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Title: Re-engineering E. coli for in vivo production of fluorometabolites
Author: Markakis, Konstantinos
ISNI:       0000 0004 8497 6675
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Fluorinated products are widely used in pharmaceutical and agrochemical applications where a large portion of the commercial compounds being developed contain at least one atom of fluorine. Most of the conventional fluorination reactions require volatile chemicals and are highly exothermic requiring controlled procedures which are rather costly, produce low yields and exhibit low selectivity of the moiety accepting fluorine. As a result, much attention has been drawn on enzymatic fluorination in a quest to ameliorate these issues. The only characterized enzyme that mediates direct fluorination is the 5'-fluoro- 5'-deoxyadenosine (5'-FDA) synthase (a.k.a. fluorinase), which uptakes an S- adenosyl-L-methionine (SAM) molecule and a fluorine atom as substrates and yields 5'-FDA and methionine. Variants of this enzyme have been extensively studied structurally and functionally with in vitro assays and they exhibit very high homology, and a similarly slow activity. In this work, a bacterial in vivo bioreactor is presented, based on a variant of the enzyme, which is found in Streptomyces sp. MA37 (FlA1). The base host that has been chosen for the implementation of modiflcations is Escherichia coli BL21(DE3), which is a host of choice for the over-expression of recombinant proteins and in some occasions utilized for the construction of novelty bioreactors. In the case of enzymatic fluorination, a few challenges had to be addressed. First of all, E. coli contains an ion channel protein, termed CrcB, which expels fluoride anions out of the cell. Secondly, SAM cannot passively enter E. coli cells rendering the intracellular molarity of SAM a bottleneck. Last but not least, the E. coli purine nucleoside phosphorylase, DeoD, has been suggested to degrade the fluorination product, 5'-FDA. Therefore, the modified E. coli strain, consists of 2 deleted genes (∆crcB∆deo∆), the expression of a SAM transporter from Rickettsia prowazekii and the over-expression of flA1. Using this strain, in vivo production of 5'-FDA to near millimolar concentrations has been confirmed using an array of chemical analysis methods including High Performance Liquid Chromatography (HPLC), Fluorine Nuclear Magnetic Resonance (F-NMR) and High Resolution Mass Spectrometry (HRMS).
Supervisor: Elfick, Alistair ; Rosser, Susan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: enzymatic fluorination ; 5'-FDA synthase ; in vivo bioreactor ; MA37 ; E. coli ; S- adenosyl-L-methionine