S.-F. Li et al.
Bioorganic Chemistry 109 (2021) 104712
However, up to date, the number of AKRs or other keto reductases with
outstanding catalytic performance suitable for the industrial applica-
tions is still limited [19].
Max Super-Fidelity DNA polymerase from Vazyme Biotech Co., Ltd
(Nanjing, China), and the PCR products were treated by the restriction
enzyme Dpn I from ThermoFisher Scientific Co., Ltd (Beijing, China), all
other chemicals and reagents were of analytical grade or higher quality.
Besides activity and selectivity, thermostability is an obstacle to the
industrial applications of keto reductases. Also, it is well known that
raising reaction temperature increases reaction rate, lowers the viscosity
of the medium, and hence promoting mass transfer [20]. Some reports
have investigated the thermostability mechnisms of SDRs or MDRs
through analysis of the crystal structures of thermostable keto re-
ductases and the structure-thermostability relationship via protein en-
gineering methods [21,22]. The thermostability of a hyperthermophilic
archaeal carbonyl reductase (CR) was proposed to be attributed to
strong inter-subunit interaction [23]. Directed evolution was used to
enhance the thermostability of Streptomyces coelicolor (ScCR) by intro-
ducing additional hydrogen bonds, which strengthened its structural
rigidity [24].
2.2. Methods
2.2.1. Homologous modeling and molecular docking
The three-dimensional (3D) structure of KmAKRM5 was constructed
based on the crystal structure of a reductase C1 from Candida parapsilosis
were established by ChemDraw software. YASARA 16.3.5 software was
used to conduct molecular docking of (5R)-1 and NADPH into the ho-
mology modeling structure of KmAKRM5. All the structures of the
modeling and enzyme-substrate docking were visualized in PyMOL
2.3.2.
AKR has a different structure lacking a Rossmann fold like domain
that is typical in SDR and MDR, and commonly exist as a single domain
structure [25]. Moreover, there are few investigations on the thermo-
stability mechanism of AKRs. Although directed evolution, semi-rational
design and rational design have successfully applied in thermostability
enhancement [26,27], the trade-off between stability and activity is still
a challenge [28]. Therefore, strategies for improving thermostability
without compromise in activity or selectivity of AKRs are strongly
demanded.
2.2.2. Construction and screening of the mutant library
SSM was performed at position 30 and 302 with pET-28a
(+)-kmakr as the template. The reaction system (25
M5
μ
L) consisted of
ꢀ 1
12.5
μ
L 2 × Phanta buffer, 0.5
μ
L dNTP mixture (each at 10 mmol L ),
ꢀ
1
ꢀ 1
1
1
μ
L forward primer (10
μ
mol L ), 1
L ultra-pure water, and 0.5
). PCR was conducted as follows: 95 ℃ for 5 min,
μ
L reverse primer (10
μ
mol L ),
μ
L plasmid template (50 ng), 8.5
μ
μ
L DNA
ꢀ
1
Polymerase (1 U
μ
L
◦
◦
◦
Consensus method is a powerful tool to enhance thermostability,
which is based on the theory that the consensus residues at specific
positions contribute much more than the other non-conserved residues
to the stability [29,30]. However, some deviations in the final consensus
sequence were always caused by the overrepresentation of a few sub-
families of homologous sequences, and some efforts have been made
to improve the efficiency of the consensus method in thermostability
enhancement and reduce deviations [31,32].
then 30 cycles (95 C for 15 s, 50 C for 15 s, and 72 C for 6.5 min), and
◦
finally extension at 72 C for 10 min. The PCR product digested by Dpn I
was transformed into E. coli BL21(DE3) competent cells. The sequences
of the primers for recombinant plasmid construction were listed in Table
S1.
A colorimetric assay for AKR using 2,4-dinitrobenzene was used to
screen the positive mutants. Transformants from the plates were
randomly selected and picked into a 96 deep well plate containing 1 mL
ꢀ
1
In our previous work, an aldo-keto reductase (KmAKR) from a
thermotolerant yeast Kluyveromyces marxianus ZJB14056 was cloned,
and a series of mutants of KmAKR with enhanced activity and thermo-
stability were developed [17,33,34]. And the mutant KmAKRM5
LB medium supplemented with 50
μ
g mL kanamycin, and cultivated
◦
at 37 C, 200 rpm overnight. Then 50
μL broth was transferred to
◦
another sterile 96 deep well plate, and grown at 37 C, 180 rpm for 4 h.
Inducer IPTG was added at a final concentration of 0.15 mM, and further
◦
(
W297H/Y296W/K29H/Y28A/T63M) was constructed via site-
cultured at 28 C, 180 rpm for 16 h. The cells in the 96 deep well plate
saturation mutagenesis (SSM), iterative saturation mutagenesis (ISM)
were harvested by centrifugation. Each well was resuspended into
ꢀ 1
and alanine scanning methods. Although KmAKRM5 exhibited a 155-fold
150 µL PBS (100 mM, pH 7.0) containing (5R)-1 (3.3 g L ), glucose (4.0
ꢀ
1
ꢀ 1
ꢀ 1
◦
ꢀ 1
increase in catalytic activity kcat/K
showed only an increase of 22% in half-life (t1/2) at 40 C (695 vs
46 min) in comparison with the WT KmAKR, and therefore there is still
m
(210.77 vs 1.35 s
mM ), it
g L ) and EsGDH (0.8 g DCW L ). The mixtures were incubated at
◦
35 C, 200 rpm for 30 min. After that, 20
μ
L reaction mixtures were
8
transferred into the 96 well assay plate, then 20 µL 2,4-dinitrobenzene
a large room for its thermostability enhancement.
(20 mM) and 20 µL PBS (100 mM, pH 7.0) were added. After further
◦
Herein, thermostability and catalytic efficiency of KmAKRM5 were
simultaneously enhanced through stepwise evolution. Firstly, using SSM
and high-throughput screening (HTS), we identified the hotspot residues
further enhancing the catalytic efficiency by increasing the binding af-
finity between KmAKR and (5R)-1. Then, we identified the critical res-
idues responsible for thermostability enhancement without trade-off in
activity by using a novel consensus strategy in combination with
structural analysis, producing a “best” mutant KmAKRM9. Finally, pro-
cess evaluation of KmAKRM9-catalyzed synthesis of (3R,5R)-2 was con-
ducted to investigate its industrial potential.
incubation at 37 C for 15 min, 150
μ
L NaOH (6 M) was added to quench
the reaction, and a Multiskan FC instrument (Thermo Fisher Scientific,
MA, USA) was used to measure the optical density at 480 nm.
The positive mutants were further re-screened by high performance
liquid chromatography (HPLC) analysis. The positive transformants
ꢀ 1
were inoculated in 10 mL LB medium containing 50
μg mL
kana-
◦
mycin, and cultivated at 37 C, 200 rpm for 12 h, and then 1 mL pre-
cultures were transferred to 100 mL LB liquid medium with
μ
g mL 1 kanamycin at 37 C, 200 rpm. When optical density at
ꢀ
◦
50
600 nm (OD 00) reached to 0.6–0.8, IPTG (final concentration
6
ꢀ
1
0
.15 mmol L ) was added and further cultivated to induce protein
◦
2
. Materials and methods
expression at 28 C, 200 rpm for 12 h. Cells were harvested by centri-
fugation at 12,000 rpm for 10 min. The bioconversion reactions were
conducted in 10 mL phosphate buffer (100 mM, pH 7.0) containing 3.0 g
2
.1. Materials
ꢀ
1
ꢀ 1
DCW (dry cell weight) L engineered cells of KmAKR, 1.0 g DCW L
ꢀ 1
ꢀ 1
The plasmids pET-28b(+)-kmakrM5 and pET-28b(+)-esgdh (glucose
cells harboring EsGDH, 30 g L (5R)-1 and 30 g L glucose. Finally,
900 µL anhydrous ethanol was added to 100 µL reaction mixture to
terminate the reaction. After centrifugation and microfiltration, the
supernatant of the reaction mixture was then analyzed by HPLC (Agi-
lent, USA). The relative activity of KmAKRM5 was designated as 100%.
dehydrogenase) were constructed in our previous study. E. coli BL21
(
DE3) was the host for cloning and expression of the recombinant pro-
teins. (5R)-1 was presented by Zhejiang Lepu Pharmaceutical Co., Ltd.
Taizhou, China). NADPH and (3R,5R)-2 were purchased from Roche
Co., Ltd. (Basel, Switzerland) and Toronto Research Chemicals Co., Ltd.
Toronto, Canada), respectively. The PCRs were performed with Phanta
(
One unit (U) of activity was determined as the amount of KmAKR that
◦
(
synthesizes 1
μ
mol of (3R,5R)-2 per minute at 35 C, pH 7.0.
2