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Tesi sul tema "Biosynthesis"

Autore: Grafiati
Pubblicato: 4 giugno 2021
Ultima modifica: 29 luglio 2024

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1

Eyles, Tom. "Biosynthetic Lego : reprogramming RiPP biosynthesis". Thesis, University of East Anglia, 2018. https://ueaeprints.uea.ac.uk/69571/.

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Ribosomally synthesised and post translationally modified peptides (RiPPs) are a diverse class of industrially-important and clinically-relevant natural products. Reprogramming the biosynthesis of RiPPs can provide an understanding of their biosynthesis, increases in their yield, and compound derivatives. In this thesis, two RiPP biosynthetic pathways are reprogrammed to achieve these aims. Bottromycin is a potent antibiotic RiPP, however it is produced in low yields by its native producer and it is rapidly hydrolysed in blood plasma. It was hypothesised that the bottromycin gene cluster could be reprogrammed to increase the production of bottromycin and to derivatise it. Synthetic biology techniques available at the start of the project were deemed inappropriate for use in reprogramming the bottromycin gene cluster as they lacked the ability to conduct refactoring, produce gene insertions/deletions, and make targeted mutations in single steps in the high-GC bottromycin gene cluster. Here, a one-step yeast-based method that enables efficient and flexible modifications to the bottromycin gene cluster is presented. Multiple modifications are showcased, including refactoring, gene deletions and targeted mutations. This facilitated the construction of an inducible, riboswitch-controlled pathway that achieved a 120-fold increase in pathway productivity in a heterologous host. Additionally, an unexpected biosynthetic bottleneck resulted in the production of a suite of new bottromycin-related metabolites. Thiostreptamide S4 is part of a family of promising antitumor RiPPs, the thioviridamide-like molecules. The gene cluster responsible for thiostreptamide S4 production has been identified, yet the biosynthesis has not been elucidated. It was hypothesised that reprogramming the thiostreptamide S4 gene cluster could provide insights into its biosynthesis. These modified clusters were constructed, and in-depth metabolomics enabled an understanding of the biosynthetic pathway. This biosynthetic understanding could pave the way for future engineering projects and allow key biosynthetic steps to be identified for use in genome mining.
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2

Khairudin, Khairunisa. "Biosynthetic studies and combinatorial biosynthesis of pleuromutilin antibiotics". Thesis, University of Bristol, 2018. http://hdl.handle.net/1983/46271504-0b2b-457a-92d0-073885f512cd.

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Pleuromutilin has potential as a next-generation antibiotic, and many semi-synthetic pleuromutilin derivatives have been developed. Recently, characterization of individual enzymatic steps involved in the production of pleuromutilin has been carried out. A linear pathway of pleuromutilin biosynthesis was established; however, there is a possibility of alternative or shunt pathways. Thus, the first part of this thesis aimed to investigate if any other possible routes could lead to the biosynthesis of pleuromutilin. Two alternative pathways were identified from the expression of various combinations of pleuromutilin biosynthetic genes in Aspergillus oryzae. However, neither route led to mature pleuromutilin due to the lack of activity of P450-3. It was shown that most of the tailoring enzymes except for P450- 3 exhibited a more relaxed substrate specificity as they were able to catalyze reactions in different premutilin intermediates. The structure-activity of pleuromutilin and its derivatives, including one synthesized by a semi-synthetic approach, was also investigated through antibacterial assay against Bacillus subtilis. It was observed that missing the substituents, 3-ketone, 11-OH or 14-acetyl from the pleuromutilin core affected the antibacterial activity of the pleuromutilin. Thus, it was important to retain the three side groups on the pleuromutilin core to maintain the bioactivity of the pleuromutilin, although further modification can be done on the C-14 to improve its activity. The second part of this study evaluated the potential of using the A. oryzae secondary host as a platform for further in vivo derivatization of the pleuromutilin core through the expression of foreign genes. However, no accumulation of new pleuromutilin analogs could be detected, which suggested the difficulties in achieving pleuromutilin hybrid products through combinatorial biosynthesis. Further knowledge of the interaction between enzymes and their substrates may be required to find suitable hybrid enzyme combinations that could lead to the biosynthesis of compounds with diverse chemical structures and possibly improve the antimicrobial activities.
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3

Walczak, Robbie J. "Analyses of antibiotic biosyntheses in Streptomyces spp. : the molecular biology of nonactin biosynthesis and the novel biochemistry of daunorubicin biosynthesis /". The Ohio State University, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488205318510564.

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4

Gray, Jennifer A. "Biotin biosynthetic enzymes and the metabolic control of biotin biosynthesis". [Ames, Iowa : Iowa State University], 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1473213.

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5

Jackson, Catherine Mary. "Tetronasin biosynthesis". Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303274.

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6

Jacobs, Adam. "Aspyrone biosynthesis". Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241064.

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7

Ndiege, Isaiah Omolo. "Polyketide biosynthesis". Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315331.

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8

Suzuki, Shiro. "Stereochemical diversity in lignan biosynthesis and establishment of norlignan biosynthetic pathway". Kyoto University, 2002. http://hdl.handle.net/2433/78141.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第9652号
農博第1280号
新制||農||848(附属図書館)
学位論文||H14||N3684(農学部図書室)
UT51-2002-G410
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 島田 幹夫, 教授 桒原 保正, 教授 坂田 完三
学位規則第4条第1項該当
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9

Purvis, Michael Bernard. "Stereochemical aspects of virginiamycin biosynthesis: biosynthesis of antibiotic A33853". Diss., Virginia Polytechnic Institute and State University, 1989. http://hdl.handle.net/10919/54266.

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The biochemical pathways for the formation of the unusual amino acids found in virginiamycin M₁ and A33853 were investigated. Specifically tritiated and carbon 14 labeled serines were incorporated into virginiamycin M₁. (2S)-serine and (2S,3R)-[3-³H] serine were found to be precursors, thus giving evidence of stereochemical control in the formation of the oxazole moiety. This information allowed for postulation of a ring closure pathway. Stereochemical investigations were also carried out on the dehydroproline unit and it was shown that both (R) and (S) prolines were incorporated into the dehydroproline unit. (2S,3R)-[3-³H] proline was synthesized and upon incorporation lost the (3-³H) label as evidence of stereochemical control in the formation of the dehydroproline unit from a saturated precursor. The basic biosynthetic origins of A33853 were investigated by feeding of D-[U-¹⁴C] glucose, sodium [U-¹⁴C] acetate, (S)-[U-¹⁴C] lysine, (S)-[U-¹⁴C] aspartic acid, [carboxyl-¹⁴C] anthranilic acid, and (S)-[5-³H] tryptophan. D-[U-¹⁴C]. Glucose and (S)-[U-¹⁴C] lysine appeared to be the main precursors. ¹³C¹⁵N lysine was synthesized and used to examine the ring closure of the 3-hydroxypicolinic amide ring in virginiamycin S₁.
Ph. D.
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10

Milne, Keith Livingston. "Bacterial isoprenoid biosynthesis". Thesis, University of Edinburgh, 1990. http://hdl.handle.net/1842/11172.

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This thesis describes a possible alternative isoprenoid pathway in bacteria by considering some previously unpublished feeding studies in the context of the related background literature. Three synthetic routes to 2,4-dihydroxy-4-methyltetrahydropyran (63) and three synthetic strategies towards the synthesis of 2-carboxy-2,4-dihydroxy-4-methyltetrahydropyran (63) are discussed. These compounds are considered as potential intermediates in the proposed alternative bacterial isoprenoid pathway. Labelled synthesis of (63) and structural analysis of (63) and 4-hydroxy-2-methoxy-4-methyltetrahydropyran (99) by proton nmr are also described. Feeding studies including the 13C isotopically labelled tetrahydropyrans (63) and (99) are described and a revised interpretation of all of the feeding studies considered. HMGCoA synthase is assayed in Rh. capsulata after a description of its assay in bakers yeast.
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11

Milne, Claire E. "Biosynthesis and engineered biosynthesis of Calcium Dependent Antibiotic (CDA) from streptomyces coelicolor". Thesis, University of Manchester, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506586.

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Calcium Dependent Antibiotic (CDA), is a nonribosomal lipopeptide antibiotic from Streptomyces coelicolor. It is an undecapeptide with an unusual N-terminal 2,3-epoxyhexanoyl fatty acid side chain. It contains many nonproteinogenic amino acids such as D-4-hydroxyphenylglycine, D-3-phosphohydroxyasparagien, L-3-methylglutamic acid and Z-2,3-dehydrotryptophan. CDA is very similar in structure to the antibiotic daptomycin, which under the trade name Cubicin has recently been approved for the treatment of skin infections. The work described in this thesis focuses on a number of different areas, with the overall aim of delineating the biosynthetic origin of CDA and investigating the possibility of producing modified cyclic lipopeptides through a combined chemical and biochemical approach. Strategies used in engineering the biosynthesis of CDA may then be applied to other related, nonribosomal peptides of therapeutic importance, in order to improve their biological activity.
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12

Schildknecht, Stefan. "Redoxregulation of prostanoid biosynthesis". [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=976070073.

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13

Wicher, Krzysztof B. "Haptoglobin: Biosynthesis and Evolution". Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6446.

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Haptoglobin (Hp) is a serum protein known for its ability to form a tight complex with hemoglobin (Hb) and thereby inhibiting the oxidative activity of Hb.

Mammalian Hp is synthesized as a precursor (proHp) that undergoes proteolytic cleavage by a previously unidentified enzyme in the endoplasmic reticulum (ER). In this study, a proHp-cleaving enzyme was isolated from human serum and identified as complement C1r-like protein (C1rLP). Co-expression of C1rLP with proHp in mammalian cells resulted in cleavage of the latter protein in the ER. Mutation of either the active site serine residue in C1rLP or the arginine residue in the cleavage site of Hp abolished the cleavage of proHp by C1rLP. RNAi studies in mammalian cells identified the proHp-cleaving enzyme as C1rLP.

Hp has been found in all mammals studied to date but its presence in non-mammalian species has not been unambiguously shown. By searching currently available genomic DNA and cDNA sequence databases, a gene orthologous to mammalian Hp was found in bony fish. Hp-like protein expressed from this gene was demonstrated to be a major Hb protein in fish serum. Surprisingly, no Hp-like gene was found in the genomes of either frog or chicken. In chicken, a protein previously described as Hp was identified as PIT54, a member of a scavenger receptor cysteine-rich family of proteins. Interestingly, ostrich serum seemed to contain two Hb-binding proteins; one similar to PIT54 and one to mammalian Hp. We are not aware of any other case where the function of one gene has been taken over by another, completely unrelated gene

Fish Hp (fHp) is composed of a serine proteinase-related domain preceded by an extension consisting of several aminoa acids and a signal peptide. The extension contains a consensus motif for cleavage by subtilisin-like proprotein convertases (SPCs). fHp was found to be cleaved by SPCs in the Golgi complex.

Collectively, this thesis presents evidence that Hp has undergone significant changes during evolution with respect to its molecular organization and to the mechanism of its proteolytic cleavage.

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14

Kollmeyer, Jessica Elaine. "Regulation of Galactosylceramide Biosynthesis". Thesis, Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/11630.

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An important branchpoint of mammalian sphingolipid metabolism occurs at the step where ceramides are glycosylated to glucosylceramide (GlcCer) versus galactosylceramide (GalCer), which are precursors of all mammalian glycosphingolipids. Relatively few studies have focused on this branchpoint because these monohexosylceramides are somewhat difficult to resolve chromatographically and because molecular biology tools have only recently become available to follow expression of these genes. The goal of this thesis is to better understand the mechanisms of cell regulation determining galactosylceramide synthesis.
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15

Ross, Timothy Kieran. "Regulation of polyglycerophospholipid biosynthesis". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ32238.pdf.

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16

Lewis, Elizabeth A. "The biosynthesis of chloramphenicol". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ36494.pdf.

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17

Keyes, Robert F. "The biosynthesis of ravidomycin". Thesis, This resource online, 1989. http://scholar.lib.vt.edu/theses/available/etd-08252008-162246/.

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18

Lewis, C. N. "The biosynthesis of canescin". Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373266.

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19

Loughran, Mark Stephen. "The biosynthesis of erythromycin". Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307943.

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20

Andrews, Timothy Stephen. "The biosynthesis of erythromycin". Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358365.

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21

Lipscomb, Sarah. "Studies on cephalosporin biosynthesis". Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433559.

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22

Lee, Hwei-Jen. "Studies on cephalosporin biosynthesis". Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409949.

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23

Lloyd, Matthew David. "Biosynthesis of calavulanic acid". Thesis, University of Oxford, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294290.

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24

Sleeman, Mark. "Studies on carbapenem biosynthesis". Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432411.

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25

Rackham, Emma Jayne. "Elucidation of pacidamycin biosynthesis". Thesis, University of East Anglia, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539367.

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26

Hardick, David James. "The biosynthesis of nojirimycins". Thesis, University of Warwick, 1992. http://wrap.warwick.ac.uk/4419/.

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Streptomyces subrutilus ATCC 27467, when grown on a glucosecontaining soyabean medium, produces both 1-deoxymannonojirimycin (DMJ) and 1-deoxynojirimycin (DNJ) in its culture medium. When 1- or 2-[2H]-D-glucose is used, the deuterium label appears at C6 in both alkaloids and the labelling pattern suggests that the first step in the biosynthesis of both DNJ and DMJ is a glucose to fructose isomerisation. Studies with 5-[2H]-D-glucose and 6,6-[2H2]-D-glucose indicate that oxidation of the 6-position of the glucose/ fructose occurs during the biosynthesis and that mannonojirimycin is the first amino sugar to be formed. Mannonojirimycin can then undergo dehydration and reduction to DMJ. Alternatively, epimerisation of mannonojlrlmycin can occur at C2 to give nojirimycin which is then dehydrated and reduced to DNJ. Studies with another microorganism, B. subtilis var niger ATCC 9372, indicate that a similar biosynthetic pathway is in operation. DMJ, however, is not produced by this microorganism and only low levels of NJ are postulated from enzyme inhibition and deuterium labelling studies. A minor biosynthetic route is also evident from labelling studies with 1-[13C]-D-glucose and 1-[13C]-D, L-glyceraldehyde. It is suggested that the fructose at the beginning of the biosynthesis can split into two C3 trioses which are in equilibrium with each other. These can then recombine to continue in the usual biosynthetic pathway. New chemical routes to 542H]-D-glucose and 2-[2H]-D-glucose are described in Chapter 3 along with a method for introducing deuterium or tritium into DNJ or NJ. Other isotopically enriched glucoses or intermediates have also been prepared using literature methodology. Studies have been undertaken to assess whether S. subrutilus can utilise glucose analogues in the biosynthetic pathway. To this end, new or modified routes to these glucose derivatives have been investigated and some work has focused on the chemical synthesis of DNJ analogues themselves.
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27

Watson, A. J. "Aflatoxin biosynthesis in Aspergillus". Thesis, University of East Anglia, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267259.

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28

Sanders, M. "Experiments in rotenoid biosynthesis". Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376182.

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29

MacLachlan, W. S. "The biosynthesis of cryptosporiopsinol". Thesis, University of Glasgow, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375450.

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30

Lam, Wai Ho. "Biosynthesis of Mureidomycin A". Thesis, University of Warwick, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444836.

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31

Browne, M. "Carotenoid biosynthesis in bacteria". Thesis, University of Liverpool, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372685.

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32

Clark, C. A. "The biosynthesis of nonactin". Thesis, University of Southampton, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370333.

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33

Sparkes, Andrew Windsor. "Studies of archaemycin biosynthesis". Thesis, University of Southampton, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239645.

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34

Gordon, J. "The biosynthesis of aphidicolin". Thesis, University of Sussex, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375170.

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35

McAndrew, Douglas. "Studies on polyketide biosynthesis". Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337292.

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36

Demetriadou, A. K. "The biosynthesis of heptaketides". Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355259.

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37

Hill, Alison Margaret. "The biosynthesis of aspyrone". Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319492.

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38

Di, Marzo V. "Neuropeptides and leukotriene biosynthesis". Thesis, Imperial College London, 1988. http://hdl.handle.net/10044/1/47024.

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39

Gómez, Velasco Anaximandro. "Studies in mycobactin biosynthesis". Thesis, University of Birmingham, 2009. http://etheses.bham.ac.uk//id/eprint/8030/.

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Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a single bacterial pathogen, Mycobacterium tuberculosis. Current TB chemotherapy remains useful in treating susceptible M. tuberculosis strains, however, the emergence of MDR-TB and XDR-TB demand the development of new drugs. Enzymes involved in mycobactin biosynthesis, low molecular weight iron chelators, do not have mammalian homologues; therefore they are considered potential targets for the development of new anti-TB drugs. The aims of this study were to identify potential inhibitors and to investigate the function of the mbtG and AmbtE and AMbtF genes during mycobactin biosynthesis. The full length of mbtB and the ArCP domain were successfully cloned and post-translationally modified by MtaA, a broad phosphopantetheinyl transferase from Stigmatella aurantiaca, using Escherichia coli. Inhibitors identified by virtual screening as well as 13 chemically synthesised PAS analogues were initially investigated in whole-cell assay against Mycobacterium bovis BCG Pasteur. Seven of these compounds had interesting growth inhibition under ironsufficient conditions. The mbtA gene was cloned and expressed as soluble protein using Mycobacterium smegmatis mc2155. Preliminary in vitro MbtA assays provided hints of its activity, although, the KM for SAL and ATP have not been determined yet. The mbtG and ambtE genes have been cloned and expressed in E. coli to further investigate their biochemical function in mycobactin biosynthesis.
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40

Kirkpatrick, Peter Neil. "The biosynthesis of chloroeremomycin". Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621622.

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41

Filipek-Górniok, Beata. "Glycosaminoglycan Biosynthesis in Zebrafish". Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-264269.

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Proteoglycans (PGs) are composed of highly sulfated glycosaminoglycans chains (GAGs) attached to specific core proteins. They are present in extracellular matrices, on the cell surface and in storage granules of hematopoietic cells. Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) GAGs play indispensable roles in a wide range of biological processes, where they can serve as protein carriers, be involved in growth factor or morphogen gradient formation and act as co-receptors in signaling processes. Protein binding abilities of GAGs are believed to be predominantly dependent on the arrangement of the sugar modifications, sulfation and epimerization, into specific oligosaccharide sequences. Although the process of HS and CS/DS assembly and modification is not fully understood, a set of GAG biosynthetic enzymes have been fairly well studied and several mutations in genes encoding for this Golgi machinery have been linked to human genetic disorders. This thesis focuses on the zebrafish N-deacetylase/N-sulfotransferase gene family, encoding key enzymes in HS chain modification, as well as glycosyltransferases responsible for chondroitin/dermatan sulfate elongation present in zebrafish. Our data illustrates the strict spatio-temporal expression of both the NDST enzymes (Paper I) and CS/DS glycosyltransferases (Paper II) in the developing zebrafish embryo. In Paper III we took advantage of the four preexisting zebrafish mutants with defective GAG biosynthesis. We could demonstrate a relation between HS content and the severity of the pectoral fin defects, and additionally correlate impaired HS biosynthesis with altered chondrocyte intercalation. Interestingly, altered CS biosynthesis resulted in loss of the chondrocyte extracellular matrix. One of the main findings was the demonstration of the ratio between the HS biosynthesis enzyme Extl3 and the Csgalnact1/Csgalnact2 proteins, as a main factor influencing the HS/CS ratio. In Paper IV we used the newly developed CRISPR/Cas9 technique to create a collection of zebrafish mutants with defective GAG biosynthetic machineries. Lack of phenotypes linked to null-mutations of most of the investigated genes is striking in this study.
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42

Donovan, Tessa May. "Biosynthesis of fungal melanin". Thesis, University of Edinburgh, 1989. http://hdl.handle.net/1842/13689.

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43

Ramos, Tania. "Cysteine biosynthesis in Leishmania". Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5156/.

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Every year 2 million people are diagnosed with leishmaniasis and 350 million are at risk of becoming infected. Spread throughout 88 countries in the world, leishmaniasis is a group of diseases comprising visceral, mucocutaneous and cutaneous leishmaniasis as the main forms. Visceral leishmaniasis is the most severe form of the disease and is caused by Leishmania donovani. The parasite, Leishmania, is transmitted to humans by a sandfly vector. The absence of vector-control procedures and effective vaccines for humans makes chemotherapy the only weapon against leishmaniasis. Great efforts have been made to develop new drugs against these diseases, however toxic side effect and the constant cases of resistance call for new drug targets and drugs to be discovered and developed. Cysteine is a key building block of trypanothione, an antioxidant unique to trypanosomatids that plays a pivotal role for the survival of the parasites. Leishmania can obtain cysteine in two ways, using the sulphydrylation and trans-sulphuration pathways. Humans lack the sulphydrylation pathway, thus this, and especially cysteine synthase (CS), of Leishmania could be a potential drug target. In order to determine the relative importance of these pathways, the levels of thiols at different stages of promastigote growth of wild-type, mutants lacking CS (deltacs), and CS episomal re-expressor parasite lines were determined. It was found that during logarithmic phase the mutant parasites have significantly reduced levels of thiols, which is reversed in the CS re-expressing parasite line. The mRNA and protein levels of cystathionine β-synthase (CBS) were increased in deltacs L. donovani, while this decrease was reversed in the CS re-expessing line. These data suggest that the reverse trans-sulphuration pathway compensates for the loss of CS to some extent but that this is not sufficient to maintain thiol levels during logarithmic growth. It was further found that ornithine decarboxylase mRNA is up-regulated while the protein levels appear to be reduced in the deltacs parasite line. The latter was supported by the finding that the deltacs mutant parasites have low sensitivity to alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. The studies on the recombinant L. major cysteine synthase (CS) have shown that it can be inhibited by small peptides based on the C-terminal of L. major serine acetyltransferase. The CPM assay showed promising results to be used in high throughput screening of CS inhibitors. The data overall suggests that the levels of thiols are dependent on an adequate supply of cysteine through the sulphydrylation pathway. How this affects polyamine biosynthesis is yet to be investigated.
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44

Reva, Anna. "Enzymology of gentamicin biosynthesis". Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277902.

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Gentamicin C complex is a mixture of five structurally similar aminoglycoside antibiotics, gentamicins C1, C1a, C2, C2a, and C2b, produced by the actinomycete bacterium Micromonospora echinospora. It is established in clinical use and despite significant toxicity remains valuable to treat severe Gram-negative bacterial infections. There is a pressing need to develop novel versions of such antibiotics to combat the rise of resistance among pathogens. Engineering of the pathway requires a detailed knowledge of the genes, enzymes, and intermediates involved. The final steps of gentamicin biosynthesis begin at gentamicin X2, the last common intermediate of the C complex. 6'-C-Methylation generates two branches, with analogous reactions happening in both. Candidate genes and enzymes for the steps from the first 6'-C-methylated intermediate, G418, to an aminated metabolite JI-20B have already been described, but none for the subsequent loss of two hydroxyl groups from Ring II, or the N-methylation that then occurs. A novel separation method using dynamic countercurrent chromatography was successfully applied to the difficult purification of gentamicin metabolites. The results described here allowed a detailed mechanism to be proposed for almost the entire pathway from G418 to the C complex, and by analogy for the unbranched pathway, too. The last step of the pathway is 6'-N-methylation of gentamicins C1a and C2. Genome mining and cell-free assays were used by the group of Professor Yuhui Sun (Wuhan University) to identify genL, a methyltransferase gene encoded elsewhere on the M. echinospora genome and capable of this catalysis. Here, in vitro reactions with recombinant GenL confirmed its function, and its kinetic parameters were measured with its substrates. The full mechanistic pathway for the late stages of gentamicin C complex biosynthesis has therefore now been elucidated.
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45

Martins, Filipa Teixeira. "Studies of thiamine biosynthesis". Thesis, University of Southampton, 2009. https://eprints.soton.ac.uk/71854/.

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In Escherichia coli, and other prokaryotes, thiamine (vitamin B1) is assembled by coupling 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (Hmp-PP) and 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (Thz-P). The thiazole moiety is biosynthesised from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine, and at least six genes are required including thiH, thiG, thiS, and thiF. Whilst in aerobes, the C2-N3 fragment of Thz-P derives from glycine in a reaction catalysed by the flavoenzyme ThiO, in anaerobes such as E. coli, dehydroglycine is formed from tyrosine in a ThiH dependent reaction. This biosynthetic step requires the cleavage of the Cα-Cβ bond of tyrosine and a release of an aromatic side chain. ThiH shows sequence similarity with the ‘radical S-adenosylmethionine’ (AdoMet) family of proteins, including conserved cysteine ligands to the essential [4Fe-4S] cluster and has been shown to form a complex with ThiG. With the purpose of studying the mechanistic enzymology by which Thz-P is assembled it was crucial to isolate ThiH in the holo-form. Several expression systems and purification methodologies were investigated. The optimisation of the purification method, together with in vitro chemical reconstitution with exogenous iron and sulfide allowed the successful isolation of holo-ThiH. To facilitate the mechanistic investigation of Thz-P biosynthesis, an in vitro assay was developed, and the reaction products formed in vitro were elucidated and quantified. The aromatic by-product derived from the side chain of tyrosine is p-cresol and the remaining fragment yields glyoxylate, a product of hydrolysis of dehydroglycine
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46

Khaw, Lake Ee. "The biosynthesis of rapamycin". Thesis, University of Cambridge, 1995. https://www.repository.cam.ac.uk/handle/1810/272287.

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47

Singh, Neena. "Biosynthesis of glycophospholipid anchors". Case Western Reserve University School of Graduate Studies / OhioLINK, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=case1054926816.

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48

Cho, Hyeongjin. "Studies in antibiotics biosynthesis /". The Ohio State University, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487676847114716.

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49

Simpson, T. J. "Biosynthesis of fungal metabolites". Thesis, University of Edinburgh, 1986. http://hdl.handle.net/1842/11389.

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50

Fugelstad, Johanna. "Cellulose Biosynthesis in Oomycetes". Licentiate thesis, Stockholm : KTH Biotechnology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-9282.

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