Influence of antibiotics and trace salts on lysine production by arthrobacter globiformis

A hydrocarbon utilizing strain of Arthrobacter globiformis Lb isolated from local soil has been found to yield lysine 3.4 g l^?1, keeping the medium optimal for pH, C- and N-sources. Addition of antibiotics and micronutrients to that optimal media stimulated cell growth and enhanced lysine yield.     

Synthesis, tandem MS- and NMR-based characterization, and quantification of the carbon 13-labeled advanced glycation endproduct, 6-N-carboxymethyllysine

Summary. 6-N-carboxymethyllysine (CML), generated by the glycation and/or oxidation of lysine residues, has been measured in biological materials and food products using techniques such as ELISA, HPLC with fluorescence detection and mass spectrometry methods. Only limited information has been reported regarding the preparation of standards labeled with either deuterium, ¹³C or ¹⁵N atoms to be used as internal standards. In the present paper, a synthesis of carbon-13 labeled CML is described using l,2-¹³C₂-glyoxylic acid and 2-N-acetyllysine as starting materials. The resulting labeled 2-N-acetyl-CML was purified by HPLC-UV as a dibutyl ester. After a deprotection step, the yield was evaluated to be 53% when the reaction was conducted 17 h at 37°C. CML was extensively studied by ¹H- and ¹³C-NMR and the fragments observed in the collision induced dissociation (CID) spectrum were also assigned. Finally, the standards of CML and carbon-13 labeled CML were accurately quantified based on ¹H-NMR and tandem MS using lysine as an internal reference.     

Regulation of lysine decarboxylase activity in Escherichia coli K-12

The biodegradative lysine decarboxylase of E. coli has been reported to attain a higher specific activity when grown to saturation in the presence of excess lysine under conditions of low pH and absence of aeration. In order to examine possible sources of the pH and anaerobic regulation, a series of isogenic strains of E. coli K-12 were constructed. The effects of cadR^-, fnr ^-, cya ^-, crp ^-and pgl ^-mutations on lysine decarboxylase expression were examined. Cultures were grown in a lysine supplemented rich medium at pH 5.5, pH 6.8, and pH 8.0 with and without aeration and the enzyme was assayed from log phase cultures. The results suggested that the pH and air responses were independent and that these known regulatory processes are not responsible for this regulation of the biodegradative lysine decarboxylase.     

Mutation affecting the specific regulatory control of lysine biosynthetic enzymes in Saccharomyces cerevisiae

Summary A Saccharomyces cerevisiae mutant which exhibits a considerably increased cellular lysine pool has been isolated and characterized. Assay of enzymes of the lysine and arginine pathways shows that the mutation harboured by this mutant alters the specific repression of lysine but does not influence the general control of amino acid biosynthesis. Because it is recessive to the wild-type allele and acts pleiotropically on the synthesis of several lysine pathway enzymes, this regulatory mutation has been denominated lys80-1 (or lysR ^––1). It is believed to affect the synthesis or the structure of a factor which plays a negative role in the control of LYS gene expression.     

Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

Corynebacterium glutamicum, a Gram‐positive bacterium used for the production of various biochemicals, is naturally a biotin auxotroph. We introduced the biotin genes from Bacillus subtilis on a plasmid, pBIO, into a lysine‐producing derivative (termed AHP‐3) that has been described previously, and achieved biotin prototrophy. We found that AHP‐3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain was cultivated without biotin, indicating a suboptimal intracellular concentration of biotin. In an attempt to locate the potential bottleneck, we added pimelic acid, an early biotin precursor, and found that growth rate could be restored fully, which demonstrates that the bottleneck is in pimeloyl‐CoA (or pimeloyl‐Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin‐dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes.     

The indoleacetic acid-lysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants

Summary The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase that conjugates lysine to indoleacetic acid. A chimaeric gene consisting of the iaaL coding region under the control of the 35S RNA promoter from cauliflower mosaic virus (35SiaaL) has been used to test if iaaL gene expression leads to morphological alterations in tobacco and potato. Transgenic tobacco plantlets bearing this construct have been shown to synthesize IAA-[¹⁴C]lysine when fed with [¹⁴C]lysine. In late stages of development, their leaves show an increased nastic curvature (epinasty) of the petiole and midvein, a finding suggestive of an abnormal auxin metabolism. The alteration is transmitted to progeny as a dominant Mendelian trait cosegregating with the kanamycin resistance marker. Transgenic potato plants harbouring the construct are also characterised by petiole epinasty. Moreover, 35SiaaL transgenic plants have an increased internode length in potato and decreased root growth in both tobacco and potato. An increased content of IAA-conjugates in leaf blade was found to correlate with the epinastic alterations caused by iaaL gene expression in tobacco leaves. These data provide evidence that IAA conjugation is able to modulate hormone action, suggesting that the widespread endogenous auxin-conjugating activities are of physiological importance.     

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

Summary 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, dihydrodipicolinate 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.     

The Effect of Several Protein Amino Acids and Some Inorganic Nitrogen Sources on the Growth of Sphagnum nemoreum

The nitrogen metabolism of a bog moss, Sphagnum nemoreum Scop., has been studied in aseptic cultures. The effect of several protein amino acids, especially those found in peat, has been investigated. NH₄NO₃ (1.25 mM) was the best nitrogen source but NH₄⁺ ions were more effectively utilized than NO₃⁻ ions when given as the only nitrogen source. Some of the amino acids (2.5 mM) allowed fairly satisfactory growth (arginine and alanine) when given as the only nitrogen source, but some of them were not utilized at all (leucine, lysine, isoleucine and methionine). Given at low concentrations (0.001 and 0.25 mM) together with NH₄NO₃ (2.5 mM), most of the protein amino acids failed to reveal any growth-promoting or -inhibiting effect. Only lysine (0.25 mM) clearly inhibited growth under these conditions. The nitrogen metabolism of Sphagnum nemoreum seems to be rather flexible and this species is more tolerant of organic nitrogen, especially hydroxyproline, than the higher plants.     

Lysine and glutamate transport in the erythrocytes of Common Brushtail Possum, Tammar Wallaby and Eastern Grey Kangaroo

It was recently coincidentally discovered, using ¹H NMR spectroscopy, that the erythrocytes of two species of Australian marsupials, Tammar Wallaby (Macropus eugenii) and Bettong (Bettongia penicillata), contain relatively high concentrations of the essential amino acid lysine (Agar NS, Rae CD, Chapman BE, Kuchel PW. Comp Biochem Physiol 1991;99B:575–97). Hence, in the present work the rates of transport of lysine into the erythrocytes from the Common Brushtail Possum (Dactylopsila trivirgata) and Eastern Grey Kangaroo (Macropus giganteus) (which both have low lysine concentrations), and Tammar Wallaby were studied, to explore the mechanistic basis of this finding. The concentration-dependence of the uptake was studied with lysine alone and in the presence of arginine, which may be a competitor of the transport in some species. In relation to GSH metabolism, glutamate uptake was determined in the presence and absence of Na⁺. The data was analysed to yield estimates of the maximal velocity (V_max) and the K_m in each of the species. Erythrocytes from Tammar Wallaby lacked saturable lysine transport in contrast to the other two species. The glutamate uptake was normal in all three animals for adequate GSH biosynthesis.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

C3-Carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylase-deficient Corynebacterium glutamicum

Phosphoenolpyruvate carboxylase (PEPCx) has recently been found to be dispensable as an anaplerotic enzyme for growth and lysine production of Corynebacterium glutamicum. To clarify the role of the glyoxylate cycle as a possible alternative anaplerotic sequence, defined PEPCx- and isocitrate-lyase (ICL)-negative double mutants of C. glutamicum wild-type and of the l-lysine-producing strain MH20-22B were constructed by disruption of the respective genes. Analysis of these mutants revealed that the growth on glucose and the lysine productivity were identical to that of the parental strains. These results show that PEPCx and the glyoxylate cycle are not essential for growth of C. glutamicum on glucose and for lysine production and prove the presence of another anaplerotic reaction in this organism. To study the anaplerotic pathways in C. glutamicum further, H¹³CO₃ ^–-labeling experiments were performed with cells of the wild-type and a PEPCx-negative strain growing on glucose. Proton nuclear magnetic resonance analysis of threonine isolated from cell protein of both strains revealed the same labeling pattern: about 37% ¹³C enrichment in C-4 and 3.5% ¹³C enrichment in C-1. Since the carbon backbone of threonine corresponds to that of oxaloacetate, the label in C-4 of threonine positively identifies the anaplerotic pathway as a C₃-carboxylation reaction that also takes place in the absence of PEPCx. Received: 27 December 1995 / Accepted: 20 March 1996     

Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose

Summary. We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N^ε-(carboxymethyl)lysine; CML) were analyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N^ε-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.     

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.     

Extracellular lysine production from hydrocarbons byArthrobacter globiformis

A bacterium isolated from Burdwan (India) soil was found to accumulatel-lysine in the growth medium and was identified asArthrobacter globiformis. The strain grew and accumulated lysine in a purely synthetic medium. Supplementation of the synthetic medium stimulated growth but did not improve the yield. The entire fermentation period could be divided into a growth phase and a production phase, which could be prolonged by adjustment of pH to the neutral range. Among the different hydrocarbon and nitrogen sources tested SR gas oil at 4 % and ammonium sulphate at 0.4 %, respectively, were found most to be suitable. Different vitamins and antibiotics stimulated growth and lysine yield; inoculum of 7 % (V/V) of the medium was found to be optimal. The yield of lysine under optimal conditions was 3.4 g per litre medium. Lysine was isolated in crystalline form from the fermented broth by IEC and found to be a purel-isomer.     

Synthesis of 4-thia-[6-13C]lysine from [2-13C]glycine: access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine

4-Thialysine (S-(2-aminoethyl)-l-cysteine) is an analog of lysine. It has been used as an alternative substrate for lysine in enzymatic reactions. Site-directed isotopomers are often needed for elucidation of mechanism of reactions. 4-Thialysine can be synthesized by reacting cysteine with 2-bromoethylamine, an important reagent in chemical-modification rescue (CMR) of proteins. Here, we present the synthesis of 4-thia-[6-¹³C]lysine, one of the isotopomers of 4-thialysine, from commercially available starting material [2-¹³C]glycine via formation of five intermediates including 2-amino[2-¹³C]ethanol and 2-bromo[1-¹³C]ethylamine. The compounds were characterized using various spectroscopic techniques. Moreover, we discuss that our strategy would provide access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine. Biological activity of 4-thia-[6-¹³C]lysine was tested in the enzymatic reaction of lysine 5,6-aminomutase.     

Improving lysine production through construction of an Escherichia coli enzyme-constrained model

Microbial cell factories are widely used for the production of high-value chemicals. However, maximizing production titers is made difficult by the complicated regulatory mechanisms of these cell platforms. Here, k_cat values were incorporated to construct an Escherichia coli enzyme-constrained model. The resulting ec_iML1515 model showed that the protein demand and protein synthesis rate were the key factors affecting lysine production. By optimizing the expression of the 20 top-demanded proteins, lysine titers reached 95.7 ± 0.7 g/L, with a 0.45 g/g glucose yield. Moreover, adjusting NH₄⁺ and dissolved oxygen levels to regulate the synthesis rate of energy metabolism-related proteins caused lysine titers and glucose yields to increase to 193.6 ± 1.8 g/L and 0.74 g/g, respectively. The ec_iML1515 model provides insight into how enzymes required for the biosynthesis of certain products are distributed between and within metabolic pathways. This information can be used to accurately predict and rationally design lysine production.     

Methylation of Bovine Rhodopsin

METHYLATED basic amino-acids have been detected in many proteins¹ and we report here the methylation of rhodopsin^2–5. Bovine rhodopsin has been found to contain MML (?-N-monomethyl lysine), DML (?-N-dimethyl lysine) and 3-MH (3-methyl histidine). Residues of TML (?-N-trimethyl lysine) and DMA (dimethyl arginine) were also detected in an un-characterized protein association with rhodopsin which is solubilized from outer segments by 1 % nonionic detergent ‘Ammonyx LO’. In vitro methylation of the basic residues in bovine rhodopsin was also detected after dissected retinas had been incubated in a nutrient medium containing ³H-(methyl) methionine.     

Lysine production from hydrocarbon by micrococcus species

A bacterium isolated from Assam (India) soil was found to accumulate L-lysine in the mineral salt-hydrocarbon medium and identified to be a strain of Micrococcus luteus. The strain is able to grow and accumulate l-lysine in a purely synthetic medium, but supplementation of the synthetic medium with casamino acid or yeast extract or both, improves the yield. The entire fermentation period can be divided into a growth phase and a production phase, which can be prolonged by adjustment of the pH to the neutral range. Among the different hydrocarbon and nitrogen sources tested straight run gas oil (47percnt;) and ammonium sulphate (0.4%), respectively, were found most suitable. Erythromycin at 1 μg/ml level inhibited growth bu¸t stimulated lysine excretion. An inoculum level of 10% (v/v) of the medium was optimal for lysine production. The yield of lysine under optimal conditions was found to be 3.25 g per litre of the medium. Lysine has been isolated in crystalline form from the fermented broth by ion exchange resin chromatography and found to be pure sample of L-isomer.     

Arginine and Lysine Aminopeptidase Activities in Chromaffin Granules of Bovine Adrenal Medulla: Relevance to Prohormone Processing

Abstract: Conversion of prohormones and neuropeptide precursors to smaller, biologically active peptides requires specific proteolytic processing at paired basic residues, which generates intermediate peptides with NH₂ and COOH termini extended with Lys or Arg residues. These basic residues are then removed by aminopeptidase and carboxypeptidase activities, respectively. Among the proteases involved in prohormone processing, the basic residue aminopeptidase activity has not been well studied. This report demonstrates arginine and lysine aminopeptidase activities detected with Arg-methylcoumarinamide (Arg-MCA) and Lys-MCA substrates in neurosecretory vesicles of bovine adrenal medulla [chromaffin granules (CG)], which contain endoproteolytic processing enzymes co-localized with [Met]-enkephalin and other neuropeptides. These arginine and lysine aminopeptidase activities showed many similarities and some differences. Both arginine and lysine aminopeptidase activities were stimulated by the reducing agent β-mercaptoethanol (β-ME) and inhibited by p-hydroxymercuribenzoate, suggesting involvement of reduced cysteinyl residues. The arginine aminopeptidase activity was stimulated by NaCl (150 mM), but the lysine aminopeptidase activity was minimally affected. Moreover, characteristic β-ME/NaCl-stimulated Arg-MCA cleaving activity and β-ME-stimulated Lys-MCA cleaving activity were detected only in CG and not in other subcellular fractions; these findings indicate the localization of these particular basic residue aminopeptidase activities to secretory vesicles. The arginine and lysine aminopeptidase activities showed pH optima at 6.7 and 7.0, respectively. K_m(app) values for the arginine and lysine aminopeptidase activities were 104 and 160 µM, respectively. Inhibition by the aminopeptidase inhibitors bestatin, amastatin, and arphamenine was observed for Arg-MCA and Lys-MCA cleaving activities. Inhibition by the metal ion chelators indicated that metalloproteases were involved; Co²⁺ stimulated the arginine aminopeptidase activity but was less effective in stimulating lysine aminopeptidase activity. In addition, the lysine aminopeptidase activity was partially inhibited by Ni²⁺ and Zn²⁺ (1 mM), whereas the arginine aminopeptidase activity was minimally affected. These results demonstrate the presence of related arginine and lysine thiol metalloaminopeptidase activities in CG that may participate in prohormone processing.