Tai-Boong Uhm

Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain

BackgroundThe phenylpropanoid metabolites are an extremely diverse group of natural products biosynthesized by plants, fungi, and bacteria. Although these compounds are widely used in human health care and nutrition services, their availability is limited by regional variations, and isolation of single compounds from plants is often difficult. Recent advances in synthetic biology and metabolic engineering have enabled artificial production of plant secondary metabolites in microorganisms.ResultsWe develop an Escherichia coli system containing an artificial biosynthetic pathway that yields phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, from simple carbon sources. These artificial biosynthetic pathways contained a codon-optimized tal gene that improved the productivity of 4-coumaric acid and ferulic acid, but not caffeic acid in a minimal salt medium. These heterologous pathways extended in E. coli that had biosynthesis machinery overproducing tyrosine. Finally, the titers of 4-coumaric acid, caffeic acid, and ferulic acid reached 974 mg/L, 150 mg/L, and 196 mg/L, respectively, in shake flasks after 36-hour cultivation.ConclusionsWe achieved one gram per liter scale production of 4-coumaric acid. In addition, maximum titers of 150 mg/L of caffeic acid and 196 mg/L of ferulic acid were achieved. Phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, have a great potential for pharmaceutical applications and food ingredients. This work forms a basis for further improvement in production and opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from phenylpropanoic acids.

Production of high-content inulo-oligosaccharides from inulin by a purified endoinulinase

Inulo-oligosaccharides were produced from inulin with high yield by using a purified endoinulinase from a commercial inulinase preparation. The maximum yield of oligosaccharide achieved was around 96% irrespective of substrate concentrations ranged from 50 to 150 g inulin/l. A wide range of degradation products from inulin, varying in their DP (degree of polymerization) 2 to 6, were obtained where the major oligosaccharides were DP3 and 4. The reaction pH gave rise to a significant difference in yield and sugar composition of inulo-oligosaccharides.

Cloning and Nucleotide Sequence of the Endoinulinase-Encoding Gene, inu2, from Aspergillus ficuum

A 2.3 kb DNA fragment that contains a gene encoding endoinulinase, inu2, from Aspergillus ficuum ATCC 16882 was isolated and analyzed. It includes an open reading frame of 1,551 bp, coding for a polypeptide with calculated molecular weight of 55,790 Da, including a putative signal peptide of 22 amino acids. Alignment of amino acid sequences revealed 73.3% identity and 93.9% similarity between A. ficuum and Penicillium purpurogenum endoinulinase.

Expression of the INU2 gene for an endoinulinase of Aspergillus ficuum in Saccharomyces cerevisiae

The INU2 gene encoding an endoinulinase of Aspergillus ficuum was expressed by the Kluyveromyces marxianus INU1 promoter in a SUC2-deleted Saccharomyces cerevisiae to produce the endoinulinase preparation free of an exoinulinase and an extracellular invertase in the culture medium. A recombinant yeast strain produced the sufficient amount of the enzyme to make a halo around its colony, when inulin was included in the medium.

Evaluation of the therapeutic efficacy of a VEGFR2-blocking antibody using sodium-iodide symporter molecular imaging in a tumor xenograft model.

PURPOSE Vascular endothelial growth factor receptor 2-blocking antibody (DC101) has inhibitory effects on tumor growth and angiogenesis in vivo. The human sodium/iodide symporter (hNIS) gene has been shown to be a useful molecular imaging reporter gene. Here, we investigated the evaluation of therapeutic efficacy by molecular imaging in reporter gene transfected tumor xenografts using a gamma imaging system. METHODS The hNIS gene was transfected into MDA-MB-231 cells using Lipofectamine. The correlation between the number of MDA-MB-231-hNIS cells and the uptake of (99m)Tc-pertechnetate or (125)I was investigated in vitro by gamma imaging and counting. MDA-MB-231-hNIS cells were injected subcutaneously into mice. When the tumor volume reached 180-200 mm(3), we randomly assigned five animals to each of three groups representing different tumor therapies; no DC101 (control), 100 μg, or 150 μg DC101/mouse. One week and 2 weeks after the first injection of DC101, gamma imaging was performed. Mice were sacrificed 2 weeks after the first injection of DC101. The tumor tissues were used for reverse transcriptase-polymerase chain reaction (RT-PCR) and CD31 staining. RESULTS Uptake of (125)I and (99m)Tc-pertechnetate into MDA-MB-231-hNIS cells in vitro showed correlation with the number of cells. In DC101 treatment groups, the mean tumor volume was smaller than that of the control mice. Furthermore, tumor uptake of (125)I was lower than in the controls. The CD31 staining and RT-PCR assay results showed that vessel formation and expression of the hNIS gene were significantly reduced in the tumor tissues of treatment groups. CONCLUSION This study demonstrated the power of molecular imaging using a gamma imaging system for evaluating the therapeutic efficacy of an antitumor treatment. Molecular imaging systems may be useful in evaluation and development of effective diagnostic and/or therapeutic antibodies for specific target molecules.

Thermal stability of an acidic inulinase from Scytalidium acidophilum

Microbial inulinases (EC 3.2.1.7 or EC 3.2.1.80) offer interesting perspectives in view of the production of the high fructose syrup and fructooligosaccharides from inulin. Among them, Heat stable inulinases are advantageous in view of controlling microbial contamination and permit the use of higher soluble inulin concentrations. Recently, we found that the acid-resistant fungus Scytalidium acidophilum showed exoinulinase (EC 3.2.1.80) activity. To evaluate its industrial potential, the thermal stability of this enzyme was investigated

Trp17 and Glu20 residues in conserved WMN(D/E)PN motif are essential for Aspergillus ficuum endoinulinase (EC 3.2.1.7) activity.

The importance of the WMN(D/E)PN motif, which is well conserved among β-fructofuranosidases grouped in the glycosylhydrolase family 32, in Aspergillus ficuum endoinulinase was accessed. Each mutant enzyme generated by site-directed mutagenesis of Trp17 in the conserved motif to Gln, Leu, Ser, Pro, Thr, or Met had an activity of less than 1% of the wild type. Another mutant enzyme obtained by mutation of Glu20 in the motif to Ser, Leu, Thr, Gln, Ala, or Val had an enzyme activity of less than 1% of the wild type. Furthermore, the E20D mutant enzyme, in which Glu20 in the conserved motif was replaced with Asp, had 1.1% of the wild type activity. These results clearly indicated that Trp17 and Glu20 are essential for the enzyme activity.

A peptide antibody for rapid screening of Streptomyces species producing phospholipase D

By examining the conserved regions in the protein sequences of eight different Streptomyces phospholipase Ds (PLD) reported so far and the X-ray crystallographic structure of a Streptomyces PLD, we designed a peptide sequence, DPANRGAVGSGGYSQIKSL, for the screening of microorganisms producing PLD. In the enzyme-linked immunosorbent assay using a mouse antibody raised against the designed peptide, we recovered seven producing strains out of 128 soil isolates.

The haplotypes of TNFRSF17 polymorphisms are associated with colon cancer in a Korean population

PurposeWe previously found that the haplotypes of TNFRSF17 single nucleotide polymorphisms (SNPs) were associated with the susceptibility to inflammatory bowel disease on Korean population. The present study aimed to investigate whether the polymorphisms in the TNFRSF17 gene are associated with susceptibility to colorectal cancer (CRC).MethodsGenotype analysis in the TNFRSF17 SNPs was performed by high-resolution melting and TaqMan probe analysis, and the genotype and allele frequencies of TNFRSF17 SNPs were compared between the CRC patients and the healthy controls. The haplotype frequencies of TNFRSF17 for multiple loci were estimated using the expectation maximization algorithm.ResultsAlthough, the genotype and allelic frequencies of these SNPs, in the colon cancer and rectal cancer patients, were not significantly different from those in the healthy controls, the genotype and allele frequency of g.2493G>A was significantly different between the healthy controls and the right colon cancer patients (P = 0.014 and 0.004, respectively). Moreover, the haplotypes frequencies in the healthy controls were significantly different from those in the colon cancer patients.ConclusionOur results suggest that TNFRSF17 may be a candidate gene associated with the pathogenesis of colon cancer, and the haplotypes of the TNFRSF17 polymorphisms might be one of the markers for colon cancer susceptibility.

Effect of Molecular Imaging on Validation of Developed Anti-hVEGFR2 Therapeutic Antibody

Vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted tumor treatment is an antiangiogenic therapeutic strategy. The human sodium iodide symporter (hNIS) gene is a useful reporter gene for tumor imaging and radiotherapy. In this study, we investigated the evaluation of therapeutic efficacy in hNIS gene-transfected tumor xenografts using a gamma imaging system after treatment with an anti-VEGFR2 antibody. Human breast cancer MDA-MB-231 cells transfected with the hNIS gene were injected subcutaneously into the right flanks of BALB/c nude mice. Therapy was initiated when the tumor volume reached approximately 130-180 mm(3). The animals were intravenously injected with 50, 100, or 150 μg of antibody every 3 days for 16 days. Gamma imaging was performed 1 and 2 weeks after the first injection to monitor the effects of tumor therapy. Mice were sacrificed 2 weeks after the first injection of antibody and the tumors were removed for CD31 staining and reverse transcription-polymerase chain reaction (RT-PCR) assay. All groups of mice that were treated with anti-hVEGFR2 antibody showed markedly reduced tumor growth compared to control mice. In vivo gamma imaging results showed that, at 1 week after the first injection of the anti-hVEGFR2 antibody, (125)I uptake of a tumor treated with 150 μg of antibody was 24.5% lower than that in the controls. At 2 weeks, (125)I uptake in the tumor treated with 150 μg of antibody was as low as 44.3% of that in the controls. CD31 staining and RT-PCR assays showed that blood vessel formation and expression of the hNIS gene were reduced with increased treatment doses. This study demonstrated the feasibility of molecular imaging and the therapeutic efficacy of developing therapeutic antibody anti-hVEGFR2 using a gamma imaging system in hNIS gene-transfected tumor xenograft mice.