Influence of B-vitamins and trace elements on lysine production by micrococcus varians 2Fa

A hydrocarbon utilizing strain of Micrococcus varians 2Fa isolated from local soil has been found to yield lysine 2.6 g l^?1. Addition of B-vitamins and trace elements to the optimal media, has been found to stimulate growth and enhance lysine yield.     

Phenotypic characterization of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using elementary modes towards synthesis of amino acids

Elementary flux mode (EFM) analysis is a powerful tool to represent the metabolic network structure and can be further utilized for flux analysis. The method enables characterization and quantification of feasible phenotypes in microbes. EFM analysis was employed to characterize the phenotype of Corynebacterium glutamicum to yield various amino acids. The metabolic network of C. glutamicum yielded 62 elementary modes by incorporating the accumulation of amino acids namely, lysine, alanine, valine, glutamine and glutamate. The analysis also allowed us to compute the maximum theoretical yield for the synthesis of various amino acids. These 62 elementary modes were further used to obtain optimal phenotypic space towards accumulation of biomass and lysine. The study indicated that the optimal solution space from 62 elementary modes forms a super space which incorporates various mutants including lysine producing strain of C. glutamicum. The analysis was also extended to obtain sensitivity of the network to variation in the stoichiometry of NADP in the definition of biomass.     

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.     

Identification and characterization of a novel GNAT superfamily Nα-acetyltransferase from Salinicoccus halodurans H3B36

N^α-acetyl-α-lysine was found as a new type of compatible solutes that acted as an organic cytoprotectant in the strain of Salinicoccus halodurans H3B36. A novel lysine N^α-acetyltransferase gene (shkat), encoding an enzyme that catalysed the acetylation of lysine exclusively at α position, was identified from this moderate halophilic strain and expressed in Escherichia coli. Sequence analysis indicated ShKAT contained a highly conserved pyrophosphate-binding loop (Arg-Gly-Asn-Gly-Asn-Gly), which was a signature of the GNAT superfamily. ShKAT exclusively recognized free amino acids as substrate, including lysine and other basic amino acids. The enzyme showed a wide range of optimal pH value and was tolerant to high-alkali and high-salinity conditions. As a new member of the GNAT superfamily, the ShKAT was the first enzyme recognized free lysine as substrate. We believe this work gives an expanded perspective of the GNAT superfamily, and reveals great potential of the shkat gene to be applied in genetic engineering for resisting extreme conditions.     

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.     

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.     

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.     

Production of Microbial Cells from Hydrocarbons

Hydrocarbon-assimilating yeasts and bacteria were isolated from soil and sewage. The optimal conditions of cell yield from liquid paraffine by a Torulopsis yeast and a Pseudomonas strain were studied. A Torulopsis yeast gave, in optimal condition, 70 percent cell yield on a weight conversion basis from light oil fraction. In a strain of Pseudomonas the additions of amino acids, Fe^{+ +}, Mg^{+ +} and Ca^{+ +} ions were effective for cell production. This strain showed, in optimal condition, 80 percent cell yield (wt%) from kerosene.     

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.     

Bisphenol derivative of allysine for high-performance liquid chromatographic analysis of allysine residue of proteins

Allysine is the most important precursor of physiologically essential cross-links formation in collagen and elastin and is formed by enzymatic oxidative deamination of lysine residues. Because it is a highly reactive aldehyde, many cross-linking amino acid residues may arise from its reaction with other allysine residues or lysine or even histidine residues. We purified and isolated an allysine bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxyphenylpentane (ACPP), from the reaction products of phenol and allysine residue of bovine ligamentum nuchae by acid hydrolysis in 6 M HCl. The structure of ACPP was verified by UV, fast atom bombardment-MS, ¹H- and ¹³C-nuclear magnetic resonance spectroscopies. The optimal reaction condition for ACPP synthesis accompanied by hydrolysis of such proteins was investigated and an ion-paired high-performance liquid chromatographic method for determination of allysine as ACPP was also developed.     

Atypical incorporation of single amino acids into myelin proteins.

—Purified myelin incorporated l -[¹⁴C]leucine and l -[¹⁴C]lysine into myelin proteins in an enzymatic process similar to that of renal brush border membranes. The system was not inhibited by cycloheximide or puromycin or by pretreatment with ribonuclease; the reaction was inhibited by cetophenicol. ATP was an effector, shifting the optimal pH from 7.2 to 8.3. In the presence of ATP, myelin was less dense in a sucrose gradient. Ammonia was released from the membrane during the incorporation of amino acids. Myelin preloaded with cold leucine did not incorporate [¹⁴C]leucine but did incorporate [¹⁴C]lysine; there was no cross inhibition between the two amino acids. The incorporation was into or onto proteins of the Wolfgram proteolipid fraction of myelin. The incorporation was of the high affinity type with a K_m of 10^?7m and was restricted to the natural amino acids.     

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.     

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.     

Selective control of Microcystis using an amino acid – a laboratory assay

An amino acid (L-lysine) was screened against eighteen strains ofCyanophyceae, Bacillariophyceae and Chlorophyceae. OnlyMicrocystis strains (Cyanophyceae) were sensitive tolysine; other strains were not affected. Cells of sevenMicrocystis strains (10⁶ cellsmL^–1) were completely killed within 48h by lysine at concentrations between 0.6 and 5.0 mgL^–1. Two Microcystis strains wereinhibited by 92 and 98 %. Similar results were obtained when lysine malonateandlysine copper were used as algicides. Microcystis specieswere killed by lysine malonate at concentrations between 0.6 and 5 mgL^–1, and by lysine copper at concentrations between 0.5and 20 mg L^–1.     

Enhanced cadaverine production from <Emphasis Type="SmallCaps">l</Emphasis>-lysine using recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> co-overexpressing CadA and CadB

The effect of fusing the PelB signal sequence to lysine/cadaverine antiporter (CadB) on the bioconversion of l-lysine to cadaverine was investigated. To construct a whole-cell biocatalyst for cadaverine production, four expression plasmids were constructed for the co-expression of lysine decarboxylase (CadA) and lysine/cadaverine antiporter (CadB) in Escherichia coli. Expressing CadB with the PelB signal sequence increased cadaverine production by 12 %, and the optimal expression plasmid, pETDuet-pelB-CadB-CadA, contained two T7 promoter-controlled genes, CadA and the PelB-CadB fusion protein. Based on pETDuet-pelB-CadB-CadA, a whole-cell system for the bioconversion of l-lysine to cadaverine was constructed, and three strategies for l-lysine feeding were evaluated to eliminate the substrate inhibition problem. A cadaverine titer of 221 g l^?1 with a molar yield of 92 % from lysine was obtained.     

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.     

Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus

Recent analysis of prokaryotic N^ε‐lysine‐acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N^ε‐lysine‐acetylated proteome of gram‐positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl‐lysine‐specific antibodies followed by LC‐MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism.     

Specificity of Lysine:N-Hydroxylase: A Hypothesis for a Reactive Substrate Intermediate in the Catalytic Mechanism

The recombinant cytoplasmic preparation of lysine:N⁶-hydroxlase catalyzes the conversion ofL-lysine to itsN⁶-hydroxy derivative when supplemented with the cofactors NADPH and FAD. A number of lysine analogs reflecting minor alterations in the inherent structural features of the amino acid as well compounds with relatively high affinity for lysine binding domains in other proteins were examined for their ability to serve as substrates of lysine:N⁶-hydroxylase. These studies have revealed that the enzyme does not tolerate any change in the structural features ofL-lysine, its preferred substrate, with the exception of the replacement of the C_γH₂-methylene group by sulfur, as in (S)-2-aminoethyl-L-cysteine.L-Norleucine is a potent inhibitor of the enzyme whileL-norvaline andL-α-aminobutyric acid do not exhibit such effect, indicating the importance of a C₄hydrophobic side chain for effective interaction with the enzyme. Among theN-alkyl amides of hydrophobic amino acids, onlyL-norleucine methylamide andL-α-aminobutyric acid ethylamide serve as moderate inhibitors of lysine:N⁶-hydroxylase. Based on the enzyme's stringent substrate specificity, a mechanism involving the conversion ofL-lysine to 2-aminocaprolactam prior to its oxygenation by the 4a-peroxyflavin intermediate in the catalytic cycle is proposed.     

Lysine Requirements of Rats of Various Body Weights

The lysine requirements of rats of various body weights were estimated using the feeding and isotope tests.

The regression equation obtained by the feeding test was Y= 1.03 – 0.58 log X. Where Y is lysine percentage of the diet and X is the mean of initial and final body weights (g) of rats achieving optimal growth gains during the feeding period.

The regression equation obtained by the isotope test was 7=0.90 – 0.49 log X, where Y and X are lysine percentage in the diet and body weights (g) of rats achieving optimal growth gains at the injection time respectively.