• Title/Summary/Keyword: Metabolic Engineering

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Screening and Characterization of a Novel Cellulase Gene from the Gut Microflora of Hermetia illucens Using Metagenomic Library

  • Lee, Chang-Muk;Lee, Young-Seok;Seo, So-Hyeon;Yoon, Sang-Hong;Kim, Soo-Jin;Hahn, Bum-Soo;Sim, Joon-Soo;Koo, Bon-Sung
    • Journal of Microbiology and Biotechnology
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    • v.24 no.9
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    • pp.1196-1206
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    • 2014
  • A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at $50^{\circ}C$ and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of $20{\sim}50^{\circ}C$ and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thin-layer chromatography suggested that CS10 is an endo-${\beta}$-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.

Current status of CRISPR/Cas9 base editor technologies and their applications in crop precision breeding

  • Kim, Rigyeong;Song, Jaeeun;Ga, Eunji;Min, Myung Ki;Lee, Jong-Yeol;Lim, Sun-Hyung;Kim, Beom-Gi
    • Korean Journal of Agricultural Science
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    • v.46 no.4
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    • pp.885-895
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    • 2019
  • Plant biotechnologists have long dreamed of technologies to manipulate genes in plants at will. This dream has come true partly through the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, which now has been used to edit genes in several important crops. However, there are many restrictions in editing a gene precisely using the CRISPR/Cas9 technology because CRISPR/Cas9 may cause deletions or additions in some regions of the target gene. Several other technologies have been developed for gene targeting and precision editing. Among these, base editors might be the most practically and efficiently used compared to others. Base editors are tools which are able to cause a transition from cytosine into thymine, or from adenine into guanine very precisely on specific sequences. Cytosine base editors basically consist of nCas9, cytosine deaminase, and uracil DNA glycosylase inhibitor (UGI). Adenine base editors consist of nCas9 and adenine deaminase. These were first developed for human cells and have since also been applied successfully to crops. Base editors have been successfully applied for productivity improvement, fortification and herbicide resistance of crops. Thus, base editor technologies start to open a new era for precision gene editing or breeding in crops and might result in revolutionary changes in crop breeding and biotechnology.

Applications of Metabolic Modeling to Drive Bioprocess Development for the Production of Value-added Chemicals

  • Mahadevan, Radhakrishnan;Burgard, Anthony P.;Famili, Iman;Dien, Steve Van;Schilling, Christophe H.
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.10 no.5
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    • pp.408-417
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    • 2005
  • Increasing numbers of value added chemicals are being produced using microbial fermentation strategies. Computational modeling and simulation of microbial metabolism is rapidly becoming an enabling technology that is driving a new paradigm to accelerate the bioprocess development cycle. In particular, constraint-based modeling and the development of genome-scale models of industrial microbes are finding increasing utility across many phases of the bioprocess development workflow. Herein, we review and discuss the requirements and trends in the industrial application of this technology as we build toward integrated computational/experimental platforms for bioprocess engineering. Specifically we cover the following topics: (1) genome-scale models as genetically and biochemically consistent representations of metabolic networks; (2) the ability of these models to predict, assess, and interpret metabolic physiology and flux states of metabolism; (3) the model-guided integrative analysis of high throughput 'omics' data; (4) the reconciliation and analysis of on- and off-line fermentation data as well as flux tracing data; (5) model-aided strain design strategies and the integration of calculated biotransformation routes; and (6) control and optimization of the fermentation processes. Collectively, constraint-based modeling strategies are impacting the iterative characterization of metabolic flux states throughout the bioprocess development cycle, while also driving metabolic engineering strategies and fermentation optimization.

In Silico Analysis of Lactic Acid Secretion Metabolism through the Top-down Approach: Effect of Grouping in Enzyme kinetics

  • Jin, Jong-Hwa;Lee, Jin-Won
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.10 no.5
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    • pp.462-469
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    • 2005
  • A top-down approach is known to be a useful and effective technique for the design and analysis of metabolic systems. In this Study, we have constructed a grouped metabolic network for Lactococcus lactis under aerobic conditions using grouped enzyme kinetics. To test the usefulness of grouping work, a non-grouped system and grouped systems were compared quantitatively with each other. Here, grouped Systems were designed as two groups according to the extent of grouping. The overall simulated flux values in grouped and non-grouped models had pretty similar distribution trends, but the details on flux ratio at the pyruvate branch point showed a little difference. This result indicates that our grouping technique can be used as a good model for complicated metabolic networks, however, for detailed analysis of metabolic network, a more robust mechanism Should be considered. In addition to the data for the pyruvate branch point analysis, Some major flux control coefficients were obtained in this research.

DsLCYB Directionally Modulated β-Carotene of the Green Alga Dunaliella salina under Red Light Stress

  • Yanhong Lan;Yao Song;Yihan Guo;Dairong Qiao;Yi Cao;Hui Xu
    • Journal of Microbiology and Biotechnology
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    • v.32 no.12
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    • pp.1622-1631
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    • 2022
  • Carotenoids, which are natural pigments found abundantly in wide-ranging species, have diverse functions and high industrial potential. The carotenoid biosynthesis pathway is very complex and has multiple branches, while the accumulation of certain metabolites often affects other metabolites in this pathway. The DsLCYB gene that encodes lycopene cyclase was selected in this study to evaluate β-carotene production and the accumulation of β-carotene in the alga Dunaliella salina. Compared with the wild type, the transgenic algal species overexpressed the DsLCYB gene, resulting in a significant enhancement of the total carotenoid content, with the total amount reaching 8.46 mg/g for an increase of up to 1.26-fold. Interestingly, the production of α-carotene in the transformant was not significantly reduced. This result indicated that the regulation of DsLCYB on the metabolic flux distribution of carotenoid biosynthesis is directional. Moreover, the effects of different light-quality conditions on β-carotene production in D. salina strains were investigated. The results showed that the carotenoid components of β-carotene and β-cryptoxanthin were 1.8-fold and 1.23-fold higher than that in the wild type under red light stress, respectively. This suggests that the accumulation of β-carotene under red light conditions is potentially more profitable.

Analytical Techniques for Vancomycin - A Review

  • Avinash P. Sattur;Lee, Je-Hyuk;Song, Ki-Bang;T. Panda;Kim, Chul-Ho;Rhee, Sang-Ki;B. Gokul
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.5 no.3
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    • pp.153-158
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    • 2000
  • Vancomycin belongs to the vancomycin-risocetin family of glycopeptides, and is a subclass of linear sugar containg peptides composed of seven amino acids. Its strochemical configuration forms the basic of a peptidoglycon monomer. The glycosylated hexapeptide chainconsists of chloro-$\beta$-hydroxytyrosines, p-hytidoglycines, N-anthylleucine and aspartic acid forms a rigid molecular frame work and gives the difficulty in the analysis. Voncomycin in the serum samples is usually estimated by liquid chromatography and the bacterial sensitivity was genereally tested by the microbiological assay. The pressent review deals with the qualitative, quantutative, microbioligical and immunological assays and the comparison of the quantitative methods. Clinical implications of vancomycin have also been cited in the review.

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MRI Content-Adaptive Finite Element Mesh Generation Toolbox

  • Lee W.H.;Kim T.S.;Cho M.H.;Lee S.Y.
    • Journal of Biomedical Engineering Research
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    • v.27 no.3
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    • pp.110-116
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    • 2006
  • Finite element method (FEM) provides several advantages over other numerical methods such as boundary element method, since it allows truly volumetric analysis and incorporation of realistic electrical conductivity values. Finite element mesh generation is the first requirement in such in FEM to represent the volumetric domain of interest with numerous finite elements accurately. However, conventional mesh generators and approaches offered by commercial packages do not generate meshes that are content-adaptive to the contents of given images. In this paper, we present software that has been implemented to generate content-adaptive finite element meshes (cMESHes) based on the contents of MR images. The software offers various computational tools for cMESH generation from multi-slice MR images. The software named as the Content-adaptive FE Mesh Generation Toolbox runs under the commercially available technical computation software called Matlab. The major routines in the toolbox include anisotropic filtering of MR images, feature map generation, content-adaptive node generation, Delaunay tessellation, and MRI segmentation for the head conductivity modeling. The presented tools should be useful to researchers who wish to generate efficient mesh models from a set of MR images. The toolbox is available upon request made to the Functional and Metabolic Imaging Center or Bio-imaging Laboratory at Kyung Hee University in Korea.

Fabrication of a Partial Genome Microarray of the Methylotrophic Yeast Hansenula polymorpha: Optimization and Evaluation of Transcript Profiling

  • OH , KWAN-SEOK;KWON, OH-SUK;OH, YUN-WI;SOHN, MIN-JEONG;JUNG, SOON-GEE;KIM, YONG-KYUNG;KIM, MIN-GON;RHEE, SANG-KI;GERD GELLISSEN,;KANG, HYUN-AH
    • Journal of Microbiology and Biotechnology
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    • v.14 no.6
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    • pp.1239-1248
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    • 2004
  • The methylotrophic yeast Hansenula polymorpha has been extensively studied as a model organism for methanol metabolism and peroxisome biogenesis. Recently, this yeast has also attracted attention as a promising host organism for recombinant protein production. Here, we describe the fabrication and evaluation of a DNA chip spotted with 382 open reading frames (ORFs) of H. polymorpha. Each ORF was PCR-amplified using gene-specific primer sets, of which the forward primers had 5'-aminolink. The PCR products were printed in duplicate onto the aldehyde-coated slide glasses to link only the coding strands to the surface of the slide via covalent coupling between amine and aldehyde groups. With the partial genome DNA chip, we compared efficiency of direct and indirect cDNA target labeling methods, and found that the indirect method, using fluorescent-labeled dendrimers, generated a higher hybridization signal-to-noise ratio than the direct method, using cDNA targets labeled by incorporation of fluorescence-labeled nucIeotides during reverse transcription. In addition, to assess the quality of this DNA chip, we analyzed the expression profiles of H. polymorpha cells grown on different carbon sources, such as glucose and methanol, and also those of cells treated with the superoxide­generating drug, menadione. The profiles obtained showed a high-level induction of a set of ORFs involved in methanol metabolism and oxidative stress response in the presence of methanol and menadione, respectively. The results demonstrate the sensitivity and reliability of our arrays to analyze global gene expression changes of H. polymorpha under defined environmental conditions.