Influence of β-alanine on ultrastructure,tanning, and melanization of Drosophila melanogaster cuticles

In Drosophila melanogaster, the chitinous microfibrils arising from the tips of the epidermal villi in adult cuticles remain irregular and loose in the mutant ebony (which fails in cuticular incorporation of -alanine) but closely knit and regular in normal flies. Addition of -alanine to cuticles from which nonchitinous materials have been removed with alkali converts the loose arrangement of the microfibrils to a compact and sharply delineated arrangement. -alanine also accelerates tyrosinase-catalyzed oxidation of N-acetyldopamine by reacting with the oxidized product of the reaction to produce an orange-red complex. Similarly, -alanine accelerates oxidation of N-acetyldopamine when these two substances are added to fluids from the hemocoel, to lead to tanning instead of normal blackening. These findings may help explain why -alanine induces tanning while inhibiting melanization in insects.This study was supported by Grant GM-18680 from the National Institutes of Health.     

Metabolic engineering of E. coli for β-alanine production using a multi-biosensor enabled approach

β-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of β-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo β-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in β-alanine yield (0.85 mol β-alanine/mole glucose) and specific β-alanine production (0.74 g/L/OD₆₀₀), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol β-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L β-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of β-alanine.     

Role of β-alanine in pupal cuticle coloration in the tobacco hornworm,Manduca sexta: Effects of β-alanine analogues on melanization and catecholamine levels

Hydrazino and aminooxy derivatives of β-alanine were found to cause blackening of Manduca sexta pupal cuticle when they were injected into pharate pupae at the onset of pre-ecdysial tanning. One of these compounds, ethyl hydrazinoacetate (EHA), was used for further study. It was effective if injected up to about 4 hr before pupal ecdysis. These melanized cuticles contained excessive amounts of dopamine and decreased amounts of N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA). Furthermore, EHA induced elevated dopamine and lowered β-alanine levels in the hemolymph. Similar blackening occurred when 20 mg/animal dopamine was injected. Injection of excess β-alanine rescued the normal brown color, irrespective of the concentration of EHA. Also, EHA caused melanization in vitro in the presence of dopamine, whereas the addition of β-alanine and NBAD allowed normal pupal coloration in vitro. These hydrazino and aminooxy compounds likely interfere with β-alanine synthesis or mobilization and thus with N-acylation of the catecholamines to form NBAD and N-β-alanylnorepinephrine.     

The role of β-alanine in the biosynthesis of nitrate byAspergillus flavus

d-Serine (0.05m) inhibited nitrification byAspergillus flavus in media containing either peptone, aspartate,a-alanine or -alanine as the sole nitrogen source. A similar inhibition was observed in an aspartate + peptone medium, but nitrate was formed in a -alanine + peptone medium in the presence of the inhibitor. Exceptionally high yields of nitrate were obtained in the -alanine + peptone medium. In replacement cultures,d-serine inhibited nitrification of aspartate but not of -alanine. Manometric studies indicated that aspartate was decarboxylated byA. flavus and that the reaction was inhibited byd-serine. In view of these results, it is suggested that aspartate is a precursor and -alanine is an intermediate in nitrification by this fungus.     

Transport of β-alanine in renal brush border membrane vesicles

The findings that the equilibrium uptake of β-alanine decreased with increasing medium osmolarity and preincubation with β-alanine increased uptake of the amino acid indicate that the uptake of β-alanine by rabbit renal brush border membranes represents transport into membrane vesicles. A Na⁺ electrochemical gradient (extravesicular > intravesicular) stimulated the initial rate of β-alanine uptake about three times and effected a transient accumulation of the amino acid twice the equilibrium value. Stimulation of the uptake was specific for Na⁺. Gramicidin abolished the overshoot, presumably by dissipating the gradient by accelerating the electrogenic entrance of Na⁺ into the vesicle via a pathway not coupled to uptake of β-alanine. In K⁺-loaded vesicle, valinomycin enhanced the Na⁺ gradient-dependent uptake of β-alanine. These findings indicate that the Na⁺ gradient-dependent transport of β-alanine is an electrogenic process and suggest that the membrane potential is a determinant of β-analine transport. Uptake of β-aniline, at a given concentration, reflected the sum of contributions from Na⁺ gradient-dependent and -independent transport systems. The dependent system saturated at 100 μM. The independent system did not saturate. At physiological concentrations the rate of the Na⁺ gradient-dependent uptake was four times that in the absence of the gradient. The Na⁺ gradient-dependent rate of β-alanine uptake was strongly inhibited by taurine, suggesting that β-amino acids have a common transport system, α-Amino acids, i.e. l-arginine, l-glutamate, l-proline, and glycine, representing previously reported specific α-amino acid transport systems in the brush border membrane, did not inhibit the uptake of β-alanine. These findings indicate that the brush border membrane has a distinct transport system for β-amino acids.     

Similar effects ofβ-alanine and taurine in cholesterol metabolism

β-Alanine, though producing a deficiency of taurine in the tissues, had a similar effect on cholesterol metabolism as taurine. Both caused increased activity of hepatic hydroxymethylglutaryl coenzyme A reductase and increased incorporation of 1, 2 of [¹⁴C]-acetate into liver cholesterol. Both caused increased concentration of biliary cholesterol and bile acids. There was increased activity of lipoprotein lipase in heart, but decreased activity in the adipose tissue in both cases. Release of lipoproteins into circulation was decreased in both cases.     

Effect of β-alanine administration on carbon tetrachloride-induced acute hepatotoxicity

Summary. Mice were supplemented with β-alanine (3%) in drinking water for one week. β-Alanine intake reduced hepatic taurine levels, but elevated cysteine levels significantly. Hepatotoxicity of CCl₄ in mice fed with β-alanine was decreased as determined by changes in serum enzyme activities. Hepatic glutathione and taurine concentrations after CCl₄ challenge were increased markedly by β-alanine intake. The enhanced availability of cysteine for synthesis of glutathione and/or taurine appears to account for the hepatoprotective effects of β-alanine against CCl₄-induced liver injury.     

High-affinity uptake of taurine and β-alanine in primary cultures of rat astrocytes

The kinetics and specificity of taurine and -alanine uptake were studied in primary cultures of rat astrocytes under identical experimental conditions. The uptake consisted of nonsaturable penetration and saturable high-affinity transport that was strictly sodium dependent. The cells accumulated taurine more effectively than -alanine, both the affinity and uptake capacity being greater for taurine. Taurine uptake was competitively inhibited by -alanine and GABA, the former being more potent. Also, hypotaurine and 2-guanidinoethanesulphonic acid strongly reduced taurine uptake, but L-2,4-diaminobutyric acid had no significant effect. -Alanine uptake was also competitively inhibited by GABA, but the most potent inhibitors were hypotaurine and 2-guanidinoethanesulphonic acid.l-2,4-Diaminobutyric acid was moderately active. The uptake systems for taurine and -alanine were thus in principle similar, and they exhibited certain characteristics typical for a neurotransmitter amino acid. The inhibition studies further suggest the existence of only one common transport system for taurine, -alanine, and GABA in cultured primary astrocytes. The same uptake system may also be used for hypotaurine.     

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of β-alanine

β-Alanine is an important β-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of β-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of β-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of β-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient β-alanine biosynthesis was used to construct a β-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative β-alanine exporter was newly identified, and its overexpression further improved the β-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of β-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.     

Simultaneous determination of F-β-alanine and β-alanine in plasma and urine with dual-column reversed-phase high-performance liquid chromatography

F-β-Alanine and β-alanine were detected in plasma and urine samples with fluorescence detection of orthophthaldialdehyde derivatives of F-β-alanine and β-alanine after separation with dual-column reversed-phase HPLC. The detection limits of F-β-alanine and β-alanine in the HPLC system were approximately 0.3 and 0.7 pmol, respectively. The procedure proved to be very reproducible with intra-assay RSDs and inter-assay RSDs being less than 8%. The usefulness of the method was demonstrated by the analysis of the F-β-alanine and β-alanine concentrations in plasma and urine samples from tumor patients treated with S-1 (Tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in a molar ratio of 1:0.4:1).     

β-Glutamyl Peptides, 3-(2-Furoyl)-alanine and Ascorbalamic Acid in Roots of Rumex obtusifolius L. (Ezo-no-Gishigishi)

We found a potent platelet aggregation inhibitor in the culture broth of Streptomyces strain M-193. The inhibitor was purified as white crystals and investigated; it was identified as staurosporine. It strongly inhibited the platelet aggregation induced by collagen or ADP with IC₅₀ values of 3.4 μm, and 11.6 μm, respectively. The inhibitor had no effect on thrombin-induced platelet aggregation or membrane stabilization against heat-induced hemolysis.     

Synthesis of Nα,Nδ-dicarbobenzoxy-L-ornithyl-β-alanine benzyl ester,a derivative of salty peptide,in 1,1,1-trichloroethane with polyethylene glycol-modified papain

Summary N,N-Dicarbobenzoxy-L-ornithyl--alanine benzyl ester, a derivative of salty peptide, was synthesized from N,N-dicarbobenzoxy-L-ornithine ethyl ester and -alanine benzyl ester in 1,1,1-trichloroethane using papain modified with polyethylene glycol. The peptide bond formation proceeded in a transparent organic solvent at room temperature and the product was obtained as precipitates from the reaction system.