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Lee H.C.,BioNgene Co. | Kim J.S.,BioNgene Co. | Jang W.,Dongguk University | Kim S.Y.,BioNgene Co.
Journal of Biotechnology | Year: 2010

Thymidine is a commercially important precursor in the production of antiviral drugs, including azidothymidine for the treatment of AIDS. In order to produce thymidine in a large scale, we previously developed a thymidine-overproducing Escherichia coli strain BLdtug24 by engineering pathways. To further enhance thymidine yield, we increased the availability of a cofactor (NADPH) in thymidine biosynthesis by disrupting phosphoglucose isomerase in BLdtug24 to construct BLdtugp24. Additionally, NAD+ kinase or soluble transhydrogenase was overexpressed in BLdtugp24, which can reroute glucose metabolic flow from the EMP pathway to the PP pathway, to construct BLdtugp34N or BLdtugp34U, respectively. In chemostat cultures, BLdtugp24 had an increased NADPH availability and a 4-fold enhancement in thymidine yield for glucose compared with BLdtug24. BLdtugp34N and BLdtugp34U had increased thymidine yields for glucose by 1.2- and 2-fold compared with BLdtugp24, respectively. The NADPH/NADP+ ratios at steady-state had overall positive correlations with thymidine yields in these strains. Real-time RT-PCR analysis revealed that the transcriptional regulations of NAD(P)H-related enzymes and transhydrogenases affected the redox balance and shifted reaction equilibrium toward increasing NADPH. In fed-batch fermentation, BLdtugp34U with the highest NADPH/NADP+ ratio in chemostat experiment produced 1.9gl-1 of thymidine with 29.7mgl-1h-1 of thymidine productivity. © 2010 Elsevier B.V.


Koo B.S.,BioNgene Co. | Koo B.S.,Korea University | Hyun H.H.,Hankuk University of foreign Studies | Kim S.Y.,BioNgene Co. | And 2 more authors.
Biotechnology Letters | Year: 2011

Purpose of Work: Thymidine is an important precursor in antiviral drugs. We have enhanced thymidine production in E. coli by eliminating the repressors in the transcription of the gene coding for carbamoyl phosphate synthetase. The operon for carbamoyl phosphate synthetase (CarAB) in the thymidine biosynthesis regulatory pathway was derepressed by disrupting three known repressors (purR, pepA and argR). Combinatorial disruption of three repressors increased CarA expression levels in accordance with degree of disruption, which had a positive correlation with thymidine production. By simultaneous disruption of three repressors (BLdtugRPA), CarA expression level was increased by 3-fold compared to the parental strain, leading to an increased thymidine yield from 0.25 to 1.1 g thymidine l-1. From BLdtugRPA, we established BLdtugRPA24 by transforming two plasmids expressing enzymes in the thymidine biosynthetic pathway and obtained 5.2 g thymidine l-1 by Ph-stat fed-batch fermentation. © 2010 Springer Science+Business Media B.V.


Koo B.-S.,BioNgene Co. | Koo B.-S.,Korea University | Gong Y.-J.,BioNgene Co. | Kim S.-Y.,BioNgene Co. | And 2 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2010

Previously screened CoQ10-overproducing Agrobacterium tumefaciens A603-35 showed a relatively high NADH/NAD+ ratio (1.1), as compared to parental strain C58 (0.2) when we increased the expression levels of NADH-generating enzymes. Also, the intracellular NADH/NAD+ ratio showed a positive correlation with the CoQ10 content in A603-35. Overexpression of glyceraldehyde 3-phosphate dehydrogenase in A603-35 shifted the NADH/NAD+ ratio at 48 h from 0.8 to 1.2, and thus the CoQ 10 content in flask culture increased from 2.16 to 3.63 mg/g DCW. Due to the addition of hydroxybutyrate to the culture media, the intracellular NADH/NAD+ ratio in A603-35-gapA shifted from 1.2 to 1.4, which led to an increase CoQ10 content (5.27 mg/g DCW).


Kim T.-S.,Konkuk University | Jung H.-M.,BioNgene Co Ltd | Kim S.-Y.,BioNgene Co Ltd | Zhang L.,Konkuk University | And 5 more authors.
Journal of Microbiology and Biotechnology | Year: 2015

Acetate and lactate in growth media are detrimental to the production of Thermus maltogenic amylase (ThMA), a heterologous protein, as well as to the growth of recombinant Escherichia coli. Only 50 mM of acetate or 10 mM of lactate reduced 90% of specific ThMA activity. In this study, mutant E. coli strains blocked in the ackA-pta or ackA-pta and ldh pathways were created, characterized, and assessed for their culture performace in 300 L-scale fermentation. The ackApta and ldh double-mutant strain formed significantly less lactate and acetate, and produced a concomitant increase in the excretion of pyruvate (17.8 mM) under anaerobic conditions. The ackA-pta mutant strain accumulated significant acetate but had an approximately 2-fold increase in the formation of lactate. The ackA-pta and ldh double-mutant strain had superior overall performance in large-scale culture under suboptimal conditions, giving 67% higher cell density and 66% higher ThMA activity compared with those of the control strain. The doublemutant strain also achieved a 179% improvement in volumetric ThMA production. © 2015 by The Korean Society for Microbiology and Biotechnology.


Singh R.,Konkuk University | Singh R.K.,Konkuk University | Kim S.-Y.,BioNgene Co. | Sigdel S.,Konkuk University | And 4 more authors.
Biochemical Engineering Journal | Year: 2016

D-Ribulose, a potentially valuable rare sugar and an excellent building block in asymmetric synthesis, is usually produced by chemical synthesis, which unfortunately does not meet the increasing demand. In addition, ribitol oxidization for the production of d-ribulose has not yet become an industrial reality because of the lack of efficient biocatalysts, resulting in higher production costs as well as a poor yield. In this study, we have employed the Enterobacter aerogenes ribitol: NAD+ 2-oxidoreductase (EaRDH), which could efficiently and selectively convert ribitol to D-ribulose. The purified EaRDH enzyme and the recombinant Escherichia coli strain (as a whole-cell catalyst) were used to produce d-ribulose. Ribitol was efficiently converted to d-ribulose by EaRDH with a yield of ~85%, suggesting the usefulness of this enzyme for the in vivo and in vitro production of d-ribulose from ribitol. The oxidation of ribitol to D-ribulose by EaRDH was accomplished in the presence of stoichiometric amounts of NAD+; therefore, D-ribulose production was further enhanced by the incorporation of diaphorase for continuous NAD+ regeneration. The biocatalytic process presented should be a promising route for the biotechnological production of d-ribulose (and related branched pentoses) at an efficient and industrially relevant scale. © 2016 Elsevier B.V.


Kim J.-H.,BioNgene Co. | Prabhu P.,Konkuk University | Jeya M.,Konkuk University | Tiwari M.K.,Konkuk University | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2010

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k cat of 14,504 min-1 and a k cat/K m of 121 min-1mM-1 for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose. © 2009 Springer-Verlag.


PubMed | BioNgene Co. and Konkuk University
Type: Journal Article | Journal: Journal of microbiology and biotechnology | Year: 2015

Acetate and lactate in growth media are detrimental to the production of Thermus maltogenic amylase (ThMA), a heterologous protein, as well as to the growth of recombinant Escherichia coli. Only 50 mM of acetate or 10 mM of lactate reduced 90% of specific ThMA activity. In this study, mutant E. coli strains blocked in the ackA-pta or ackA-pta and ldh pathways were created, characterized, and assessed for their culture performace in 300 L-scale fermentation. The ackApta and ldh double-mutant strain formed significantly less lactate and acetate, and produced a concomitant increase in the excretion of pyruvate (17.8 mM) under anaerobic conditions. The ackA-pta mutant strain accumulated significant acetate but had an approximately 2-fold increase in the formation of lactate. The ackA-pta and ldh double-mutant strain had superior overall performance in large-scale culture under suboptimal conditions, giving 67% higher cell density and 66% higher ThMA activity compared with those of the control strain. The doublemutant strain also achieved a 179% improvement in volumetric ThMA production.


Kim J.-S.,ForBioKorea Co. | Jeong M.-K.,BioNgene Co. | Koo B.-S.,ForBioKorea Co. | Lee H.-C.,ForBioKorea Co. | Lee H.-C.,BioNgene Co.
Applied and Environmental Microbiology | Year: 2015

A novel thymidine-producing strain of Escherichia coli was prepared by genome recombineering. Eleven genes were deleted by replacement with an expression cassette, and 7 genes were integrated into the genome. The resulting strain, E. coli HLT013, showed a high thymidine yield with a low deoxyuridine content. DNA microarrays were then used to compare the gene expression profiles of HLT013 and its isogenic parent strain. Based on microarray analysis, the pyr biosynthesis genes and 10 additional genes were selected and then expressed in HLT013 to find reasonable candidates for enhancing thymidine yield. Among these, phage shock protein A (PspA) showed positive effects on thymidine production by diminishing redox stress. Thus, we integrated pspA into the HLT013 genome, resulting in E. coli strain HLT026, which produced 13.2 g/liter thymidine for 120 h with fedbatch fermentation. Here, we also provide a basis for new testable hypotheses regarding the enhancement of thymidine productivity and the attainment of a more complete understanding of nucleotide metabolism in bacteria. © 2015, American Society for Microbiology.


PubMed | BioNgene Co. and ForBioKorea Co.
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2015

A novel thymidine-producing strain of Escherichia coli was prepared by genome recombineering. Eleven genes were deleted by replacement with an expression cassette, and 7 genes were integrated into the genome. The resulting strain, E. coli HLT013, showed a high thymidine yield with a low deoxyuridine content. DNA microarrays were then used to compare the gene expression profiles of HLT013 and its isogenic parent strain. Based on microarray analysis, the pyr biosynthesis genes and 10 additional genes were selected and then expressed in HLT013 to find reasonable candidates for enhancing thymidine yield. Among these, phage shock protein A (PspA) showed positive effects on thymidine production by diminishing redox stress. Thus, we integrated pspA into the HLT013 genome, resulting in E. coli strain HLT026, which produced 13.2 g/liter thymidine for 120 h with fed-batch fermentation. Here, we also provide a basis for new testable hypotheses regarding the enhancement of thymidine productivity and the attainment of a more complete understanding of nucleotide metabolism in bacteria.


PubMed | Institute of SK KU Biomaterials, BioNgene Co. and Konkuk University
Type: Journal Article | Journal: Journal of microbiology and biotechnology | Year: 2016

Methane (CH) is the most abundant component in natural gas. To reduce its harmful environmental effect as a greenhouse gas, CH can be utilized as a low-cost feed for the synthesis of methanol by methanotrophs. In this study, several methanotrophs were examined for their ability to produce methanol from CH; including Methylocella silvestris, Methylocystis bryophila, Methyloferula stellata, and Methylomonas methanica. Among these methanotrophs, M. bryophila exhibited the highest methanol production. The optimum process parameters aided in significant enhancement of methanol production up to 4.63 mM. Maximum methanol production was observed at pH 6.8, 30C, 175 rpm, 100 mM phosphate buffer, 50 mM MgCl as a methanol dehydrogenase inhibitor, 50% CH concentration, 24 h of incubation, and 9 mg of dry cell mass ml(-1) inoculum load, respectively. Optimization of the process parameters, screening of methanol dehydrogenase inhibitors, and supplementation with formate resulted in significant improvements in methanol production using M. bryophila. This report suggests, for the first time, the potential of using M. bryophila for industrial methanol production from CH.

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