Asymmetric synthesis of l-6-hydroxynorleucine from 2-keto-6-hydroxyhexanoic acid using a branched-chain aminotransferase

Article abstract of DOI:10.3109/10242422.2011.638373

l-6-Hydroxynorleucine was synthesized from 2-keto-6-hydroxyhexanoic acid using branched-chain aminotransferase from Escherichia coli with l-glutamate as an amino donor. Since the branched-chain aminotransferase was severely inhibited by 2-ketoglutarate, the branched-chain aminotransferase reaction was coupled with aspartate aminotransferase and pyruvate decarboxylase. Aspartate aminotransferase converted the inhibitory 2-ketoglutarate back to l-glutamate by using l-aspartate as an amino donor. On the other hand, pyruvate decarboxylase further shifted the reaction equilibrium towards l-6-hydroxynorleucine through decarboxylation of pyruvate to acetaldehyde. The concerted action of the three enzymes significantly enhanced the yield compared to that of branched-chain aminotransferase alone. In the coupled reaction, 90.2 mM l-6-hydroxynorleucine (> 99% ee) was produced from 100 mM 2-keto-6-hydroxyhexanoic acid, whereas in a single branched-chain aminotransferase reaction only 22.5 mM l-6-hydroxynorleucine (> 99% ee) was produced.

Full text of DOI:10.3109/10242422.2011.638373

Biocatalysis and Biotransformation, March–April 2012; 30(2): 171–176
ORIGINAL ARTICLE
Asymmetric synthesis of L-6-hydroxynorleucine from 2-keto-6-
hydroxyhexanoic acid using a branched-chain aminotransferase
YOUNG-MAN SEO1, ARAN KIM1, HAN-SEOP BEA1, SANG-HYEUP LEE2∗ &
HYUNGDONYUN1∗
1School of Biotechnology,Yeungnam University, Gyeongsan, Republic of Korea and 2Department of Life Chemistry,
Catholic University of Daegu, Gyeongsan, Republic of Korea
Abstract
L-6-Hydroxynorleucine was synthesized from 2-keto-6-hydroxyhexanoic acid using branched-chain aminotransferase from
Escherichia coli with L-glutamate as an amino donor. Since the branched-chain aminotransferase was severely inhibited by
2-ketoglutarate, the branched-chain aminotransferase reaction was coupled with aspartate aminotransferase and pyruvate
decarboxylase. Aspartate aminotransferase converted the inhibitory 2-ketoglutarate back to L-glutamate by using L-aspar-
tate as an amino donor. On the other hand, pyruvate decarboxylase further shifted the reaction equilibrium towards L-6-
hydroxynorleucine through decarboxylation of pyruvate to acetaldehyde. The concerted action of the three enzymes
significantly enhanced the yield compared to that of branched-chain aminotransferase alone. In the coupled reaction, 90.2
mM L-6-hydroxynorleucine (Ͼ99% ee) was produced from 100 mM 2-keto-6-hydroxyhexanoic acid, whereas in a single
branched-chain aminotransferase reaction only 22.5 mM L-6-hydroxynorleucine (Ͼ99% ee) was produced.
Keywords: aminotransferase, branched-chain aminotransferase, coupling reaction, L-6-hydroxynorleucine, unnatural amino
acid
Introduction
L-norleucine (Kaufman & Mason 1982) and gluta-
Enantiomerically pure amino acids are fundamental
building blocks for the preparation of pharmaceuti-
cal and agrochemical target molecules, such as pep-
tides, proteins and many other natural products.
L-6-Hydroxynorleucine (L-2) is a chiral intermediate
useful in the synthesis of omapatrilat, indospicines,
siderophores and peptide hormone analogs (Bodan-
szky et al. 1978, Dreyfuss 1974, Maurer & Miller
1981, 1982, 1983, Patel 2001). It has been used in
the synthesis of C-7 substituted azepinones, which
are potential intermediates for the antihypertensive
metalloprotease inhibitors (Robl & Cimarusti 1994,
Robl et al. 1997). So far, several biocatalytic
approaches have been explored to synthesize L-2,
including D-amino acid oxidase catalysed kinetic
resolution of rac-2 (Kern & Reitz 1978), L-amino
acid acylase catalysed kinetic resolution of rac-N-
acetylhydroxynorleucine (Robl et al. 1997), pheny-
lalanine hydroxylase catalysed hydroxylation of
mate dehydrogenase catalysed L-selective reductive
amination of 2-keto-6-hydroxyhexanoic acid, 1
(Hanson et al. 1999).
Aminotransferases have been extensively studied
for the production of chiral amino acids due to their
rapid reaction rates, broad substrate specificity and
high enantioselectivity (Li et al. 2002, Hwang et al.
2005, Steward 2001, Taylor et al. 1998, Zhu & Hua
2009, Hong et al. 2010). Among them, branched-
chain aminotransferase (BCAT) has been used to
prepare aliphatic unnatural amino acids, such as
L-tert-leucine and L-3-hydroxyadamantylglycine (Li
et al. 2002, Hwang et al. 2005). However, BCAT
catalysed L-2 production has not previously been
reported. In this study, we attempted to synthesize
L-2 asymmetrically from 1 using Escherichia coli
BCAT, which is severely inhibited by the by-product,
2-ketoglutarate. To overcome product inhibition by
2-ketoglutarate and to drive the reaction equilibrium
∗ These authors are co-corresponding authors.
Correspondence: H. Yun, School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, 712-749, Republic of Korea. Fax: ϩ82-53-810-4769.
E-mail: hyungdon@ynu.ac.kr, S. H. Lee, Department of Life Chemistry, Catholic University of Daegu, Gyeongsan, Gyeongbuk, 712-702, Republic of
Korea. Fax:ϩ82-53-850-3781. E-mail: leeshh@cu.ac.kr
(Received 12 May 2011; revised 5 September 2011; accepted 2 November 2011)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2012 Informa UK, Ltd.
DOI: 10.3109/10242422.2011.638373

172
Y.-M. Seo et al.
NH2
CO2H
O
BCAT
2-Ketoglutarate
L-Asp
HO
HO
CO2H
L-2
1
L-Glu
Oxaloacetate
AspAT
CO2
PDC
CO2
Pyruvate
Acetaldehyde
Figure 1. Schematic diagram of the asymmetric synthesis of L-6-hydroxynorleucine, L-2 from 2-keto-6-hydroxyhexanoic acid, 1 using
BCAT/AspAT/PDC.
in the forward direction, BCAT was coupled with
aspartate aminotransferase (AspAT) and pyruvate
decarboxylase (PDC) (Figure 1).
containing 20 mM imidazole, and the C-terminal
His6-tagged protein was eluted with phosphate buf-
fer (pH 8.0) containing 200 mM imidazole. The
eluted solution containing the purified protein was
dialysed against 50 mM potassium phosphate buffer
(pH 7.0) containing 20 μM PLP and then concen-
trated using an Amicon PM-10 ultrafiltration unit.
Finally, glycerol was added to the purified enzyme
solution (25% glycerol), which was then stored at
–20°C for further study.
The AspAT gene was amplified from the genomic
DNA of E. coli K12 by PCR using the primers (5′-
AAAAAACATATGTTTGAGAACATTACCGC-
CGCT-3′ and 5′-AAAAAAGAATTCTTACAGCA
CTGCCACAATCGC-3′), digested with NdeI and
EcoRI, and inserted into the pET22b(ϩ) (Novagene,
USA).The crude extract of recombinant E. coli cells
expressing AspAT was prepared as described earlier,
with ampicillin (100 μg mL–1) as the antibiotic. Each
crude extract was directly used for the coupling reac-
tion without further purification.
The PDC gene was amplified from the genomic
DNA of Zymomonas mobilis by PCR using the prim-
ers (5′-AAAAAACATATGAGTTATACTGTCGG
TACCTATTTAG-3′ and 5′-AAAAAAGGATCCC
TAGAGGAGCTTGTTAACAGGCTTAC-3′),
digested with NdeI and BamHI, and inserted into the
pET24ma vector.The crude extract of recombinant
E. coli cells was prepared by sonication of the cell
pellet as described elsewhere (Yun & Kim 2008).
The crude extract was directly used for the coupling
reaction without further purification.
Materials and methods
Materials
Isopropyl-β-D-thiogalactopyranoside (IPTG), 2, 3,
4, 6-tetra-O-acetyl-β-D-glucopyranosyl isothiocya-
nate (GITC), 4-methyl-2-oxovaleric acid, 3-methyl-
2-oxobutyric acid and antibiotics were purchased
from Sigma-Aldrich (MO, USA). All other chemi-
cals used in the current study were of analytical or
reagent grade.
Enzyme preparation
To express BCAT from E. coli with a His-tagged
polypeptide, the coding region of BCAT was ampli-
fied from the genomic DNA of E. coli K12 by PCR
using the primers (5′-AAAAAAGGATCCATGACC
ACGAAGAAAGCT-3′ and 5′-AAAAAACTCGAG
TTGATTAACTTGATC-3′). The PCR product
was digested with BamHI and XhoI, and inserted
into the plasmid pET24ma (Yun et al. 2005). The
plasmid was then introduced into E.coli (BL21).The
transformants were grown at 37°C in 1 L LB broth
containing 50 μg mL–1 of kanamycin. When the
OD600 reached 0.25, IPTG was added to a final con-
centration of 0.5 mM. After 6 h of induction, the
cells were harvested and washed twice with 50 mM
phosphate buffer (pH 8.0). After centrifugation, the
cell pellet was resuspended in two volumes of 20
mM phosphate buffer (pH 7.0) containing 20 μM
pyridoxal 5′-phosphate (PLP), 2 mM EDTA, 1 mM
PMSF and 10% glycerol. Then, ultrasonic disrup-
tion was performed for 15 min. The protein was
purified at 4°C on an Ni-NTA agarose resin obtained
from Qiagen (Hilden, Germany). Briefly, the crude
extract was passed directly over a column containing
3 mL of Ni-NTA agarose resin.The column was then
washed with 50 mL of phosphate buffer (pH 8.0)
Synthesis of 1 and 2
The substrate 1 was synthesized as its sodium salt
from 4-chlorobutan-1-ol and diethyl oxalate, as pre-
viously reported by Hanson et al. (1997). The race-
mic 2 was synthesized from 2,3-dihydropyran using
a published method (Morales et al. 2006) as a white
crystalline solid after recrystallisation (from water-
ethanol). The NMR data were in accordance with
reported data (Morales et al. 2006).

Asymmetric synthesis of L-6-hydroxynorleucine 173
Enzyme assay and enzyme reaction
For the analysis of L-Glu, L-Leu, L-2, D-2 and tert-
Leu, an appropriate amount of L-Val was added into
the reaction mixture from 2 mM L-Val stock solution
and 5 mM GITC was added into the mixture for the
derivatization. Here, derivatized L-Val was used as an
internal standard, whereas for the analysis of L-Val,
L-Leu was used as an internal standard. The pyru-
vate (retention time, 15.5 min) was analysed on an
Aminex HPX-87H HPLC column (Bio-Rad, CA,
USA) with an isocratic elution of 5 mM H2SO4 at
0.6 mL min–1.
One unit of BCAT was defined as the amount of
enzyme catalysing the formation of 1 μmol of L-2 per
minute in 100 mM phosphate buffer (pH 7.5) con-
taining 10 mM 1 and 10 mM L-Glu. The relative
activities of BCAT towards 1, 4-methyl-2-oxovaleric
acid (keto form of Leu) and 3-methyl-2-oxobutyric
acid (keto form of Val) were examined in 100 mM
phosphate buffer (pH 7.5) containing 10 mM L-Glu
with 10 mM α-keto acid (substrate). For the kinetic
analysis of BCAT, the reaction rates were evaluated
with 10 mM L-Glu, BCAT (0.05 U mL–1) and vari-
ous concentrations of 1 (0–15 mM) in 100 mM
phosphate buffer (pH 7.5) at 25°C.The reaction was
terminated after 30 min by adding two volumes of
acetonitrile and the initial rates were determined by
analysing the L-2 produced after GITC derivatiza-
tion.The AspAT activity assay was carried out using
a crude extract of recombinant E. coli expressing
AspAT in 100 mM phosphate buffer (pH 7.0) con-
taining 10 mM 2-ketoglutarate and 10 mM L-Asp
at 25°C (Hong et al. 2010). The initial rates were
determined by analysing the L-Glu produced after
GITC derivatization. The PDC activity assay of the
crude extract of recombinant E. coli expressing PDC
was carried out in 100 mM phosphate buffer (pH
6.5) containing 10 mM pyruvate, 5 mM MgSO4 and
0.1 mM thiamine diphosphate (ThDP) at 25°C and
the consumed pyruvate was analysed by HPLC.The
assays were carried out in capped polypropylene
1.5-mL Eppendorf tubes in a water bath. The stan-
dard coupling reactions were carried out in 10 mL
capped glass vials placed in a shaking incubator at
250 rpm and 37°C. The reactions were initiated on
addition of an enzyme into the 100 mM phosphate
buffer (pH 7.5) containing the substrates (the final
volume was 5 mL). For the 100 mL-scale reaction,
reactions were carried out in a 250 mL flask in a
shaking incubator at 250 rpm and 37°C.
Results and discussion
Expression, purification and kinetic analysis of BCAT
The BCAT enzyme was effectively expressed in E.
coli BL21 (DE3) and then purified on an Ni-NTA
affinity column as described elsewhere (Hong et al.
2010). The size of the over-expressed protein band
matched the corresponding molecular weight (36.5
kDa) and the purified enzyme appeared as a single
polypeptide band on SDS-PAGE (Figure 2).
Analytical methods
Quantitative analysis and determination of the
enantiomeric purity of amino acids were carried out
by reverse-phase HPLC with a Shimadzu LC-VP
system (Shimadzu, Kyoto, Japan) equipped with a
C18 Symmetry column (Waters, MA, USA), after
derivatization with GITC (Yun et al. 2005). The
mobile phase was methanol-water (50:50 by vol,
0.1% trifluoroacetic acid was added) at a flow rate
of 1.0 mL min–1, and the effluent was monitored by
absorbance at 254 nm. After derivatization, the
retention times for L-Glu, L-Val, L-Leu, L-2 and D-2,
L-tert-Leu and D-tert-Leu were observed to be 5.2,
12.0, 20.5, 5.9, 6.9, 15.8 and 26.5 min, respectively.
Figure 2. Expression and purification of BCAT. Lane M, standard
protein marker; lane 1, crude extract of E. coli BL21; lane 2,
crude extract of cells expressing BCAT; lane 3, purified BCAT
(36.5 kDa).

174
Y.-M. Seo et al.
The relative activities of BCAT towards 1,
4-methyl-2-oxovaleric acid (keto form of Leu) and
3-methyl-2-oxobutyric acid (keto form of Val) were
examined with 10 mM L-Glu with 10 mM α-keto
acid (substrate). The enzyme activity towards 1 was
57% and 145% of that towards 3-methyl-2-oxobu-
tyric acid and 4-methyl-2-oxovaleric acid, respec-
tively.The specific activity of the purified BCAT was
18 U mg–1 in the presence of 10 mM 1 and 10 mM
L-Glu at 25°C.The enzyme showed its highest activ-
ity at pH 7.5 (data not shown). For kinetic analysis,
the initial reaction rates were analysed with various
concentrations of 1 (0–15 mM) in the presence of
10 mM L-Glu. The apparent Km and kcat for 1 were
2.6 mM and 9.6 s–1, respectively. When asymmetric
synthesis was carried out with 10 mM 1, 10 mM
L-Glu and BCAT (0.5 U mL–1) in 1 mL of 100 mM
phosphate buffer (pH 7.5) at 37°C, 4.6 mM L-2
(Ͼ99% ee) was produced, which matches the
reported result that the equilibrium constant of ami-
notransferase is close to unity (Taylor et al. 1998).
inhibition by L-Glu was not observed up to a concen-
tration of 200 mM. In the case of 1, the reaction rate
increased as the concentration of 1 was increased up
to 20 mM. However, when the concentration of 1
exceeded 20 mM, the reaction rate decreased, and
the reaction rate at 200 mM 1 was 55% that of 20
mM 1. Further, product inhibition by 2-ketoglutarate
was examined with 20 mM L-Glu and 20 mM 1.The
enzyme lost 83% of its activity in the presence of 10
mM 2-ketoglutarate. Product inhibition of BCAT by
2-ketoglutarate has previously been reported (Hong
et al. 2010, Taylor et al. 1998).
These results indicate that continuous removal of
2-ketoglutarate could improve the production of L-2
by BCAT. Asymmetric syntheses were carried out
with different amounts of BCAT (1.0 or 2.5 U mL–1)
in 1 mL of 100 mM phosphate buffer (pH 7.5) con-
taining 50 (or 100 or 200) mM 1, and 100 (or 200)
mM L-Glu for 24 h. In all cases, the amount of L-2
produced (Ͼ99% ee) did not exceed 25 mM.
Asymmetric synthesis of L-2 using a coupled system
Asymmetric synthesis of L-2 with only BCAT
To overcome product inhibition of BCAT by 2-ke-
toglutarate, AspAT was coupled with BCAT. The
produced 2-ketoglutarate is converted back to L-Glu
using L-Asp as the amino donor by AspAT, then
spontaneous decarboxylation of oxaloacetate forms
pyruvate. Thus, AspAT drives the reaction beyond
equilibrium (Taylor et al. 1998). PDC, which can
further shift the equilibrium of the reaction, was also
introduced into the BCAT/AspAT coupled reaction
(Figure 1). PDC performs the non-oxidative thia-
mindiphosphate-mediated decarboxylation of pyru-
vate to acetaldehyde (Yun & Kim 2008). AspAT and
PDC genes were cloned into pET22b(ϩ) (Novagene,
USA) and vector pET24ma, respectively, and effec-
tively expressed in E. coli BL21 (DE3) (Figure 4A).
The AspAT and the PDC activity of each of the
crude extracts of recombinant E. coli were found to
be 35 and 8.5 U mg–1, respectively. Each crude
extract was directly used for the coupling reaction
without further purification.
When the asymmetric synthesis was carried out
in 100 mM phosphate buffer (pH 7.5) with 100 mM
1, 100 mM L-Glu and BCAT (1.5 U mL–1), 22.5
mM L-2 (Ͼ99% ee) was produced after 24 h (the
conversion of 1 reached a plateau after 4-h reaction)
(Figure 4B).The BCAT/AspAT and BCAT/AspAT/
PDC coupling reaction produced 65 and 88 mM L-2
(Ͼ99% ee) after 24 h, respectively (Figure 4B).The
BCAT/AspAT/PDC coupling reaction gave about
3.9-fold higher yield than the BCAT reaction, as well
as 1.4-fold higher yield than the BCAT/AspAT cou-
pling reaction. It is notable that pyruvate itself did
not have any inhibitory effect on the BCAT reaction
Since the aminotransferase reaction is greatly influ-
enced by the product and substrate concentrations,
product and substrate inhibition of BCAT was exam-
ined (Figure 3). To measure substrate inhibition by
L-Glu and 1, the initial reaction rates were analysed
under the conditions described in Figure 3. Substrate
100
80
60
40
20
0
0
0
50
100
150
200
10
L-Glu or 2-keto-6-hydroxyhexanoic acid (mM)
2
4
6
8
2-Ketoglutarate (mM)
Figure 3. Substrate and product inhibition of BCAT.The reaction
was carried out with 1 mL of 100 mM phosphate buffer (pH 7.5)
containing BCAT (0.05 U mL–1) at 37°C. Substrate inhibition by
L-Glu ( ), reaction conditions: 20 mM 1, L-Glu (0–200 mM);
°
substrate inhibition by 1 (•), reaction conditions: 20 mM L-Glu
and 1 (0–200 mM); product inhibition by 2-ketoglutarate (Δ),
reaction conditions: 20 mM 1, 20 mM L-Glu and 2-ketoglutarate
(0–10 mM).

Asymmetric synthesis of L-6-hydroxynorleucine 175
100
80
60
40
20
0
BCAT/AspAT/
PDC
BCAT
BCAT/AspAT
Figure 4. Asymmetric synthesis of L-2 using coupling reaction. (A) Expression of AspAT and PDC. Lane M, standard protein marker;
lanes 1 and 3, crude extract of E. coli BL21; lane 2, crude extract of cells expressing AspAT (43.6 kDa); lane 4, crude extract of cells
expressing PDC (60.7 kDa). (B) The coupling reaction for the production of L-2. The reaction was carried out in 5 mL of 100 mM
phosphate buffer (pH 7.5) at 37°C for 24 h. BCAT reaction conditions: 100 mM 1, 100 mM L-Glu, BCAT (1.5 U mL–1). BCAT/AspAT
reaction conditions: 100 mM 1, 50 mM L-Glu, 200 mM L-Asp, BCAT (1.5 U mL–1) and AspAT (2.0 U mL–1). BCAT/AspAT/PDC
reaction conditions: 100 mM 1, 50 mM L-Glu, 200 mM L-Asp, BCAT (1.5 U mL–1), AspAT (2.0 U mL–1) and PDC (2.0 U mL–1).
contrast, if the whole-cell reaction is carried out with
recombinant E. coli co-expressing BCAT, AspAT and
PDC, the PDC can reduce L-Ala formation by con-
verting pyruvate to acetaldehyde. Therefore, the tar-
get product can be easily purified from the reaction
mixture. Currently, we are developing the asymmet-
ric synthesis of L-2 by a whole-cell reaction system
with recombinant E.coli co-expressing BCAT,AspAT
and PDC, which will reduce the labour and costs as
it eliminates the process of enzyme extraction from
the cells and also minimises by-product formation.
(data not shown). The enhanced yield of BCAT/
AspAT/PDC coupling compared to that of BCAT/
AspAT coupling was due to an equilibrium shift
caused by the reduction in pyruvate concentration.
Finally, reaction scale-up has been carried out using
crude extracts of enzymes in 100 mL of 100 mM
phosphate buffer (pH 7.5) containing 100 mM 1, 50
mM L-Glu, 200 mM L-Asp, BCAT (1.5 U mL–1),
AspAT (1.0 U mL–1) and PDC (2.5 U mL–1) at
37°C. After 24 h reaction, 90.2 mM L-2 (Ͼ99% ee)
was produced based on HPLC analysis.
In addition, to examine the applicability of the
coupled reaction system for other aliphatic unnatural
amino acids, L-tert-Leu was chosen, which is an
important precursor for the synthesis of pharmaceu-
ticals (Hong et al. 2010). When the asymmetric syn-
thesis of L-tert-Leu was carried out in 1 mL of 100
mM phosphate buffer (pH 7.5) containing 100 mM
trimethylpyruvate, 100 mM L-Glu and BCAT (1.0 U/
mL), 21.5 mM L-tert-Leu (Ͼ99% ee) was produced
after 24 h. On the other hand, 87.5 mM L-tert-Leu
(Ͼ99% ee) was produced from 100 mM trimeth-
ylpyruvate by BCAT/AspAT/PDC coupled reaction
in the presence of 50 mM L-Glu, 200 mM L-Asp,
BCAT (1.0 U/mL), AspAT (1.5 U/mL) and PDC
(1.5 U/mL) after 24 h. The concerted action of the
three enzymes significantly enhanced the yield about
4.1-fold higher compared to that of BCAT alone.
When the whole-cell reaction is carried out with
recombinant E. coli co-expressing BCAT/AspAT,
the final by-product pyruvate is converted into L-Ala
by endogenous aminotransferases. L-Ala formation
prevents simple isolation of L-2 (Li et al. 2002). By
Conclusion
Enzyme inhibition by the by-product and the equi-
librium constant of the reaction were major hurdles
to overcome in order to carry out an efficient BCAT
reaction for the synthesis of aliphatic unnatural amino
acids from their corresponding α-keto acid. To over-
come the problems of product inhibition by 2-keto-
glutarate and reaction equilibrium,we have developed
an efficient BCAT coupled reaction system for the
asymmetric synthesis of L-2 using AspAT and PDC.
In the coupled reaction, 90.2 mM L-2 (Ͼ99% ee) was
produced from 100 mM 1, whereas in a single BCAT
reaction only 22.5 mM L-2 (Ͼ99% ee) was produced.
The BCAT/AspAT/PDC coupled reaction signifi-
cantly enhanced the yield to about 4-fold higher com-
pared to that of BCAT alone. Interestingly, this
coupling reaction system was applicable to the asym-
metric synthesis of other useful aliphatic unnatural
amino acids such as L-tert-Leu.

176
Y.-M. Seo et al.
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Declaration of interest: This research was supported
by the Basic Science Research Program (no.
20100028158) through the National Research Foun-
dation of Korea and the 21C Frontier Microbial
Genomics & Applications Center Program (no.
1120081800300) funded by the Ministry of Educa-
tion, Science andTechnology. It was also supported by
the cooperative R&D Program (no. B551179100300)
through the Korea Research Council for Industrial
Science and Technology. The authors report no con-
flicts of interest.The authors alone are responsible for
the content and writing of the paper.
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