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University of London Thesis REFERENCE ONLY UNIVERSITY OF LONDON THESIS Degree Year^^0^ Name of Author C O P Y R IG H T This is a thesis accepted for a Higher Degree of the University of London. It is an unpublished typescript and the copyright is held by the author. All persons consulting the thesis must read and abide by the Copyright Declaration below. COPYRIGHT DECLARATION I recognise that the copyright of the above-described thesis rests with the author and that no quotation from it or information derived from it may be published without the prior written consent of the author. LOANS Theses may not be lent to individuals, but the Senate House Library may lend a copy to approved libraries within the United Kingdom, for consultation solely on the premises of those libraries. Application should be made to: Inter-Library Loans, Senate House Library, Senate House, Malet Street, London WC1E 7HU. REPRODUCTION University of London theses may not be reproduced without explicit written permission from the Senate House Library. Enquiries should be addressed to the Theses Section of the Library. Regulations concerning reproduction vary according to the date of acceptance of the thesis and are listed below as guidelines. A. Before 1962. Permission granted only upon the prior written consent of the author. (The Senate House Library will provide addresses where possible). B. 1962- 1974. In many cases the author has agreed to permit copying upon completion of a Copyright Declaration. C. 1975 - 1988. Most theses may be copied upon completion of a Copyright Declaration. D. 1989 onwards. Most theses may be copied. ------------------------------------------- I I This copy has been deposited in the Library of --------------------:— This copy has been deposited in the Senate House Library, Senate House, □ Malet Street, London WC1E 7HU. C:\Documents and Settings\lproctor\Local Settings\Temporary Internet Files\OLK8\Copyright - thesis (2).doc Biocatalytic Synthesis of Amino-Alcohols Using a de novo Designed Transketolase-P-Alanine: Pyruvate Transaminase Pathway inEscherichia coli A Thesis Submitted for the Degree of Doctor of Philosophy to the University of London Christine U. Ingram Department of Biochemical Engineering University College London UMI Number: U592924 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592924 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 For my Grandpa, Maurice Ingram, who departed before I arrived. Abstract Biocatalysis continues to emerge as a powerful technique for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The power of a biocatalytic stage can be enhanced if two or more reactions can be achieved by a single whole cell biocatalyst containing a pathway designed de novo to facilitate a required synthetic sequence. This will allow higher space-time yields and reduce the number of intermediate process steps required. The enzymes transketolase (TK) and transaminase (TAm) respectively catalyse asymmetric carbon-carbon bond formation and amine group addition to suitable substrate molecules. The ability of a transaminase to accept the product of the transketolase reaction can allow the two catalysts to be employed in series to create chiral amino alcohols from achiral substrates. These compounds are synthetically very useful in the production of a range of compounds with pharmaceutical application. The p-alanine: pyruvate aminotransferase (p-A: P TAm) from Pseudomonas aeruginosa has been cloned, to create plasmid pQR426, for over-expression in E.coli strain BL21gold(DE3). The aromatic transaminase (ArTAm) from E.coli was also cloned, to create plasmid pQR416. The substrate specificity of the transaminases has been explored in order to assess the feasibility of functioning downstream of TK and the potential to access a wider range of TK-TAm pathway products. Both directed evolution and site directed mutagenesis have also been applied in order to facilitate acceptance of transketolase products. Over-expression of the p-A: P TAm alongside the native transketolase (from plasmid pQR411), in a single E.coli host, has created a novel bacterial biocatalyst capable of the synthesis of chiral amino alcohols via a synthetic two-step pathway. The feasibility of using the biocatalyst has been demonstrated by the formation of 2-amino-1,3,4-butanetriol (ABT) product, in up to 21 % yield, by the p-alanine: pyruvate transaminase, via transamination of L- erythrulose synthesised by transketolase, from glycolaldehyde (GA) and p- hydroxypyruvate (p-HPA) substrates. The degradation of the amino alcohol product and the low activity of the transaminase, initial rate 0.1 mM.hr'1, relative to TK, initial rate 2.1 mM.min'1, are found to be limiting the process requiring further investigation. 3 Acknowledgements To all those people who have assisted me in the course of my PhD, thank you. In particular I would like to thank my supervisor Professor Gary Lye and advisor Dr Paul Dalby. Additionally Dr John Ward and Dr John Woodley both of whom gave me extensive advice and support. Thank you to Dr Mark Smith to whom I am extremely grateful for not only helping me with my chemistry quandaries but who brought with him enthusiasm and inspired motivation as well as lending a sympathetic ear in the closing months of my project. Additionally to Martin Bommer and Kirsty Smithies for lending their molecular biology and analytical skills respectively. Thank you to Sarah Horton with whom I travelled through this journey sharing experiences and frustrations throughout and to Brenda Parker and Ryan McCoy for their support. A special thank you to my parents, Jan and Clarice, and my sister Pauline for their love, encouragement and patience and continuing to show interest in my work when I either gave endless intricate details of exciting results or refused to tell them anything. 4 Abbreviations ABT 2-amino-1,3,4-butanetriol ACN Acetonitrile AmpR Ampicillin resistance AQC 6-aminoquinolyl-A-hydroxysuccinimidyl carbamate p-A: P TAm P-alanine: pyruvate transaminase ArTAm Aromatic transaminase AspTAm Aspartic acid transaminase Axnm Absorbance at x nm DNA Deoxynucleic acid dNTP Deoxynucleotidetriphosphate DoxP synthase 1 -deoxy-D-xylulose-5- phosphate synthase EBA Ethylbenzylamine E.C. Enzyme commission E.coli Escherichia coli EDTA ethylenediaminetetraacetic acid E-PLP Transaminase -PLP complex E-PMP Transaminase-PMP complex GA Glycolaldehyde p-HPA P-hydroxypyruvate HPLC High performance liquid chromotography IPTG Isopropyl-P-D-thiogalactosidase KanR Kanamycin resistance a-KG a-ketoglutarate Km Michaelis constant MBA Methylbenzylamine NADH Nicotinamide adenine dinucleotide OD Optical density P.aeruginosa Pseudomonas aeruginosa PCR Polymerase chain reaction PHA Polyhydroxlalkanoates PLP Pyridoxal phosphate PMP Pyridoxamine phosphate P.putida Pseudomonas putida RBS Ribosome binding site SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis TAm Transaminase TFA Trifluoroacetic acid TK Transketolase TK-TAm Co-expressed transketolase and transaminase pathway TLC Thin layer chromatography TPP Thiamine pyrophosphate Tris Tris(hydroxymethyl)aminomethane UV Ultraviolet Umax Maximal velocity pmax Maximal specific growth rate wpepPCR Whole plasmid error prone PCR 6 Contents Abstract................................................................................................................................ 3 Acknowledgements............................................................................................................4 Abbreviations ..................................................................................................................... 5 Contents............................................................................................................................... 7 List of Figures................................................................................................................... 13 List of Tables.....................................................................................................................15 Chapter 1 - Introduction and Project Aims............................................................... 16 1.1 Development of chiral pharmaceuticals ..........................................................16 1.2 Industrial Biocatalysis .......................................................................................20 1.3 Multi-step Biocatalysis and Pathway Engineering ........................................21 1.4 Chiral amines .....................................................................................................27 1.4.1 Chemical synthesis routes to chiral amines............................................ 27 1.4.2 Enzymatic approaches to chiral amines................................................. 29 1.5 Asymmetric carbon-carbon bond formation ...................................................30 1.5.1 Enzymatic synthesis o f C-C bonds..........................................................
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