Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H₂O₂) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA ^D210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsA ^D210Y mutation into a wild-type strain resulted in lower H₂O₂ concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H₂O₂ concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H₂O₂ concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H₂O₂ concentrations. Among the ROS generated by fatty-acid β-oxidation, H₂O₂ critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.     

Aerobic metabolism of l-β-lysine in a Pseudomonas. Coenzyme A-dependent acetylation of l-β-lysine

An unidentified Pseudomonas, strain B4, capable of using l-β-lysine as a sole carbon, nitrogen and energy source was isolated from soil. The enzyme system required for the oxidation of this substrate is induced by l-β-lysine and to a lesser degree by 4-aminobutyrate. Extracts of the bacterium contain an enzyme catalyzing a reaction between acetyl-CoA and l-β-lysine to form 6-N-acetyl-l-β-lysine. Lysine, ornithine and other diamino and monoamino acids are not acetylated by this enzyme. The relatively high activity of the acetylating enzyme and the absence of enzymes catalyzing direct oxidation or deamination of l-β-lysine, indicate that acetylation is the first step in β-lysine metabolism in this organism. Acetyl-β-lysine is readily deaminated by extracts in the presence of an α-keto acid to form a second product that is uncharged in acid solution.     

Circular dichroism and proton magnetic resonance studies of random chain poly-L-lysine

The 218-nm peak, characteristic of the circular dichroism of randomly coiled poly-α-amino acids can be demonstrated in solutions of penta-L -lysine, α-glycyl-L -lysine, as well as poly-L -lysine. The thermal stability of the particular state that gives rise to this 218-nm band in the CD is similar for all three peptides. These results eliminate the possibility that poly-L -lysine forms a structure with long-range order in acidic aqueous solution since the stability of such a structure would be expected to be greater for a higher molecular weight polymer than for a pentamer. The intrinsic viscosity of poly-L -lysine of molecular weight 180,000 varies only slightly between 25 and 60°C. The proton magnetic resonance spectra of poly-L -lysine and penta-L -lysine are indistinguishable on the basis of the chemical shift of all resonances, their line widths, and the exchange rates of the N? H protons. This demonstrates that poly-L -lysine does not possess a cooperatively formed ordered structure in acidic solutions. A weak band at 238 nm is observed in the circular dichroism of poly-L -lysine and other peptides. It is suggested that the effects of change in temperature, salt concentration, or polymer on both the magnitude and position of the 238-nm band may be explained if it is assumed that it is a shoulder of a lower wavelength peak.     

The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-β-lysine

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts containing non-α-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of β-lysine, a substrate also predicted by genomic analyses. EF-P was efficiently functionally modified with (R)-β-lysine but not (S)-β-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.     

β-Lysine discrimination by lysyl-tRNA synthetase

Elongation factor P is modified with (R)-β-lysine by the lysyl-tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate α- and β-lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site guided by the PoxA structure. A233S LysRS behaved as wild type with α-lysine, while the G469A and A233S/G469A variants decreased stable α-lysyl-adenylate formation. A233S LysRS recognized β-lysine better than wildtype, suggesting a role for this residue in discriminating α- and β-amino acids. Both enantiomers of β-lysine were substrates for tRNA aminoacylation by LysRS, which, together with the relaxed specificity of the A233S variant, suggest a possible means to develop systems for in vivo co-translational insertion of β-amino acids.     

Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) mediates the first unique reaction of (S)-lysine biosynthesis in plants and microbes-the condensation of (S)-aspartate-beta-semialdehyde ((S)-ASA) and pyruvate. It has been shown that DHDPS is partially feedback inhibited by (S)-lysine; it is suggested that this mechanism regulates flux through the DAP biosynthetic pathway. Others have characterised DHDPS from Escherichia coli with respect to (S)-lysine inhibition. They have concluded that, with respect to pyruvate, the first substrate of the reaction, DHDPS shows uncompetitive inhibition: as such, they further suggest that (S)-lysine inhibits DHDPS via interaction with the binding site for the second substrate, (S)-ASA. Yet, this finding is based on the assumption that (S)-lysine is a fully uncompetitive inhibitor. In light of crystallographic studies, which lead to the proposal that (S)-lysine affects the putative proton-relay of DHDPS, we re-evaluated the inhibition mechanism of DHDPS with respect to (S)-lysine by incorporating the observed hyperbolic inhibition. Our data showed that lysine is not an uncompetitive inhibitor, but a mixed inhibitor when pyruvate and (S)-lysine concentrations were varied. Thus, consistent with the crystallographic data, (S)-lysine must have an effect on the initial steps of the DHDPS reaction, including the binding of pyruvate and Schiff base formation.     

Absorption des acides amines par les lutoïdes du latex d'Hevea brasiliensis

Absorption of Amino Acids by Lutoids from the Latex of Hevea brasiliensis. The latex of Hevea brasiliensis is a fluid protoplasm containing organelles with a single membrane that are called lutoids. They are microvacuoles of a lysosomal character. The intact lutoids can easily be isolated in an isotonic medium by the usual procedures of cellular fractionation. Previous studies have shown the existence of a compartmentation of the free amino acids in latex. While acidic components dominate in the cytoplasmic serum, the lutoid serum contains mainly basic components. These findings, as well as the part played by the lutoids in the latex flow and in the process of latex coagulation, led the authors to study the penetration of acidic, neutral and basic amino acids into the lutoids. Labelled amino acids were used for the purpose. Special interest was given to the uptake of lysine, which is transported into the lutoids against a concentration gradient in the absence of a metabolic energy source. The process is linear for at least 30 min and follows Michaelis–Menten kinetics as a function of the lysine concentration. The uptake is strictly temperature and pH dependent. It increases in the presence of ATP. Lysine uptake by lutoids is also increased by application of Ethrel, ethylene generator, to the bark of tapped trees. 2,4-Dinitrophenol (2,4-DNP), ammonium chloride and N-ethylmaleimide (NEM) inhibit the influx. Neutral l -amino acids do not affect the uptake of l -lysine, while l arginine inhibits lysine transport competitively. d -lysine is without effect on l -lysine uptake by lutoids. Under conditions used in this study no lysine efflux is observed. A mechanism governing the penetration of basic amino acids against a concentration gradient is discussed, and the possible role of a proton gradient is suggested.     

Amino Acid Analyses of Desert Varnish from the Sonoran and Mojave Deserts

There has long been a debate as to whether desert varnish deposits are microbially mediated or are deposited by inorganic processes. Several researchers have cultured bacteria from the surface of desert varnish suggesting that bacteria are intimately associated with varnish coatings and may play a role in their formation. To test this hypothesis, we have collected scrapings of desert varnish from the Sonoran Desert in Arizona and the Mojave Desert in California and analyzed them for amino acids. Thirteen amino acids were found in desert varnish indicating a biogenic component of these varnishes. Two protein amino acids that were not detected in any of the varnishes are cysteine and tryptophan. Two nonprotein amino acids,β-alanine andγ-amino butyric acid, were found. These are known to be formed by enzymatic decarboxylation, thereby indicating possible organismal activity in varnish. Some D -enantiomers of the amino acids were also found. In addition to small amounts of the D -enantiomer of aspartic acid, which is rapidly formed by racemization and was present in most samples, D -alanine and D -glutamic acid were found. These latter two amino acids are components of the peptidoglycan cell wall material of bacteria. L -lysine was also detected, but not diaminopimelic acid. The combination of L -lysine, D -alanine, and D -glutamic acid is characteristic of the peptidoglycan from Gram-positive bacteria. Although the presence of these biomarkers does not prove that Gram-positive bacteria produce the coatings, finding them is consistent with the hypothesis that they may play a role in desert varnish formation.     

<Emphasis Type="Italic">Bacillus methanolicus</Emphasis>: a candidate for industrial production of amino acids from methanol at 50°C

Amino acids are among the major products in biotechnology in both volume and value, and the global market is growing. Microbial fermentation is the dominant method used for industrial production, and today the most important microorganisms used are Corynebacteria utilizing sugars. For low-prize bulk amino acids, the possibility of using alternative substrates such as methanol has gained considerable interest. In this mini review, we highlight the unique genetics and favorable physiological traits of thermotolerant methylotroph Bacillus methanolicus, which makes it an interesting candidate for overproduction of amino acids from methanol. B. methanolicus genes involved in methanol consumption are plasmid-encoded and this bacterium has a high methanol conversion rate. Wild-type strains can secrete 58 g/l of L: -glutamate in fed-batch cultures at 50 degrees C and classical mutants secreting 37 g/l of L: -lysine have been selected. The relative high growth temperature is an advantage with respect to both reactor cooling requirements and low contamination risks. Key genes in L: -lysine and L: -glutamate production have been cloned, high-cell density methanol fermentation technology established, and recently a gene delivery method was developed for this organism. We discuss how this new knowledge and technology may lead to the construction of improved L: -lysine and L: -glutamate producing strains by metabolic engineering.     

Production of l-Lysine by Leucine Auxotrophs Derived from AEC Resistant Mutant of Brevibacterium lactofermentum

The effect of amino acids was examined on the production of l-lysine by AEC resistant mutant of B. lactofermentum. Among amino acids tested, only leucine showed strong specific inhibition. In order to release the production of l-lysine from this negative effect of leucine, leucine auxotrophs were derived from AEC resistant strain of B. lactofermentum. Most of these leucine auxotrophs produced larger amount of l-lysine (maximally 41 mg/ml) than the parental strain which produced about 18 mg/ml of l-lysine. It was confirmed that leucine auxotrophs derived from AEC resistant mutant of other glutamate producing bacteria, B. saccharolyticum and Corynebacterium glutamicum. These results suggested that leucine might directly or indirectly affect the biosynthesis of lysine.

However, this increase in lysine productivity of leucine auxotrophs could not be explained by the alteration of aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3). These enzymes are key enzymes in lysine and threonine biosynthesis, respectively.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.     

Immunochemical approach to characterize advanced glycation end products of the Maillard reaction. Evidence for the presence of a common structure

Reaction of protein amino groups with glucose (the Maillard reaction) leads from early stage products such as Schiff base and Amadori products to advanced glycation end products (AGE), structures implicated in diabetic complications and the aging process. We have prepared the polyclonal anti-AGE antibody and the monoclonal anti-AGE antibody against AGE-bovine serum albumin and made an immunochemical approach to characterize AGE structures. Both polyclonal and monoclonal antibodies reacted with AGE-proteins such as AGE-bovine serum albumin, AGE-human serum albumin, and AGE-hemoglobin but not with unmodified counterparts. Treatments of these AGE-proteins with borohydride had no effect on the immunoreactivity. Moreover, fructosyl-epsilon-caproic acid, a synthetic Amadori compound, did not serve as an antigen, indicating that these antibodies were specific for AGE products but not for early stage products of the Maillard reaction. In addition, these antibodies were also able to recognize AGE products prepared either from alpha-tosyl-1-lysine, alpha-tosyl-1-lysine methyl ester, monoaminocarboxylic acid such as epsilon-aminocaproic acid, gamma-amino-n-butyric acid, and beta-alanine. Thus, these results strongly suggest the presence of a common structure in AGE preparations, regardless of whether AGE products are generated from proteins, amino acids, or monoaminocarboxylic acids.     

Identification and estimation of some uncommon diamino acids

Application of the amino acid analyzer is described for the separation, identification, and estimation of several uncommon dibasic amino acids that are eluted with a 0.35 m, pH 5.28, sodium citrate buffer between lysine and ammonia, namely, 2,5-diaminohexanoate, threo- and erythro-3,5-diaminohexanoate, 3,6-diaminohexanoate (β-lysine) and 2,4-diaminopentanoic acid. Conditions are also given for estimating d- and l-β-lysine as their l-glutamyl peptides, and for estimating the enantiomers of threo- and erythro-3,5-diaminohexanoate as the l-glutamyl derivative of the corresponding lactams.     

An enzyme electrode for specific determination of L-lysine: A real-time control sensor

Lysine decarboxylase (L-lysine carboxylyase, E.C.4.1.1.18) is immobolized on a carbon dioxide gas-sensing electrode, by copolymerization with gelatin using the bifuncitional agent glutaraldehyde. The enzyme electrodes thus prepared are used in a continuous flow system to measure the concentration of L-lysine in a mixture of amino acids. The measuring time for each sample is about 3 min, including response and rinsing times. The electrode response is linear between 0.01-1 g/L and has a high specificity for L-lysine. The enzyme electrode response to lysine at concentrations below 0.5 g/L is stable on repeated use for at least 500 assays.     

Subcellular organization of neurophysins, oxytocin, [8-lysine]-vasopressin and adenosine triphosphatase in porcine posterior pituitary lobes

Posterior pituitary lobes from young pigs were fractionated by differential and sucrose-density-gradient centrifugation. The distributions of oxytocin and [8-lysine]-vasopressin were measured by bioassay and the distributions of neurophysin-I and -II by radioimmunoassays specific for each of these two proteins. Most of the hormone and neurophysin applied to the density gradient was localized in particles with the density expected of neurosecretory granules. However, the neurosecretory granules were separated into two bands (D and E). A close statistical correlation between the distributions of [8-lysine]-vasopressin and neurophysin-I, and of oxytocin and neurophysin-II on the gradients, suggested that in vivo porcine neurophysin-I binds [8-lysine]-vasopressin within one population of granules and porcine neurophysin-II binds oxytocin within another type of granule. However, there was no significant separation of oxytocin and vasopressin in fractions D and E. The molar ratios of hormones and neurophysins indicated that there was insufficient of either neurophysin to bind the [8-lysine]-vasopressin in the granule fractions or in the whole gland. Polyacrylamide-gel electrophoresis showed that only bands corresponding in mobility to porcine neurophysins-I, -II and -III were present in large amounts in the whole gland and in the granule fractions. The component with the mobility of neurophysin-III was, however, relatively enriched in whole young glands and granule fractions compared with adult gland extracts. It is suggested that the vasopressin that cannot be assigned to neurophysin-I may occur in (a) vesicles containing vasopressin but no neurophysin, (b) vesicles containing vasopressin and a protein that cannot be quantified by the radioimmunoassays used, such as porcine neurophysin-III, or (c) normal vasopressin–neurophysin granules which have accumulated extra vasopressin. Band E of the gradient was rich in adenosine triphosphatase activity, whereas band D possessed very little of this enzyme.     

Nature of amino acid side chain and alpha-helix stability.

In order to investigate the ability of neutral amino acids to support the α-helix conformation, the coil–helix transition of poly(L -lysine) and of lysine copolymers with these amino acids was studied in water/methanol using circular dichroism. The transtions were recorded at constant pH adding buffer to the methanol/water mixtures. With poly(L -lysine), experiments were performed at several constant pH's; the transition midpoint on the water (methanol) concentration scale was found to depend strongly upon pH; the helix stability region is shifted towards higher water concentrations, when the pH is increased. Copolymers of lysine and several neutral amino acids revealed the same effect in that increasing amounts of, for example, norleucine also shifted the transition midpoint to higher water concentrations. A series of copolymers containing L -lysine as the host and different hydrophobic amino acids were synthesized and the helix–coil transition in water/methanol was observed at constant pH. Different copolymers of equal composition showed significant differences with respect to the nature of the amino acid incorporated into polylysine. From these studies an α-helix-philic scale (in decreasing order): Leu, Nle, Ile, Ala, Phe, Val, Gly is deduced and discussed; the results obtained were compared with those of different procedures.     

Utilization of organic nitrogen compounds by yeasts of the genusSaccharomyces

Yeasts belonging to 27 species ofSaccharomyces were tested for their ability to used-amino acids,l-lysine and various amines and amides as nitrogen source. Most yeasts capable of growing onl-lysine or amines could utilized-amino acids.Saccharomyces (sensu strictu) have a very limited ability to grow on the organic nitrogen compounds tested. However, there is no obvious relationship between the utilization of these compounds and the proposed divisions of the genusSaccharomyces.Issued as N.R.C. No. 9845.     

Metabolic pathways and fermentative production of L-aspartate family amino acids

The L -aspartate family amino acids (AFAAs), L -threonine, L -lysine, L -methionine and L -isoleucine have recently been of much interest due to their wide spectrum of applications including food additives, components of cosmetics and therapeutic agents, and animal feed additives. Among them, L -threonine, L -lysine and L -methionine are three major amino acids produced currently throughout the world. Recent advances in systems metabolic engineering, which combine various high-throughput omics technologies and computational analysis, are now facilitating development of microbial strains efficiently producing AFAAs. Thus, a thorough understanding of the metabolic and regulatory mechanisms of the biosynthesis of these amino acids is urgently needed for designing system-wide metabolic engineering strategies. Here we review the details of AFAA biosynthetic pathways, regulations involved, and export and transport systems, and provide general strategies for successful metabolic engineering along with relevant examples. Finally, perspectives of systems metabolic engineering for developing AFAA overproducers are suggested with selected exemplary studies.     

Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of l-lysine biosynthesis in bacteria and plants and is allosterically regulated by l-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to l-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the l-lysine-sensitive DHDPS from Escherichia coli and l-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor’s binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by l-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of l-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of l-lysine production yield by 46 %.