Browsing by Author "Chiu, Hsien-Tai"

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Analysis of circular genome rearrangement by fusions, fissions and block-interchanges
(2006-06-12)
Lu, Chin Lung
;
Huang, Yen Lin
;
Wang, Tsui Ching
;
Chiu, Hsien-Tai
;
生物科技學系
Background: Analysis of genomes evolving via block-interchange events leads to a combinatorial problem of sorting by block-interchanges, which has been studied recently to evaluate the evolutionary relationship in distance between two biological species since block-interchange can be considered as a generalization of transposition. However, for genomes consisting of multiple chromosomes, their evolutionary history should also include events of chromosome fusions and fissions, where fusion merges two chromosomes into one and fission splits a chromosome into two. Results: In this paper, we study the problem of genome rearrangement between two genomes of circular and multiple chromosomes by considering fusion, fission and block-interchange events altogether. By use of permutation groups in algebra, we propose an O(n(2)) time algorithm to efficiently compute and obtain a minimum series of fusions, fissions and block-interchanges required to transform one circular multi-chromosomal genome into another, where n is the number of genes shared by the two studied genomes. In addition, we have implemented this algorithm as a web server, called FFBI, and have also applied it to analyzing by gene orders the whole genomes of three human Vibrio pathogens, each with multiple and circular chromosomes, to infer their evolutionary relationships. Consequently, our experimental results coincide well with our previous results obtained using the chromosome-by-chromosome comparisons by landmark orders between any two Vibrio chromosomal sequences as well as using the traditional comparative analysis of 16S rRNA sequences. Conclusion: FFBI is a useful tool for the bioinformatics analysis of circular and multiple genome rearrangement by fusions, fissions and block-interchanges.
Biochemical characterization and substrate specificity of the gene cluster for biosyntheses of K-252a and its analogs by in vitro heterologous expression system of Escherichia coli
(2009)
Chiu, Hsien-Tai
;
Lin, Yu-Chin
;
Lee, Meng-Na
;
Chen, Yi-Lin
;
Wang, Mei-Sin
The indolocarbazole family of natural products has attracted great attention because of their unique structural features and potential therapeutic applications. Structurally distinct in the family, K-252a is characterized by an unusual dihydrostreptose moiety cross-bridged to K-252c aglycone with two C-N linkages. K-252a has served as a valuble lead for treatments of various cancers and neurodegenerative disorders. Recent cloning of the nok gene cluster for biosyntheses of K-252a and its analogs from Nocardiopsis sp. K-252 (NRRL15532) has revealed the nokABCD genes indispensible for K-252c biosynthesis and the key gene (nokL) coding for N-glycosylation. Herein, we report the first, successful demonstration of in vitro sugar transferase activity of indolocarbazole N-glycosyltransferase (NokL) by use of soluble protein expressed from Escherichia coli. Notably, NokL was found to exhibit peculiar mode of substrate promiscuity. Moreover, NokA and NokB reactions were biochemically characterized thoroughly by natual and alternative (e. g. fluoro-) substrates and by ammonium hydroxide (NH(4)OH). Interestingly, the in vitro expression of NokA revealed high substrate stereoselectivity, giving several indole-3-pyruvic acid-derived compounds, including indol-3-carboxaldehyde (ICA) and indole-3-acetic acid. The use of NH(4)OH successfully dissected the in vitro NokA/NokB coupled reaction, revealing mechanistic insight into the enzymes and their cross-talking relationship. Also, a simple, useful method to synthesize K-252d, ICA and chromopyrrolic acid (the NokB product) was developed by the E. coli expression systems of NokL, NokA and NokA/NokB, respectively. Together with NokA and NokB, NokL may serve as a useful tool for combinatorial engineering of K-252a and its analogs for improved therapeutic values.
BIOL 68-Identification, functional expression and kinetic characterization of two GDP-L-fucose biosynthetic enzymes from Ganoderma lucidum
(2008-04-06)
Chiu, Hsien-Tai
;
Wu, Chih Chien
;
Deng, Shi-Jie
;
Huang, Pei-Ning
;
生物科技學系
CARB 126-Combinatorial biosynthesis of NDP-deoxysugars and spinosyn glycosides by in vitro recombination of secondary metabolite biosynthetic pathways
(2007-03-25)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
生物科技學系
;
Department of Biological Science and Technology
CARB 83-In vitro enzymatic generation of GDP-L-fucose and GDP-D-rhamnose by GDP-D-mannose 4,6-dehydratase and GDP-L-fucose synthetase from medicinal fungus Ling-Zhi
(2008-04-06)
Chiu, Hsien-Tai
;
Deng, Shi-Jie
;
Wu, Chih Chien
;
生物科技學系
;
Department of Biological Science and Technology
Effect of Pulsed Electromagnetic Field on the Proliferation and Differentiation Potential of Human Bone Marrow Mesenchymal Stem Cells
(2009-05-01)
Sun, Li-Yi
;
Hsieh, Dean-Kuo
;
Yu, Tzai-Chiu
;
Chiu, Hsien-Tai
;
Lu, Sheng-Fen
Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 Pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the 2 PEMF-exposed Cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm(2) respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20-60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results Suggest that PEMF exposure Could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF-exposed BMMSC showed multi-lineage differentiation potential similar to the control group. Bioelectromagnetics 30:251-260, 2009. (C) 2009 Wiley-Liss. Inc.
Exploring combinatorial potential of K-252c biosynthesis by precursor-directed enzyme-coupling reactions
(2011-08-28)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
Lin, Yu-Chin
;
Lee, Meng-Na
;
生物科技學系
Molecular cloning, sequence analysis and functional characterization of the gene cluster for biosynthesis of K-252a and its analogs
(2009)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
Chen, Chien-Yu
;
Jin, Chyn
;
Lee, Meng-Na
Among the indolocarbazole alkaloids of antitumor antibiotics, K-252a represents a structurally unique indolocarbazole glycoside and exhibits potent neuroprotective and broad anticancer activities. K-252a consists of K-252c and the unusual dihydrostreptose moiety, linked together with oxidative and glycosidic C-N bonds. Herein, we reported a complete sequence of an approximately 45 kb genomic fragment harboring the gene cluster for the biosynthesis of indolocarbazole alkaloids in Nocardiopsis sp. K-252 (NRRL15532). The sequence of 35 open reading frames discovered several new, critical genes, hence shedding new light on biosynthesis, resistance and regulation of K-252a and its analogs. To functionally characterize the gene cluster in vitro and in enzyme level, a multigene expression cassette containing the K-252c biosynthetic genes was constructed and successfully overexpressed in Escherichia coli to yield soluble proteins for cell-free tandem enzymatic assays. Consequently, the heterologous expression with soluble NokA and NokB led to in vitro production of chromopyrrolic acid (CPA), thereby providing functional evidence for K-252c biosynthesis. Moreover, a facile production of CPA in culture broth was successfully accomplished by using an in vivo biotransformation of L-tryptophan with E. coli harboring the gene cassette. Importantly, by sequence analysis and the functional characterization here and in the companion paper, biosynthetic pathways leading to formation of K-252a and its analogs were hence proposed. Together, the results provide critical information and materials useful for combinatorial biosynthesis of K-252a and its analogs for therapeutic applications.
Molecular insight into catalytic mechanism of spinosyn rhamnosyltransferase by in vitro combinatorial reactions with NDP-sugar-modifying enzymes
(2009-08-16)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
Lin, Yu-Chin
;
Chen, Yi-Hsine
;
生物科技學系
Pulsed Electromagnetic Fields Accelerate Proliferation and Osteogenic Gene Expression in Human Bone Marrow Mesenchymal Stem Cells During Osteogenic Differentiation
(2010-04-01)
Sun, Li-Yi
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Hsieh, Dean-Kuo
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Lin, Po-Cheng
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Chiu, Hsien-Tai
;
Chiou, Tzyy-Wen
Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMISCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi-quantitative RT-PCR indicated that PEMFs significantly altered temporal expression of osteogenesis-related genes, including a 2.7-fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in B M MSC-mediated osteogenesis, and accelerate the osteogenesis. Bioelectromatmetics 31:209-219, 2010. (C) 2009 Wiley-Liss, Inc.
R3D-BLAST: a search tool for similar RNA 3D substructures
(2011-07-01)
Liu, Yun-Chen
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Yang, Chung-Han
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Chen, Kun-Tze
;
Wang, Jyun-Rong
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Cheng, Mei-Ling
R3D-BLAST is a BLAST-like search tool that allows the user to quickly and accurately search against the PDB for RNA structures sharing similar substructures with a specified query RNA structure. The basic idea behind R3D-BLAST is that all the RNA 3D structures deposited in the PDB are first encoded as 1D structural sequences using a structural alphabet of 23 distinct nucleotide conformations, and BLAST is then applied to these 1D structural sequences to search for those RNA substructures whose 1D structural sequences are similar to that of the query RNA substructure. R3D-BLAST takes as input an RNA 3D structure in the PDB format and outputs all substructures of the hits similar to that of the query with a graphical display to show their structural superposition. In addition, each RNA substructure hit found by R3D-BLAST has an associated E-value to measure its statistical significance. R3D-BLAST is now available online at http://genome.cs.nthu.edu.tw/R3D-BLAST/ for public access.
Reconstructing genome trees of prokaryotes using overlapping genes
(2010-02-24)
Cheng, Chih-Hsien
;
Yang, Chung-Han
;
Chiu, Hsien-Tai
;
Lu, Chin Lung
;
生物科技學系
Background: Overlapping genes (OGs) are defined as adjacent genes whose coding sequences overlap partially or entirely. In fact, they are ubiquitous in microbial genomes and more conserved between species than nonoverlapping genes. Based on this property, we have previously implemented a web server, named OGtree, that allows the user to reconstruct genome trees of some prokaryotes according to their pairwise OG distances. By analogy to the analyses of gene content and gene order, the OG distance between two genomes we defined was based on a measure of combining OG content (i.e., the normalized number of shared orthologous OG pairs) and OG order (i.e., the normalized OG breakpoint distance) in their whole genomes. A shortcoming of using the concept of breakpoints to define the OG distance is its inability to analyze the OG distance of multi-chromosomal genomes. In addition, the amount of overlapping coding sequences between some distantly related prokaryotic genomes may be limited so that it is hard to find enough OGs to properly evaluate their pairwise OG distances. Results: In this study, we therefore define a new OG order distance that is based on more biologically accurate rearrangements (e. g., reversals, transpositions and translocations) rather than breakpoints and that is applicable to both uni-chromosomal and multi-chromosomal genomes. In addition, we expand the term "gene" to include both its coding sequence and regulatory regions so that two adjacent genes whose coding sequences or regulatory regions overlap with each other are considered as a pair of overlapping genes. This is because overlapping of regulatory regions of distinct genes suggests that the regulation of expression for these genes should be more or less interrelated. Based on these modifications, we have reimplemented our OGtree as a new web server, named OGtree2, and have also evaluated its accuracy of genome tree reconstruction on a testing dataset consisting of 21 Proteobacteria genomes. Our experimental results have finally shown that our current OGtree2 indeed outperforms its previous version OGtree, as well as another similar server, called BPhyOG, significantly in the quality of genome tree reconstruction, because the phylogenetic tree obtained by OGtree2 is greatly congruent with the reference tree that coincides with the taxonomy accepted by biologists for these Proteobacteria. Conclusions: In this study, we have introduced a new web server OGtree2 at http://bioalgorithm.life.nctu.edu.tw/ OGtree2.0/ that can serve as a useful tool for reconstructing more precise and robust genome trees of prokaryotes according to their overlapping genes.
Reconstructing genome trees of prokaryotes using overlapping genes
(2010-02-24)
Cheng, Chih-Hsien
;
Yang, Chung-Han
;
Chiu, Hsien-Tai
;
Lu, Chin Lung
;
生物科技學系
Background: Overlapping genes (OGs) are defined as adjacent genes whose coding sequences overlap partially or entirely. In fact, they are ubiquitous in microbial genomes and more conserved between species than nonoverlapping genes. Based on this property, we have previously implemented a web server, named OGtree, that allows the user to reconstruct genome trees of some prokaryotes according to their pairwise OG distances. By analogy to the analyses of gene content and gene order, the OG distance between two genomes we defined was based on a measure of combining OG content (i.e., the normalized number of shared orthologous OG pairs) and OG order (i.e., the normalized OG breakpoint distance) in their whole genomes. A shortcoming of using the concept of breakpoints to define the OG distance is its inability to analyze the OG distance of multi-chromosomal genomes. In addition, the amount of overlapping coding sequences between some distantly related prokaryotic genomes may be limited so that it is hard to find enough OGs to properly evaluate their pairwise OG distances. Results: In this study, we therefore define a new OG order distance that is based on more biologically accurate rearrangements (e. g., reversals, transpositions and translocations) rather than breakpoints and that is applicable to both uni-chromosomal and multi-chromosomal genomes. In addition, we expand the term "gene" to include both its coding sequence and regulatory regions so that two adjacent genes whose coding sequences or regulatory regions overlap with each other are considered as a pair of overlapping genes. This is because overlapping of regulatory regions of distinct genes suggests that the regulation of expression for these genes should be more or less interrelated. Based on these modifications, we have reimplemented our OGtree as a new web server, named OGtree2, and have also evaluated its accuracy of genome tree reconstruction on a testing dataset consisting of 21 Proteobacteria genomes. Our experimental results have finally shown that our current OGtree2 indeed outperforms its previous version OGtree, as well as another similar server, called BPhyOG, significantly in the quality of genome tree reconstruction, because the phylogenetic tree obtained by OGtree2 is greatly congruent with the reference tree that coincides with the taxonomy accepted by biologists for these Proteobacteria. Conclusions: In this study, we have introduced a new web server OGtree2 at http://bioalgorithm.life.nctu.edu.tw/ OGtree2.0/ that can serve as a useful tool for reconstructing more precise and robust genome trees of prokaryotes according to their overlapping genes.
Tandem enzymatic syntheses of TDP-deoxysugars and spinosyn analogs by in vitro recombination of natural product biosynthetic pathways
(2007-03-25)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
Wu, Chih Chien
;
生物科技學系
;
Department of Biological Science and Technology
Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and connic enzymes
(2016-01-01)
Chiu, Hsien-Tai
;
Weng, Chien-Pao
;
Lin, Yu-Chin
;
Chen, Kuan-Hung
;
生物科技學系
Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-L-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis.
Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and connic enzymes
(2016)
Chiu, Hsien-Tai
;
Weng, Chien-Pao
;
Lin, Yu-Chin
;
Chen, Kuan-Hung
;
生物科技學系
Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-L-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis.
Trichodiene synthase: Synthesis and inhibition kinetics of 12-fluoro-farnesylphosphonophosphate for sesquiterpene cyclases
(2006-10-01)
Chen, Yi-Lin
;
Chiu, Hsien-Tai
;
生物科技學系
;
Department of Biological Science and Technology
trans, trans-Farnesyl diphosphate (FPP) serves as a universal substrate for a large family of sesquiterpene cyclases that are responsible for biosynthesis of more than 300 structurally diverse sesquiterpenes in nature. A new FPP substrate analogue, 12-fluoro-farnesylphosphonophosphate (12-F-F-CH2PP), was synthesized in this paper for applications on kinetic and mechanistic studies of the enzyme family. Trichodiene synthase (TS), a sesquiterpene cyclase, catalyzes the conversion of trans,transfarnesyl diphosphate (FPP) to trichodiene. 12-F-F-CH2PP was tested as a potential inhibitor of TS. Inactivation and inhibition kinetic experiments showed that 12-F-F-CH2PP was not a mechanism-based inactivator for TS; instead, a mixed-type reversible inhibition was observed with inhibition constants K-i1 = 2.33 +/- 0.50 mu M and K-i2 25.80 +/- 7.70 mu M, values close to those previously determined for farnesylphosphonophosphate, K-i1 3.25 mu M and K-i2 = 9-10 mu M Although 12-F-F-CH2PP did not irreversibly inactivate TS, this new analogue serves as a potential active-site directed inactivator and mechanistic probe of other sesquiterpene cyclases and FPP-utilizing enzymes, which utilize FPP as a common acyclic substrate.