• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1235-1244
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• 2008

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylase-like proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5$\alpha$. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.

• Journal of Life Science
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• v.25 no.11
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• pp.1290-1297
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• 2015

Studies of the inactivation of a gene encoding pyruvate decarboxylase, pdc, in an ethanol-producing bacterium, Zymomonas mobilis, identified a mutant strain with 50% reduced PDC activity. To evaluate the possibility of a carbon-flux shift from an ethanol pathway toward higher value fermentation products, including pyruvate, succinate, and lactate, fermentation studies were carried out. Despite attempts to silence pdc expression in the wild-type strain ZM4 using cat-inserted pdc and pdc-deleted homologs by electroporation, the strain isolated showed partial gene activation. Fermentation experiments with the PDC mutant strain showed that the reduced expression level of PDC activity resulted in decreased rates of substrate uptake and ethanol production, together with increased pyruvate accumulation of 2.5 g l^-1 , although lactate and succinate concentrations were not significantly enhanced in these modified strains. Despite numerous attempts, no strains were isolated in which complete pdc inactivation occurred. This result indicates that the ethanol fermentation pathway of this bacterium is totally dependent on the activity of the PDC enzyme. To ensure a redox balance of intracellular NAD and NADH levels, other enzymes, such as lactate dehydrogenase for lactate, and enzymes involved in the production of succinic acid, such as pyruvate dehydrogenase (PDH) and malic enzymes, may be needed for their increased end-product production.

• BMB Reports
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• v.32 no.1
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• pp.39-44
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• 1999

Pyruvate decarboxylase (PDC) catalyzes the conversion of pyruvate to acetaldehyde as the penultimate step in alcohol fermentation. The enzyme requires two cofactors, thiamin diphosphate (ThDP) and $Mg^{2+}$, for activity. Zymomonas mobilis PDC shows a strong preference for pyruvate although it will use the higher homologues 2-ketobutyrate and 2-ketovalerate to some extent. We have investigated the effect of mutagenesis of valine 111 and leucine 112 on the substrate specificity. V111 was replaced by glycine, alanine, leucine, and isoleucine while L112 was replaced by alanine, valine, and isoleucine. With the exception of L112I, all mutants retain activity towards pyruvate with $k_{cat}$ values ranging from 40% to 139% of wild-type. All mutants show changes from wild-type in the affinity for ThDP, and several (V111A, L112A, and L112V) show decreases in the affinity for $Mg^{2+}$. Two of the mutants, V111G and V111A, show an increase in the $K_m$ for pyruvate. The activity of each mutant towards 2-ketobutyrate and 2-ketovalerate was investigated and some changes from wild-type were found. For the V111 mutants, the most notable of these is a 3.7-fold increase in the ability to use 2-ketovalerate. However, the largest effect is observed for the L112V mutation which increases the ability to use both 2-ketobutyrate (4.3-fold) and 2-ketovalerate (5.7-fold). The results suggest that L112 and, to a lesser extent, V111 are close to the active site and may interact with the alkyl side-chain of the substrate.

• Applied Biological Chemistry
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• v.38 no.3
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• pp.278-282
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• 1995

The effect of Schizandrae fructus extract on the physiological properties of Saccharomyces cerevisiae was investigated. S. cerevisiae was inoculated into glucose broth, added with Schizandrae fructus extract, 0, 0.01, 0.1, 0.5 and 1%(w/v), respectively. And a 96 hours incubation was followed to investigate the changes in the growth, alcohol production, alcohol dehydrogenase and pyruvate decarboxylase activities of S. cerevisiae. The growth of S. cerevisiae was more pronounced in the broth containing 0.1 and 0.OlfS Schizandrae fructus extract than in the control. The growth was, however, inhibited in the broth containing 0.5 and 1% of the extract. The content of alcohol produced by S. cerevisiae also showed very similiar results with those of the yeast growth by addition of Schizandrae fructus extract. Alcohol dehydrogenase activities of S. cerevisiae cultured in broth treated with the extract of 0.1% and 0.01% increased by 25% and 18% than those in control group. Pyruvate decarboxylase activities in 0.1% and 0.01% treatments increased to 1.32 and 1.26 times. The activities in 0.5% and 1% treatments, however, decreased by 30% and 44%.

• Microbiology and Biotechnology Letters
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• v.47 no.2
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• pp.242-249
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• 2019

Biosynthesis of indole-3-acetate (IAA) from L-tryptophan via indole-3-pyruvate pathway requires three enzymes including aminotransferase, indole-3-pyruvate decarboxylase, and indole-3-acetate dehydrogenase. To establish a bio-based production of IAA, the aspC, ipdC, and iad1 from Escherichia coli, Enterobacter cloacae, and Ustilago maydis, respectively, were expressed under control of the tac, ilvC, and sod promoters in C. glutamicum. Cells harboring ipdC produced tryptophol, indicating that the ipdC product is functional in this host. Analyses of SDS-PAGE and enzyme activity revealed that genes encoding AspC and Iad1 were efficiently expressed from the sod promoter, and their enzyme activities were 5.8 and 168.5 nmol/min/mg-protein, respectively. The final resulting strain expressing aspC, ipdC, and iad1 produced 2.3 g/l and 7.3 g/l of IAA from 10 g/l L-tryptophan, respectively, in flask cultures and a 5-L bioreactor.

• Journal of Microbiology and Biotechnology
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• v.29 no.10
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• pp.1636-1643
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• 2019

Two hydroxybenzoyl amines, 4-hydroxybenzoyl tyramine (4-HBT) and N-2-hydroxybenzoyl tryptamine (2-HBT), were synthesized using Escherichia coli. While 4-HBT was reported to demonstrate anti-atherosclerotic activity, 2-HBT showed anticonvulsant and antinociceptive activities. We introduced genes chorismate pyruvate-lyase (ubiC), tyrosine decarboxylase (TyDC), isochorismate synthase (entC), isochorismate pyruvate lyase (pchB), and tryptophan decarboxylase (TDC) for each substrate, 4-hydroxybenzoic acid (4-HBA), tyramine, 2-hydroxybenzoic acid (2-HBA), and tryptamine, respectively, in E. coli. Genes for CoA ligase (hbad) and amide formation (CaSHT and OsHCT) were also introduced to form hydroxybenzoic acid and amine conjugates. In addition, we engineered E. coli to provide increased substrates. These approaches led to the yield of 259.3 mg/l 4-HBT and 227.2 mg/l 2-HBT and could be applied to synthesize diverse bioactive hydroxybenzoyl amine conjugates.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.572-576
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• 1997

Effect of anoxia treatment on alcohol fermentation in the placenta of oriental melons (Cucumis melo) at different developmental stages was studied. Results showed that fruits at the rapid growth stage (stage III) contained the lowest amount of acetaldehyde and ethanol as compared with fruits at other developmental stages. During anoxia treatment, a steady increase in ethanol content was observed in the placenta of oriental melons, regardless of their developmental stages, while the increment of acetaldehyde content was relatively small. Alcohol dehydrogenase in growing and maturing stage fruits showed increased activity with the maximum value at one day after the onset of anoxia treatment and then decreased gradually. An increase in the activity of pyruvate decarboxylase was also observed during anoxia.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.81-84
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• 2000

• Journal of Ginseng Research
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• v.10 no.2
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• pp.200-208
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• 1986

It was attempted in this study to investigate the effect of ginseng saponin on several glycolytic enzymes of yeast cell and the following results were obtained. The amount of $CO_2$formed during the incubation of yeast cells in medium containing saponin fraction of Panax ginseng C.A. Meyer was greater than that of control cells and found that the $CO_2$ formation was greatest when the cells were grown in the medium containing 10$^{-3}$% of the saponin fraction, at which the uptake of inorganic phosphate and glucose consumption were also increased. Radioactivity study of several glycolytic intermediates of yeast cells cultured in the medium containing [U-$^{14}$C]-glucose showed that the radioactivity of fructose 6-phosphate of test cells was as much as 1.6times that of control group. On the other hand, the radioactivity of pyruvate of test cells was considerably decreased compared to control. Investigation of the effect of ginseng saponin on yeast hexokinase, phosphoglucose isomers, pyruvate kinase and perverted decarboxylase in vitro showed that the maximum activities of the above enzymes were observed when the concentration of ginseng saponin was 10-$^{-5}$% in the reaction mixture. It seemed that the ginseng saponin stimulated both glycolytic enzymes such as hexokinase, phosphoglucose isomers and perverted decarboxylase significantly.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.56-59
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• 2000

Thermophilic bacterium, Bacillus stearothermophilus NUB3621, was engineered to produce ethanol from glucose by introducing cloned thermostable pyruvate decarboxylase and alcohol dehydrogenase genes. A novel promoter sequence was screened and used for the enhancement of these two enzymes. Successful redirection of metabolic flux into ethanol was obtained. In addition, gene expression profiling using Bacillus subtilis DNA microarray was analyzed to overcome the intrinsic low glucose utilization of B.stearothermophilus. Many known and unknown genes were identified to be up or down regulated under glucose-containing media.