A Reconstructed Common Ancestor of the Fatty Acid Photo-decarboxylase Clade Shows Photo-decarboxylation Activity and Increased Thermostability

Article abstract of DOI:10.1002/cbic.202000851

Light-dependent enzymes are a rare type of biocatalyst with high potential for research and biotechnology. A recently discovered fatty acid photo-decarboxylase from Chlorella variabilis NC64A (CvFAP) converts fatty acids to the corresponding hydrocarbons only when irradiated with blue light (400 to 520 nm). To expand the available catalytic diversity for fatty acid decarboxylation, we reconstructed possible ancestral decarboxylases from a set of 12 extant sequences that were classified under the fatty acid decarboxylases clade within the glucose-methanol choline (GMC) oxidoreductase family. One of the resurrected enzymes (ANC1) showed activity in the decarboxylation of fatty acids, showing that the clade indeed contains several photo-decarboxylases. ANC1 has a 15 °C higher melting temperature (T_m) than the extant CvFAP. Its production yielded 12-fold more protein than this wild type decarboxylase, which offers practical advantages for the biochemical investigation of this photoenzyme. Homology modelling revealed amino acid substitutions to more hydrophilic residues at the surface and shorter flexible loops compared to the wild type. Using ancestral sequence reconstruction, we have expanded the existing pool of confirmed fatty acid photo-decarboxylases, providing access to a more robust catalyst for further development via directed evolution.

Full text of DOI:10.1002/cbic.202000851

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A Reconstructed Common Ancestor of the Fatty Acid
Photo-decarboxylase Clade Shows Photo-decarboxylation
Activity and Increased Thermostability
Yue Sun,[a] Elia Calderini,[a] and Robert Kourist*[a]
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°
Light-dependent enzymes are a rare type of biocatalyst with
high potential for research and biotechnology. A recently
discovered fatty acid photo-decarboxylase from Chlorella varia-
bilis NC64A (CvFAP) converts fatty acids to the corresponding
hydrocarbons only when irradiated with blue light (400 to
520 nm). To expand the available catalytic diversity for fatty
acid decarboxylation, we reconstructed possible ancestral
decarboxylases from a set of 12 extant sequences that were
classified under the fatty acid decarboxylases clade within the
glucose-methanol choline (GMC) oxidoreductase family. One of
the resurrected enzymes (ANC1) showed activity in the
decarboxylation of fatty acids, showing that the clade indeed
contains several photo-decarboxylases. ANC1 has a 15 C higher
melting temperature (Tm) than the extant CvFAP. Its production
yielded 12-fold more protein than this wild type decarboxylase,
which offers practical advantages for the biochemical inves-
tigation of this photoenzyme. Homology modelling revealed
amino acid substitutions to more hydrophilic residues at the
surface and shorter flexible loops compared to the wild type.
Using ancestral sequence reconstruction, we have expanded
the existing pool of confirmed fatty acid photo-decarboxylases,
providing access to a more robust catalyst for further develop-
ment via directed evolution.
Introduction
While previously known oxidative fatty acid decarboxylases
such as OleT[8] from Jeotgallicoccus and UndA/B[9] from
Pseudomonas produce terminal olefins; CvFAP can produce
industrially attractive compounds with low consumption of
energy using renewable starting material. Furthermore, it can
not only convert carboxylic acids into hydrocarbons but also
can recognize cis/trans isomers and stereoisomers.[10] Addition-
ally, the range of compounds that can be converted to alkanes
could be increased in cascade reactions. For example, Holl-
mann’s group reported the combination of CvFAP with lipases
to convert waste material to more valuable products.[11,12] The
photoenzymatic cascades comprise CvFAP to transform unsatu-
rated fatty acid in alkenes and subsequently an hydratase
converts them into alcohol. Here, CvFAP lacks the chloroplast
targeting sequence at the enzyme’s Nterminus (sCvFAP), as it
was shown to increase both conversion and enzyme’s
production.[13]
Light is essential for sustaining life on earth: It is essential to
generate carbohydrates and oxygen in plants and microalgae.[1]
Photochemical reactions are fundamental for photosynthesis,
vision and the biosynthesis of vitamin D,[2] among the most
relevant. Nevertheless, if we do not consider photosynthesis, a
surprisingly small number of photoenzymatic reactions have
been identified, these being the photolyases involved in the
repair of DNA damage[3] and the photo-biocatalytic reaction for
the reduction of C=C double bonds by protochlorophyllide
reductases.[4] The successful exploitation of coupling photo-
system with biocatalysis spun into the new research field
photo-biocatalysis.[5,6] Recently, a new type of light-dependent
enzymes has been discovered in the microalgae C. variabilis: the
fatty acid photo-decarboxylase (CvFAP).[7] The enzyme belongs
to the glucose-methanol choline (GMC) oxidoreductase family,
which comprises a wide range of oxidases and dehydrogenases.
The conversion of (waste) carboxylic acids and oils into
biofuels and corresponding synthetic applications has been the
focus of research for decades.[14,15]
Unfortunately, synthetic applications of CvFAP are limited
by its relatively poor production in Escherichia coli, its light
stability and its sensitivity to solvents.[11,16,17] All this results in a
limited product formation, which cannot pay off the effort for
the production of the enzyme. On top of the low stability,
production yields remain low, which is an obstacle for both the
enzyme’s biochemical characterization and industrial applica-
tion – even though the purification under red light was
beneficial.[10,11,13] Engineering of enzymes is a proficient method
to increase their operational stability.[18–20] Despite its recent
discovery, several groups reported enzyme engineering studies
[a] Y. Sun, Dr. E. Calderini, Prof. Dr. R. Kourist
Institute of Molecular Biotechnology
Graz University of Technology
Petersgasse 14, 8010 Graz (Austria)
E-mail: kourist@tugraz.at
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbic.202000851
This article is part of a joint Special Collection with ChemCatChem on
PhotoBioCat – Light-Driven Biocatalysis. Please see our homepage for
more articles in the collection.
© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
to
improve
CvFAP’s
substrate
spectrum
and
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enantioselectivity.[10,21] While changes in catalytic activity or
selectivity can be achieved with a small number of mutations,
improvements in stability and production in bacteria often
require a higher number of mutations. So far, predicting
combinations of many mutations is out of our understanding or Results and Discussion
current computational power. Ancestral sequence reconstruc-
tion (ASR) is the probabilistic reconstruction of ancient protein
sequences based on extant genes. It has emerged as a
proficient method to produce functional proteins that keep the
aspects of the protein family.[22,23] Reconstructed enzymes may
not only have a broader substrate range compared to their
modern descendants,[24] but often show improved thermo-
stability and solvent tolerance.[25,26] Therefore, we hypothesized
that the common ancestor in the FAP clade identified by
Sorigue et al.[7] would still show photo-decarboxylation but with
improved stability and/or soluble production in E. coli as
demonstrated previously.[27,28] To verify this, we set out to
reconstruct three possible ancestors of this clade and inves-
tigate their photo-decarboxylative activity.
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ASR of photo-decarboxylases produces functional enzymes
The typical first step of any ancestral sequence reconstruction is
the collection of a set of recent homologous sequences. The
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most straightforward way to gather
a large number of
sequences is a similarity search. The major downfall is that
public databases contain more sequences than needed for a
successful ASR. In our case, a second risk was present as CvFAP
is a member of the glucose-methanol-choline (GMC) super-
family. Thus, a BLAST search would produce a highly unbal-
anced dataset with the risk to lose the light-dependent activity
and produce an ancestral oxidoreductase. Giving the extremely
rare frequency of photoenzymes, we decided to only include 12
sequences that were classified in the fatty acid photo-decarbox-
ylases clade by Sorigue and co-workers where only two
enzymes had confirmed photo-decarboxylation activity at the
time of the reconstruction, lately during the experimental
characterization more photo-decarboxylases were experimen-
tally verified (Figure 1).[7,29] All sequences belong to the algal
genus. As ancestral enzymes have shown before to have an
improved soluble production in E. coli, the expectation was that
the same could be achieved for the photo-decarboxylases.[27,28]
Aware of the limitation of our dataset, we performed three
different reconstructions named ANC1, ANC2 and ANC3 using
different sequence alignment tools and evolutionary models to
increase the probability to obtain an active variant. The
sequences of the node 0 elements from the three different
reconstructions were ordered as codon-optimized synthetic
genes. The sequence identity with the wild type was deter-
mined to be 76% for ANC1 and ANC2 and 54% for ANC3,
respectively (a multiple sequence alignment is available in
Figure S8 in the Supporting Information). Figure 2 visually
illustrates how ANC1 and ANC2 are more similar to WT (more
black bars) whereas ANC3 has a lower sequence identity (more
Figure 1. Phylogenetic relationship of the 12 sequences of extant FAP
enzymes used to reconstruct the ancestral sequences; * enzymes with
confirmed photo-decarboxylation activity at the time of the reconstruction.
Figure 2. Pairwise identity between each ancestor and sCvFAP. The residue number is given on the x-axis; the y-axis denotes pairwise identity at each site.
Black bars denote the pairwise identity of 100% at each site. The portion of ANC1 corresponding to sANC1 is highlighted at the top of the figure. (pChTS:
predicted chloroplast targeting sequence.)
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white bars). Despite the differences, they all share the
conserved amino acid positions that characterize FAPs: C432,
R451 and A576 (numbering corresponding to CvFAP).[29,30] A576
is possibly one of the most striking difference between FAPs
and other members of the GMC superfamily. At this position,
most members of the GMC superfamily have a histidine.[30]
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ANC1 showed better production yield and stability
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So far sCvFAP production in E. coli has shown limited yields of
soluble protein in cell-free extract and, particularly, low enzyme
production after purification.[17] In general, efforts to regulate
the solubility of the protein have included the use of weak
promoters, low cultivation temperature during protein produc-
tion, modified growth media, and fusion with solubility
enhancing tags.[31–33] A recently published study demonstrated
that higher expression levels could be achieved by removing
the predicted chloroplast targeting sequence in CvFAP[11] and
that loss of catalytic activity could be reduced by performing
the in vitro purification under red light.[17]
Figure 3. Comparison of sCvFAP, ANC1 and sANC1 production under two
different IPTG concentrations. The plots on the left report production yield
as mg/L culture, the plots in the centre report units per litre culture, and the
plots on the right show pentadecane production from palmitic acid as
substrate after 1 h using 4 μM of purified enzymes. Units are given per mg of
FAD-loaded enzyme.
As already shown by several studies, the concentration of
the inductor IPTG is crucial for soluble production of the CvFAP
variant lacking the chloroplast targeting sequence (sCvFAP)
when using the T7 system.[34–36] This was confirmed with an
increase of the production yield from 2 mg/L culture (with
0.1 mM IPTG) to 13 mg/L culture (with 0.5 mM IPTG). Interest-
ingly, a similar purification yield (Table S2) was achieved using
low copy plasmid such as pASK or pBAD, whose gene
expression is under control of anhydrotetracycline or arabinose,
respectively (Table S2, Figure S2). Testing the expression of
sCvFAP under different promoters was crucial to obtain a
reliable melting temperature curve (Figure 4). Unfortunately,
ANC2 and ANC3 showed extremely low soluble production
(Figure S1) and no activity was detected (data not shown).
Therefore, the higher expression level and loss of catalytic
activity for sCvFAP is not only influenced by in vitro purification
but also by the intracellular protein folding (e.g., removing the
predicted chloroplast targeting sequence, promoter strength or
decreasing IPTG concentration; Table S2, Figure S2). In contrast,
using the same approaches for ANC1 (e.g., removing the
predicted chloroplast targeting sequence or increasing IPTG
concentration) further increased soluble expression and produc-
tion yield of ANC1. After one-step purification, sANC1 showed a
20-fold higher production yield compared to sCvFAP when
using 0.1 mM IPTG, and still a three-fold higher yield when
using 0.5 mM (Figure 3). Here, the final yield of purified ANC1
was 32.2�2.5 mg/L culture, sANC1 reached 33.3�4.7 mg/L
culture, whereas sCvFAP purification only reached 13.3�
2.3 mg/L culture (Figure 3, bottom left). For the three variants
we determined the FAD content to be approximatively 22% for
sCvFAP, 42% for ANC1 and 30% for sANC1. An higher FAD
loading is possibly another benefit of the enzyme purification
under red light as this resulted in 69% FAD loaded enzyme.[17]
Nevertheless, FAD integration seems to be a slow process as
supplementing FAD at different ratios (1:10, 1:1 and 10:1) did
not show significant improvements (Figure S7).
Surprisingly, a higher concentration of IPTG resulted in an
extremely high improvement in sCvFAP total and specific
activity (Figure 3, Table S3); however, the thermostability de-
creased significantly (see below). This resulted in a 40-fold
improvement in the volumetric activity of cell-free extracts
compared to expression with 0.1 mM IPTG. For ANC1, the
volumetric activity only increased 1.7 times whereas sANC1
showed comparable performance across both conditions. It
appears that ANC1 and sANC1 production is more consistent at
different IPTG concentrations. In contrast, the functional
production of sCvFAP is favoured at higher IPTG concentrations.
The poor solubility of free fatty acids in water limit the load
of the substrate, and furthermore, a co-solvent is always needed
to increase the substrate solubility further. Organic solvents can
interact with the hydrophobic patches of proteins perturbing
their folding/unfolding equilibrium in aqueous solutions.[37]
Deep eutectic solvents (DESs) have often been used as an
alternative to organic solvents.[38,39] DES and DES/water mixtures
have been shown to partially preserve enzyme activity leading
to improved performances compared to similar setups using
organic solvents.[40] Several attempts were also made in
biocatalysis where DES have been used as (co-)solvents
resulting in increased substrate solubility and enzyme
stability.[41,42]
In a previous work, switching to a DES system allowed us to
increase the substrate concentration from 10 mM to 300 mM
using a phenolic acid decarboxylase as biocatalyst.[42] Thus, we
envisioned that DES could be beneficial.
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Table 1. Conversion of palmitic acid to pentadecane[a] using different
mixtures of DES and water.
Table 2. Decarboxylation of ANC1 and sANC1 of various fatty acid
substrates compared to wild-type sCvFAP (5 mM substrate, 3 h reaction
time).
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Reaction medium
sCvFAP[b]
ANC1[a]
Fatty acid
Product [μM]
ANC1
1
2
3
4
DMSO (30%)
24%
16%
9%
1.6%
16%
6%
CvFAP
sANC1
DES/H2O[c] (50%)
DES/H2O[c] (60%)
DES/H2O[c] (70%)
C18:0
C18:1 Δ9
C18:2 Δ9,12
1146.9
651.2
29.6
128.1
0
0
278.8
25.9
16.5
8%
4%
[a] Reaction conditions: Palmitic acid=10 mM, purified enzymes=10 μM,
Reaction time=20 h. [b] Conversion determined by GC-FID. [c] DES ChCl:
Gly in a 1:2 molar ratio mixed in different ratios in H2O.
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ute to the higher ANC1 chemoselectivity towards unsaturated
fatty acids compared to wild type.
Despite both sCvFAP and ANC1 could perform photo-
decarboxylation in DES, no considerable improvement was
observed compared to the aqueous system with 30% DMSO as
co-solvent. It is interesting to note that the activity of sCvFAP in
ChCl:Gly DES decreased. ANC1 activity was nine times higher in
the DES/water mixture than in aqueous solution but did not
exceed that of sCvFAP under the same conditions (Table 1).
ANC1 and sANC1 have higher Tm and thermostability
In many cases, ancestral proteins are more thermostable than
modern proteins. This is often explained by a higher average
earth temperature in prehistoric times.[19] We anticipated that
our ancestral proteins would show higher thermostability
compared to sCvFAP. Therefore, we determined the melting
temperatures (Tm) for sCvFAP and the ancestral proteins using
the ThermoFAD method.[44] This method relies on the fact that
the flavin-containing proteins display different fluorescent
properties between the folded and denatured state. In partic-
ular, during the denaturation of flavoproteins, the secondary
and tertiary structure of the protein is disrupted, and
interactions with the flavin break down. The released free flavin
usually results in a large increase in fluorescence at 530 nm.[45]
The denaturation of sCvFAP[17] and its resurrected ancestor
ANC1 yielded a very pronounced decreasing fluorescence
intensity. This was in contrast to the known increasing
fluorescence observed with a flavin-dependent monooxygenase
CahJ that was used as control (Figure S3).[44] Thus, in this case,
the Tm is identified by plotting the negative of the first
derivative of the fluorescence emission as a function of temper-
ature (-dF/dT).[44] At first, the fluorescent signal of sCvFAP was
low and presented undefined peaks. To resolve this, we
performed the thermoFAD experiment with sCvFAP expressed
under different promoters to obtain more defined melting
temperature curves of sCvFAP for better comparison with the
profiles observed for ANC1 and sANC1 (Figure 4). In particular,
we used pASK and pBAD where the heterologous gene is under
the control of anhydrotetracycline (Ptet) and arabinose (araBp).
In all cases, a profile with two peaks in the derivative curve
was found, indicating that two distinct domains unfold at
different temperatures. This is in agreement with observations
from Sorigue et al.,[7] who described a structure with two
distinct domains, one for binding FAD and one for the
substrate, respectively. We hypothesized that for the expression
of the sCvFAP gene under the control of the T7 promoter, we
obtained a mixed population of correctly folded and unfolded
sCvFAP that resulted in the undefined profile (Figure 4C).
Producing sCvFAP from vectors with a weaker promoter gave
similar production yields, but resulted in a more homogeneous
enzyme population with defined melting temperature profiles
(Figure 4A and B).
Influence of the putative chloroplast targeting sequence on
the properties of ANC1
As reported earlier, removing the predicted chloroplast target-
ing sequence in sCvFAP increased the solubility and production
yield significantly, and could slightly improve catalytic
activity.[11] Even though the ASR already resulted in a shorter
N terminus compared to wild type, we attempted to further
improve ANC1 production and activity by investigating whether
the resurrected enzyme presented the predicted chloroplast
targeting sequence and if removing it would have similar
effects observed for wild type.
Thus, the shorter variant comprising residues 62–623 and
thus lacking the predicted chloroplast targeting sequence of
ANC1 (sANC1) was recombinantly produced in E. coli BL21(DE3)
and purified. As seen for ANC1, sANC1 showed similar soluble
overproduction, production yield (Figure S1) and slightly better
decarboxylation activity with palmitic acid as the substrate
compared to the extended version ANC1 (Figure 3).
In previous studies, sCvFAP showed different substrate
preference for oleic acid, linoleic acid, and saturated counter-
part stearic acid as a substrate.[11] Therefore, we selected these
three substrates of ANC1 and sANC1 to investigate if the
resurrected enzymes displayed a different preference compared
to wild type. Interestingly, ANC1 showed no activity for the
unsaturated substrates whereas, sANC1 showed conversion for
all three but with a high preference for the unsaturated
counterpart (Table 2). It appears that the chloroplast targeting
sequence influences ANC1 substrate preference as increasing
the amount of enzyme did not show an increased conversion
for the unsaturated substrate in ANC1 (data not shown).
Furthermore, ANC1 and sANC1 with their increased thermo-
stability are possibly more rigid than wild type; thus we believe
that unsaturated fatty acids are less likely to fit in the active
tunnel since they are also more rigid due to the double bond
compared to their saturated counterparts.[43] This might contrib-
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Figure 5. Comparison of sCvFAP, ANC1 and sANC1 residual activities at
different temperatures and IPTG concentrations. The photoenzymatic
decarboxylation reactions were catalysed by sCvFAP, ANC1, and sANC1
produced using different IPTG concentrations (0.1 and 0.5 mM). Purified
enzymes (4 μM) were incubated at different temperatures for 10 min, and
Figure 4. First derivative curves of the melting temperature curves obtained
in fluorescence measurements. A) sCvFAP produced in E. coli TOP10 (pBAD-
sCvFAP); B) sCvFAP produced in E. coli BL21(DE3) (pASK-sCvFAP); C) sCvFAP
produced in E. coli BL21(DE3) (pET28a-sCvFAP); D) ANC1 produced in E. coli
BL21(DE3) (pET28a-ANC1); E) sANC1 produced in E. coli BL21(DE3) (pET28a-
sANC1).
°
then the reactions were performed with gentle magnetic stirring at 30 C in
a total volume of 200 μL Tris·HCl buffer (pH 8.5, 100 mM) containing 30%
DMSO.
Here, ANC1 and sANC1 showed a significantly higher
melting temperature for both domains with an increase of 15
production had a more significant effect on sCvFAP compared
to the resurrected enzymes. In particular, sCvFAP produced
with 0.5 mM IPTG seems to be approximately three times less
thermostable compared to sCvFAP produced with 0.1 mM IPTG.
For some reason, a higher concentration of IPTG contributes to
producing a more active enzyme, which is far more prone to
aggregation and thermal denaturation (Figure 5). ANC1 and
sANC1 seem more robust to changes in expression conditions
and maintained similar residual activity at both IPTG concen-
trations. It is striking that when produced with 0.1 mM
concentration ANC1 showed a twofold improvement compared
°
and 13 C, respectively (Table 3). Furthermore, the addition of
30% DMSO (Figure 4, orange curves) seems to accelerate the
denaturation of the second peak, as a faster denaturation
results in a higher -dF/dT. Possibly, the second peak is the
substrate-binding domain as it should be more hydrophobic to
interact with fatty acids. Thus, while interacting more easily
with DMSO, it unfolds at a faster rate.
To validate this data, we further determined the thermo-
stability of ANC1, sANC1, and sCvFAP by measuring the residual
°
°
activity at 30 C after incubating the purified enzymes at a range
to wild-type in residual activity at 50 C, whereas it displayed
°
of temperatures (Figure 5). The plots confirm that ANC1 and
sANC1 are more thermostable photo-decarboxylases compared
to sCvFAP. Again, different IPTG concentrations in protein
almost 20-fold improvement when produced at 0.5 mM at 50 C
compared to wild type produced under the same conditions.
All in all, the higher thermostability determined with the
ThermoFAD method could be validated with activity measure-
ments confirming that ANC1 and sANC1 are significantly more
thermostable than the wild-type enzyme.
Unfortunately, the increase in thermostability did not trans-
late in higher resistance to light inactivation. Here we found
that ANC1 is slightly more photosensitive than wild type
(Figure S6).
Lakavath and co-workers described the radical based photo-
inactivation as an intrinsic property of sCvFAP which is due to
localised modification of protein residues around FAD and in
the active site.[17] It is unclear why the chloroplast targeting
sequence should have such effect on photoinactivation giving
Table 3. Comparison of Tm of both peaks determined by ThermoFAD for
sCvFAP, ANC1 and sANC1 in aqueous buffer with or without 30% DMSO.
°
Expression
vector
Tm[ C]
Tris·HCl buffer
Tris·HCl buffer+30%DMSO
pBAD-sCvFAP
pASK-sCvFAP
pET28a-sCvFAP
pET28a-ANC1
pET28a-sANC1
14/36
16/36.5
n.d.
36/44
36/49.5
18/36
24/35.5
n.d.
31/49
31/49
n.d. not determined.
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that ANC1 and sANC1 share the same active site and FAD
biding domain, however elucidating this was out of the scope
of this study.
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Homology modelling and structural considerations
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The availability of the three-dimensional structure of the
enzyme from C. variabilis (PDB ID: 5NCC) made it possible to
generate homology models for all ancestors using the Robetta
server and try to infer possible reasons for the higher thermo-
stability and production.[46] Aligning the homology models to
the experimental crystal structures showed few structural
variations, primarily characterized by shorter loop regions,
whereas ANC3 seems to have more differences compared to
sCvFAP structure (Figure 6).
As the homology models were built based on the wild type
structure, no dramatic structural changes were visible, the most
striking difference, especially in the active ANC1, is the length
of a few flexible loops which seem to be shorter. Shorter flexible
loops in the ancestor might have contributed to a higher
enzyme’s rigidity resulting in increased thermostability.
One proven method to increase enzyme stability is to
increase the packing at the enzyme’s core with hydrophobic
interaction and to increase the hydrophilicity of surface residues
for better interaction with aqueous systems.[19,47] Thus, we
investigated whether we could observe significant changes in
surface residues. As anticipated, most of the substitutions on
the outer surface of ANC1 were towards more hydrophilic
amino acids (Figure 7). In contrast, we could not identify
variants in the protein core that may explain the improvement
in the production yield of ANC1 without compromising the
functional activity.
Figure 7. ProtScale hydrophobicity graph for sCvFAP (pink) and ANC1
(orange). A positive score is associated with higher hydrophobicity. Arrows
designate the surface residues where appreciable changes are visible.
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clade containing CvFAP, CreFAP and 10 enzymes that had been
so far annotated as oxidases confirms the assumption that all
members of this clade might be photo-decarboxylases. This
assumption was later further confirmed by Moulin and co-
workers that confirmed FAP activity for additional enzymes.[29]
Two additional ancestors could not be characterized due to
problems with the soluble production after expression of their
genes in E. coli. ANC1 shows higher production of soluble
protein in E. coli, which could be further increased by N-
terminal truncation. Moreover, we were pleased to observe that
purification yields were higher than with the extant decarbox-
ylase, which is an important advantage for the biochemical
characterization of the enzyme. Interestingly, the N-terminal
truncation influenced the substrate spectrum as well, which is
difficult to explain with the currently available homology
models of ANC1. It is a common notion that ancestral enzymes
often show improved thermostability. Indeed, besides improved
production yield, ANC1 and its shorter version sANC1 showed a
°
remarkable increase in melting temperature of 15 and 13 C for
the two protein domains, respectively.
Homology modelling and analysis of surface residues high-
lighted that most of the surface residues substitutions were
towards more hydrophilic residues and most flexible loops in
ANC1 were a few residues shorter. Possibly, this contributed to
the higher thermostability observed for ANC1 by decreasing its
flexibility. Common protein engineering methods do not take
Conclusion
The photo-decarboxylase from C. variabilis CvFAP shows unique
activity in the conversion of fatty acids to alkanes. The
successful reconstruction of the common ancestor ANC1 from a
Figure 6. Homology models of ancestors A) ANC1 (green), B) ANC2 (sand) and C) ANC3 (purple) superimposed on the sCvFAP structure (turquoise; PDB ID:
5NCC). Black arrows highlight the main differences visible between WT and the ancestors’ homology models. Images generated in PyMOL 2.3.5.
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into account the length of flexible loops, and often thermo-
stability is achieved by mutagenesis; however, it seems that
optimizing loop length could be a viable option when more
stability is desired without significant changes in catalytic
activity or substrate scope.
All in all, ancestral sequence reconstruction proved to be a
valuable tool even with a limited amount of extant sequences
giving the extremely rare nature of photoenzymes. Despite the
trade-off in activity, ANC1 and sANC1 have superior handling
compared to wild type; thus it could also be used as a scaffold
for further improvements and for industrial applications.
Table 4. Buffers for purification.
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NaH2PO4
[mM]
NaCl
[mM]
Glycerol
(w/v)
Imidazole
[mM]
pH
lysis buffer
wash buffer
elution buf-
fer
desalting
buffer
50
50
50
100
100
100
10%
10%
10%
10
20
250
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8
8
50
–
10%
–
7.4
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Preparation of DES: DESs ChCl/Gly was prepared by gently heating
and stirring with choline chloride and glycerol in a molar ratio of
°
1:2 at a temperature of 80 C until a clear and homogenous liquid
was formed.
Experimental Section
Photocatalytic setup: The photoenzymatic decarboxylation reac-
tions catalysed by sCvFAP and ancestral reconstructed proteins
were performed at 30 C in a total volume of 200 μL Tris-HCl buffer
Ancestral sequence reconstruction (ASR) of the photo-decarbox-
ylase was done using the online tool GRASP.[48] In particular, the 12
sequences classified in the photo-decarboxylase clade were sub-
mitted to the stated different sequence alignment tools (see below)
available from the EMBL-EBI website. Subsequently, the alignments
were inspected for possible artefacts or sequences out place and
afterwards the phylogenetic trees were inferred by maximum
likelihood using RAxML. Finally, different evolutionary models
readily available in the GRASP suite were selected to increase the
probability to obtain an active variant. All algorithms were
performed under default settings. More in detail ANC1 was
generated using T-coffee[49] for the sequence alignment, and the
JTT[50] evolutionary model. ANC2 was generated by changing the
evolutionary model to LG,[51] whereas ANC3 was generated by using
MAFFT[52] for the sequence alignment and the JTT evolutionary
model.
°
(pH 8.5, 100 mM) containing 30% DMSO or DES as co-solvents. The
reaction system was added into a transparent glass vial (total
volume 5 mL). The vial was sealed and exposed to blue LED light
under gentle magnetic stirring with speed 200 rpm. At intervals,
aliquots were withdrawn and the reagents were extracted with
ethyl acetate (containing 1 mM of cyclohexanol as internal
reference) in a 1:1 ratio (v/v). The remaining organic phase was
analysed using GC-FID.
Melting temperature (Tm) determination using ThermoFAD: Melting
temperatures for all enzymes were determined using the Thermo-
FAD method as first described by Forneris et al.[44] While this
method does not assess the unfolding equilibrium, it is valuable for
establishing the thermostability of a protein. In a real-time PCR (RT-
PCR) machine (Eppendorf) fitted with a 470–543 nm excitation filter
and an SYBR Green emission filter (523–543 nm), 20 μL of 1 mg/mL
protein were loaded. A temperature gradient from 10 C to 95 C
was applied (0.5 C/min), and fluorescence data were recorded
every 0.5 C. A sigmoidal curve was obtained after plotting the
The ancestral FAPs were modelled using Robetta.[46] The surface
residues selection and Images of protein structures were prepared
using PyMOL.[53] The hydropathic scale were computed and
represented using ProtScale.[47,54]
°
°
°
°
Sequences, plasmid, strains, and growth conditions: All DNA
sequences used are collected in Table S1. All the ancestral sequence
reconstructions were synthesized and cloned into pET28a with C-
terminal His tag. E. coli DH5α was used for the propagation of
plasmids. E. coli BL21(DE3) was used for the high-level expression of
the recombinant photo-decarboxylases.
fluorescence against the temperature. The unfolding temperature,
Tm, is then determined as the maximum of the derivative of this
sigmoidal curve.
Thermostability determination assay with purified enzyme: All
enzymatic assays were performed in transparent glass vials sealed
with caps having a septum. Reaction mixtures contained 200 μL of
each purified enzyme (4 μM), 5 mM fatty acid (50 mM stock in
DMSO), and 30% DMSO as co-solvent. Thermal stability was
measured by incubating the purified enzymes at various temper-
atures for 10 min with occasional shaking. Activities were deter-
Protein expression and purification: For detailed analysis, the
ancestral variants, E. coli BL21(DE3) harbouring the appropriate
°
plasmid were grown at 37 C in LB supplemented with 50 μg/mL of
kanamycin, until an OD600 nm of approximately 0.8 was reached.
Overexpression was induced by adding IPTG (0.1 mM or 0.5 mM);
°
mined by assaying the residual activity at 30 C under standard
°
the cultures were grown for another 20 h at 18 C and harvested
reaction conditions.
°
(2900×g, 15 min, 4 C). The cells were resuspended in lysis buffer as
Photoinactivation determination assay with purified enzyme: All
purified enzymes were incubated under operational conditions but
without substrate for 5, 10 and 15 min. Activities were determined
by assaying the residual activity at 30 C under standard reaction
conditions comparing with a control enzymatic reaction without
pre-incubation.
shown in Table 4, and then the cells were disrupted by sonication.
°
The sonicated solution was then centrifuged (30000×g, 4 C,
60 min) to remove any insoluble parts. The soluble fraction was
mixed with Ni-NTA (nitrilotriacetic acid) and was incubated at 4 C
°
°
for 60 min with low speed shaking. The column was then washed
by gravity flow with the wash buffer (Table 4). The bound protein
was eluted with the elution buffer (Table 4). The fractions were
collected and analysed by SDS-PAGE to select the ones containing
the target enzyme. The enzyme solution was desalted with the
desalting buffer (Table 4) to remove imidazole. Finally, the enzyme
samples were concentrated using Amicon® Ultra-15 centrifugal filter
device (50 kDa cutoff, Millipore). The final enzyme solution was
FAD content determination: FAD content of purified enzymes using
NanoDrop 2000 (Thermo Fisher Scientific). In particular, the
concentrations were determined with the following extinction
coefficients at 280 nm: 79300 MÀ 1cmÀ 1 for sCvFAP, and
61310 MÀ 1cmÀ 1 for ANC1 and sANC1 (ProtParam, ExPaSy). All
protein signals were corrected for the FAD absorbance at 280 nm
using the extinction coefficient 24300 MÀ 1cmÀ 1. The concentration
°
aliquoted, frozen in liquid nitrogen, and stored at À 20 C.
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Acknowledgements
We thank Frank Hollmann for providing the gene of sCvFAP and
Dr. Tea Pavkov-Keller for the useful advice on the ThermoFAD
experiments.
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Enzyme resurrection strikes again:
Ancestral sequence reconstruction
was applied to a putative photo-de-
carboxylases clade. One common
ancestor showed up to 12-fold pro-
Y. Sun, Dr. E. Calderini, Prof. Dr. R.
Kourist*
1 – 9
A Reconstructed Common Ancestor
of the Fatty Acid Photo-decarboxy-
lase Clade Shows Photo-decarboxy-
lation Activity and Increased Ther-
mostability
°
duction yield, a 15 C increase in ther-
mostability and up to 20-fold im-
provement in residual activity when
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°
incubated at 50 C. Ancestral
sequence reconstruction proved to
be a valuable tool to introduce valid
mutations to a little understood fatty
acid photo-decarboxylase.