Thin layer chromatography and high performance liquid chromatography were used to compare pigments of the wild type streptomycin resistant strain of C. poinsettiae with those of mutants derived from it. Possible biosynthetic pathways in carotenoid biosynthesis of the wild type were postulated on the basis of observed blocks in pigment synthesis.

No / Bacterial lipoproteins are proteins that are post-translationally modified with a diacylglyceride at an N-terminal cysteine, which serves to tether these proteins to the outer face of the plasma membrane or to the outer membrane. This paper reviews recent insights into the enzymology of bacterial lipoprotein biosynthesis and localization. Moreover, we use bioinformatic analyses of bacterial lipoprotein signal peptide features and of the key biosynthetic enzymes to consider the distribution of lipoprotein biosynthesis at the phylum level. These analyses support the important conclusion that lipoprotein biosynthesis is a fundamental pathway utilized across the domain bacteria. Moreover, with the exception of a small number of sequences likely to derive from endosymbiont genomes, the enzymes of bacterial lipoprotein biosynthesis appear unique to bacteria, making this pathway an attractive target for the development of novel antimicrobials. Whilst lipoproteins with comparable signal peptide features are encoded in the genomes of Archaea, it is clear that these lipoproteins have a distinctive biosynthetic pathway that has yet to be characterized.

Lipids are stored in a cell for providing energy. The main advantages of storing lipids over carbohydrates like glycogen is that, lipids yield more energy after oxidation because they represent the highly reduced form of carbon, needs less space and water for storage. Conservation of chemical energy in the form of biologically inert form is by storing molecules like triacylglycerol (TAG) and Steryl esters (SE). Triacylglycerol is the major storage form of energy in all eukaryotic cells. During the periods of nutritional excess and nutritional stress, all organisms like bacteria, yeast, animals, and plants can able to do the critical function of synthesizing the triacylglycerol. TAG is an energy store and a repository of essential and non-essential fatty acids and precursors for phospholipid biosynthesis. TAG synthesis mainly takes place in endoplasmic reticulum in mammals and in plants, it takes place in plastid and mitochondria. Triacylglycerol synthesis discovered by Kennedy starts with glycerol 3- phosphate. Glycerol 3-phosphate gets acylate to form lysophosphatic acid (LPA), which in turn acylate to form phosphatic acid (PA) and the reactions are catalyzed by glycerol 3-phosphate acyltransferase (GPAT) and LPA acyltransferase (LPAT) respectively. PA undergoes phosphorylation by PA phosphatase enzyme to give diacylglycerol (DAG). Further acylation of DAG gives rise to TAG and the reaction is catalyzed by diacylglycerol acyltransferase (DGAT). There are several DGAT classes were identified they are DGAT1, DGAT2, PDAT and bifunctional TAG/wax ester synthase. However all the enzymes involved in Kennedy TAG biosynthetic pathway as well as the enzymes of all different DGAT classes are membrane bound enzymes. Through our studies an another DGAT class that is soluble and cytosolic DGAT was first identified in peanut and also in yeast, Rhodotorula glutinis in which a soluble cytosolic complex itself has been identified. The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in R. glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of alternate TAG biosynthetic pathway in oleaginous yeasts. A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX4D acyltransferase motif at the N-terminal end and a lipid binding motif VX2GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

Carotenoid pigments were extracted from Corynebacteriuma poinsettiae (wild type) ATCC 9682, and from 108 mutants obtained by exposure of a streptomycin resistant strain of C. poinsettiae to ultra-violet light irradiation and N-methyl- N'-nitro-N-nitrosoguanidine. The pigments were characterized by their absorption maxima, Rf-values, and partition ratios in petroleum ether and methanol. Thin layer chromatography was used to compare pigments of the wild type with those of the mutants. Possible biosynthetic pathways in carotenoid synthesis of the wild type were postulated on the basis of the observed genetic blocks. Mutants were found which suggested the existence of a linear pathway in carotenoid synthesis from the aliphatic C4 0 molecule to the bi-cyclic C50-diol. Other mutants suggested possible alternative pathways in the biosynthesis of these pigments or the presence of intermediates not detectable by thin layer chromatography.

by Stanton Hon-Lung Kok. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 142-147). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The continued propagation of antibiotic resistance requires the development of new therapeutics. The lipopeptide antibiotic enduracidin has demonstrated high activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus. In addition to a lack of cross-resistance with existing antibiotic classes, enduracidin has no known transferrable resistance mechanism. The development of enduracidin as a human therapeutic is hampered by its poor solubility in plasma. Utilizing chemical and genetic techniques, analogs of enduracidin have been produced and evaluated for biological activity. Making use of the hydroxyphenylglycine (Hpg) biosynthetic pathway, fluorine was incorporated into enduracidin with minimal to no loss of bioactivity. The semisynthetic chemical modification of enduracidin proved to be challenging. The chemical nitration of the Hpg residues was unsuccessful. Modifications to the lipid tail by cleavage at the C2-olefin with ozone and the use of Diels-Alder reagents to react with the lipid tail diene also proved unsuccessful. However, the reduction and dihydroxylation modifications of the lipid tail diene were successful. Introduction of polar hydroxyl groups onto the alkyl tail reduced bioactivity while reduction of the diene had no significant effect. Analysis of the biosynthetic pathways involved in producing the lipid tail and the unusual amino acid enduracididine yielded some insights into the formation of the antibiotic. Through complementation of mutants having disruptions in the biosynthetic gene cluster and crystallographic data, the function of EndR as a cyclase was established. Additionally, the use of 4-hydroxyarginine as an intermediate in enduracididine biosynthesis was demonstrated. The ability of EndQ to function as a transaminase on both 4-hydroxyarginine and 2-ketoenduracididine was also established. The specific functions of EndP and EndQ have not been determined. The introduction of the lipid tail diene by the three enzymes Orf39, Orf44 and Orf45 was confirmed. Orf45 functions as a CoA ligase and a dehydrogenase to introduce the C2 double bond. The functions of Orf39 and Orf44 appear to be the introduction of the C4 double bond and isomerization of the C2 olefin. / Graduation date: 2013

Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.

<p> In four separate experiments, DL-[5-3H]/DL-[5-14C]ornithine, L-[5-3H]/DL- [5- 14C]ornithine, D-[5-3H]/DL- [5- 14C]ornithine and L-[2-3H]/L-[5- 14c]ornithine were administered to intact tobacco plants (Nicotiana tabacum). Nicotine, ornithine and proline were isolated in each of these experiments. In another experiment, R-[1-2H][l ,4-14C] putrescine was administered to intact tobacco plants and nicotine was isolated. The results of these experiments are consistent with the accepted mode of biosynthesis of nicotine from ornithine via putrescine (1,4-diaminobutane), N-methylputrescine, N-methyl-4-aminobutanal and N- methyl-1-pyrrolinium ion. The 3H:14c ratios of nicotine, the distribution of tritium within nicotine as established by chemical degradation and the distribution of deuterium within nicotine as established by 2H NMR are interpreted as showing that L-ornithine is the preferred enantiomer for nicotine biosynthesis, that the decarboxylation of L-ornithine to yield putrescine proceeds with retention of configuration at the reaction site, and that the oxidation of N-methylputrescine to N-methyl-4-aminobutanal proceeds with loss of the 4(S)hydrogen. </p> <p> Contrary to earlier reports, ornithine isolated in the 3H, 14C experiments had a changed 3:14c ratio from the ornithine which was fed. These results are interpreted as showing that L-ornithine is metabolised more rapidly than is D-ornithine in the tobacco plant. </p> <p> In all 3H, 14c experiments, proline was found to contain at least a small amount of tritium. In particular, when L-[2- 3H]/L-[5-14C] ornithine served as substrate, proline was found to contain 40 + 1% of the tritium, relative to 14C, that had been present in the feeding material. This result is interpreted as showing that, contrary to earlier reports, L-ornithine can be converted into proline via either a-keto-s-aminovaleric acid or glutamic semialdehyde. Together with the 3H: 14C ratios of proline in the other experiments, the results of this work are interpreted as showing that, when DL-ornithine serves as the substrate for proline biosynthesis in tobacco, 88 + 1% of the proline arises from D-ornithine via a-oxidation, 7 + 1% of the proline comes from L-ornithine via a-oxidation and 5 + 1% of the proline is produced from L-ornithine via s-oxidation. </p> / Thesis / Master of Science (MS)

BACKGROUND:Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.RESULTS:A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.CONCLUSIONS:The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

No description available.