Aromatic Halogenation by Using Bifunctional Flavin Reductase–Halogenase Fusion Enzymes

Article abstract of DOI:10.1002/cbic.201700391

The remarkable site selectivity and broad substrate scope of flavin-dependent halogenases (FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts have demonstrated that FDHs can be tuned for non-native substrate scope and site selectivity. FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous organisms. In both cases, reduced flavin cofactor, usually supplied by a separate flavin reductase (FR), is required. Herein, we report functional synthetic, fused FDH-FR proteins containing various FDHs and FRs joined by different linkers. We show that FDH-FR fusion proteins can increase product titers compared to the individual components for in vivo biocatalysis in Escherichia coli.

Full text of DOI:10.1002/cbic.201700391

Accepted Article
Title: Aromatic Halogenation Using Bifunctional Flavin Reductase-
Halogenase Fusion Enzymes
Authors: Mary C. Andorfer, Ketaki D. Belsare, Anna M. Girlich, and
Jared Lewis
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
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To be cited as: ChemBioChem 10.1002/cbic.201700391
Link to VoR: http://dx.doi.org/10.1002/cbic.201700391
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ChemBioChem
10.1002/cbic.201700391
Aromatic Halogenation Using Bifunctional Flavin Reductase-Halogenase Fusion Enzymes
‡
‡
*
Mary C. Andorfer, Ketaki D. Belsare, Anna M. Girlich, and Jared C. Lewis
Department of Chemistry, University of Chicago, Chicago IL 60637
‡
Authors contributed equally to this work.
Keyword: Halogenase, Fusion enzymes, directed evolution, in vivo halogenation, RebH
Abstract
The remarkable site-selectivity and broad substrate scope of flavin dependent halogenases
(FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts
have demonstrated that FDHs can be tuned for non-native substrate scope and site-selectivity.
FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated
into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous
organisms. In both cases, reduced flavin cofactor, usually supplied by a separate flavin reductase
(FR), is required. Here, we report functional synthetic, fused FDH-FR proteins, containing
various FDHs and FRs, joined by different linkers. We show that FDH-FR fusion proteins can
increase product titers compared to the individual components for in vivo biocatalysis in E. coli.
Introduction
Halogenated aromatic compounds often exhibit unique biological activities and are thus
[
1]
commonly used as pharmaceutical drugs and agrochemicals. Aryl halides are also valuable
building blocks for synthetic chemistry, particularly due to their centrality to a range of powerful
[
2,3]
cross-coupling reactions. Despite the importance of aromatic halogenation, however, common
methods of aromatic halogenation, perhaps most notably electrophilic aromatic substitution,
[
4]
often suffer from poor regioselectivity. More recent efforts have therefore explored the ability
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ChemBioChem
10.1002/cbic.201700391
of directing groups to enable selective halogenation of proximal C-H bonds on suitably pre-
[
5,6]
functionalized substrates.
Complementing these traditional synthetic methods, flavin-dependent halogenases
FDHs) have been shown to halogenate a range of electron rich hetero(arenes) with high
(
[
7-9]
selectivity (Scheme 1). FDH catalysis proceeds via an electrophilic halogen species (both a
[
10]
[11]
lysine-derived haloamine and HOX have been proposed), which, due to its orientation
relative to bound substrate, can override electronic biases of different substrates to catalyze
[
12,13]
aromatic halogenation with novel regioselectivity.
Notably, FDH catalysis proceeds in
aqueous solution at ambient temperature and requires only reduced flavin cofactor (FADH2),
sodium chloride as a halide source, and oxygen from air as a terminal oxidant. A cofactor
regeneration system (CRS) comprised of a flavin reductase, a NAD(P) oxidoreductase (e.g.
glucose dehydrogenase), FAD, NAD(P), and a terminal reductant (e.g. glucose, the only
stoichiometric reagent in the CRS) can be used to supply FADH2.
H
Cl
A
FDH, NaCl, O2
FADH2
R
R
B
COOH
OH
O
OMe
O OMe
O
NH2
O
O
HO
O
MeO
N
H
Cl
Cl
Cl
1
2
3
O
Cl
H
O
Cl
N
Cl
NH
NH
N
H
H
H N
N
H
O
2
H
O
OH
N
4
5
6
N
Scheme 1. A) General scheme for chlorination by FDHs. B) Representative products from
[
14-17]
chlorination of native (1-3) and non-native FDH substrates (4-6).
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ChemBioChem
10.1002/cbic.201700391
A number of efforts involving directed evolution and targeted mutagenesis have been
[
18,19]
[16,20]
used to engineer FDH variants with increased stability,
expanded substrate scope,
and
[
21,22]
altered regioselectivity
. In all of these efforts, a CRS analogous to that described above was
used to ensure maximum product formation, which necessitates the purification of a suitable
flavin reductase. Typically, E. coli flavin reductase, Fre, or the native RebH (a 7-tryptophan
FDH from the native organism Lechevalieria aerocolonigenes) partner RebF have been used for
[
20]
in vitro halogenation assays. Due to low solubility of RebF when over-expressed in E. coli, a
[
23]
fusion of maltose binding protein and RebF (MBPF) is often used in place of RebF. The
requirement of flavin reductase (FR) can be tedious for directed evolution efforts, since sufficient
reductase for thousands of reactions must be regularly prepared, purified, and quality tested. Of
course, this requirement could be eliminated by co-(over)expressing genes for the reductase and
halogenase either individually or as fusion enzymes. Genetic fusion of the flavin reductase and
halogenase could also improve halogenation efficiency, particularly for in vivo applications
where a high local concentration of reduced FADH cannot necessarily be guaranteed.
2
The utility of in vivo FDH catalyzed halogenation has now been established in a number
[
15,24-29]
of different organisms.
For example, Rdc2 has been used to halogenate phenolic
compounds in E. coli without co-expressing a flavin reductase, since this organism contains
[
15,30]
naturally occurring flavin reductases.
Likewise, targeting FDH expression to plant
chloroplasts (which have high levels of FADH ) is sufficient to enable FDH catalysis in planta,
2
[
26]
but co-expression of a reductase is required if cytosolic expression is desired. Regardless of
whether endogenous reductases may be able to supply FADH , many studies have shown that
2
increasing the local concentration of enzymes can increase flux through multistep enzymatic
[
31,32]
pathways.
Previous work has demonstrated that Baeyer-Villiger monooxygenases can be
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ChemBioChem
10.1002/cbic.201700391
+
[33]
genetically fused with NADP reductases, simplifying cofactor regeneration. Ferrodoxin and
flavodoxin reductase type domains can also be fused to cytochrome P450 heme domains to
[
34-38]
generate self-sufficient hydroxylation catalysts.
We therefore envisioned that an FDH-FR
fusion enzyme could be useful for a wide range of in vitro and in vivo applications.ꢀ
In vitro Characterization of FDH-FR Fusion Enzymes
The genes encoding wild-type RebH and RebF were genetically fused using three linkers
based on sequences used to create the functional P450-reductase fusion enzymes noted
[
34,35]
above.
These linkers consisted of 10, 16, and 22 amino acid residues (Figure 1A), and the
corresponding fusion enzymes are referred to as H-10-F, H-16-F, and H-22-F. The fusion
-
1
constructs were co-expressed with the pGro7 chaperone system in E. coli to afford 10-30 mg L
soluble protein following purification (Fig. S2). These yields are similar to those observed with
-
1
the MBP-RebF fusion (33 mg L ) often used in FDH bioconversions; however, higher yields are
[
39]
observed when expressing RebH with the chaperone system.
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ChemBioChem
10.1002/cbic.201700391
Figure 1. A) Fusion constructs encoded on a pET28 vector. B) Overview of cofactor
regeneration using fusion enzymes.
The activities of both the reductase and halogenase domains of the fusion enzymes were
next examined. Halogenase activity was established by comparing the yield of L-tryptophan
chlorination catalyzed by RebH in the presence of MBPF to the yields for the same reaction
catalyzed by H-10-F, H-16-F, and H-22-F (Figure 1B). All three fusion enzymes retained
substantial halogenase activity (Table 1, entries 2-4, 38-55% yield); however, lower chlorination
yields were observed relative to RebH/MBPF (Table 1, entry 1, 90% yield). Chlorination yields
were unaffected by the difference in linker length and amino acid composition between linkers
1
0 and 16 (55% and 56% yield, respectively), but the longest linker, 22, led to a lower yield
[
40]
(
38%). Reductase activity was established by measuring NADH oxidation, which occurred at
-
1
a similar rate for H-16-F and MBPF (k = 206 and 197 min , respectively, Fig. S3-4). ꢀ
cat
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ChemBioChem
10.1002/cbic.201700391
The apparent melting temperature (T ) of H-16-F was also compared with that of RebH
M
to determine if decreased stability, in addition to modestly decreased activity, might hinder its
performance. Apparent T values of 49.5 °C and 44.9 °C were obtained for RebH and H-16-F,
m
respectively, indicating that reduced stability of the fusion could indeed be compromising H-16-
F performance (Fig. S5). Previously, a thermostable flavin reductase from Bacillus subtilis (Fre)
[
41]
had been found compatible with FDH halogenation systems. This thermostable Fre was
therefore fused to RebH using the 16 amino acid linker with the goal of generating a fusion
enzyme with increased stability. This resulting fusion enzyme, RebH-16-Fre, also expressed as a
soluble protein, but it provided a lower yield for L-tryptophan chlorination (Table 1, entry 5,
3
0% yield) and had a comparable melting temperature (apparent T = 45 °C, Fig. S5) relative to
M
H-16-F.
[
42]
[16]
[22]
Three RebH variants previously engineered in our laboratory, 1K, 3SS, and 10S
were also fused to RebF via the 16 amino acid linker described above. These variants were
engineered for altered substrate scope (1K-E461K+R231K and 3SS-
S2P+M71V+G112S+K145M+N467T+N470S) and site selectivity (10S-
I52H+L380F+F465C+N470S+Q494R+R509Q). Soluble protein was obtained for all FDH fusion
enzymes, and bioconversions were conducted with the purified enzymes. As observed for H-16-
F, all three FDH fusion enzymes retained activity for their respective substrates (Table 1, entries
7
, 9, 11), but lower conversions were observed relative to the individual enzymes.
Bioconversions using H-16-F, 1K-F, 3SS-F, and 10S-F were scaled up, and the site-selectivity of
chlorination was found to be the same as the corresponding two-component FDH system in all
cases (see supporting information).
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ChemBioChem
10.1002/cbic.201700391
H
Cl
0.2-5 mol % FDH
cofactor regeneration system
1
0-100 mM NaCl, 25 mM HEPES
R
R
(5% DMSO), pH = 7.4
COOH
NH2
NH2
NH2
NH
COOH
N
H
N
H
N
H
7
8
9
10
Scheme 2. General reaction scheme for in vitro reactions on substrates 7-10.
Table 1. Aromatic chlorination catalyzed by different FDH or FDH-FR fusion enzymes.
a
Entry
FDH
RebH
H-22-F
H-16-F
H-10-F
H-16-Fre
3SS
3SS-16-F
1K
1K-16-F
10S
Substrate [FDH]
Yield
(%)
90.3
38.3
56.2
54.6
30.3
23.4
14.8
38.2
22.5
26.3
17.2
(
μM)
1.5
1.5
1.5
1.5
1.5
1
1
2
3
4
5
6
7
8
9
7
7
7
7
7
8
8
1
9
1
9
1
1
1
0
1
10
10
25
25
10S-16-F
a
-1
0
.5 mM substrate, 1-25 μM FDH, 9 U mL GDH, 10-100 mM NaCl, 20 mM glucose, 100 μM
NAD and FAD, 25 mM HEPES buffer pH = 7.4, 25 °C, 75 μL final reaction volume. 2.5 μM
reductase was added to reactions that did not contain a fusion enzyme. Reactions were quenched
with one volume MeOH. 0.5 mM phenol (for 7, 8, 10) or 0.5 mM benzoic acid (for 9) was added
as an internal standard, and reactions were analyzed by HPLC.
We sought to further understand the reduced yields of the fusion enzyme in vitro. No
significant change in reductase activity was observed (see above), and only a slight decrease in
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ChemBioChem
10.1002/cbic.201700391
the apparent melting temperature of H-16-F was observed relative to RebH. Although this
difference in T could be responsible for decreased yield, time courses revealed that decreased
m
catalyst lifetime was not observed. Based on these observations, we hypothesized that reduced
kinetic parameters of halogenase activity of H-16-F was primarily responsible for the its reduced
yield. We therefore measured the steady state halogenase kinetics for RebH and H-16-F. Because
[
40]
the K of RebH-catalyzed chlorination of L-tryptophan is low (~2 μM) , low substrate
m
concentrations and correspondingly low FDH concentrations (0.007 μM) were required to ensure
that rates were measured at <10% conversion. For preparative FDH-catalyzed reactions, we have
generally found that 2.5 μM FR provides satisfactory product yields regardless of FDH
[
16-18,22,39]
concentration on the reaction scales we have investigated.
While this concentration
represents an enormous excess of FR relative to the FDH concentrations noted above, we
reasoned that supplementing bioconversions involving either RebH or H-16-F with 2.5 μM FR
would allow for analysis of halogenase activity under conditions in which halogenase activity
[
40,43]
(
rather than FADH supply) limits overall product formation.
Under these conditions, similar
and Km-H-16-F = 0.7 μM); however, a nearly
m-RebH
2
K values were observed for both halogenases (K
m
two-fold decrease in k is observed for the fusion H-16-F compared to that of RebH (k
=
cat
cat-RebH
-
1
-1
4
.26 min and k
= 2.34 min , Table 2, entries 1 and 2).
cat-H-16-F
Kinetic parameters were also obtained for H-16-F with no added reductase, conditions
under which H-16-F is the only source of FADH . Interestingly, a much lower k was observed
2
cat
-
1
under these conditions (0.14 min , Table 2, entry 4) relative to those noted above. Presumably,
because the concentration of FR was >80-fold lower than previous experiments, the supply of
FADH to RebH, which involves both the rate of FAD reduction and non-catalyzed FADH2
2
[
40,43]
oxidation prior to RebH binding, could become rate limiting.
It is possible that the lower
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ChemBioChem
10.1002/cbic.201700391
concentration of FADH is insufficient to saturate RebH under the conditions used, leading to the
2
lower rates of chlorination observed. To test whether the k of the two-component system is also
cat
significantly affected by the concentration of reductase, steady state kinetic parameters were
obtained for RebH under analogous conditions 1:1 RebH/MBPF. A lower k was again
cat
-
1
observed (0.18 min , Table 2, entry 3). Surprisingly, at this lower concentration of reductase
(
and thus lower concentration of FADH ), there appears to be little difference between the
2
chlorination rates of RebH and H-16-F. This would suggest that as the local concentration of
reductase is important in the comparative halogenase activity between the single and two-
component systems, however, further detailed analysis will be necessary to fully understand this
observation.
a
Table 2. Kinetic parameters for aromatic chlorination catalyzed by RebH and H-16-F.
Entry FDH
[H-16-F] [RebH] [RebF]
kcat
(min )
4.26±0.13 0.7±0.1
2.34±0.07 0.7±0.1
0.18±0.01 0.7±0.2
0.14±0.01 0.9±0.3
Km
(μM)
-
1
(μM)
(μM)
0.007
-
(μM)
2.5
2.5
0.03
-
1
2
3
4
RebH
H-16-F
RebH
-
0.007
-
0.03
0.03
-
H-16-F
a
-1
0
.5-15 μM L-tryptophan, 0.007-0.03 μM FDH, 0.03-2.5 μM reductase, 9 U mL GDH, 10 mM
NaCl, 20 mM glucose, 100 μM NAD and FAD, 25 mM HEPES buffer pH = 7.4, 25 °C, 75 μL
final reaction volume. Reactions were quenched with MeOH 5-20 minutes after reaction
initiation. 0.5 mM phenol was added as an internal standard, and reactions were analyzed by
HPLC. Saturation plots used to calculate values can be found in the supporting information (Fig.
S8-11).
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ChemBioChem
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In addition to using FDH-FR fusion enzymes to facilitate directed evolution efforts, we
also envisioned that they could provide higher product yields in whole cell bioconversions. Our
kinetic data support the hypothesis that as reductase concentration decreases, the use of fusion to
generate higher local concentration of FADH becomes more relevant. Within a cell, the local
2
concentration of FADH could greatly impact FDH activity. To test whether the fusion would
2
outperform the two-component system in vivo, we examined the halogenase activity of E. coli
BL21 pGro7 cells transformed with H-16-F, RebH, RebH+RebF, and RebH+MBPF (note that
RebH+RebF and RebH+MBPF cells contain these two enzymes on two separate plasmids).
Cultures were grown to an OD600 of 0.85-0.9, IPTG (100 µM) and L-arabinose (2 mg/mL) were
added to induce halogenase expression, and solutions of L-tryptophan (7, 1 mM) and NaCl (100
mM) were added to initiate the halogenation reactions. High titers of 7-chlorotryptophan were
obtained following incubation at 30 °C for 24 h, and a 2.5-fold increase in product concentration
was observed for cells containing H-16-F relative to RebH, RebH+RebF, or RebH+MBPF
(Figure 2). Excited by this result, we sought to demonstrate in vivo chlorination on a non-native
substrate. Because E. coli cells contain significant quantities of L-tryptophan, we used an
engineered FDH that does not halogenate L-tryptophan. Variant 1K was found to have greatly
reduced activity on L-tryptophan, and in competition reactions between L-tryptophan and
anthranilic acid (9), no conversion of tryptophan is observed (see supporting information).
-
1
-1
Significantly higher titers (55.01 mg L with 1K-16-F, 15.21 mg L with 1K+MBPF) were
obtained with this enzyme in vivo (Figure 2).
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ChemBioChem
10.1002/cbic.201700391
1
60
40
20
00
COOH
NH2
COOH
NH2
1
1
1
N
H
N
H
Cl
8
6
4
2
0
0
0
0
0
H-16-F
RebH
RebH+RebF RebH+MBPF
7
0
NH2
NH2
6
5
4
3
2
1
0
0
0
0
0
0
0
COOH
Cl
COOH
1
K-16-F
1K
1K+RebF
1K+MBPF
ꢀ
Figure 2. In vivo biocatalysis with H-16-F and 1K-16-F to afford chlorinated L-tryptophan (7)
and anthranilic acid (9), respectively. Upon induction of expression of 50 mL cultures in TB
media, 1 mM substrate and 100 mM NaCl were added. Cultures were expressed for 24 hours at
3
0 °C, and aliquots of the supernatant were analyzed by HPLC. Three independent trials of
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ChemBioChem
10.1002/cbic.201700391
triplicate cultures were performed for each cell line, and resulting standard deviations are shown
as error bars (n = 9).
Because lower yields of purified, soluble enzyme were produced for in vitro studies using
the fusion FDH-FRs, we anticipated that the observed increase in product formation in vivo was
the result of higher local concentration of FADH , not increased fusion expression relative to the
2
corresponding FDHs. An SDS-PAGE gel and a western blot for the soluble fractions of in vivo
bioconversions confirmed low levels of soluble fusion expression for H-16-F compared to RebH
(see supporting information, Fig. S12-13). While this does not unequivocally confirm our
hypothesis, it does qualitatively suggest that increased enzyme expression is not responsible for
high product titers in vivo using the fusion system.
In summary, we have demonstrated that functional FDH-FR fusion enzymes can be
engineered using different linkers, FDHs, and reductases. Although a slight reduction in activity
is observed for these enzymes compared with their corresponding two-component systems in
vitro, the use of fusion enzymes could simplify FDH engineering efforts by eliminating the need
for added FR. In addition, higher product titers are observed when FDH-FR fusion enzymes are
used for in vivo biocatalytic transformations. These systems could therefore serve as valuable
tools for in vivo chlorination in several different organisms, and efforts are currently underway in
our laboratory to engineer systems that provide increased product titers for large-scale
halogenation in E. coli.
Acknowledgements
This work was supported by the NIH (1R01GM115665). MCA was supported by
an NSF predoctoral fellowship (DGE-1144082).
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ChemBioChem
10.1002/cbic.201700391
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