades by classic mutagenesis for high productivity. We here report that seven biosynthetic enzymes were assayed and oversynthesis of the dihydrodipicolinate synthase resulted in an increase of lysine accumulation from 220 mM to 270 mM. The synthase, encoded by dapA, is located at the branch point of metabolite distribution to either lysine or threonine and competes with homoserine dehydrogenase for the common substrate aspartate semialdehyde. When graded dapA expression was used, as well as quantification of enzyme activities, intracellular metabolite concentrations and flux rates, a global response of the carbon metabolism to the synthase activity became apparent: the increased flux towards lysine was accompanied by a decreased flux towards threonine. This resulted in a decreased growth rate, but increased intracellular levels of pyruvate-derived valine and alanine. Therefore, modulating the flux at the branch point results in an intrinsically introduced growth limitation with increased intracellular precursor supply for lysine synthesis. This does not only achieve an increase in lysine yield but this example of an intracellularly introduced growth limitation is proposed as a new general means of increasing flux for industrial metabolite overproduction. Received: 8 August 1997 / Received revision: 2 October 1997 / Accepted: 14 October 1997     

Studies on Mechanisms for Lysine Production by Pyruvate Kinase-Deficient Mutants of Brevibacterium flavurn

Methionine-insensitive revertants with normal homoserine dehydrogenase (HD) derived from Brevibacterium flavum mutant No. 1-231, a lysine producer with S-(2-aminoethyl)-l-cysteine (AEC) resistance, methionine sensitivity, a low HD level and a pyruvate kinase (PK) defect, were still AEC-resistant and PK-deficient similar to No. 1-231. But they did not produce more lysine than the original strain, No. 15-8, from which strain No. 1-231 was derived. A high lysine producing mutant, No. 22, which was derived from strain No. 1-231, selected by sensitivity to β-fluoropyruvate (FP), and was defective in HD, produced more lysine than HD-defective mutants which were derived by two-step mutation from strain No. 1-231, selected by homoserine auxotrophy. Strain No. 22 did not show FP sensitivity under the conditions tested. Among various lysine-biosynthetic enzymes examined, it had a higher level of aspartate-β-semialdehyde dehydrogenase than did its parent and the latter HD-defective mutants. Strain No. 22 produced 50 g/liter of lysine as the HC1 salt when cultured for 72 hr in a medium containing soybean-meal hydrolysate, methionine and 100 g/liter of glucose.     

Segregation for endosperm lysine in F2, F3 and F4 progeny from a cross of in vitro-selected and unselected cultivar of rice

Summary Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F₂ populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R² 0.52 and 0.56). The F₂ segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F₃ and F₄ data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility.Research done under the auspices of the USDA, ARS, Plant Sciences Institute, Plant Molecular Biology Laboratory, Beltsville, MD 20705, USA     

Irradiation induced physiological and morphological variation in Colletotrichum capsici,incitant of anthracnose of chillies

With the aid of radioactive phosphorus and gamma rays, a number of morphological and biochemical mutants ofColletotrichum capsici have been induced and these showed wide range of variability. Biochemical mutants mostly induced by means of p³² irradiation showed deficiency of amino acids or vitamins. Specific deficiency analysis of some of the biochemical mutants revealed requirements for lysine, methionine, glycine or serine and biotin. These biochemical mutants could not be distinguisbed from the wild type in morphological characters when grown on complete or otherwise suitable supplemented media.     

The histone methyltransferase SDG8 regulates shoot branching in Arabidopsis

Histone lysine methylation is an evolutionally conserved modification involved in determining chromatin states associated with gene activation or repression. Here we report that the Arabidopsis SET domain group 8 (SDG8) protein is a histone H3 methyltransferase involved in regulating shoot branching. Knockout mutations of the SDG8 gene markedly reduce the global levels of histone H3 trimethylation at lysines 9 and 36 as well as dimethylation at lysine 36. The sdg8 mutants produce more shoot branches than wild-type plants. The expression of SPS/BUS (supershoot/bushy), a repressor of shoot branching, is decreased in sdg8 mutants, while UGT74E2 (UDP-glycosyltransferase 74E2), a gene associated with increased shoot branching, is up-regulated in sdg8 mutants. The altered expression of SPS/BUS and UGT74E2 correlates with changed histone H3 methylation at these loci. These results suggest that SDG8 regulates shoot branching via controlling the methylation states of its target genes.     

Effect of dimethyl sulfoxide on lysine production by a mutant ofBacillus subtilis with low homoserine dehydrogenase activity

SomeBacillus subtilis mutants with different levels of homoserine dehydrogenase were described. Strains that do not accumulate methionine have a high homoserine dehydrogenase activity. Low activity was detected in mutants where cell growth was completely inhibited by 0.7 mmol/L methionine. A low concentration of dimethyl sulfoxide had a stimulatory effect on lysine production by the methionine-sensitive mutant ofBacillus subtilis.     

Undermodification in the first position of the anticodon of supG-tRNA reduces translational efficiency

Summary Two mutants of Escherichia coli, trmC1 and trmC2, which are both defective in the synthesis of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) were utilized to study the function of this complex modified nucleoside. Transfer RNAs specific for glutamine, glutamic acid and lysine as well as a specific ochre suppressor derived from lysine tRNA (tRNA _UAA ^lys encoded by the supG allele), contain this modified nucleoside at position 34 (the wobble position). It was found that two different undermodified derivatives of mnm⁵s²U were present in the two trmC mutants, which suggests that the two mutations affect two different enzymatic activities. Using the lacI-Z fusion system (Miller and Albertini 1983), we found that the efficiency of supG-mediated suppression was reduced to 30%–90% of the wild-type value in the trmC mutants. The modificationdeficient supG-tRNA in the mutants showed a higher sensitivity to codon context than the normal tRNA _UAA ^lys .     

The fungal α‐aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate

Fungi produce α‐aminoadipate, a precursor for penicillin and lysine via the α‐aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.     

Production of N-Succinyl-l-diaminopimelic Acid by Auxotrophic Mutants of Aerobacter aerogenes and Serratia marcescens

α,ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl-l-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.

The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP^?) and S. marcescens KY 8921 (DAP^?/Lys^?) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for l-lysine. High levels of DAP (2000~4000 μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200 μg/ml at 200 μg/ml of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i.e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000 μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50~100μg/ml.     

Lysine metabolism in higher plants

Summary. The essential amino acid lysine is synthesised in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine. Enzyme kinetic studies and the analysis of mutants and transgenic plants that overaccumulate lysine, have indicated that the major site of the regulation of lysine synthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, there is strong evidence that lysine is also subject to catabolism in plants, specifically in the seed. The two enzymes involved in lysine breakdown, lysine 2-oxoglutarate reductase (also known as lysine α-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/dephosphorylation. Received December 21, 1999 Accepted February 7, 2000     

Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The Arabidopsis KRYPTONITE gene encodes a member of the Su(var)3-9 family of histone methyltransferases. Mutations of kryptonite cause a reduction of methylated histone H3 lysine 9, a loss of DNA methylation, and reduced gene silencing. Lysine residues of histones can be either monomethylated, dimethylated or trimethylated and recent evidence suggests that different methylation states are found in different chromatin domains. Here we show that bulk Arabidopsis histones contain high levels of monomethylated and dimethylated, but not trimethylated histone H3 lysine 9. Using both immunostaining of nuclei and chromatin immunoprecipitation assays, we show that monomethyl and dimethyl histone H3 lysine 9 are concentrated in heterochromatin. In kryptonite mutants, dimethyl histone H3 lysine 9 is nearly completely lost, but monomethyl histone H3 lysine 9 levels are only slightly reduced. Recombinant KRYPTONITE can add one or two, but not three, methyl groups to the lysine 9 position of histone H3. Further, we identify a KRYPTONITE-related protein, SUVH6, which displays histone H3 lysine 9 methylation activity with a spectrum similar to that of KRYPTONITE. Our results suggest that multiple Su(var)3-9 family members are active in Arabidopsis and that dimethylation of histone H3 lysine 9 is the critical mark for gene silencing and DNA methylation.