Re-engineering E. coli for in vivo production of fluorometabolites
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Date
28/11/2019Author
Markakis, Konstantinos
Metadata
Abstract
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).