3. Results and discussion
A cascade reaction enables fluorogenic determination of ADH
activity
As shown in Figure 1, the catalytic activity of ADHs in an oxidation
reaction can be expressed as the rate of forming
NAD(P)H. Although it is possible
to quantitate NAD(P)H by directly measuring its light absorbance (UV 340
nm), this method suffers from low sensitivity and high background noise
(R. Zhang et al., 2015). Alternatively, measuring the fluorescence of
NAD(P)H Ex/Em=340/445 nm) can be used to quantify NAD(P)H and has shown
a greater sensitivity compared to the absorbance-based approach (Held,
2007). However, our experiments showed that although the linear
correlation between NADH concentration and its fluorescence can be
obtained when NADH was serially diluted with deionized water, buffer or
fresh media (Figure S1 A-D), such a correlation cannot be established
when spent media was used as the diluent (Figure S1 E-F). These results
suggest that metabolites released by growing cells can substantially
interfere with the fluorescence detection of NADH, and therefore such
fluorescence assay is not suitable for determining the NADH level in
crude samples such as cell lysate or culture supernatant.
To tackle this issue, we employed an indicator enzymatic reaction in
which NADH was used by diaphorase to reduce resazurin into resorufin,
which is highly fluorescent (Figure 1). The main advantage of this
system is that the long excitation/emission wavelengths of resorufin
(Ex/Em=535/588 nm) can reduce the
interferences from cellular metabolites, because most fluorescent
molecules in cell lysates are excited in the UV range (Simeonov &
Davis, 2004). As expected, our results showed that fluorescence of the
formed resorufin responded linearly to NADH concentration across
different medium backgrounds, including spent media (Figure S1 G-L).
Meanwhile, this reaction system offers a much more extended linear
detection range (0.1-700 μM) compared to NADH fluorescence-based
approach (0.1-200 μM when the medium background is deionized water).
Additionally, through transforming the unstable NADH into the more
stable resorufin, this reaction system provides a more durable and
accurate fluorescent signal, allowing a reliable and continuous
fluorogenic determination of NADH concentration.
Given these advantages, we may couple an ADH-catalysed reaction with
this indicator system to form a coupled enzyme assay for the ADH (Figure
1). To examine whether the ADH activity can be determined by this
cascade reaction, we employed an enantioselective ADH,
(R )-2-octanol dehydrogenase (PfODH) as the model enzyme. In the
cascade reaction mixture, the purified enzyme PfODH was supplied with
cofactor NAD+ and substrate to initiate target
reactions. Concurrently, excessive amount of resazurin and diaphorase
were provided in the mixture to stoichiometrically convert NADH into
fluorescent resorufin. The enzymatic activity of ADH was thus
proportional to the rate at the red fluorescence increased. As shown in
Figure 2, the catalytic activity of PfODH toward various substrates,
including its native substrate ((R )-2-octanol) and other
non-native substrates (4-Fluoro-α-methylbenzyl alcohol,
1-(3-Methylphenyl)ethanol and 1-Phenylethanol), were determined by
following the respective increases in fluorescence of resorufin over
time using a plate reader. In contrast, the negative controls without
adding PfODH all exhibited no fluorescence increase over time. These
results showcased that the coupled enzyme assay can enable a sensitive
and continuous, time-dependent monitoring of the ADH-catalysed reaction.
Incorporating the coupled enzyme assay with a protein
secretion system
High-throughput screening used in enzyme evolution has relied heavily on
the use of cell-based approach (Zeymer & Hilvert, 2018). However, there
are two major defects with this method: (i) It requires cell lysis that
could introduce interference to the screening assay; (ii) It relies on
the false assumption that the amount of enzyme being assayed is uniform
across all mutants, therefore resulting in an unfair comparison that
consequently increases false positive or negative rates. To further
develop our coupled enzyme assay into a better screening method, we set
out to address these two limitations.
In particular, we proposed to combine the cascade reaction with a
protein secretion system which is based on MsfGFP. The addition of such
a secretion system can potentially offer two unique features to our
assay. First, it has been reported that when recombinant proteins were
fused with MsfGFP, it can lead to the auto-secretion of the fusion
protein without disturbing their conformation and function (Z. Zhang et
al., 2017). Such a non-specific auto-secretion system is highly
desirable because cell lysis can therefore be exempted, and the secreted
target enzyme can be assayed in the culture broth, which is a much
simpler matrix than cell lysate. Second, MsfGFP can serve as a protein
expression reporter for its passenger protein. It has been demonstrated
that the fluorescence of sfGFP (non-mutated superfolder GFP) fusion
protein is linearly proportional to the protein quantity (Pédelacq,
Cabantous, Tran, Terwilliger, & Waldo, 2006). This feature can
potentially be used to normalize the enzyme activity of each mutant
during high throughput screening, since the fluorescence of MsfGFP
fusion protein can be easily and efficiently measured using fluorescence
plate reader.
To evaluate whether the MsfGFP-guided secretion system is generally
applicable to ADHs, we fused the MsfGFP or eGFP (enhanced GFP, a control
which would not lead to protein secretion) to N-terminus of five ADHs,
including PfODH from Pichia finlandica, (S )-1,3-butanediol
dehydrogenase (CpSADH) from Candida parapsilosis, glycerol
dehydrogenase (GlyDH) from E. coli , Meso-2,3-butanediol
dehydrogenase (BDH_KP) from Klebsiella pneumoniae and
2,3-butanediol dehydrogenase (BDH_BS) from Bacillus subtilis .
The protein expression of the MsfGFP/eGFP-ADH fusion proteins was
induced by IPTG. The grown cell cultures were then centrifuged to obtain
the supernatants for examining whether the fusion proteins were secreted
out of the cells. As shown in Figure 3, all the supernatants of the
cells expressing the MsfGFP fusion proteins exhibited fluorescence to
various degrees, whereas all the controls did not show any fluorescence.
Meanwhile, the presence of secreted MsfGFP fusion proteins and the
absence of eGFP fusion proteins in supernatant were further confirmed by
SDS-PAGE analysis (Figure 3). Consistent with the fluorescence
observation, the respective protein bands of MsfGFP fusion proteins
showed varying degrees of staining, further indicating different levels
of secretion for the MsfGFP fusion proteins. These results suggest that
secretion of five ADHs can be realized by using this MsfGFP-guided
auto-secretion system, although with various degree of secretion
efficiency.
After we confirmed MsfGFP can enable the secretion of ADHs, we moved
forward to examine whether the secreted ADHs’ activity can be determined
by the coupled enzymatic assay. Assay reaction components were directly
added to the culture supernatants of each fusion protein. As shown in
Figure 4, the supernatant samples of most MsfGFP-guided ADHs (GlyDH,
CpSADH, PfODH, and BDH_KP) generated various fluorescence increases
over time through the assay reaction, whereas there was no fluorescence
change for all the supernatant sample of eGFP fusion proteins. However,
unlike other ADHs, the rate of red fluorescence increase was low for
MsfGFP-BDH_BS, which may be mainly due to its low secretion level.
Nonetheless, these results demonstrated that the MsfGFP-guided secretion
system can be combined with the coupled enzyme assay to establish a
secretion-based assay for determining activity of some ADHs.
Normalizing ADH activity by the fluorescence of MsfGFP
We next assessed the feasibility of using the fluorescence of MsfGFP to
normalize the catalytic activity of ADHs among different mutants in
high-throughput screening. To mimic the actual screening condition of
the secreted ADHs, we purified the fusion protein MsfGFP-PfODH and
serially diluted it into different concentrations with cell-free spent
medium. The green fluorescence (Ex/Em=488/525 nm) of the diluted enzyme
samples were first determined by fluorescence plate reader. The green
fluorescence intensity exhibits a strong linear correlation to the
concentration of MsfGFP-PfODH (Figure S2A). Subsequently, the catalytic
activity of the MsfGFP-PfODH samples with various concentrations were
determined by measuring their respective red fluorescence (Ex/Em=488/525
nm) after adding coupled enzyme assay reagents. When
(R )-2-octanol was used as the substrate, the samples with higher
concentrations of MsfGFP-PfODH exhibited higher red fluorescence
intensities (Figure 5). Apparently, without considering the different
amounts of enzyme in the assay, the specific activity of these enzyme
samples, which was supposed to be the same, can be misrepresented based
on the red fluorescence measurement. To address this limitation, we used
the green fluorescence intensity, which reflected the total amount of
the ADH in the assay, to normalize the red fluorescence intensity of
each sample. The normalized signal of each sample was very close to each
other (Figure 5), indicating similar specific activity. Moreover, Figure
S3 showed that the same normalizing effect of MsfGFP’s fluorescence can
also be applied when a different substrate ((R )-2-butanol) was
used in the assay reaction. In addition, our further experiments also
proved that this normalizing feature of MsfGFP also worked for another
ADH (Figure S4). These results demonstrated that the apparent activity
of ADHs can be normalized by the fluorescence of MsfGFP fusion proteins
to enable a fairer comparison of enzyme mutants, thus reducing the false
positive and false negative rates during screening.
Using the secretion-based dual fluorescent assay in protein
directed evolution
Considering the aforementioned advantages, we combined the
resorufin-based cascade reaction system and the MsfGFP-guided secretion
system to establish the secretion-based dual fluorescent assay (SDFA)
for directed evolution of ADHs. On the one hand, the cascade reaction
system offers a sensitive and
continuous determination of ADHs
activity through a red fluorescence measurement. On the other hand,
MsfGFP-guided secretion system provides an easy and efficient way to
translocate the target ADHs to a
simpler reaction matrix and to normalize the enzyme’s activity through a
green fluorescence measurement.
In the workflow of SDFA (Figure 6), the mutated genes of an ADH were
first fused to the C-terminus of msfgfp to create a plasmid
library. The host cells (E. coli ) were then transformed with the
constructed plasmids and were spread on selection agar plates. Fully
grown mutant colonies were inoculated in liquid culture medium and
induced for enzyme expression. Subsequently, the cell cultures were
centrifuged down to obtain the supernatants that contained the secreted
enzymes for further enzyme screening. The green fluorescence of the
supernatants from the enzyme mutants was first determined by a plate
reader. Afterwards, assay reagents were added to the supernatant samples
to monitor the red fluorescence. Finally,
the catalytic activity of each
mutant was normalized (red/green fluorescence) to identify the true
positive mutant hits.
As a case study for demonstrating the
applicability
of SDFA in high-throughput screening, we employed PfODH as the model
enzyme and sought to engineer its stereoselectivity. In oil industry,
alcohol oxidation is one of the most extensively studied reactions as it
plays a key role in converting petroleum-based feedstock into essential
building blocks for a wide variety of chemicals (Parmeggiani, Matassini,
& Cardona, 2017). PfODH belongs to the short chain
dehydrogenase/reductase (SDR) superfamily. It has a very broad substrate
specificity and can catalyse the oxidation of alcohols to the
corresponding aldehydes or ketones, which are the ubiquitous chemical
precursors in pharmaceutical and fine chemical industries (H. Yamamoto
& Kudoh, 2013). However, PfODH has a substrate preference towardR -enantiomer of alcohols, thus resulting in incomplete conversion
of racemic alcohol, i.e. S -enantiomer of alcohols cannot be fully
utilized (H. Yamamoto & Kudoh, 2013). From an atom economy point of
view, enzymes that can oxidize both enantiomers of alcohol are more
desirable for this application.
Therefore, we set out to engineer the stereoselectivity of PfODH through
screening enzyme variants with enhanced activity towardS -enantiomer of alcohol. To achieve this goal, we constructed a
combinatorial library, PfODH-G99VWA-H150WTA-Y193RBT
(containing
60 genotypes), on critical sites G99, H150, and Y193 (Figure 7), which
are
involved
in the formation of the active cavity and extend their side chains into
the cavity of PfODH. These corresponding sites were suggested as the
switch to tune the stereoselectivity of the enzymes in the superfamily
of SDRs (Qin et al., 2018). Variation of steric hindrance and polarity
on sites of G99, H150, and Y193 might influence the stereo-preference of
PfODH (Qin et al., 2018). Therefore, the mentioned library was
constructed as the demonstration to test the applicability of SDFA as a
high throughput screening method in directed evolution of ADHs.
To ensure full coverage of the design space for our constructed library,
307 clones were picked for enzyme expression. These 307 mutants were
assayed by SDFA in 96 well plate format (Figure S5). Through screening,
top 10 variants that exhibited the highest catalytic activity against
(S )-2-octanol were selected for sequencing to identify their
genome type. Sequence analysis revealed that 8 out of the 10 selected
mutants were identified as PfODHG99L/A155L/Y193A,
whereas the remaining two mutants are
PfODHG99Q/A155L/Y193A and
PfODHG99V/A155L/Y193A. Given the fact that
PfODHG99L/A155L/Y193A was dominant in the top 10
positive hits and also showed higher catalytic activity than the other
two variants, PfODHG99V/A155L/Y193A was identified as
the best mutant from this library screening. To cross-validate the
screening result of SDFA, PfODHG99L/A155L/Y193A was
expressed in shaking flask cultures and purified for further determining
its kinetic parameter. Our results showed that
PfODHG99L/A155L/Y193A had a 197-fold higher
kcat/km value toward
(S )-2-octanol in comparison with wild-type PfODH. These results
demonstrated the capacity of SDFA to identify improved enzyme variants
from a library of mutants, illustrating the usefulness of this assay in
high-throughput screening of ADH mutants.