Transporter engineering and enzyme evolution for pyruvate production from d/l-alanine with a whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis

Article abstract of DOI:10.1039/c6ra16507a

Pyruvate is an essential metabolite in the central metabolism of microbes, and it has been widely used in the food, pharmaceutical, and agrochemical industries. Both chemical and biological processes have been used for industrial pyruvate production. In this study, one-step pyruvate production from d/l-alanine with a whole-cell E. coli biocatalyst expressing l-amino acid deaminase (pm1) from Proteus mirabilis was investigated. Alanine uptake transporters (cycA, amaP) and a pyruvate uptake transporter (lldP) were knocked out to prevent substrate and product utilization by the biocatalyst. The pyruvate production titer from d/l-alanine increased from 1.14 g L^-1 under control conditions to 5.38 g L^-1 with the mutant whole-cell biocatalyst. Directed evolution was used to engineer pm1 and improve the catalytic activity with d/l-alanine. Three rounds of error-prone polymerase chain reaction generated the mutant pm1ep3, which showed improved affinity (6.76 mM for l-alanine) and catalytic efficiency (0.085 s^-1 mM^-1 and 0.027 s^-1 mM^-1 for l- and d-alanine, respectively). The final pyruvate titer was increased to 14.57 g L^-1 and the conversion ratio was increased to 29.14% by using the engineered whole-cell biocatalyst containing the evolved pm1ep3.

Full text of DOI:10.1039/c6ra16507a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Transporter engineering and enzyme evolution for
pyruvate production from D/L-alanine with a whole-
cell biocatalyst expressing L-amino acid deaminase
from Proteus mirabilis†
Cite this: RSC Adv., 2016, 6, 82676
ab
c
ab
ab
ab
Gazi Sakir Hossain, Hyun-dong Shin, Jianghua Li, Guocheng Du, Jian Chen
and Long Liu*
ab
Pyruvate is an essential metabolite in the central metabolism of microbes, and it has been widely used in the
food, pharmaceutical, and agrochemical industries. Both chemical and biological processes have been used
for industrial pyruvate production. In this study, one-step pyruvate production from D/L-alanine with
a whole-cell E. coli biocatalyst expressing L-amino acid deaminase (pm1) from Proteus mirabilis was
investigated. Alanine uptake transporters (cycA, amaP) and a pyruvate uptake transporter (lldP) were
knocked out to prevent substrate and product utilization by the biocatalyst. The pyruvate production
ꢀ1
ꢀ1
titer from D/L-alanine increased from 1.14 g L under control conditions to 5.38 g L with the mutant
whole-cell biocatalyst. Directed evolution was used to engineer pm1 and improve the catalytic activity
with D/L-alanine. Three rounds of error-prone polymerase chain reaction generated the mutant pm1ep3,
Received 27th June 2016
Accepted 24th August 2016
ꢀ1
ꢀ1
which showed improved affinity (6.76 mM for L-alanine) and catalytic efficiency (0.085 s mM and
ꢀ1
ꢀ1
ꢀ1
0
.027 s mM for L- and D-alanine, respectively). The final pyruvate titer was increased to 14.57 g L
DOI: 10.1039/c6ra16507a
and the conversion ratio was increased to 29.14% by using the engineered whole-cell biocatalyst
containing the evolved pm1ep3.
www.rsc.org/advances
chemical processes are not only energy intensive but also
require the use of heavy metals.
Introduction
1
Pyruvate plays key roles in central carbon metabolism. It is
There are three processes for biotechnological pyruvate
currently used as a nutraceutical, antioxidant, dietary supple- production: direct fermentation, enzymatic production, and
2
–4
8
ment, and weight-control supplement. Currently, pyruvate is whole-cell biocatalysis. In the fermentative production of
produced through chemical or biotechnological processes. The pyruvate, two kinds of microorganisms—the multivitamin
chemical synthesis of pyruvate is a multistep process starting auxotroph Torulopsis glabrata and the lipoic acid auxotroph
9
from either tartaric acid or lactate and its derivatives. In one Escherichia coli—have been using for decades. A NaCl-tolerant
ꢀ1
route, the dehydration and decarboxylation of tartaric acid is mutant, T. glabrata RS23, can produce 94.3 g L pyruvate in
10
required, which is energy intensive owing to the pyrolysis of 82 h. A pyruvate decarboxylase disrupted T. glabrata strain
ꢀ
1
11
5
tartaric acid. In another route, ethyl pyruvate is produced from accumulates 82.2 g L pyruvate in 52 h. A mutant E. coli
ethyl lactate by using various MoO
-based mixed oxides. This YYC202 strain, in which the conversion pathways of pyruvate to
3
route requires the esterication of lactic acid as well as the acetyl-CoA, acetate, and lactate are blocked, has been used to
6
hydrolysis of alkyl pyruvate. In another chemical synthesis increase pyruvate production to 40% above that obtained with
12
process, pyruvic acid is produced from lactic acid via oxidative the wild-type. However, the recovery of pyruvate from complex
7
dehydrogenation to form CO through C–C bond ssion. These fermentation broth is generally difficult and expensive.
2
Various enzymes, such as D-amino acid oxidase, pyruvate
synthase, formaldehyde dehydrogenase, tartrate dehydratase, and
glycolate oxidase, have been used for pyruvate synthesis. Among
these enzymes, a D-amino acid oxidase of Rhodotorula gracilis has
a
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan
University, Wuxi 214122, China. E-mail: longliu@jiangnan.edu.cn; Fax: +86-510-
8
b
5918309; Tel: +86-510-85918312
been used to convert D/L- or D-amino acid to pyruvate, with
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education,
13
conversion of more than 90% of the D-alanine into pyruvate. In
Jiangnan University, Wuxi 214122, China
c
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, another enzymatic synthesis process, pyruvate is produced from
14
Atlanta, GA 30332, USA
2
acetaldehyde and CO by using pyruvate decarboxylase. This
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c6ra16507a
process does not produce pyruvate in large quantities, however.
1
82676 | RSC Adv., 2016, 6, 82676–82684
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
A Pseudomonas sp. g31 whole-cell biocatalyst has also been rst to integrate a transporter engineering strategy and enzyme
used for pyruvate production from fumarate, producing 94 mM evolution for the improvement of pyruvate production by
of pyruvate from 100 mM fumarate in 24 h under optimized a whole-cell biocatalyst.
15
conditions. Resuspended whole-cell biocatalysts of E. coli
12
YYC202 produce 0.93 g of pyruvate per gram of glucose.
A
Materials and methods
Microorganisms, plasmids, chemicals, and cultivation
recombinant Pichia pastoris whole-cell biocatalyst containing
glycolate oxidase from spinach has been used to produce
16
conditions
pyruvate from lactate. However, hydrogen peroxide is a by-
product of this reaction and causes the breakdown of pyru- The plasmids and bacterial strains used in this study are listed
vate to acetate and CO , drastically decreasing the nal yield. in Table 1. Enzymes (PrimeSTAR HS DNA polymerase, 2ꢁ Taq
2
Therefore, genetically modied strains of Hansenula poly- PCR mix) and kits (Genomic Extraction Kit, DNA Purication
morpha and P. pastoris that express both glycolate oxidase and Kit, and Competent Cell Preparation Kit) were supplied by
catalase have been used to produce pyruvate from L-lactic TaKaRa (Dalian, China). With the exception of pyruvate (Sigma-
17,18
acid.
A Pseudomonas stutzeri SDM whole-cell biocatalyst has Aldrich, Shanghai, China), all chemical reagents were
been used to produce pyruvate from D/L-lactate, with titers purchased from Shanghai Sangon Biological Engineering
ꢀ
1
19
reaching 22.6 g L
within 24 h. Compared with these Technology and Services Co. Ltd. (Shanghai, China). All
ꢂ
processes, the production of pyruvate at high concentrations microorganisms were grown at 37 C in Luria-Bertani broth (LB;
from the cheaper substrate D/L-alanine is a more valuable 10 g L tryptone, 5 g L yeast extract, 10 g L NaCl) or on LB
process on an industrial scale because racemic alanine is much agar plates.
ꢀ1
ꢀ1
ꢀ1
cheaper than puried L-alanine. Therefore, in this study, D/L-
alanine was chosen as a substrate for pyruvate production with Deletion of alanine and pyruvate uptake transporters in E. coli
whole-cell biocatalysis. However, uptake transporters have
For the gene knockout, a one-step disruption protocol was fol-
considerable roles in the utilization of substrates or products
by whole-cell biocatalysts. Hence, we turned to transporter
engineering to improve the implemented biotechnological
processes.
The present study investigated one-step pyruvate production
from D/L-alanine with a whole-cell E. coli biocatalyst expressing
L-amino acid deaminase (pm1) from Proteus mirabilis (Fig. 1A).
Substrate and product utilization by the biocatalyst were
initially blocked by deleting two alanine uptake transporter
genes (cycA, amaP) and one pyruvate uptake transporter gene
20
lowed. First, the cycA gene (gene accession number gi
53778409) was knocked out. A DNA fragment containing
7
r
a kanamycin resistance marker (Kan ) anked by sequences
homologous to cycA genes was amplied from plasmid pKD13
using PCR with the cycA-Knock primer pair (Table S1†). The cycA
knockout mutant was constructed by transducing this DNA
fragment into E. coli BL21 cells by electroporation and using
kanamycin resistance as a positive selection. Gene knockout
strains were veried by PCR using the cycA-ver primer pair
(Table S1†) and DNA sequencing. The resistance marker
(lldP). Then, three rounds of error-prone polymerase chain
cassettes were removed from the chromosome with FLP
recombinase by using the temperature-inducible plasmid
pCP20. Aer that the other two genes amaP (gene accession
number gi 753774961) and lldP (gene accession number gi
reaction (ep-PCR) were performed to obtain an evolved mutant,
pm1ep3. The newly evolved mutant containing the whole-cell
ꢀ
1
biocatalyst increased the pyruvate titer to 14.57 g L and the
conversion ratio to 29.14%. To our knowledge, this study is the
753777805) were knocked out, veried and removed the
Fig. 1 (A) The reaction for the production of pyruvate from alanine by L-amino acid deaminase containing whole cell biocatalyst. (B) Schematic
representation for the substrate and product uptake and their metabolism in the bacterial cell.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 82676–82684 | 82677

View Article Online
RSC Advances
Paper
Table 1 Strains and plasmids used in this study
Characteristics
Reference
Strains
0
Escherichia coli JM109 recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, D(lac-proAB)/F (traD36, proAB+, lacIq, lacZDM 15) Takara, Otsu, Japan
Parent strain
AlaU-mut
AlaPyrU-mut
E. coli BL21(DE3) harboring the recombinant pm1
E. coli BL21(DE3) DcycADamaP
E. coli BL21(DE3) DcycADamaPDlldP
22
This study
This study
Plasmids
pET20b(+)-pm1
pKD13
pKD46
pCP20
Wild type pm1
oriRg bla FRT-kan -FRT
oriR101 repA101(Ts) bla araC gam-bet-exo (araBp)
pSC101 replicon (Ts) bla cat Flp(gRp)cI857
22
20
20
48
+
+
+
+
resistance marker cassettes similarly using corresponding
For mutant library generation, the ligation products were
primers (Table S1†). The alanine uptake transporters (DcycA used to transform competent E. coli JM109 cells via electro-
and DamaP) mutant was designated E. coli BL21 (DE3) (AlaU- poration. All of the resulting colonies were washed with sterile
mut) and the pyruvate and alanine uptake transporter (DlldP, water, and then plasmid DNA was extracted from the pooled
DcycA and DamaP) mutant was designated E. coli BL21 (DE3) E. coli library and subsequently transformed into the engi-
(AlaPyrU-mut). The recombinant plasmid (pm1ep3) was then neered E. coli (BL21) and plated on solid LB media containing
transformed into the engineered strains, and whole-cell kanamycin. Single colonies from the mutant library were picked
biotransformation was performed with various concentrations with colony picker (QPix 420, Molecular Devices, Sunnyvale, CA,
ꢀ
1
of D/L-alanine (5, 10, 20, and 50 g L ) to produce pyruvate. The USA) and incubated in separate wells of a 96-deep-well, at-
ꢀ
1
uptake of alanine and pyruvate by the wild-type E. coli BL21 bottom block containing 600 mL LB and 10 mg mL kana-
DE3) and its mutants was determined under the optimal mycin. The block was removed aer overnight incubation at
(
ꢀ
1
ꢀ1
ꢂ
biotransformation conditions of 50 g L D/L-alanine, 20 g L
cells (dry cell weight [DCW]), pH 8.0, 40 C, 220 rpm, and 4 h. 600 mL Terric broth media (12 g L tryptone, 24 g L yeast
The residual amounts of pyruvate and alanine were measured extract, and 4 mL L glycerol). Then, the plates were incubated
with high-performance liquid chromatography (HPLC) aer the for 5 h at 28 C with shaking at 700 rpm.
37 C and the cells were transferred to 96-well plates containing
ꢂ
ꢀ1
ꢀ1
ꢀ1
ꢂ
removal of cells via centrifugation to measure the uptake
quantities.
The cells were collected via centrifugation (5000 ꢁ g for 20
min), the media was removed, and the cells were washed with
20 mM Tris–HCl buffer. To start the reaction, 50 mL of 100 mM
alanine in Tris–HCl buffer (pH 8.0) was added. The reaction was
stopped by adding 45 mL of 20% trichloroacetic acid. Then, the
mixture was kept at room temperature for 30 min, and 20 mL of
Mutant library construction with ep-PCR and screening
Ep-PCR was performed by using Mutazyme II DNA polymerase
and a GeneMorph II Random Mutagenesis Kit (Agilent Tech-
nologies, Santa Clara, CA, USA) as recommended by the
manufacturer. Each amplication reaction (50 mL) contained
20 mM 2,4-dinitrophenylhydrazine was added. The mixture was
kept at room temperature for 15 min, and the reaction was
stopped by adding 400 mL of 0.8 M NaOH. Finally, the mixture
was kept at room temperature for 15 min to complete the
reaction and then centrifuged at 5000 ꢁ g for 20 min for clari-
0.5 mM each of the primers pm1_F1 and pm1_R1 (see Table
S1†), 200 mM deoxyribonucleotide triphosphates, and 2.5 U
Mutazyme II DNA polymerase in Mutazyme II reaction buffer.
PCR was performed with an initial 2 min incubation at 95 C, 30

cation. The absorbance of the supernatant was measured at
ꢂ
520 nm with a Multiskan Spectrum spectrophotometer (Thermo
ꢂ
ꢂ
cycles of amplication (1 min at 95 C, 1 min at 55 C, and 2.5
Scientic, Vantaa, Finland) aer appropriate dilution.
ꢂ
ꢂ
min at 72 C), and a nal 10 min elongation step at 72 C. Two
separate mutagenic amplications were conducted. The
amount of template amplicon varied in the rst set of experi-
ments (100, 10, 1, 0.1, or 0.001 ng). In the second set of exper-
Biocatalyst preparation and assay of whole-cell biocatalytic
activity
iments, 100 ng of template amplicon was used, but the number For seed preparation, recombinant E. coli cells were grown at
ꢂ
of amplication cycles varied (one, three, ve, seven, 10, 15, or 37 C on a rotary shaker (200 rpm) in LB medium containing
ꢀ1
2
1
0 cycles). Aer the process optimization, it was detected that ampicillin (10 mg L ) for 12 h. A seed culture was inoculated at
.0 ng of template and 15 cycles of amplication were suitable a concentration of 1% (v/v) into Terric broth containing
condition for the production of two or three amino acids con- ampicillin for cultivation. When the optical density at 600 nm
taining mutants. Aer the amplication under optimal condi- (OD600) reached 0.6, 0.4 mM isopropyl b-D-1-thiogalactopyr-
tions, the PCR products were digested with BamHI and XhoI, anoside was added to initiate pm1 production. The agitation
puried, and ligated into the expression vector pET20b (+), speed, aeration rate, and temperature were controlled at 400
ꢂ
which was digested with the same enzymes.
rpm, 1.0 vvm, and 28 C, respectively, to avoid the formation of
82678 | RSC Adv., 2016, 6, 82676–82684
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
inactive inclusion bodies. Aer induction for 5 h, the cells were was performed with an Ultra-4 Centrifugal Filter Device (Ami-
ꢂ
harvested via centrifugation at 8000 ꢁ g for 10 min at 4 C and con, Shanghai, China). The protein concentration was deter-
then washed twice with 20 mM Tris–HCl buffer (pH 8.0). The mined with a BCA protein assay kit (TianGen, Beijing, China).
cell pellet was resuspended in 20 mM Tris–HCl buffer (pH 8.0)
The kinetic analysis of pyruvate production with the evolved
ꢂ
and maintained at 4 C for further experiments. Biomass pm1ep1, pm1ep2, and pm1ep3 was performed by measuring
concentrations were analyzed spectrophotometrically (UV-2450 pyruvate titers with various concentrations of L-alanine and D-
PC; Shimadzu Co., Kyoto, Japan) at OD620 and converted to alanine (1, 40, 80, 120, 160, 200, 240, and 280 mM) as the
46
ꢂ
DCW by using eqn (1):
substrate at 40 C for 30 min. The kinetic parameters K
m
and
V
max were determined with the Michaelis–Menten plotting by
ꢀ1
DCW (g L ) ¼ 0.4442 ꢁ OD600 ꢀ 0.021
(1)
using the method shown in eqn (2):
Whole-cell biocatalytic activity was assayed by measuring the
pyruvate titer in the reaction solution. For measuring the initial
production rate, the reaction solution (50 g L D/L-alanine and where V is the reaction rate (the amount of pyruvate produced
m
V ¼ {Vmax[S]/(K + [S])}
(2)
ꢀ1
ꢀ
1
2
0.0 g [DCW] per L whole-cell biocatalyst in 20 mM Tris, pH 8.0) by 1 mg of evolved mutant per minute; mmol pyruvate min
ꢂ
ꢀ1
ꢀ1
was incubated at 40 C on a rotary shaker for 4 h. To investigate mg ), Vmax is the maximum reaction rate (mmol pyruvate min
ꢀ
1
the time prole for pyruvate production, we performed mg protein), Km is the Michaelis constant (mM), and [S] is the
biotransformation in a 50 mL ask with 10 mL reaction solution concentration of L-alanine and D-alanine (mM). The activities of
for 36 h. The agitation speed, pH, and temperature were mutants were measured in the presence of equal amount (1.5
ꢂ
maintained at 220 rpm, 8.0, and 40 C, respectively. Samples mg of total membrane proteins) of exogenous membranes
were collected at various times and centrifuged at 8000 ꢁ g for prepared from E. coli (BL21). Paleontological statistics soware
10 min to stop the reaction, and the pyruvate concentration in (PAST) was used to analyze Michaelis–Menten graphs and non-
the supernatant was measured with HPLC. The biotransfor- linear curve tting (Fig. S1 and S2†).
mation ratio was determined by using following equation:
Isothermal titration calorimetry
Conversion ratio (%) ¼ M
1
ꢀ M
2
/M
1
ꢁ 100%
Isothermal titration calorimetry (ITC) was performed with
represents the moles of alanine before transformation iTC200 Microcalorimeter from MicroCal (GE Healthcare, MA,
and M represents the moles of alanine remaining aer the USA). Titrations were accomplished by injecting 2 mL aliquots of
where M
1
2
conversion.
different concentrations of the alanine solution into the ITC
sample cell (volume of 198 mL) containing different enzyme
HPLC analysis of pyruvate and alanine concentration
samples (with similar amount of membrane of E. coli DE21) at
ꢂ
4
0
C. The stirring speed was set at 400 rpm and reference
The pyruvate concentration in the reaction mixture was deter-
mined with HPLC (Agilent 1100 series; Agilent Technologies)
combined with an Aminex HPX-87H column (300 mm ꢁ 7.8
mm; Bio-Rad, Hercules, USA). The mobile phase was 5 mM
sulfuric acid in distilled and deionized water, and the ow rate
ꢀ
1
power was set at 10 mcal s . Similar conditions were used to
measure the heat background by dropping the buffer only and
no ligand was used in the cell. Data were acquired by the so-
d
ware supplied by MicroCal. Then, the dissociation constant (K )
and the binding enthalpy change (DH) were determined and
gibbs free energy change (DG) was calculated according to the
ꢀ
1
was 0.6 mL min . The column temperature was maintained at
ꢂ
35
C, and the injection volume was 10 mL. Pyruvate was
following equation: DG ¼ ꢀRT ln K
d
, where R and T represent
the gas constant and the absolute temperature, respectively.
detected at a wavelength of 210 nm with a ultraviolet detector.
Alanine was quantied by using an HPLC system (Agilent 1200
series; Agilent Technologies) aer derivation with 1-uoro-2,4-
21
dinitrophenyl-5-L-alanine amide. The pure substances were
used as standards.
Molecular modeling and substrate docking
The hypothetical conformation of the protein was predicted by
the Swiss Model Automatic Modeling Mode (http://
Purication of the evolved mutants and determination of
kinetic parameters
37
swissmodel.expasy.org/). The PmaLAAD structure (PDB code:
JMI), which showed the highest identity (55%), was used as
5
The solubilization and purication of the evolved mutants were
performed as follows. The induced cells were centrifuged at
modeling template. All the enzyme-substrate docking analyses
were performed by using of Patchdock server (http://
8
000 ꢁ g for 10 min. Then, the cell pellets were suspended in
38
bioinfo3d.cs.tau.ac.il/PatchDock/). The visualization of the
purication buffers containing n-dodecyl-b-D-maltoside
three dimensional model of the enzyme was performed by using
(0.01%). Aer disruption with ultrasonication for 20 min on ice
(sonication for 1 s and intermission for 2 s) with a Vivra-Cell
(Sonics, Newtown, CT), the detergent solubilization solution
41
PYMOL (Schrondinger, LLC, Version 1.2).
Statistical analysis
was ltered through a membrane (0.45 mM pore size), and the

ltrate was puried on a HisTrap™ FF 5 mL column with an All experiments were performed at least three times, and the
AKTA Explorer (GE Healthcare, Piscataway, NJ). Then, desalting results are expressed as means ꢃ standard deviation (n ¼ 3).
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 82676–82684 | 82679

View Article Online
RSC Advances
Paper
Data were analyzed by using the Student's t-test. P values of less pyruvate transporter mutant (AlaPyrU-mut) whole-cell bio-
than 0.05 were considered statistically signicant.
catalyst containing pm1 further increased the biotransforma-
ꢀ
1
ꢀ1
tion yield from 3.67 g L to 5.38 g L (see Fig. 2C). Because
monocarboxylate pyruvate is a key metabolite that can also
serve as sole carbon source for E. coli, the knockout of these
uptake transporters eventually increased pyruvate yields as well
as biotransformation efficiency. Similar phenomenon was
Results and discussion
Effects of transporter gene knockout on the production of
pyruvate
For value-added chemical production, whole-cell biocatalysts observed in case of g-aminobutyric acid (GABA) production by
are sustainable and superior to biocatalytic routes that use using Corynebacter glutamicum, where a GABA uptake trans-
44
puried enzymes because puried enzymes can denature porter (GabPCg) was knocked out. As a result, the uptake of
during purication as well as during incubation with the GABA by the transporter engineered strain was reduced and
substrate. However, in whole-cell biocatalysis, substrate and productivity was increased by 12.5%. In another example,
product consumption by the whole cell itself is a major draw- during the tryptophan production by a tryptophan-producing
back. In addition, if the substrate or product, such as pyruvate, strain of C. glutamicum, three mutants with lower levels
is more functional for the cell, then consumption has an even uptake of tryptophan showed improved production kinetics and
45
greater effect on yield. Therefore, this consumption must be accumulated about 20% more tryptophan than the wild type.
prevented for efficient chemical production by whole-cell bio- However, bacterial cells are efficient in taking up diverse
catalysts. Substrate or product consumption is usually pre- carboxylic acids from the surrounding system across the
vented by regulating the disruption of metabolic pathways. bacterial membrane via several transport mechanisms such as
28
However, L-amino acid deaminase from P. mirabilis is localized active transport, vectorial acylation, and facilitative diffusion.
22
in the membrane and outwardly facing as well. Therefore, it Therefore, the uptake of some portion of the substrate or
can catalyze reactions when substrates are present outside of product via other transport processes can still occur in the
the cell. For this reason, disruption of the cell uptake system or reaction mixture. In addition, the pyruvate concentration
uptake transporters (Fig. 1B) can strongly inuence whether increases in a linear fashion initially under the highest
cellular utilization of a substrate or product occurs. Although substrate concentration (Fig. S3†). Furthermore, the effect of
membrane transporters do not involve in the chemical changes addition of different concentration of FAD (avin adenine
directly, but uptake type transporters contribute signicantly dinucleotide) on the enzyme's activity was examined. As shown
and conrm the accumulation of product in the much greater in Fig. S4,† the addition of FAD had no effect on the enzyme's
content at the extracellular space. Therefore, different strategies activity, indicating that the further addition of that cofactor is
for rening biotechnological processes might give greater not essential for the reaction. It also indicates that the enzyme
attention to transporters and offer enormous opportunities for expressed as holoenzyme with FAD. Such deaminase enzyme
increasing the extracellular product formation by transporter has a benecial effect from the industrial point of view, since
engineering. Because D/L-alanine was used in the present study the biotransformation will be occurred without supply of costly
as the substrate for pyruvate production, one aim of the study cofactor. In case of L-amino acid oxidase (L-AAO) of Rhodococcus
4
7
was to evaluate whether uptake transporter knockout increased opacus, similar phenomenon was also observed.
the production titer by minimizing uptake and subsequent
utilization (see Fig. 1B).
Ep-PCR-based directed evolution and the inuence of
Generally, transporters are involved not only in the efflux but
mutation on pyruvate production
also in the uptake of various metabolites and chemicals across
cellular membranes. They compose an enormous protein family Directed evolution is useful in creating enzymes with novel
and are widely distributed from prokaryotic bacterial cells to specicity, enantioselectivity, and stereoselectivity. For
23
eukaryotic plants and human beings. Three transporters in E. example, combinatorial mutagenesis of galactose oxidase has
coli are generally responsible for the uptake of alanine and been used to change the substrate specicity to glucose, and
pyruvate. Among them, cycA and amaP are responsible for a variant with three point mutations showed activity against D-
2
4,25,33
29
alanine uptake
and lldP is responsible for pyruvate glucose. Directed evolution has been used to change the
26,42,43
uptake. D-Alanine is essential for bacterial cell wall enantioselectivity of D-hydantoinase toward L-5-(2-methyl-
synthesis and is one of the subunits in peptidoglycan cross- thioethyl)hydantoin. The stereospecicity of tagatose-1,6-
linking. L-Alanine is used in cellular protein synthesis and bisphosphate aldolase has been altered 100-fold via three
other cellular metabolic processes. Knockout of alanine uptake rounds of directed evolution and screening, which resulted in
transporters resulted in alanine uptake lower than that of the an enzyme that can catalyze the formation of carbon–carbon
30
27
31
wild type (Fig. 2A), and the alanine transporter mutant (AlaU- bonds with unnatural diastereoselectivity.
mut) whole-cell biocatalyst containing the amino acid deami-
In the present study, ep-PCR was used to produce a regular
mutation rate of not more than two or three amino acids
ꢀ1
nase pm1 increased the biotransformation yield from 1.14 g L
ꢀ
1
to 3.67 g L (Fig. 2C). Based on the transport and competition changes per protein. Subsequently, a library of mutants
experiments with vesicles, it was found that lldP transporter is produced by ep-PCR was screened in a 96-well plate format to
42
responsible lactate and pyruvate uptake. Knockout of this identify mutants with high biotransformation efficacy. Only
uptake transporter lower pyruvate uptake (see Fig. 2B), and the mutants with biotransformation efficacy from D/L-alanine to
82680 | RSC Adv., 2016, 6, 82676–82684
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
Fig. 2 Effect of transporter gene knockout on the uptake of alanine and pyruvate by the wild type and mutants. (A) Uptake of D/L-alanine by AlaU-
mut strain; (B) uptake of pyruvate by AlaPyrU-mut strain. (C) Time profile for the biotransformation of D/L-alanine by the wild type and uptake
transporter mutants. Wild type (round sign); D/L-alanine uptake mutant, AlaU-mut (rectangular sign) and D/L-alanine & pyruvate uptake mutant,
AlaPyrU-mut (triangular sign). (D) Time profile for the biotransformation of D/L-alanine by the engineered whole cell biocatalyst containing
epPCR mutant of L-amino acid deaminase, pm1ep3. (E) Biotransformation ration by the wild type whole cell biocatalyst containing pm1 and by
the engineered whole cell biocatalyst containing evolved pm1ep3.
pyruvate higher than that of the wild-type enzyme were selected respectively (Table 2). In addition, substrate affinities increased
for further conrmation. Three rounds of ep-PCR were per- simultaneously for both D- and L-alanine (see Table 2). ITC
4
formed, and aer the screening of approximately 3 ꢁ 10
experiments also have revealed the increased affinity (2.25-fold
compared to wild type) for L-alanine and
mutant clones, an evolved mutant, pm1ep3, with a considerably decrease in K
d
improved titer aer the biotransformation of D/L-alanine was a negative enthalpy changes suggesting the binding is enthalpy
identied. Pm1 engineering resulted in the mutation of resi- driven (Table S2†). Interestingly, the equilibrium binding
dues E122G, Q265G, A276V, L282R, G291N, N364D, E366K, affinities for alanine (K values) measured by ITC for wild type
d
D380N, and D423E. The mutant Pm1ep3 increased Vmax from and mutants are similar to the apparent kinetic affinities (K
m
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
1
.83 mM min mg to 3.1 mM min mg and from 0.50 mM values), suggesting that the apparent affinity approaches the
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
min
mg
to 0.81 mM min
mg
for L- and D-alanine, binding affinity for the substrate under catalytic turnover
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 82676–82684 | 82681

View Article Online
RSC Advances
Paper
conditions. The affinities reported in other similar enzymes are
also in the mM range (Table S3†), which seems very weak for an
enzyme-substrate interaction. Nevertheless, the expression level
of the pm1 and other mutants including pm1ep3 in the engi-
neered whole cell biocatalyst were similar. Directed evolution
usually improves the desired enzyme properties by changing the
amino acid residues located close to the active center or at the
32
substrate-binding pockets.
Occasionally both sites are
changed owing to random mutagenesis, which affects not only
34
the active site structure but also the catalytic reaction. Finally,
ꢀ
1
the titer had increased to 14.57 g L (Fig. 2D) and biotrans-
formation ratio was increased to 29.14% by using the engi-
neered whole-cell biocatalyst (Fig. 2E) containing the evolved
pm1ep3.
A docking model was constructed of the wild type pm1 and
evolved mutant (pm1ep3) with alanine based on the homology
model using the PatchDock soware to identify the potential
molecular basis for the improvement of catalytic efficiency. pm1
is a FAD-containing enzyme and produces a-keto acid and
ammonia without forming hydrogen peroxide. Therefore, this
enzyme is different from known oxidases. Due to its broad
35
substrate specicity, the substrate-binding pocket is usually
lined by hydrophobic residues. The entrance site of this type
enzyme mostly occupied with negatively charged amino acids
(
Glu108, Glu145, Glu149, Asp156, Glu340, Asp416, & Glu417) and
˚
36
unusually wide (15–20 A) (Fig. S5†). In addition, the substrate-
˚
binding pocket of this type of enzyme is also about 20 A deep,
36
and mostly hydrophobic. In the evolved mutant, three amino
acids (Q265G, A276V, and D423E) were changed in the FAD
binding domain and six amino acids were changed (E122G,
L282R, G291N, E364D, E366K, and D380N) in the substrate
binding domain (Fig. 3A). In addition, the substrate docking
showed that the atomic distances between residues 97Y/311A
˚
˚
and the substrate alanine were increased from 21.4 A/16.4 A to
˚
˚
2
1.6 A/16.5 A, respectively (Fig. 3B and C). As a result, epPCR
have changed the distances between the substrate and actives
sites comparatively in a progressive manner. Consequently,
substrate binding affinity and catalytic efficiency was improved
in the evolved pm1ep3 (Table 2). Similar phenomena was
observed in case of trehalase from Zunongwangia sp. where the
shape of the binding pocket was enlarged in the epPCR mutant
of the enzyme and the variant C4 with two altered sites (Y227H,
and R442G) displayed a 3.3 fold increase in catalytic efficiency
39
compared with the wild type. In addition, the binding affinity
of that C4 variant with the substrate was improved as well as the
product releasing capacity was promoted. Although random
mutagenesis is targeting the entire coding sequence of the pm1,
however, the distances of mutated residues form the substrate
˚
˚
ligand were within 15 A to 31 A (Fig. S6†). Therefore, the local
environment close to the active site appeared to be signicant for
the improvement of the enzyme performance. It was observed
that mutations near the substrate-binding site affectedly raise
40
the success rate in many directed evolution experiments.
Directed evolution has produced numerous mutants that are
benecial in the manufacture of signicant industrial products.
Compared with applications of fermentative or enzymatic
methods, those of whole-cell biocatalysts are modest owing to
82682 | RSC Adv., 2016, 6, 82676–82684
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
Fig. 3 (A) The model structure of evolved mutant (pm1ep3) was constructed with the crystal structure of pmaLAAD (5FJM) as a template. The
colour in yellow and red are for substrate binding domain and FAD binding domain, respectively. The mutant residues are shown in a “CPK”
(
Corey–Pauling–Koltun) representation. Molecular distances of alanine (substrate) from the active sites: (B) in the wild type pm1; (C) in the mutant
pm1ep3.
substrate scope, operational stability, and reproducibility. With investigated transporter engineering for improved extracellular
the help of powerful enzyme discovery methods such as pyruvate production with whole-cell biocatalysis. Furthermore,
directed-evolution techniques, we obtained more selective and the engineering of transporters to reduce the cellular utilization
appropriate biocatalysts in this study. Moreover, our results of substrate and products has great potential to enhance nal
demonstrate a pyruvate production method that is more envi- yields and titers.
ronmentally friendly and sustainable than previously reported
methods.
Acknowledgements
In conclusion, the results of our study show that transporter
engineering that blocks substrate and product uptake is an This work is nancially supported by the Fundamental
effective metabolic engineering method with which to construct Research Funds for the Central Universities (JUSRP51612A) and
efficient whole-cell biocatalysts for the production of pyruvate 111 project (111-2-06).
from cheap substrate such as D/L-alanine. This approach may be
used to increase the production of a variety of value-added
products from relatively cheap and renewable substrates. In
Notes and references
previous decades, numerous efforts have been made to produce
pyruvate by using whole-cell biocatalysts with substrates such as
lactate and fumarate. These efforts have focused primarily on
the intracellular conversion of substrate to product with
enzymes related to metabolic pathways. No studies have
1 D. A. Fell and A. Wagner, Nat. Biotechnol., 2000, 18, 1121–
1122.
2 P. K. Koh-Banerjee, M. P. Ferreira, M. Greenwood,
R. G. Bowden, P. N. Cowan, A. L. Almada and R. B. Kreider,
Nutrition, 2005, 21, 312–319.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 82676–82684 | 82683

View Article Online
RSC Advances
Paper
3
4
M. F. McCarty, Med. Hypotheses, 2000, 54, 483–487.
X. Wang, E. Perez, R. Liu, L. J. Yan, R. T. Mallet and
S. H. Yang, Brain Res., 2007, 1132, 1–9.
26 V. J. Lang, C. Leystra-Lantz and R. A. Cook, J. Bacteriol., 1987,
169, 380–385.
27 B. A. Ellsworth, N. J. Tom and P. A. Bartlett, Chem. Biol.,
1996, 3, 37–44.
28 W. W. Kay, Bacterial transport, Marcel Dekker, Inc., New
York, 1978, pp. 341–411.
29 L. Sun, T. Bulter, M. Alcalde, I. P. Petrounia and F. H. Arnold,
ChemBioChem, 2002, 3, 781–783.
5
6
J. W. Howard and W. A. Fraser, Organic Syntheses, 1932, 1,
475–480.
H. Hayashi, N. Shigemoto, S. Sugiyama, N. Masaoka and
K. Saitoh, Catal. Lett., 1993, 19, 273–277.
M. Ai and K. Ohdan, Appl. Catal., A, 1997, 150, 13–20.
7
8
H. E. Schoemaker, D. Mink and M. G. Wubbolts, Science, 30 O. May, P. T. Nguyen and F. H. Arnold, Nat. Biotechnol., 2000,
003, 299, 1694–1697. 18, 317–320.
T. B. Causey, K. T. Shanmugam, L. P. Yomano and 31 G. J. Williams, S. Domann, A. Nelson and A. Berry, Proc. Natl.
2
9
L. O. Ingram, Proc. Natl. Acad. Sci. U. S. A., 2003, 101, 2235–
240.
0 L. M. Liu, Q. L. Xu, Y. Li, Z. P. Shi, Y. Zhu, G. C. Du and
Acad. Sci. U. S. A., 2003, 100, 3143–3148.
32 G. S. Hossain, J. Li, H. D. Shin, L. Liu, M. Wang, G. Du and
J. Chen, J. Biotechnol., 2014, 187, 71–77.
2
1
1
1
1
1
1
1
1
J. Chen, Biotechnol. Bioeng., 2007, 97, 825–832.
33 S. D. Cosloy, J. Bacteriol., 1973, 114, 679–684.
1 Q. H. Wang, P. He, D. J. Lu, A. Shen and N. Jiang, Enzyme 34 M. T. Reetz, M. Bocola, J. D. Carballeira, D. Zha and A. Vogel,
Microb. Technol., 2005, 36, 832–839. Angew. Chem., Int. Ed., 2005, 44, 4192–4196.
2 B. Zeli ´c , T. Gerharz, D. Bott, D. Vasi ´c -Racki, C. Wandrey and 35 J. O. Baek, J. W. Seo, O. Kwon, S. I. Seong, I. H. Kim and
R. Takors, Eng. Life Sci., 2003, 3, 299–305. C. H. Kim, J. Basic Microbiol., 2011, 51, 129–135.
3 S. Buto, L. Pollegioni, L. Angiurl and M. S. Pilone, Biotechnol. 36 P. Motta, G. Molla, L. Pollegioni and M. Nardini, J. Biol.
Bioeng., 1994, 44, 1288–1294. Chem., 2016, 291, 10457–10475.
4 M. Miyazaki, M. Shibue, K. Ogino, H. Nakamura and 37 T. Schwede, J. Kopp, N. Guex and M. C. Peitsch, Nucleic Acids
H. Maeda, Chem. Commun., 2001, 18, 1800–1801. Res., 2003, 31, 3381–3385.
5 J. Ogawa, C. L. Soong, I. Masashi and S. Shimizu, J. Mol. 38 D. Schnidman-Duhovny, Y. Inbar, R. Nussinov and
Catal. B: Enzym., 2001, 11, 355–359. H. Wolfson, Nucleic Acids Res., 2005, 33, W363–W367.
6 M. S. Payne, K. L. Petrillo, J. E. Gavagan, L. W. Wagner, R. Di 39 Q. Cheng, H. Gao and N. Hu, BMC Biotechnol., 2016, 16, 9.
Cosimo and D. L. Anton, Gene, 1995, 167, 215–219.
7 G. Gellissen, M. Piontek, U. Dahlems, V. Jenzelewski,
40 K. L. Morley and R. J. Kazlauskas, Trends Biotechnol., 2005,
23, 231–237.
J. E. Gavagan, R. DiCosimo, D. L. Anton and 41 L. L. C. Schrodinger, The PyMOL Molecular Graphics System.
Z. A. Janowicz, Appl. Microbiol. Biotechnol., 1996, 46, 46–54. Version 1.3r1, 2010.
8 M. S. Payne, K. L. Petrillo, J. E. Gavagan, R. DiCosimo, 42 A. Matin and W. N. Konings, Eur. J. Biochem., 1973, 34, 58–
L. W. Wagner and D. L. Anton, Gene, 1997, 194, 179–182. 67.
9 J. R. Hao, C. Q. Ma, C. Gao, J. H. Qiu, M. Wang, Y. N. Zhang, 43 H. L. Kornberg and J. Smith, Biochim. Biophys. Acta, Gen.
X. Cui and P. Xu, Biotechnol. Lett., 2007, 29, 105–110. Subj., 1967, 148, 591–592.
0 K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. U. S. 44 Z. Zhao, J. Y. Ding, W. H. Ma, N. Y. Zhou and S. J. Liu, Appl.
A., 2000, 97, 6640–6645. Environ. Microbiol., 2012, 78, 2596–2601.
1 A. Ozaki, H. Kawasaki, M. Yagasaki and Y. Hashimoto, 45 M. Ikeda and R. Katsumata, Biosci., Biotechnol., Biochem.,
Biosci., Biotechnol., Biochem., 1992, 56, 1980–1984. 1995, 59, 1600–1602.
2 G. S. Hossain, J. Li, H. D. Shin, R. R. Chen, G. Du, L. Liu and 46 G. Bratbak and I. Dundas, Appl. Environ. Microbiol., 1984, 48,
1
1
2
2
2
J. Chen, J. Biotechnol., 2014, 169, 112–120.
755–777.
2
2
2
3 C. F. Higgins, Annu. Rev. Cell Biol., 1992, 8, 67–113.
4 J. C. Robbins and D. L. Oxender, J. Bacteriol., 1973, 116, 12–18.
5 F. Schneider, R. Kramer and A. Burkovski, Appl. Microbiol. 48 P. P. Cherepanov and W. Wackernagel, Gene, 1995, 158, 9–
47 B. Geueke and W. Hummel, Protein Expression Purif., 2003,
28, 303–309.
Biotechnol., 2004, 65, 576–582.
14.
82684 | RSC Adv., 2016, 6, 82676–82684
This journal is © The Royal Society of Chemistry 2016