Abstract 5-Aminolevulinic Acid Research Articles

Multi-enzymatic recycling of ATP and NADPH for the synthesis of 5-aminolevulinic acid using a semipermeable reaction system.

5-Aminolevulinic acid (ALA) is an important cellular metabolic intermediate that has broad agricultural and medical applications. Previously, attempts have been made to synthesize ALA by multiple enzymes in cell free systems. Here we report the development of a semi-permeable system for ALA production using stable enzymes. Glucose, sodium polyphosphate, ATP, tRNA, glutamate and NADPH were used as substrates for ALA synthesis by a total of nine enzymes: adenylate kinase, polyphosphate kinase, glucose-6-phosphate dehydrogenase, phosphogluconolactonase, 6-phosphogluconate dehydrogenase, glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase from E. coli, hexokinase from yeast, as well as glutamyl-tRNA reductase and its stimulator protein glutamyl-tRNA reductase binding protein (GBP) from Arabidopsis in a semi-permeable system. After reaction for 48h, the glutamate conversion reached about 95%. This semi-permeable system facilitated the reuse of enzymes, and was helpful for the separation and purification of the product. The ALA production could be further improved by process optimization and enzyme engineering.Abbreviations: PPK: polyphosphate kinase; ADK: adenylate kinase; ALA: 5-Aminolevulinic acid; HK: hexokinase; ZWF: glucose-6-phosphatedehydrogenase; PGL: phosphogluconolactonase; GND: 6-phosphogluconate dehydrogenase; GTS: glutamyl-tRNA synthetase; GTR: glutamyl-tRNA reductase; GBP: GTR binding protein; GSAAT: glutamate-1-semialdehyde aminotransferase.

A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield.

5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host,Corynebacterium glutamicum The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future.

Cellular Levels of Glutamyl-tRNA Reductase and Glutamate-1-Semialdehyde Aminotransferase Do Not Control Chlorophyll Synthesis in Chlamydomonas reinhardtii

5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole biosynthesis pathway. In plants, algae, and most bacteria, ALA is generated from glutamate. First, glutamyl-tRNA synthetase activates glutamate by ligating it to tRNA(Glu). Activated glutamate is then converted to glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR). Finally, GSA is rearranged to ALA by GSA aminotransferase (GSAT). In the unicellular green alga Chlamydomonas reinhardtii, GTR and GSAT were found in the chloroplasts and were not detected in the mitochondria by immunoblotting. The levels of both proteins (assayed by immunoblotting) and their mRNAs (assayed by RNA blotting) were approximately equally abundant in cells growing in continuous dark or continuous light (fluorescent tubes, 80 micromol photons s(-1) m(-2)), consistent with the ability of the cells to form chlorophyll under both conditions. In cells synchronized to a 12-h-light/12-h-dark cycle, chlorophyll accumulated only during the light phase. However, GTR and GSAT were present at all phases of the cycle. The GTR mRNA level increased in the light and peaked about 2-fold at 2 h into the light phase, and GTR protein levels also increased and peaked 2-fold at 4 to 6 h into the light phase. In contrast, although the GSAT mRNA level increased severalfold at 2 h into the light phase, the level of GSAT protein remained approximately constant in the light and dark phases. Under all growth conditions, the cells contained significantly more GSAT than GTR on a molar basis. Our results indicate that the rate of chlorophyll synthesis in C. reinhardtii is not directly controlled by the expression levels of the mRNAs for GTR or GSAT, or by the cellular abundance of these enzyme proteins.

Size and Surface Charge Effect of 5-Aminolevulinic Acid-Containing Liposomes on Photodynamic Therapy for Cultivated Cancer Cells

5-Aminolevulinic acid (ALA)-containing liposomes having various average diameters and/or positive surface charges were prepared, and their photodynamic therapy (PDT) efficacy for murine thymic lymphoma cells, EL-4 cells, cultivated in vitro was investigated. The PDT efficacy for EL-4 cells and the accumulation of ALA-induced protoporphyrin IX (PpIX) in the cells increased with a decrease in the average diameter of liposomes. In particular, the ALA-containing liposomes smaller than 63.5 nm in diameter promoted the PDT efficacy in comparison with that of ALA alone. We also found no significant changes in PDT efficacy and PpIX accumulation with increasing positive surface charges of liposomes.

H2 15O translocation in rice was enhanced by 10 μm 5-aminolevulinic acid as monitored by positron emitting tracer imaging system (PETIS)

5-Aminolevulinic acid (ALA) acts to increase chlorophyll biosynthesis, photosynthesis, cold stress tolerance, and salt tolerance at low concentrations. We studied the effects of ALA on H2 15O translocation from the roots to the shoots of rice plants (Oryza sativa L. cv. Nipponbare) in real time by a positron-emitting tracer imaging system (PETIS). When the plant was treated with 10 μm ALA, the velocity of the H2 15O translocation from 2 to 12 min after absorption increased to 126, 137, 140% that of the control at 1.5, 2.5, and 3.5 h after ALA treatment, respectively. However, ALA did not affect the H2 15O translocation within 0.5 h of treatment. When the plant was treated with 0.1 mM ABA at 4 h after 10 μm ALA treatment, the velocity of the H2 15O translocation decreased at 0.5 h after ABA treatment. Those observations suggested ALA might be absorbed and transported to the guard cells within 1.5 h and functioned to expand the stomatal aperture.

Antisense HEMA1 RNA expression inhibits heme and chlorophyll biosynthesis in arabidopsis.

5-aminolevulinic acid (ALA) is a precursor in the biosynthesis of tetrapyrroles including chlorophylls and heme. The formation of ALA involves two enzymatic steps which take place in the chloroplast in plants. The first enzyme, glutamyl-tRNA reductase, and the second enzyme, glutamate-1-semialdehyde-2,1-aminomutase, are encoded by the nuclear HEMA and GSA genes, respectively. To assess the significance of the HEMA gene for chlorophyll and heme synthesis, transgenic Arabidopsis plants that expressed antisense HEMA1 mRNA from the constitutive cauliflower mosaic virus 35S promoter were generated. These plants exhibited varying degrees of chlorophyll deficiency, ranging from patchy yellow to total yellow. Analysis indicated that these plants had decreased levels of chlorophyll, non-covalently bound hemes, and ALA; their levels were proportional to the level of glutamyl-tRNA reductase expression and were inversely related to the levels of antisense HEMA transcripts. Plants that lacked chlorophyll failed to survive under normal growth conditions, indicating that HEMA gene expression is essential for growth.

Urinary 5-aminolevulinic acid (ALA) adjusted by creatinine: a surrogate for plasma ALA?

5-Aminolevulinic acid (ALA) is the first intermediate substrate in the heme synthetic pathway and is the substrate of aminolevulinic acid dehydratase (ALAD, porphobilinogen synthase). Because lead effectively inhibits ALAD activity, resulting in accumulation of ALA in urine and blood, urinary ALA (ALAU) has been used as a biomarker for lead exposure or early biologic effect of lead. Intraindividual variation in urinary excretion of ALA requires the use of 24-hour urine samples or adjustment of single urine samples by other normalizing variables, such as urinary creatinine concentration. Previous studies of ALAU concentration have used various adjustment methods; however, few have compared creatinine-adjusted ALAU concentration with ALA concentration in plasma (ALAP) from subjects with low (< 30 micrograms/dL) to moderate (< 60 micrograms/dL) levels of blood lead. To determine if creatinine-adjusted ALAU is associated with ALAP, we measured ALAU, ALAP, and urinary creatinine in 65 Korean lead workers with blood lead concentrations in the range of 14-60 micrograms/dL. ALAU, ALAU/creatinine, or ALAU/log creatinine all correlated with ALAP. However, ALAU/creatinine correlated more closely with ALAP based on Spearman's r (rs = 0.40, P, = 0.0009), supporting the use of ALA/creatinine in single urine samples as a surrogate for ALAP.