1. Introduction
Many drugs and value-added chemicals need to meet stringent
enantiopurity standards in pharmaceutical and fine chemical industries.
During the last decade, there has been a growing interest in using
enzymes for producing chiral chemicals. This is mainly driven by the
inherent advantages of biocatalysis over chemical synthesis, such as
high chemo-, regio- and enantio-selectivities, mild reaction conditions
and using less toxic reagents or solvents (Nealon, Musa, Patel, &
Phillips, 2015; Zheng et al., 2017).
In particular, alcohol dehydrogenases (E.C. 1.1.1.x; x=1 or 2, ADHs) are
among the most investigated redox enzymes. ADHs are a group of
NAD(P)(H)-dependent enzymes that catalyze the interconversion between
alcohols and ketones or aldehydes. Because of their strict
stereoselectivity, ADHs have been employed in the production of various
chiral products with high optical purity (Chen, Liu, Lin, & Zheng,
2016; Kara et al., 2014; Savile, Gruber, Mundorff, Huisman, & Collier,
2014). However, the performance of wild-type ADHs is rarely adequate for
industrial processes. In general, tailoring enzyme properties, such as
catalytic activity, stereoselectivity, substrate specificity,
thermostability and tolerance of organic solvent is often required to
realize the practical application of ADHs (R. Zhang, Xu, & Xiao, 2015).
Directed evolution is one of the most effective methods to engineer
ADHs. An ADH may be randomly mutated by using error-prone PCR to
identify beneficial mutations. Putative critical residues of an ADH may
also be identified based on docking the substrate/product into
experimental-determined or predicted tertiary structure of the ADH.
Beneficial mutations at these putative residues would be characterized
through mutagenesis. Often, multiple beneficial mutations need to be
combined and the aforementioned process may be iterated.
In directed evolution, screening the mutants is usually the
rate-limiting unit operation, because the rate of hitting a beneficial
mutation is low. The assay commonly used to quantify activity of ADH is
to monitor the production or depletion of NAD(P)H based on absorbance at
340 nm as these enzymes couple their enzymatic reactions with the
nicotinamide cofactors (Forrest & Gonzalez, 2000). Usually cells
expressing an ADH was lysed prior to the assay. Unfortunately, this
approach is not suitable for high-throughput screening, because of the
strong absorption of UV light by cell lysates (Mayer & Arnold, 2002).
Alternatively, colorimetric assays that combine the redox reaction with
a dye-forming reaction have been developed to reduce the noise, because
the absorption of visible light by cell lysates was weaker than that of
UV light. One of the most widely adopted colorimetric approaches is the
nitroblue tetrazolium/phenazine methosulfate (NBT/PMS) assay (Fibla &
Gonzàlez-Duarte, 1993). In the presence of the transferring agent PMS,
NBT can be reduced by the NAD(P)H regenerated by an ADH-catalyzed
reaction to produce an insoluble blue-purple formazan dye, whose
absorbance can be measured at 580 nm. This colorimetric assay has been
used in high-throughput screening to identify novel or improved ADHs,
such as 6-phosphogluconate dehydrogenase (Huang, Chen, Zhong, Kim, &
Zhang, 2016), glycerol dehydrogenase (H. Zhang, Lountos, Ching, &
Jiang, 2010), halohydrin dehydrogenase (Fox et al., 2007), and
phenylacetaldehyde reductase (Makino, Dairi, & Itoh, 2007). However,
this colorimetric assay still needs improvement due to its background
noise and limited detection range. Moreover, this assay also fails to
enable a continuous measurement of ADHs activity as the final colour
product is insoluble, resulting in precipitation that interferes with
the absorbance quantification.
In addition, the aforementioned assays generally suffer from two common
issues when they are used to screen cells that express enzyme. First, in
order to release target enzyme from host cells for determining catalytic
activity, cell lysis is often required by these methods. However, once
the cells are lysed, together with the enzyme of interest, all cytosolic
components are simultaneously released, resulting in a complex matrix
that could reduce the sensitivity and accuracy of the assay implemented
(Contreras-Llano & Tan, 2018; Mayer & Arnold, 2002). For example,
cytosolic components, such as nicotinamide cofactor, autofluorescent or
chromogenic metabolites may miscount for the catalytic activity of the
target enzyme. Non-specific interactions with endogenous enzymes (e.g.
endogenous ADHs) could also interfere with the target enzyme reaction if
they share similar substrate specificity.
Furthermore, when these assays were applied in high-throughput format,
such as microtiter plate screening, the catalytic activity was usually
evaluated without normalizing by the amount of enzyme interrogated.
Because, it is time-consuming and tedious to quantify the enzyme amount
for each variant in a large-scale screening effort. Consequently, this
inevitably leads to an unfair comparison among the mutants. The quantity
of the target enzyme in each mutant sample could vary due to fluctuating
protein expression level and nonhomogeneous cell growth rate. Overall,
this may not only produce a considerable number of false positives, but
also miss out some of the true positives. It will be desirable to have a
simple and easy way to quantify the enzyme amount for each mutant so
that their catalytic activity can be normalized for a fairer screening.
To tackle these limitations in this study, we developed a
secretion-based dual fluorescence assay (SDFA) for high-throughput
screening of mutated ADHs. This assay is based on a cascade reaction
leading to the formation of a red fluorescent molecule. It also uses a
protein secretion system translocating the target enzyme to a simpler
reaction matrix than cell lysate.
As shown in Figure 1, in the
presence of an enzyme (diaphorase), resazurin reacts with the NAD(P)H
generated from the ADHs-catalysed reaction, leading to the generation of
the highly fluorescent molecule—resorufin—whose emission signal can
be easily detected at 588 nm when excited at 535 nm. Since the emission
falls within the range of red light, in which fewer Escherichiacoli endogenous molecules emit fluorescence, there is less
interference from the reaction mixture and spent culture media.
Meanwhile, we combined the cascade reaction with a protein secretion
system which was enabled by a mutated superfolder green fluorescent
protein (MsfGFP) (Z. Zhang et al., 2017). By fusing ADHs to MsfGFP, the
fusion proteins can be efficiently secreted out of the host cells for
enzymatic activity quantification. Therefore, cell lysis was not
required by this assay. This led to a much simpler reaction matrix that
was free of complex cytosolic components. As a result, the interference
caused by intracellular metabolites and endogenous enzymes was
eliminated. Also, this secretion
system simplified the screening procedure and avoided the potential
inconsistency of lysis efficiency.
Additionally, another merit of this assay is that the green fluorescence
of the MsfGFP fusions was linearly proportional to its protein amount.
This provides a simple and efficient way to quantitate the amount of
target enzyme in the reaction matrix. Therefore,
normalization of the catalytic
activity of different mutants can be easily achieved, further improving
the accuracy of the assay.
In this study, we have collected experimental data to support the
claimed advantages of SDFA, and successfully demonstrated the usefulness
of SDFA through engineering of an ADH, which led to substantial
improvement of its catalytic efficiency.