Discovery of Novel Bacterial Chalcone Isomerases by a Sequence-Structure-Function-Evolution Strategy for Enzymatic Synthesis of (S)-Flavanones

Article abstract of DOI:10.1002/anie.202107182

Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHI_era) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence-Structure-Function-Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHI_era according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)-selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)-flavanones in high yields.

Full text of DOI:10.1002/anie.202107182

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Accepted Article
Title: Discovery of Novel Bacterial Chalcone Isomerases by a
Sequence–Structure–Function–Evolution Strategy for Enzymatic
Synthesis of (S)-Flavanones
Authors: Hannes Meinert, Dong Yi, Bastian Zirpel, Eva Schuiten,
Torsten Geißler, Egon Gross, Stephan I. Brückner, Beate
Hartmann, Carsten Röttger, Jakob P. Ley, and Uwe
Bornscheuer
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COMMUNICATION
Discovery of Novel Bacterial Chalcone Isomerases by a
Sequence–Structure–Function–Evolution Strategy for Enzymatic
Synthesis of (S)-Flavanones
Hannes Meinert[a]†, Dong Yi*[a]†, Bastian Zirpel[b], Eva Schuiten[a], Torsten Geißler[b], Egon Gross[b],
Stephan I. Brückner[b], Beate Hartmann[b], Carsten Röttger[b], Jakob P. Ley*[b], and Uwe T.
Bornscheuer*[a]
[a]
B. Sc. Hannes Meinert, Dr. Dong Yi, M. Sc. Eva Schuiten, Prof. Dr. Uwe T. Bornscheuer
Department of Biotechnology & Enzyme Catalysis
Institute of Biochemistry, Greifswald University
Felix-Hausdorff-Straße 4, 17489 Greifswald, Germany
E-mail: dong.yi@uni-greifswald.de; uwe.bornscheuer@uni-greifswald.de
Dr. Bastian Zirpel, Dr. Torsten Geißler, Dr. Egon Gross, Dr. Stephan I. Brückner, Beate Hartmann, Carsten Röttger, Dr. Jakob P. Ley
Symrise
P.O. Box 1253, 37603 Holzminden, Germany
E-mail: jakob.ley@symrise.com
[b]
†
Equal contribution
Supporting information for this article is given via a link at the end of the document.
Abstract: Chalcone isomerase (CHI) is a key enzyme in the
biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera
chemical messengers, physiological regulators, cell cycle
inhibitors, and antibiotics against plant diseases.[1] In our long
history of using medicinal herbs, there is substantial evidence that
flavonoids show potential beneficial pharmacological properties
for humans, such as anti-infection, anti-inflammatory, anti-oxidant,
anti-tumor, anti-aging and immune response regulation.[2]
Recently, some flavonoids have been highlighted as potential
medicine with anti-coronavirus activity.[3] Due to their various
potential physiological benefits to humans, the study of the
biosynthesis of flavonoids has been a research hotspot for several
decades.[4] The synthesis of flavonoids in plants is derived from
the phenylpropanoid pathway (Scheme 1a).[4] Chalcone
isomerase (CHI) cyclizes chalcones to form (S)-naringenin which
is one of the central intermediates for the diversification of
flavonoids. As one of the key enzymes in the natural pathways of
flavonoid biosynthesis, plant CHIs have been well studied[5] with
respect to their catalytic mechanism,[6] protein structure,[6b]
classification,[7] and evolution.[8]
)
was identified from Eubacterium ramulus, but its distribution,
evolutionary source, substrate scope, and stereoselectivity are still
unclear. Here, we describe the identification of 66 novel bacterial CHIs
from Genbank using
a
novel Sequence–Structure–Function–
Evolution (SSFE) strategy. These novel bacterial CHIs show diversity
in substrate specificity towards various hydroxylated and
methoxylated chalcones. The mutagenesis of CHIera according to the
substrate binding models of these novel bacterial CHIs resulted in
several variants with greatly improved activity towards these
chalcones. Furthermore, the preparative scale conversion catalyzed
by bacterial CHIs has been performed for five chalcones and revealed
(S)-selectivity with up to 96% ee, which provides an alternative
biocatalytic route for the synthesis of (S)-flavanones in high yields.
Nature produces an enormous variety of biomolecules, among
which flavonoids are a large class of secondary metabolites
mainly derived from plants. Flavonoids have many functions in
plants, such as flower coloration, UV filtration, symbiotic nitrogen
fixation, and floral pigmentation. In addition, they also act as
The flavonoids contained in plants need to be degraded by
microorganisms. Especially for some flavonoids with antibacterial
activity, microorganisms have
a
O
O
O
O
evolved
biodegradation
ACC
3
3
HO
OH
3 CoA
HO
SCoA
CHS
pathways to resist such
flavonoids. Therefore, studying
the pathway of microbial
degradation of flavonoids can
provide more clues about the
biosynthesis and structural
modification of flavonoids.[10]
The first bacterial CHI activity in
Eubacterium ramulus for the
OH
OH
O
O
Plant
CHI
HO
HO
OH
O
HO
HO
O
O
4CL
OH
SCoA
*
S
HO
HO
HO
CoA
OH
OH
OH
OH
b
OH
OH
OH
OH
Bacterial
CHI
?
O
O
OH
O
HO
O
O
OH
O
Reductase
R
*
*
S
OH
OH
anaerobic
degradation
of
Predicted stereoselectivity
of bacterial CHI
flavonoids was proposed by
Schneider et al. in 2000.[10a] The
Scheme 1. Biosynthesis (a)[4] and predicted biodegradation pathways (b)[9] of flavanones. 4CL: 4-coumarate:CoA
ligase, ACC: acetyl-CoA carboxylase, CHS: chalcone synthase, CHI: chalcone isomerase, Reductase: enoate
reductase or flavanone- / flavanonol-cleaving reductase, and CoA: coenzyme A.
enzyme (CHIera
isolated from the cell-free
extracts of E. ramulus, which
)
was then
1
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10.1002/anie.202107182
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presented activity towards naringenin chalcone, isoliquiritigenin
and butein.[11] After ten years without further reports, the gene of
this bacterial CHI was identified from the
genome of E. ramulus and cloned into
Escherichia coli for recombinant expression
in our group.[12] Furthermore, we determined
its crystal structure and suggested the
reaction mechanism as reversible Michael
addition catalyzed by His33 as a catalytic
base abstracting the proton on C3, as well as
its evolutionarily uniqueness according to the
homology search within protein databases.[13]
This bacterial CHI was then revealed to be a
key enzyme catalyzing the conversion of the
and this would shine light on the evolutionary origin of bacterial
CHIs. The original host sources included in the dataset show that
flavanone
dihydrochalcone, together with an enoate
reductase/flavanone- and flavanonol-
and
flavanonol
to
the
cleaving reductase (ERED/Fcr) (Scheme
12]
1b).[9,
Surprisingly, this is still the only
bacterial CHI reported so far. However, the
flavonoid structures are highly diverse due to
the extensive hydroxylation, methylation, and
glycosylation modifications on the C6-C3-C6
carbon framework. In addition to the huge
amount of plant spoilage created every year,
if the flavonoid degradation pathway involves
a CHI as an important microbial degradation
step, it is hard to imagine that microbial CHIs
are rare. Besides, the substrate scope,
stereoselectivity, evolution and physiological
role of bacterial CHIs in the degradation of
flavonoids are also still unclear. These
Figure 1. Phylogenetic tree of potential bacterial CHIs. A full version is included in Figure S2. The branch
where CHIera locates is highlighted in black.
bacterial CHI-like proteins are widespread, with some intestinal
properties are not only important to understand the function and
substrate scope of CHIs, but at the same time allow access to a
toolbox of synthetically useful biocatalysts to make a range of
important flavonoids relevant for the flavor market. Herein, we
report the discovery of novel bacterial CHIs and their substrate
specificity differentiation as well as stereoselectivity. We have
bacteria, such as Clostridium, Eubacterium, Roseburia,
Lachnoclostridium, Mogibacterium, Hologhaga, Butyrivibrio and
Mobilibacterium, but also some environmentally derived bacteria,
including Vibrio and Aminicella, the thermophilic bacteria
Tepidanaerobacter and some acetogenic bacteria such as
Acetoanaerobium and Halophaga. These bacteria include Gram-
negative and Gram-positive strains, but are mostly anaerobic or
facultatively anaerobic bacteria. This indicates that the metabolic
pathway involved in bacterial CHIs might require an anaerobic
environment.
designed
a Sequence–Structure–Function–Evolution (SSFE)
strategy to identify novel and interesting candidate CHIs from
protein databanks to expand the substrate scope of known CHIs
for the biosynthesis of flavanones.
Gene mining based on a known protein sequence is an
effective strategy to find novel enzymes from gene or protein
databases. Phylogenetic analysis allows us to determine highly
probable evolutionary relations in datasets of protein or DNA
sequences based on the maximum likelihood method.[14] This can
be used to identify sequences with potentially similar or related
activities (SSFE, Sequence step). First, we searched the non-
redundant protein sequence database of Genbank with the
protein sequence of CHIera and the fragment sequences of its
catalytic and solvent-exposed domains[13] as query to collect the
protein sequences of bacterial CHI-like proteins and further
constructed a phylogenetic tree (Figure 1, a full version is included
in Figure S2). The phylogenetic tree consists of two main
branches, where most sequences are included in branch A (67
proteins), with sub-branches 1-13, including the sub-branch 7
(highlighted in black) with our starting sequence CHIera. The other
branch (branch B) consists of an enzyme cluster with relatively
lower sequence similarity to CHIera. However, it is still possible
that one may find related activities and sequences in this branch
In the phylogenetic analysis, the bacterial CHI-like proteins
are divided into clusters based on their sequence similarity.
However, enzymatic properties can be predicted not only from
homology of the sequences and residues in the catalytic site, but
also from the substrate binding pockets and protein folding
structures. These attributes are poorly reflected in the alignment
of protein sequences due to the flexibility of peptide folding and
our general lack of understanding of protein folding. Therefore,
systematic analysis of the sequence and structural characteristics
of proteins on each branch can effectively predict the activity and
partial structure-related enzymatic properties of these proteins
(SSFE, Structure step). We submitted all 88 protein sequences of
the phylogenetic tree to SWISS-MODEL[15] to model their three-
dimensional structures. The alignment of each simulated
structure to the crystal structure of the CHIera shows that all
proteins in the phylogenetic tree have quite similar folds,
especially the ones located on branch A. CHIera contains two
unique ferredoxin-like domains; the catalytic domain and the
solvent-exposed domain.[13] From the structure alignment (Figure
2
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2a), it is clear that the folding of proteins in branch A is conserved
and consistent with CHIera. However, the catalytic domain and the
solvent-exposed domain are slightly different for the proteins in
branch 14, as well as the branches 15 to 18. Extra residues
between the α-helix (α6) and β-sheet (β6)[13] in the solvent-
confusion (Table S1). From this list, it becomes obvious that all
proteins in branch A contain the key catalytic residue His33, which
can be considered as a clear indication of CHI activity. Tyr48 is
also highly conserved, which stabilizes 9=O.[13] Moreover, the
Gln69, Thr71 and Gln101 residues responsible for interactions
exposed domain form loops with high flexibility (Figure 2a, Loop1). with the A-ring of the substrate are also highly conserved, except
The α-helixes (α3, α4 and α5)[13] in the catalytic domain are
completely restructured as flexible loops (Figure 2b, Loop2). Due
to this significant difference in the catalytic domain structures to
CHIera, we predicted that the proteins in branch B might have no
CHI activity, which has been proved further by activity screening
(data not shown). Therefore, further analysis mainly focused on
the proteins in branch A.
for the proteins in the branches 1 and 2 distantly located from
CHIera in branch A. Regarding to the relatively few structural
modifications on the A-ring of flavanones, such a conservative A-
ring binding pocket is in line with our expectations to fit the limited
variety. In contrast, the binding and positioning of amino acids in
the B-ring binding are much less conserved. The most important
differences are found in the binding amino acids Asn122, Asp79,
Lys87, and Gln40. The variety of amino acids at these sites
includes polar and non-polar amino acids with long and short side
chains, which seems to suggest that both hydroxyl and methoxy
modifications on the B-ring of chalcones are accepted. As we
speculated, there might exist more bacterial CHIs in nature
adapted to the diversity of flavonoid structures. The diverse active
pockets indicate that these potential bacterial CHIs very likely
have different substrate specificities.
Scheme 2. Bioconversion of chalcones to (S)-flavanones by novel bacterial
CHIs and CHI mutants.
Several sequences containing representative substrate binding
pockets from each branch (13 in total) were selected for gene
synthesis and characterization (SSFE, Function step). All
candidate proteins (wild-type CHIs from phylogenetic analysis, all
recombinantly expressed in E. coli and used as purified enzymes)
were tested for the conversion of a range of chalcones with
modifications in their A- and B-rings, including naringenin
chalcone (1a), eriodictyol chalcone (2a), homoeriodictyol
chalcone (3a), hesperetin chalcone (4a) and 4-O-methylbutein
(5a) (Scheme 2). The results are shown in Figure 3 and Table S2.
Among the 14 wild-type bacterial CHIs (including CHIera), CHIera
presented surprisingly high activity towards 1a (270 U/mg). CHI6
belongs to the same branch with the same active pocket as CHIera
However, its activity towards 1a is only 68.3 U/mg, but 123.6 U/mg
towards 2a. Therefore, their activity differences might be caused
by the entire enzyme sequence and structure, rather than just a
more suitable substrate binding model. 3a contains a meta-
methoxy moiety in the B-ring, leading to an approx. 50% decrease
in activity for most wild-type CHIs in comparison to 2a. CHI2 and
CHI4 have relatively good activity towards 4a (93.5 U/mg and 84.5
U/mg, respectively). Both have a proline (P79) instead of an
aspartate (D79) as in CHIera. This residue causes less steric
hindrance, allowing the active site to accept the para-methoxy
moiety present in the B-ring. Compared to 4a, 5a does not contain
a hydroxyl moiety at C6’. Although the hydroxyl group at C2’ can
form a C6’ hydroxyl group due to the rotation of the C1’-C9 bond,
this is not the energy minimum structure of 5a. Therefore, the
Figure 2. (a) Examples of the overall structure analysis of potential bacterial
CHIs. Green: catalytic domain of CHIera. Red: solvent-exposed domain of CHIera
.
Blue: (R)-naringenin. Orange: simulated protein structure of WP_077390608.1
from branch 11. Cyan: simulated protein structure of WP_105292327.1 from
branch 17. (b) Active pockets of CHIera. Orange: naringenin. Magenta:
mutagenesis sites of CHIera
.
Next, we focused on the catalytic domains, particularly the active
pockets, to assign potential activities and substrates to the
sequences in each sub-branch. According to the crystal structure
of CHIera[13], the substrate binding pocket contains 21 residues
(Figure 2b), where Gln101 binds 7-OH, Glu69 and Thr71 have co-
hydrogen binding to 6’-OH in the chalcone, Arg125 probably
stabilizes the 2’-OH together with Tyr48 binding to 9=O, His33 is
the key residue for Michael addition, and Asn122, Asp79, Lys87,
and Gln40 are corresponding residues for 4-OH binding. In
addition, Phe135, Ile29, Val14, Phe50 and Ile12 are involved in
the binding pocket for the A-ring in the flavanone/chalcone, while
Arg125, His73, Ser37, Glu91, Trp75 and Phe41 are related to the
B-ring binding. We made a list of residues in the active pockets of
the unknown sequences corresponding to these catalytically
relevant residues. The residues are described by numbers that
refer to the respective amino acid positions in CHIera to avoid
.
3
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specific activity of most wild-type CHIs towards 5a is less than
1 U/mg (Table S2). CHI7 shows the highest activity of up to 1.2
U/mg. The activity screening of potential bacterial CHIs confirmed
CHI activity for all candidate enzymes. Thus, it can be inferred
that the proteins present in branch A are likely all CHIs (66 novel
bacterial CHIs plus CHIera). The relatively low activity of some
enzymes might be improved by fine-tuning of the reaction
conditions. For example, in order to improve the stability of
chalcones in aqueous solution, the reaction was performed at
alkaline conditions, pH 8, which might not be the optimal pH for
bacterial CHIs.[12] In addition, CHI1 is close to the root of the
phylogenetic tree, but shows almost the same activity to various
chalcones. Therefore, it can be speculated that the CHI activity
was obtained at the root of the phylogenetic tree and has
continued to differentiate into various substrate specificities.
The systematic analysis of the CHI sequences and
structures and activity screening above provides useful hints
about the natural evolution of active pockets for bacterial CHIs.
We believe that this natural diversity is most likely a shortcut to
guide the mutagenesis of bacterial CHIs leading to extended
substrate scope (SSFE, Evolution step). We introduced mutations
in the active pocket of CHIera (12 variants in total) to cover most
substrate pocket types as identified from the structure analysis
and the phylogenetic tree. Among the 12 CHIera variants, all lost
the high activity towards 1a (Figure 3). However, the activity of
CHIeraMut5 towards 2a (181.3 U/mg) is doubled compared to the
wild-type and this variant also has improved activity towards 3a
(62.2 U/mg). From simulations of the active pocket of CHIera-Mut5
with the substrate (Figure S4), it seems that K87R and D47E offer
hydrogen bonding to the para-hydroxyl group and slightly shift the
binding position of the B-ring in the active pocket, which probably
results in better binding of the meta-hydroxyl group to E91.
Compared with the wild-type CHIera, CHIera-Mut11 shows greatly
improved activity towards 4a (107.1 U/mg), similar to CHIera-Mut8
(96.4 U/mg), CHIera-Mut1 (93.2 U/mg), CHIera-Mut6 (83.9 U/mg),
and CHIera-Mut9 (75.5 U/mg). CHIera-Mut6, 8, 9 and 11 contain
either a proline or isoleucine at the 79 position like CHI2, which
has similar activity towards 4a (93.5 U/mg). However, CHIera-Mut1
and 3 have a D79E mutation which contains an even longer side
chain. Alternatively, R125 in CHIera-Mut1 and 3 is replaced by Gly
to offer more room to adapt the para-methoxy moiety of 4a. This
provides a new substrate binding pocket for improving the activity
of bacterial CHIs towards 4a. CHIera-Mut11 has the highest activity
towards 5a (1.4 U/mg) of all variants. Although these CHIera
variants only represent some of the substrate binding pockets
included in the phylogenetic tree, these results clearly show that
the activity towards modified B-ring chalcones can be improved
greatly by semi-rational design based on the results from
Sequence–Structure–Function analysis. In some cases, an
extensive collection of natural diversity can offer shortcuts to
obtain suitable pockets for improved substrate binding.
Figure 3. Substrate scope of bacterial CHIs (a) and CHIera mutants (b). The table of mutations shows variants in the substrate binding pocket of wild-type CHIs and
CHIera mutants. The reaction was carried out with 100 μM chalcones in phosphate buffer (50 mM, pH 8.0).
4
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Aside from the substrate scope of the novel bacterial CHIs, we
investigated the stereoselectivity. Plant-derived CHIs have been
confirmed to have a strict (S)-stereoselectivity.[5] However,
ERED/Fcr can directly reduce (R)-naringenin, which indicates that
bacterial CHIs are probably also able to isomerize (S)-naringenin
to the (R)-isomer through its chalcone in aqueous solution and
form a cascade reaction with ERED/Fcr to convert flavanones to
dihydrochalcones.[9] However, direct evidence of bacterial CHIs
with (R)-stereoselectivity is missing. Stereoselectivity was now
determined using 1a as substrate and mainly (S)-naringenin was
detected in the reaction with a ratio of approx. 9:1 (S:R) for CHIera
and the novel identified bacterial CHIs (CHI1-CHI13, Figure S5a).
Considering to the rapid self-cyclization of chalcones in aqueous
solution to generate racemic flavanones, this optical purity
measurement proved that bacterial CHIs are (S)-selective.
Furthermore, we also determined in this work the stereoselectivity
Keywords: biocatalysis • chalcone • chalcone isomerase •
flavonoid • flavanone
[1]
[2]
M. L. F. Ferreyra, S. P. Rius, P. Casati, Front. Plant Sci. 2012, 3, 222.
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T. Goris, A. Perez-Valero, I. Martinez, D. Yi, L. Fernandez-Calleja, D.
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of the CHI and mutants with the highest activity (CHIera, CHIera
-
Mut5 and CHIera-Mut11) towards 1a–5a (Figure S5b). These three
enzymes presented relatively strict (S)-stereoselectivity towards
1a and 4a, but almost no stereoselectivity for 2a and 3a, which
shows the great influence of the structure and position of the
substituents on the B-ring on the stereoselectivity of the CHIs.
CHIera-Mut11 showed high stereoselectivity (9:1) towards 4-O-
methylbutein. Although we could not identify whether it is (R)- or
(S)-stereopreference – due to the lack of standard compounds –
we speculate that CHIera-Mut11 is very likely to have (S)-
stereoselectivity for 4-O-methylbutein, referring to the same B-
ring structure of 4-O-methylbutein and hesperetin and their similar
chiral-HPLC results. These results show that the previously
assumed degradation approach of (S)-naringenin through
chalcone, (R)-naringenin, and dihydrochalcone in bacterial
(Scheme 1b)[9] is most likely incorrect. Since the natural active
flavanones are all in the (S)-configuration, these bacterial-derived
CHIs with various substrate regioselectivities provide a diverse
toolkit of enzymes to replace the plant CHIs for the heterologous
synthesis of (S)-flavanones, also keeping in mind that the
bacterial CHIs are much easier to express recombinantly.
Therefore, we have used CHIera, CHIera-Mut5 and CHIera-Mut11 as
biocatalysts and achieved the enzymatic preparative scale
synthesis of naringenin, eriodictyol, homoeriodictyol, hesperetin,
and 7,3'-dihydroxy-4'-methoxyflavanone with yields of 85-100%,
respectively.
[9]
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Lepore, T. Schwede, Nucleic Acids Res. 2018, 46, W296-W303.
In summary, we successfully identified novel bacterial CHI-
like proteins from Genbank using the SSFE strategy and
confirmed them to have CHI activity. Several wild-type CHIs as
well the designed variants show high activity towards several
chalcones. Thus, the SSFE strategy has been demonstrated as a
useful tool to mine and improve novel and useful candidate
enzymes. Finally, we confirmed that bacterial CHIs have (S)-
stereopreference, which provides an alternative biocatalytic
pathway for the synthesis of (S)-flavanones with high yields.
Acknowledgements
This work was supported by the European Union’s Horizon 2020
Research and Innovation Programme under Grant Agreement no.
814650 for the project SynBio4Flav.
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10.1002/anie.202107182
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
Novel bacterial chalcone isomerases and their variants identified by a Sequence–Structure–Function–Evolution (SSFE) strategy are
able to convert chalcones to flavanones with diverse substrate scopes and strict (S)-stereopreference enabling a biocatalytic route to
(S)-flavanones.
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This article is protected by copyright. All rights reserved.