R-3 Repository :: Browsing by Author "Matsuda, Seiichi P. T."

Browsing by Author "Matsuda, Seiichi P. T."

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A Journey through the Arabidopsis thaliana Genome: Discovering the Origins of Novel Triterpene Metabolites
(2014-08-27) Castillo-Rivera, Dorianne A; Matsuda, Seiichi P. T.; Hartgerink, Jeffrey D.; Tapia, Richard A.
Plants produce a large variety of natural products, including over 20,000 different triterpenoids. Triterpenoid functions range from roles as membrane sterols and hormones in primary metabolism, to defense compounds in secondary metabolism. Beyond these roles in the plant, triterpenoids have substantial human value as flavors, fragrances, and medicines. This thesis explores the enzymatic formation of triterpenoids through cationic cyclization of a linear precursor oxidosqualene and further metabolism by radical oxidation. Thirteen different oxidosqualene cyclases (OSCs) are encoded by the model plant Arabidopsis thaliana and collectively produce a plethora of triterpene skeletons. This structural diversity of triterpenes was investigated through a comprehensive analysis of the A. thaliana PEN6 product profile. This product profile contained 33 compounds that were found by combining genome mining, heterologous expression in yeast and HSQC analysis. Some of these compounds were novel to Arabidopsis: isoursenol, (13R,14Z,17E)-malabarica-14,17,21-trien-3β-ol, nematocyphol, (20R,S) dammarenediols, Δ8(26)-seco-β-amyrin, and 9αH-Δ8(26)-polypodatetraenol. After the cyclization, triterpenes can be modified by a number of enzymes, including cytochrome P450 monooxygenases (CYP450s). Candidate CYP450s for a given triterpene were identified by gene cluster analysis combined with microarray databases. Heterologous expression of the OSC, ATR2 and CYP450 together with GC-MS and NMR techniques allowed the elucidation of metabolic pathways and structures of a variety of oxygenated triterpenes. These experimental techniques led to the identification of new oxidized metabolites produced by the co-expression of Arabidopsis clusters: THAS1 with CYP708A2 and CYP705A5, PEN1 with CYP705A2 and MRN1 with CYP71A16. The thalianol cluster gave rise to several unexpected side chain and ring oxidized metabolites. This thesis also describes the side chain cleavage of arabidiol by CYP705A1 to give a C19 methyl ketone, and the hydroxylation of an allylic methyl of marneral/marnerol to 23-hydroxymarneral/23-hydroxymarnerol by CYP71A16. This work sheds light on the metabolic fate of some Arabidopsis triterpenes. When applied more generally, this strategy may begin to fill a large knowledge gap in metabolomics and functional genomics.
A Tale of Two Clades: Unveiling Relationships among Arabidopsis and Lactuca through Genome Mining in Triterpene Biosynthesis
(2016-04-28) Moreno Garcia, Melisa; Matsuda, Seiichi P. T.
The metabolic diversity of natural products in higher plants appears to be driven out of necessity to adapt and survive in different ecological environments. Among the vast amount of natural products found in nature, triterpenes are widely distributed among higher plants. Triterpenes serve as precursors of essential membrane sterols in primary metabolism, and as compounds involved in defense in secondary metabolism. These complex chemical structures can be leads in drug discovery. Oxidosqualene cyclases (OSCs) are enzymes that generate the ring systems of triterpenoids through cyclization of (3S)-oxidosqualene. With the arrival of the genomic era, genome mining has become a reliable strategy to give a comprehensive accounting of triterpenoids in organisms whose genome is available. Arabidopsis thaliana, a model plant organism has provided, through genome mining, the first complete picture of triterpene skeletons characteristic of plants. Now, the availability of different genomes opens up the possibility of exploring the metabolic capabilities of other plant clades. This thesis uses genome mining and heterologous expression in the yeast Saccharomyces cerevisiae to explore the biosynthetic diversity of oxidosqualene cyclases in two small clades of Brasicaceae and Asteraceae, Arabidopsis and Lactuca. OSCs have been widely studied in Arabidopsis thaliana but barely been identified in the Arabidopsis lyrata genome. Phylogenetic analysis comparing both organisms was used to discover the presence of nine putative OSCs. Three A. lyrata OSCs with interesting differences from their A. thaliana orthologs were expressed in yeast. The product profile of each enzyme was characterized by analytical techniques such as NMR and GC-MS. AlyPEN4 shows a similar product profile to its ortholog in A.thaliana, making as its major product thalianol. In the process of analysis a novel triterpene, 14(S)-25-nor-9β-methylpodioda-5(10),17,21-trienol, was discovered. Expression of AlyPEN6 illustrates the first example in secondary metabolism of orthologous genes with different function; the ratio between the two major products seco-β-amyrin and seco-α-amyrin was significantly different. Finally AlyPEN9, an OSC characteristic of A. lyrata turn out to be a β-amyrin synthase. Analysis of the genome shows that AlyPEN9 seems to be the only product specific β-amyrin of this organism. Lactuca sativa, the first organism of the Asteraceae family to have its genome available, is a promising model system for the study of triterpenes synthesis. Here, I present the progress toward the characterization of the product profile of the Lactuca genus in order to understand the evolutionary relationships among different plant clades. Manual splicing of its genome uncovered 17 putative OSC. An improved product profile of six previously characterized oxidosqualene cyclases from Lactuca is reported. Also, a novel cyclase was discovered; it makes nematocyphol as its major product and 39 other compounds, including lupane type rearranged products. One of these compounds, lup-19(21)-en-3β-ol was found for the first time as part of the product profile of an OSC. This work shows that, although Arabidopsis and Lactuca have a unique set of oxidosqualene cyclases, most of the triterpenes that have been characterized within the Brasicaceae clade are present in the Asteraceae clade. The ability to synthesize unique sets of secondary metabolites may be related to the mechanism of adaptation and defense of different species.
An Assessment of Gadonanotubes as Magnetic Nanolabels for Improved Stem Cell Detection and Retention in Cardiomyoplasty
(2013-07-24) Tran, Lesa; Wilson, Lon J.; Matsuda, Seiichi P. T.; Jacot, Jeffrey G.; Perin, Emerson C.; Cabreira, Maria da Graça
In this work, gadolinium-based carbon nanocapsules are developed as a novel nanotechnology that addresses the shortcomings of current diagnostic and therapeutic methods of stem cell-based cardiomyoplasty. With cardiovascular disease (CVD) responsible for approximately 30% of deaths worldwide, the growing need for improved cardiomyoplasty has spurred efforts in nanomedicine to develop innovative techniques to enhance the therapeutic retention and diagnostic tracking of transplanted cells. Having previously been demonstrated as a high-performance T1-weighted magnetic resonance imaging (MRI) contrast agent, Gadonanotubes (GNTs) are shown for the first time to intracellularly label pig bone marrow-derived mesenchymal stem cells (MSCs). Without the use of a transfection agent, micromolar concentrations of GNTs deliver up to 10^9 Gd(III) ions per cell, allowing for MSCs to be visualized in a 1.5 T clinical MRI scanner. The cellular response to the intracellular incorporation of GNTs is also assessed, revealing that GNTs do not compromise the viability, differentiation potential, or phenotype characteristics of the MSCs. However, it is also found that GNT-labeled MSCs exhibit a decreased response to select cell adhesion proteins and experience a non-apoptotic, non-proliferative cell cycle arrest, from which the cells recover 48 h after GNT internalization. In tandem with developing GNTs as a new stem cell diagnostic agent, this current work also explores for the first time the therapeutic application of the magnetically-active GNTs as a magnetic facilitator to increase the retention of transplanted stem cells during cardiomyoplasty. In vitro flow chamber assays, ex vivo perfusion experiments, and in vivo porcine injection procedures all demonstrate the increased magnetic-assisted retention of GNT-labeled MSCs in the presence of an external magnetic field. These studies prove that GNTs are a powerful ‘theranostic’ agent that provides a novel platform to simultaneously monitor and improve the therapeutic nature of stem cells for the treatment of CVD. It is expected that this new nanotechnology will further catalyze the development of cellular cardiomyoplasty and other stem cell-based therapies for the prevention, detection, and treatment of human diseases.
Characterization of Oxidosqualene Cyclases in Brassicaceae: the ABCs (Arabidopsis, Brassica and Capsella)
(2019-10-23) Jin, Jing; Matsuda, Seiichi P. T.
One major class of plant secondary metabolites is the triterpenes, whose biosynthesis from oxidosqualene is catalyzed by oxidosqualene cyclases (OSCs). This thesis describes the characterization of OSCs from plants within the Brassicaceae family, which include the model species Arabidopsis thaliana and Capsella rubella as well as the crop species Brassica rapa and Brassica oleracea. Detailed product profiles of those cyclases are combined with phylogenetic analysis, synteny analysis, active-site prediction, plant tissue extraction and transcriptome analysis to explore enzyme mechanisms and evolutionary relationships within the OSC family. First, detailed product profiles are constructed for four Arabidopsis thaliana OSCs. Those cyclases exemplify the range of product specificity among OSCs, ranging from the 94% specific At PEN4 to the multi-functional cyclase At LUP5 which makes two major products at similar levels. Despite their varying product specificity, all those OSCs form many minor products. Next, the characterization of three OSCs from Brassica species is detailed. Bra032185 was determined to be an astertarone A synthase, which is the first reported OSC with 6/6/6/5 20R stereospecificity. Bra039929 is the first reported euphol synthase and is another 6/6/6/5 20R specific cyclase. In addition, Bol021540 was amplified from broccoli seedling RNAs and it was determined to be a mixed-amyrin synthase. Those results were combined with Brassica plant extraction results to explore the triterpene biosynthesis in this species. Lastly, the complete characterization of all Capsella rubella OSCs is described. The triterpene biosynthetic capability of this plant includes one cycloartenol synthase, five functional OSCs in the secondary metabolism and one pseudogene. The only secondary metabolic OSC conserved between A. thaliana and C. rubella is camelliol C synthase (LUP3). In addition, three C. rubella cyclases show similarity to their A. thaliana counterparts but different products are made. Overall, the comparison of OSCs across species illustrates the fast divergence of plant secondary metabolism and explores OSC evolution among the Brassicaceae family. The OSC family showcases rapid enzyme functional evolution as evident by neofunctionalization and convergent evolution, and thus it can be a valuable model for enzyme evolution studies.
Chemical, biochemical, and bioanalytical studies of sterols and isoprenoids: Smith-Lemli-Opitz syndrome, Langer-Giedion syndrome, activation of meiosis, nuclear orphan receptor LXRalpha and cytochrome P450s
(2000) Ruan, Benfang; Schroepfer, George J., Jr.; Matsuda, Seiichi P. T.
Sterols and isoprenoids are vital cellular constituents. In studies of their formation, metabolism, and biological effects, the separation and identification of individual isomers present formidable challenges. Approximately 30 unsaturated C27 sterols were prepared and purified as authentic standards to evaluate and develop chromatographic and spectral methods. Novel chromatographic conditions were devised for silver ion HPLC (Ag+-HPLC), a technique that provided unprecedented separations of these closely related sterols. Ag+-HPLC proved very powerful in addressing several important problems in medicine and biology relating to the biochemical effects of sterols and isoprenoids; described herein. Smith-Lemli-Opitz syndrome (SLOS) is a genetic disorder of development associated with the accumulation of unsaturated C27, sterols. My analysis of SLOS and normal blood samples unequivocally demonstrated that only the Delta5,7, Delta5,8, and Delta5,7,9(11) sterols accumulate significantly in SLOS patients. The 19-nor-Delta5,7,9 sterol, which has been reported to accumulate in SLOS blood by several research groups, was not detected in my analyses and was demonstrated to be a GC artifact arising from the thermal decomposition of cholesta-5,8-dien-3beta-ol. Alternative pathways in the late stages of cholesterol biosynthesis relating to the biological origin and metabolism of the beta5,8, beta6,8, and beta6,8(14) sterols were elucidated by incubation of tritium-labeled substrates in rat liver preparations. Also, a simple and rapid colorimetric method was developed for the clinical screening of SLOS. Ag +-HPLC played a critical role in studying a possible biochemical defect in another genetic disorder, Langer-Giedion syndrome, and in isolating isomers of (20R,22R)-dihydroxycholesterol), all-trans geranylgeranoic acid, other isoprenoids, and 4,4-dimethyl-sterols, compounds obtained from novel and efficient chemical syntheses. (20R,22R)-Dihydroxycholesterol proved to be a moderately potent activator of the nuclear orphan receptor LXRalpha, whereas all trans geranylgeranoic acid was found to be an inhibitor. The synthetic 4,4-dimethyl sterols caused a resumption of meiosis in mouse oocytes. Famesoic acid was demonstrated to be an extraordinarily potent substrate inducer of cytochrome P450BM-3.
Cloning and characterization of triterpene synthases
(2000) Herrera, Jennifer Bridgette Reyna; Matsuda, Seiichi P. T.
Oxidosqualene cyclases are a group of enzymes that cyclize 2,3-oxidosqualene to any of over 80 triterpene skeletons. Sequence correlation suggests these enzymes share a common overall structure and subtle changes in active site residues are responsible for differences in the structure of the cyclization product. Further characterization of conserved residues has lead to a better understanding of the mechanisms through which regio- and stereo-selective cyclization occurs. This work has resulted in the cloning of three new oxidosqualene cyclases. By cloning triterpene synthases from far-diverged organisms, new sequence data has been added for correlation and has provided new information regarding evolution of the enzymes from a common ancestor. Arabidopsis thaliana lupeol synthase was cloned and the full-length cDNA expressed in yeast. Degenerate PCR primers designed from regions of highly conserved sequence were used to clone cycloartenol synthase and a presently uncharacterized triterpene synthase from an Abies magnifica library. Newly obtained and previously published sequence data has been used to identify residues that may correlate with catalytic activity. Site-directed mutagenesis has been performed on Arabidopsis thaliana cycloartenol synthase resulting in enzymes with different product distributions. In addition to producing cycloartenol, these mutant proteins were shown to produce a range of new triterpene alcohols, including monocyclic alcohols and a previously unknown triterpene skeleton. Sequence information as well as the mechanistic information learned from the analysis of mutant protein products has come together to reveal a complex relationship between structure and function of triterpene synthases. Using techniques of molecular biology as well as analytical and organic chemistry, aspects of the nature of oxidosqualene cyclases in terms of the mechanisms of transformation, evolution and physiological significance are presented.
Cloning of terpene synthases from plants
(2000) Hua, Ling; Matsuda, Seiichi P. T.
As part of a program to clone Artemisia annua sesquiterpene biosynthetic genes, a cDNA encoding epi-cedrol synthase was isolated from an A. annua leaf cDNA library by a homology-based strategy. The cDNA was functionally expressed in Escherichia coli and the encoded protein shown to produce (-) epicedrol and cedrol in a 96:4 ratio. The structure of epicedrol was determined by GC-MS and NMR and cedrol was identified by GC-MS and GC co-elution with the authentic sample. Neither cedrol nor epicedrol was detected in an extract of the plant from which the cDNA library was made. In addition, a sesquiterpene synthase like gene was cloned from A. thaliana ecotype Columbia by RT-PCR and the cDNA was expressed in E. coli. The second part of this thesis, describes the study of triterpene biosynthesis. A yeast strain with squalene synthase (erg9) and lanosterol synthase (erg7) double mutation was made by homologous recombination. This strain has been used by other group members to express A. thaliana cycloartenol synthase mutants and S. cerevisiae lanosterol synthase mutants. More accurate enzyme product ratios were obtained for the mutants expressed in this erg9 and erg7 double mutant yeast strain compared to the erg7 single mutant SMY8. In order to study the mechanism of oxidosqualene cyclases, A. thaliana was chosen as the model plant for cloning oxidosqualene cyclases. A triterpene synthase was isolated from the A. thaliana ecotype Landsberg young seedling cDNA library and the cloned gene had the second exon missing compared to cycloartenol synthase and lupeol synthase cloned from same organism. The cloned gene was expressed in yeast and no oxidosqualene cyclase activity was detected. An oxidosqualene cyclase like open reading frame ORF3, which is adjacent to lupeol synthase on the genomic DNA was cloned from A. thaliana ecotype Landsberg cDNA library. An open reading frame ORF1 found in the A. thaliana sequencing database, that has 71.4% identity to lupeol synthase was cloned from A. thaliana ecotype Columbia total RNA by RT-PCR. The cDNA of these two ORFs were expressed in yeast and no oxidosqualene cyclase activity was detected.
Cloning, characterization, and molecular manipulation of plant terpene synthases
(2002) Schepmann, Hala Gobran; Matsuda, Seiichi P. T.
Ginkgo biloba produces the ginkgolides, a structurally novel class of diterpenes. Part I of this thesis describes the cloning and functional characterization of levopimaradiene synthase, which catalyzes the initial cyclization step in ginkgolide biosynthesis. A G. biloba cDNA library was prepared from seedling roots and a probe was amplified using primers corresponding to conserved gymnosperm terpene synthase sequences. Colony hybridization and rapid amplification of cDNA ends yielded a full-length clone encoding a predicted protein (873 amino acids, 100,289 Da) similar to known gymnosperm diterpene synthases. The sequence includes a putative N-terminal plastid transit peptide and three aspartate-rich regions. The full-length protein expressed in Escherichia coli cyclized geranylgeranyl pyrophosphate to levopimaradiene, which was identical to a synthetic standard by GC/MS analysis. Removing 60 or 79 N-terminal residues increased levopimaradiene production, but a 128-residue N-terminal deletion lacked detectable activity. This is the first cloned ginkgolide biosynthetic gene and the first in vitro observation of an isolated ginkgolide biosynthetic enzyme. Additionally, production of abietatriene, the immediate hydrocarbon precursor of the ginkgolides, was achieved. Expression of G. biloba levopimaradiene synthase in Saccharomyces cerevisiae , metabolically engineered for geranylgeranyl pyrophosphate over-production, yielded levopimaradiene, abietadiene, abietatriene, and (+)-copalol. Part II of this thesis describes molecular manipulation of Arabidopsis thaliana cycloartenol synthase. Residues critical to enzymatic activity were identified, altered, and functionally characterized. Furthermore, cycloartenol synthase double point mutants were studied to determine the effects on catalytic function.
Directed evolution and structure-function studies of oxidosqualene cyclases
(2003) Segura, Michael Joseph Robert; Matsuda, Seiichi P. T.
Although oxidosqualene cyclase reaction mechanisms have been established, how these enzymes promote specific transformations, generate product diversity, and achieve product specificity is only partially understood. Protein mutagenesis experiments described herein have identified catalytically important residues involved in deprotonation and cyclization steps of oxidosqualene cyclase catalysis. Residues influencing specific deprotonation in lanosterol and cycloartenol synthase were identified. The Saccharomyces lanosterol synthase Thr384Tyr mutation compromised product specificity and caused the formation of the novel lanosta-24-ene-3beta,9alpha-diol, a compound previously unidentified in nature. Furthermore, the Arabidopsis thaliana cycloartenol synthase His477 position was identified by random mutagenesis. Interestingly, subtle amino acid changes at this position show dramatic and different influences on product structure; the His477Asn mutant makes predominantly lanosterol, whereas the His477Gln mutant makes mostly parkeol. The AthCAS1 His477Asn and His477Gln mutants are currently the most accurate lanosterol and parkeol synthases, respectively, generated by protein mutagenesis. AthCAS1 His477 mutations were combined with other catalytically important mutations (AthCAS1 Tyr410Thr Ile481Val) to see how the combined mutations would interact to influence product structure. Surprisingly, the His477 mutations did not influence catalysis when combined with the other mutations. The catalytic behavior of these AthCAS1 triple mutants is the first demonstration of dominant and recessive properties of catalytically important oxidosqualene cyclase mutations. DNA shuffling of cycloartenol and lupeol synthase was undertaken to determine what residues or motifs control substrate folding, a reaction step that has important consequences for product structure. Hybrid enzymes isolated after one round of DNA shuffling revealed that the N- and C-terminal ends (115 and 140 a.a., respectively) of cycloartenol synthase do not contain residues that are required for protosteryl cation formation or cyclopropyl ring formation. Those hybrids possessing lupeol synthase sequence at the termini but retaining internal cycloartenol synthase sequence maintained cycloartenol biosynthetic ability, demonstrating that catalytic components required for cycloartenol biosynthesis are located in the internal portions of the enzyme sequence. Site-directed mutagenesis and chimeragenesis experiments further defined catalytically important regions indicated by DNA shuffling experiments. In addition, this thesis describes the complete characterization of the Arabidopsis lupeol synthase and the construction of a novel yeast strain that possesses a plant sterol biosynthetic pathway.
Diterpene-producing unicellular organism
(2007-07-03) Matsuda, Seiichi P. T.; Hart, Elizabeth A.; Rice University; United States Patent and Trademark Office
The present invention is directed to a unicellular organism system, such as a yeast, for producing geranylgeranyl pyrophosphate and a diterpene in vivo. The yeast cell preferably comprises an inducible nucleic acid sequence encoding geranylgeranyl pyrophosphate synthase, an inducible nucleic acid sequence encoding a soluble form of HMG-CoA reductase, a nucleic acid sequence of an allele that confers an increase in sterol metabolic flux and, in the diterpene-producing cell, a diterpene synthase.
Elucidating the mechanism of triterpene cyclization using DNA synthesis and phylogenetic approaches
(2005) LeClair, Renee; Matsuda, Seiichi P. T.
Nearly every aspect of sterol biosynthesis has been studied or exploited as means of regulating pathway flux, inhibiting specific enzymatic reactions, elucidating product cyclization or determining the evolutionary nature of the pathway itself. Despite enormous efforts in this area pathway regulation, product formation and the evolutionary origins of sterol biosynthesis remain unknown. Described herein is the use of molecular, synthetic and phylogenetic techniques to examine how oxidosqualene cyclases (OSC) (a specific enzyme within the sterol biosynthetic pathway) have evolved over time and how this class of enzymes controls the cyclization of oxidosqualene to a variety of triterpenes. OSCs are a unique family of enzymes producing over 100 naturally occurring cyclization products ranging from mono to hexacyclic compounds. These enzymes display high sequence similarity and it is predicted that OSCs share a similar three-dimensional structure, while subtle residue changes in the active site alter catalysis. There are two subclasses of OSCs, protosteryl and dammarenyl, and they are defined by the tetracyclic intermediate cation formed during cyclization. Residues that are differentially conserved between protosteryl and dammarenyl type cyclases were identified as candidates imparting a specific catalytic function. Using DNA synthesis, libraries of chimeric enzymes were generated that blended the sequences of one protosteryl-type cyclase (cycloartenol synthase from Arabidopsis thaliana) and a dammarenyl-type cyclase (lupeol synthase from Olea europaea) and screened for the ability to produce protosteryl-type products. Using this approach we were able to narrow the number of residues imparting specific catalytic function from 759 to 15 candidate residues within the Arabidopsis thaliana cycloartenol synthase. This information prompted direct mutagenesis of several single residues to better our understanding of the active site environment. To complement the DNA synthesis experiments, the second half of this thesis describes the cloning and characterization of the Methylococcus capsulatus lanosterol synthase, the Gemmata obscuriglobus parkeol synthase and several other bacterial/ancient cyclases. Sterols, once thought to be only eukaryotic in nature, are now being isolated from many prokaryotic organisms. Whether OSCs evolved in prokaryotes or were acquired later via horizontal gene transfer remains unknown. By studying both the residue conservation patterns and the product profiles of these bacterial cyclases, we have been able to formulate a working hypothesis that supports the evolution of sterols in prokaryotes. These primitive cyclases also retain residue conservation patterns that vary from eukaryotic enzymes indicating they direct product formation differently than more modern cyclases.
Ginkgo biloba levopimaradiene synthase
(2008-05-20) Matsuda, Seiichi P. T.; Schepmann, Hala G.; Rice University; United States Patent and Trademark Office
The present invention is directed to nucleic acid sequences of Ginkgo biloba diterpene synthases, particularly of a levopimaradiene synthase. More specifically, the invention is directed to a cell of a unicellular organism, such as Saccharomyces cerevisiae or Escherichia coli, comprising levopimaradiene synthase for the metabolically engineered in vivo biosynthesis of a diterpene and a ginkgolide.
Ginkgo biloba levopimaradiene synthase
(2005-09-20) Matsuda, Seiichi P. T.; Schepmann, Hala G.; Rice University; United States Patent and Trademark Office
The present invention is directed to nucleic acid sequences of Ginkgo biloba diterpene synthases, particularly of a levopimaradiene synthase. More specifically, the invention is directed to a cell of a unicellular organism, such as Saccharomyces cerevisiae or Escherichia coli, comprising levopimaradiene synthase for the metabolically engineered in vivo biosynthesis of a diterpene and a ginkgolide.
Incorporation of Conceptual Understanding of Chemistry in Assessments, Undergraduate General Chemistry Classrooms and Laboratories, and High School Classrooms
(2012-11-02) Cloonan, Carrie; Hutchinson, John S.; Matsuda, Seiichi P. T.; Lopez Turley, Ruth N.
The novel assessment models and studies developed in this work provided new insight on effective teaching practices in chemistry classrooms and laboratories through the framework of constructivism. Each project aimed to promote greater levels of understanding and inspire interest in chemistry, both of which are great challenges within the U.S. educational system. Assessment drives learning, so appropriate tests are essential to good courses. However, large classes often make written exams impractical. A multiple-choice test of conceptual knowledge in general chemistry was created and validated to provide the chemical education community with a reliable and functional tool that correlates with open-ended General Chemistry exams. Large classes make active-learning implementation challenging, as not all students can participate. Students in a large General Chemistry course taught via active-learning were studied through surveys and interviews. The data revealed that “silent” students are engaged in the active-learning experience, yet “vocal” students outperform silent students on measures of conceptual understanding in chemistry. The motivation behind being vocal suggested students participate in order to improve their grade, and while doing so, also see the benefit to their learning. Another mixed-methods study focused on the traditionally formatted General Chemistry Laboratories. Initial data on student expectations lead to the creation of a pilot lab section and ultimately a new format of the labs with the inclusion of a discussion session. The changes resulted in the students being better prepared, focusing on the content rather than the process of the labs, and reporting better understanding of chemistry due to labs. Two novel laboratory experiences were also developed to promote conceptual understanding, and their creation and use are outlined. The impact of a professional development program on high school chemistry courses was analyzed via interviews, teacher observations and a case study. The professional development exposed teachers to novel chemistry teaching practices of inquiry-based concept development and active-learning methods. The case study showed implementation of the instructional strategies to be successful within an existing exemplary chemistry classroom. Each of these projects advanced best practices in teaching chemistry by expanding the current understanding of teaching concepts and analyzing applications of research-based pedagogies.
Metabolic engineering approaches to biosynthesize terpenoids in Saccharomyces cerevisiae
(2009) McNeil, Caroline V.; Matsuda, Seiichi P. T.
Terpenoids are the largest class of natural products and are typically isolated from natural sources. However, heterologous expression of terpene synthases in microbial hosts such as E. coli or Saccharomyces cerevisiae has become an attractive alternative. S. cerevisiae has an intact sterol biosynthetic pathway, and many of the intermediates also serve as precursors for terpene synthases. Metabolic engineering efforts focus on optimizing product yields through increasing carbon flux through the desired pathway, removing competing enzymes, or altering enzymatic activity. This work describes the metabolic engineering of S. cerevisiae to enhance terpene production by exploiting these three approaches. Diterpene synthases were expressed in a yeast strain previously reported to accumulate the diterpene precursor geranylgeranyl pyrophosphate (GGPP). The strains produced milligram amounts of GGPP hydrolysis products geranylgeraniol and geranyllinalool, as well as the GGPP cyclization products ent -copalyl pyrophosphate, ent-kaurene, and abietadiene. Because diterpene production is limited by transit peptides targeting diterpene synthases into plastids, protein expression was increased by co-expressing a chloroplast processing enzyme in two different diterpene-producing strains. The in vivo-generated mature diterpene synthases functioned more effectively, thereby increasing cyclization yield. This thesis also describes a new method for controlling farnesyl pyrophosphate (FPP) hydrolysis product profile by adjusting media pH. Hydrolysis was found to be partially controlled by a phosphatase DPP1, however a majority of FPP hydrolysis is non-enzymatic. In a squalene synthase deletion strain, FPP accumulates and hydrolyzes readily to farnesol and nerolidol, and the ratios of these products are determined by the pH of the media. Finally, a yeast strain was constructed to increase production of the 30-carbon triterpene precursors oxidosqualene (OS) and dioxidosqualene (DOS) by over-expressing the sterol biosynthesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG1) in a lanosterol synthase deletion background. This strain accumulated twenty times more OS and DOS than the strain with only the native HMG1. Over-expression of squalene epoxidase (ERG1) in a lanosterol synthase background greatly enhanced the levels of DOS compared to OS.
Metabolic engineering of Saccharomyces cerevisiae towards increased production of terpenes and characterization of sterol biosynthetic enzymes
(2001) Hart, Elizabeth A.; Matsuda, Seiichi P. T.
The yeast Saccharomyces cerevisiae possesses the endogenous precursor for diterpene production but does not biosynthesize diterpenes. Part I of this thesis describes metabolic engineering of S. cerevisiae to achieve diterpene biosynthesis at 500-fold increased production levels (5 mg/L) relative to levels observed in wild type yeast. Induced expression of S. cerevisiae genes geranylgeranyl pyrophosphate synthase (BTS1) and a truncated form of 3-hydroxy-3-methylglutaryl CoA reductase (HMG1) in yeast carrying the upc2-1 allele afforded accumulating geranylgeranyl pyrophosphate, the universal precursor for diterpene biosynthesis. The precursor demonstrated efficient cyclization to 7,13-abietadiene upon coexpression of transformed Abies grandis abietadiene synthase in a multiple copy yeast shuttle vector. Similarly, metabolic engineering of S. cerevisiae allowed investigation of achieving attenuated sesquiterpene production in vivo; those results and possible physiological implications to yeast are discussed. The recombinant strains serve as an alternative means of access to natural products via a novel in vivo production system. Part II describes the characterization of three sterol biosynthetic enzymes. Higher plants catalyze the cyclization of (S)-2,3-oxidosqualene to cycloartenol. A single point mutation demonstrated altered sterics contributing to catalytic product specificity; two point mutations are characterized and evaluated. Cycloartenol constitutes a cyclopropyl sterol structure not found in other eukaryotes. In an effort to better understand the roles served by the cyclopropyl sterols, the characterization of the gene responsible for their metabolism is described. Understanding this point of evolutionary divergence can facilitate accurate phylogenetic analysis among eukaryotes.
Model-based analysis of tissue reflectance and autofluorescence for improved cancer detection
(2009) Weber, Crystal Elaine; Matsuda, Seiichi P. T.
Improving early detection of cancer represents the most promising opportunity to reduce the mortality and morbidity of cancer. This thesis presents model-based analysis of tissue reflectance and autofluorescence which is used to improve our understanding of the biological basis of tissue spectra in the uterine cervix and oral cavity. An analytical model to extract biologically and diagnostically relevant parameters from clinical measurements of cervical tissue is developed, validated, and implemented. In addition, Monte Carlo based models are used to investigate the effect of dysplastic progression and benign inflammation on oral cavity spectra. Results show depth-sensitive spectroscopy may be able to improve cancer detection and be used as an adjunct to wide field imaging where inflammation causes decreased specificity.
Molecular biological approaches to biomedical problems in sterol and terpene biosynthesis
(2003) Xu, Ran; Matsuda, Seiichi P. T.
Molecular biological techniques represent powerful tools with applications to a wide variety of biomedical problems. Described herein is the use of metabolic engineering to manipulate sterol biosynthesis for medicinal purposes. Also presented is the cloning of terpene biosynthetic genes in plants of agricultural and pharmacological importance. Two examples of metabolic engineering of the Saccharomyces cerevisiae sterol biosynthetic pathway are illustrated in Part I. We first demonstrated the development of genetically engineered S. cerevisiae strains that efficiently produce meiosis activating sterols (MAS), which are difficult to obtain by chemical synthesis or isolation from natural sources. Homologous recombination was used to construct an erg24Deltaerg25DeltahemlDelta mutant, which accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25DeltahemlDelta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). This in vivo MAS production needs no added substrate, is technically simpler than chemical synthesis, and provides an inexhaustible source of MAS in relatively high purity and yield. In a different approach to metabolic engineering, we generated recombinant S. cerevisiae strains to screen drugs against the pathogenic parasite Trypanosoma cruzi. The native sterol Delta8-Delta7 isomerase in S. cerevisiae was replaced by the heterologous T. cruzi sterol isomerase and the corresponding human enzyme respectively. The relative effectiveness of the tested compounds on these two strains was compared using simple plate assay and validated by chromatographic methods. This differential screening method identifies inhibitors that specifically target the T. cruzi sterol isomerase with minimal side-effect on the parallel human enzyme and avoids the direct handling of deadly T. cruzi cells. Part II of this thesis describes the characterizations of two plant terpene biosynthetic genes. The saponins in model legume Medicago truncatula may negatively affect the forage digestibility of ruminants, and the aglycones of these saponins are most likely derived from triterpene beta-amyrin. A putative beta-amyrin synthase clone uncovered from M. truncatula Expressed Sequence Tag libraries was expressed in S. cerevisiae , and the recombinant enzyme cyclized 2,3-oxidosqualene cleanly into beta-amyrin. Also, a monoterpene synthase gene from Artemisia annua L., a medicinal plant with anti-malaria activity, was expressed in Escherichia coli, and the in vitro enzymatic reaction with geranyl pyrophosphate afforded (-)-beta-pinene and (-)-alpha-pinene (94:6) as the cyclization products.
Mutagenesis of cycloartenol synthase and lanosterol synthase: Broadening and narrowing product profile
(2006) Lodeiro, Silvia; Matsuda, Seiichi P. T.
This thesis describes mutagenesis experiments in cycloartenol synthase and lanosterol synthase that allowed the identification of important catalytic residues necessary to broaden and narrow product profiles. These results provided insights into factors critical for redesigning enzyme function. Mutagenesis in Arabidopsis thaliana cycloartenol synthase revealed that His477 is an essential component of the catalytic distinction between cycloartenol synthase and lanosterol synthase. Mutations at position 477 abolish cycloartenol biosynthesis, and subtle structural changes at this position dramatically alter product profile. The His477Asn mutant produces lanosterol as its major product (88%) whereas His477Gln produces primarily parkeol (73%). His477 influences deprotonation more strongly than any of the previously studied catalytic residues, but changes at this position are catalytically irrelevant in the presence of Tyr410Thr and Ile481Val mutations. The His477Asn Tyr410Thr Ile481Val and His477Gln Tyr410Thr Ile481Val triple mutants have the same product profile as the Tyr410Thr Ile481Val double mutant. Homology modeling studies established that His477 is a second-sphere residue that affects catalysis indirectly through its interactions with the active-site residue Tyr410. Changes at His477 strongly affect the location, orientation, and electronics of the Tyr410 side chain. Efforts to modify the catalytic specificity of enzymes consistently show that it is easier to broaden the substrate or product specificity of an accurate enzyme than to restrict the selectivity of one that is promiscuous. Careful examination of the homology model allowed the identification of a combination of mutations necessary to redesign cycloartenol synthase into a highly accurate lanosterol synthase. A double mutant was constructed and characterized and was shown to cyclize oxidosqualene accurately to lanosterol (99%). This catalytic change entailed both relocating polarity with a His477Asn mutation and modifying steric constraints with an Ile481Val mutation, and is among the best examples of redesigning an enzyme to accurately generate a new product. Known lanosterol synthase mutants make monocyclic or tetracyclic byproducts from oxidosqualene. Mutation of Saccharomyces cerevisiae lanosterol synthase at Tyr510 caused partial substrate misfolding and generated a tricyclic byproduct. This novel triterpene, (13alphaH)-isomalabarica-14(27),17 E,21-trien-3beta-ol, is the putative biosynthetic precursor of the isomalabaricane triterpenoids in sponges. The results suggest the facile evolution of tricyclic terpenoids as secondary metabolites in sponges.
Nanofiber Formation by Collagen Mimetic Peptides: Elucidation of Supramolecular Principles and Application of Retrosynthetic Analysis
(2015-05-14) Sarkar, Biplab; Hartgerink, Jeffrey D.; Matsuda, Seiichi P. T.; Tao, Yizhi J.
Synthetic mimics of the fibrillar protein collagen were designed by engineering specific supramolecular interactions in the triple helical building blocks. My doctoral research indicates that tri-domain collagen mimetic peptides, with positive, neutral, and negative domains, may form nanofibrous assembly through a staggered triple-helical intermediate stabilized by inter-strand charge pairs. Based on our understanding of the fibrillation pathway, I have devised a supramolecular retrosynthetic strategy to prepare collagen mimetic nanofibers. By designing an infinite triple helix with optimal number of inter-strand charge pairs, and then identifying the requisite building blocks, I was able to prepare a two-component collagen mimetic fiber with a high melting temperature. Peptide-based collagen mimetic nanofibers have great potential for future medical applications.

Browsing by Author "Matsuda, Seiichi P. T."

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