• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1136-1140
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• 2008

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'-phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1327-1332
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• 2004

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from Rhodobacter capsulatus, and its nucleotide sequence was determined. DNA sequencing data revealed one open reading frame coding for a protein with 401 amino acids that displayed high similarity to the amino acid sequences of other known ALASs. The hemA gene was then cloned and expressed under the control of constitutive promotor in E. coli. The recombinant E. coli strain was able to accumulate 5-aminolevulinic acid to 21 mM in the liquid medium supplemented with 45 mM glycine and 120 mM succinate. In addition, a marked effect of the pH of the culture medium on ALA production was observed, and the optimum pH for culture medium was determined to be 5.8-6.3.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1384-1390
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• 2018

Glyphosate inhibits the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. A mutant of EPSPS from Pantoea sp. was identified using site-directed mutagenesis. The mutant showed significantly improved glyphosate resistance. The mutant had mutations in three amino acids: Gly97 to Ala, Thr 98 to Ile, and Pro 102 to Ser. These mutation sites in Escherichia coli have been studied as significant active sites of glyphosate resistance. However, in our research, they were found to jointly contribute to the improvement of glyphosate tolerance. In addition, the level of glyphosate tolerance in transgenic Arabidopsis confirmed the potentiality of the mutant in breeding glyphosate-resistant plants.

• Weed & Turfgrass Science
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• v.6 no.1
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• pp.28-31
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• 2017

The mutation rate of proline in the position 197 (Pro197) in acetohydroxy acid synthase (AHAS) is highest among sulfonylurea (SU) herbicide-resistance mutants. Therefore, it is significant to investigate the resistance mechanism for the mutation and to develop the herbicides specific to the mutants. SU herbicide resistance mechanism of the mutants, 197Ser, 197Thr and 197Ala, in AHAS were targeted for designing new SU-herbicide. We did molecular dynamics (MD) simulation for understanding SU herbicide-resistance mechanisms of AHAS mutants and designed new herbicides with docking and MD evaluations. We have found that mutation to 197Ala and 197Ser enlarged the entrance of the active site, while 197Thr contracted. Map of the root mean square derivation (RMSD) and radius gyrations (Rg) revealed the domain indicating the conformations for herbicide resistant. Based on the enlarging-contracting mechanism of active site entrance, we designed new herbicides with substitution at the heterocyclic moiety of a SU herbicide for the complementary binding to the changed active site entrances of mutants, and designed new herbicides. We confirmed that our screened new herbicides bonded to both AHAS wild type and mutants with higher affinity, showing more stable binding conformation than the existing herbicides.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.880-886
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• 2015

Bacterial heme was produced from a genetic-engineered Escherichia coli via the porphyrin pathway and it was useful as an iron resource for animal feed. The amount of the E. coli-synthesized heme, however, was only few milligrams in a culture broth and it was not enough for industrial applications. To analyze heme biosynthetic pathways, an engineered E. coli artificially overexpressing ALA synthase (hemA from Rhodobacter sphaeroides) and pantothenate kinase (coaA gene from self geneome) was constructed as a bacterial heme-producing strain, and both the transcription levels of pathway genes and the intermediates concentrations were determined from batch and continuous cultures. Transcription levels of the pathway genes were not significantly changed among the tested conditions. Intracellular intermediate concentrations indicated that aminolevulinic acid (ALA) and coenzyme A (CoA) were enhanced by the hemA-coaA co-expression. Intracellular coproporphyrinogen I and protoporphyrin IX accumulation suggested that the bottleneck steps in the heme biosynthetic pathway could be the spontaneous conversion of HMB to coproporphyrinogen I and the limited conversion of protoporphyrin IX to heme, respectively. A strategy to increase the conversion of ALA to heme is discussed based on the results.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.517.2-517
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• 1986

The regulation of 3 ammonia assimilatory enzymes GDH(glutamate dehydrogenase), GS(glutamine synthetase) and GOGAT (glutamate synthase), have been examined in C. glutamicum for the biosynthesis of glutamate and glutmine. The cell free extracts of 3 kinds of arg, his and trp auxotrophs were investigated the activities of -ketoglutarate dehydrogenase, GDH, GS, and GOGAT on the media cultured with nitrogen excess and limiting conditions. Trp and his howed higher level of glutamate and glutamine than that of parental strain. The inhibition of GS activities by ADP suggested that GS is regulated by energy charge in C. glutamicum. The results with his, trp, glyc, ala, ser, and GMP implied that a system of feedback inhibition were effective. Three enzyme biosynthesis is repressed by nitrogen sources such as trp, pro, glyc, ala, ser and tyrosine.

• Biomolecules & Therapeutics
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• v.29 no.6
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• pp.685-696
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• 2021

In this study, we investigated the inhibitory effect of 5-aminolevulinic acid (ALA), a heme precursor, on inflammatory and oxidative stress activated by lipopolysaccharide (LPS) in RAW 264.7 macrophages by estimating nitric oxide (NO), prostaglandin E2 (PGE2), cytokines, and reactive oxygen species (ROS). We also evaluated the molecular mechanisms through analysis of the expression of their regulatory genes, and further evaluated the anti-inflammatory and antioxidant efficacy of ALA against LPS in the zebrafish model. Our results indicated that ALA treatment significantly attenuated the LPS-induced release of pro-inflammatory mediators including NO and PGE2, which was associated with decreased inducible NO synthase and cyclooxygenase-2 expression. ALA also inhibited the LPS-induced expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, reducing their extracellular secretion. Additionally, ALA abolished ROS generation, improved the mitochondrial mass, and enhanced the expression of heme oxygenase-1 (HO-1) and the activation of nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) in LPS-stimulated RAW 264.7 macrophages. However, zinc protoporphyrin, a specific inhibitor of HO-1, reversed the ALA-mediated inhibition of pro-inflammatory cytokines production and activation of mitochondrial function in LPS-treated RAW 264.7 macrophages. Furthermore, ALA significantly abolished the expression of LPS-induced pro-inflammatory mediators and cytokines, and showed strong protective effects against NO and ROS production in zebrafish larvae. In conclusion, our findings suggest that ALA exerts LPS-induced anti-inflammatory and antioxidant effects by upregulating the Nrf2/HO-1 signaling pathway, and that ALA can be a potential functional agent to prevent inflammatory and oxidative damage.

• Journal of Life Science
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• v.31 no.9
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• pp.818-826
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• 2021

5-Aminolevulinic acid phosphate (5-ALA-p) is a substance obtained by eluting 5-ALA (a natural delta amino acid) with aqueous ammonia, adding phosphoric acid to the eluate, and then adding acetone to confer properties suitable for use in photodynamic therapy applications. However, its pharmacological efficacy, including potential mechanisms of antioxidant and anti-inflammatory reactions, remains unclear. This study aimed to investigate the effects of 5-ALA-p on oxidative and inflammatory stresses in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Our data showed that 5-ALA-p significantly inhibited excessive phagocytic activity via LPS and attenuated oxidative stress in LPS-treated RAW 264.7 cells. Furthermore, 5-ALA-p improved mitochondrial biogenesis reduced by LPS, suggesting that 5-ALA-p restores mitochondrial damage caused by LPS. Additionally, 5-ALA-p significantly suppressed the release of nitric oxide (NO) and pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, which are associated with the inhibition of inducible NO synthase and respective cytokine expression. Furthermore, 5-ALA-p reduced the nuclear translocation of nuclear factor-kappa B (NF-κB) and inhibited phosphorylation of mitogen-activated protein kinases (MAPKs), indicating that the anti-inflammatory effect of 5-ALA-p is mediated through the suppression of NF-κB and MAPK signaling pathways. Based on these results, 5-ALA-p may serve as a potential candidate to reduce inflammation and oxidative stress.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.1
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• pp.97-101
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• 2006

Certain amino acids are essential precursors of a variety of important biomolecules in addition to their major function as protein building blocks. ${\delta}$-Aminolevulinic acid (ALA) is synthesized from the condensed form of succinyl-CoA with glycine after decarboxylation catalyzed by ALA synthase. The objective of the study was to determine the effects of ALA supplementation on growth performance, behavioral characteristics and hematological/immune status in nursery pigs. A total of 144 pigs weaned at 21 d of age were allotted to three dietary treatments representing (-) control (w/o antibiotics; NC), (+) control (w/carbadox at 50 mg/kg; PC), and the treatment group with ALA supplementation (0.05%; TA). Each treatment had 6 pens (replicates) with 8 pigs per pen. Pigs were fed phase 1 (21.9% CP, 1.40% Lys) and 2 (20.6% CP, 1.15% Lys) experimental diets for 3 and 2 wks, respectively. Feed intake and weight gain were measured weekly during phase 1 and at the end of phase 2. At the end of phase 2, blood samples were taken and analyzed using an automated hematology analyzer. Skin color and activity of pigs (48 h) from all pens in each treatment were measured at the second week of phase 2. Growth performance was not affected (p>0.05) by the dietary supplementation of ALA during the 5 wk nursery period. Pigs in the TA (6.46) and PC (6.68) had a higher (p<0.05) number of red blood cells ($10^6cell/{\mu}L$) than pigs in the NC (6.15). Pigs in PC (12.16) had a higher (p<0.05) hemoglobin level (g/dL) than pigs in the NC group (11.29) and the TA group (11.47). Pigs in the TA and PC had darker (p<0.05) and less (p<0.05) yellow skin color than pigs in the NC. Pigs in the PC tended (p = 0.081) to be less active than pigs in the other groups. There were no differences in behavioral characteristics between the NC and the TA. The data suggest that ALA supplementation has no adverse effects on growth performance of nursery pigs. Moreover, ALA supplementation increased red blood cell counts which may be beneficial to pigs.

• Korean Journal of Weed Science
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• v.31 no.4
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• pp.323-329
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• 2011

We investigated photodynamic stress-induced antioxidant responses in transgenic rice overexpressing Bradyrhizobium japonicum 5-aminolevulinic acid synthase (ALA-S) coding sequence lacking plastidal transit sequence. High light of $350{\mu}mol\;m^{-2}\;s^{-1}$ decreased the quantum yield in the transgenic lines, C4 and C5, compared to that of wild-type line. By contrast, non-photochemical quenching (NPQ) levels of C4 and C5 under high light were higher than those of the transgenic lines under low light of $150{\mu}mol\;m^{-2}\;s^{-1}$ as well as wild-type line under low and high light. Greater levels of NPQ in the transgenic lines exposed to high light were in a close correlation with increases in the xanthophyll pigment, zeaxanthin. Under high light, levels of neoxanthin, violaxanthin, lutein, and ${\beta}$-carotene in the transgenic lines were lower than those in wild-type line. Taken together, nonphotochemical energy dissipation and photoprotectant xanthophyll pigments play a critical role to deal with the severe photodynamic damage in the transgenic rice plants, although they could not overcome the photodynamic stress, leading to severe photobleaching symptoms.