Multi-enzyme pyruvate removal system to enhance (: R)-selective reductive amination of ketones

Article abstract of DOI:10.1039/d0ra06140a

Biocatalytic transamination is widely used in industrial production of chiral chemicals. Here, we constructed a novel multi-enzyme system to promote the conversion of the amination reaction. Firstly, we constructed the ArR-ωTA/TdcE/FDH/LDH multi-enzyme system, by combination of (R)-selective ω-transaminase derived from Arthrobacter sp. (ArR-ωTA), formate dehydrogenase (FDH) derived from Candida boidinii, formate acetyltransferase (TdcE) and lactate dehydrogenase (LDH) derived from E. coli MG1655. This multi-enzyme system was used to efficiently remove the by-product pyruvate by TdcE and LDH to facilitate the transamination reaction. The TdcE/FDH pathway was found to dominate the by-product pyruvate removal in the transamination reaction. Secondly, we optimized the reaction conditions, including d-alanine, DMSO, and pyridoxal phosphate (PLP) with different concentration of 2-pentanone (as a model substrate). Thirdly, by using the ArR-ωTA/TdcE/FDH/LDH system, the conversions of 2-pentanone, 4-phenyl-2-butanone and cyclohexanone were 84.5%, 98.2% and 79.3%, respectively.

Full text of DOI:10.1039/d0ra06140a

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Multi-enzyme pyruvate removal system to enhance
(R)-selective reductive amination of ketones†
Cite this: RSC Adv., 2020, 10, 28984
*
Jinhua Zhang, Yanshu Zhao, Chao Li and Hao Song
Biocatalytic transamination is widely used in industrial production of chiral chemicals. Here, we constructed
a novel multi-enzyme system to promote the conversion of the amination reaction. Firstly, we constructed
the ArR-uTA/TdcE/FDH/LDH multi-enzyme system, by combination of (R)-selective u-transaminase
derived from Arthrobacter sp. (ArR-uTA), formate dehydrogenase (FDH) derived from Candida boidinii,
formate acetyltransferase (TdcE) and lactate dehydrogenase (LDH) derived from E. coli MG1655. This
multi-enzyme system was used to efficiently remove the by-product pyruvate by TdcE and LDH to
facilitate the transamination reaction. The TdcE/FDH pathway was found to dominate the by-product
pyruvate removal in the transamination reaction. Secondly, we optimized the reaction conditions,
including D-alanine, DMSO, and pyridoxal phosphate (PLP) with different concentration of 2-pentanone
(as a model substrate). Thirdly, by using the ArR-uTA/TdcE/FDH/LDH system, the conversions of 2-
pentanone, 4-phenyl-2-butanone and cyclohexanone were 84.5%, 98.2% and 79.3%, respectively.
Received 14th July 2020
Accepted 28th July 2020
DOI: 10.1039/d0ra06140a
rsc.li/rsc-advances
the reaction proceed in the direction of chiral amines. One of
them is employing excess amino donors in a single enzyme
1. Introduction
catalytic system to drive the transamination reaction and push
the reaction toward the synthesis of chiral amines as much as
possible;24 the other is eliminating the substrate (i.e., by-
product) inhibition through a multi-enzyme coupled catalytic
reaction, thereby proceeding the reaction toward chiral amine
synthesis.25,26 Therefore, effective removal of the by-product,
pyruvate, is a key factor in the development of an efficient
asymmetric amination system.27–29 Several approaches to
remove pyruvate were reported to improve the conversion of
ketones to amines.29 Lactate dehydrogenase (LDH) along with
u-TA was used to convert pyruvate to lactate in transamination
reactions.30 Glucose dehydrogenase (GDH) was introduced in
the u-TA/LDH system to recycle the cofactor NADH.31 Thus, the
u-TA/LDH/GDH system has been used for conventional asym-
metric reductive amination and synthesized a series of valuable
amines.31–33 However, the conversion rate of asymmetric amines
obtained with the (R)-selective u-transaminase derived from
Arthrobacter sp. (ArR-uTA)/LDH/GDH system remained low.
Therefore, the conversion rate of amines from the correspond-
ing ketones needs further improvement.
Here, we developed a novel system to enhance the conver-
sion of ketones to amines. Firstly, we overexpressed four
formate acetyltransferases (i.e., Ybiw, PB, PD and TdcE)
derived from Escherichia coli MG1655 to convert pyruvate to
formate, and we found TdcE had the highest specic activity.
Formate dehydrogenase (FDH) was introduced to decompose
formate and produce NADH. LDH was introduced to recycle
NADH and remove pyruvate. Thus, the ArR-uTA/TdcE/FDH/
LDH system was constructed for transamination reaction
Chiral amines are a class of compounds containing amino
groups in the chiral center of small molecule compounds. They
are widely used in pharmaceutical and agricultural elds, such
as neurological, cardiovascular, antihypertensive, anti-infective
drugs, and vaccines, which play an important role in the
national economy.1–5 At present, the main methods for the
preparation of chiral amines6–9 are chemical synthesis, biolog-
ical resolution and biological asymmetric synthesis. Chiral
amines synthesized by chemical methods have many short-
comings, such as the need to use expensive metal catalysts, high
production costs, low enantioselectivity, and environmental
pollution.10–14 The maximum theoretical conversion of chiral
amine prepared by biological resolution is only 50%.15 Thus,
chemical methods and biological resolution cannot meet the
needs of industrial production. Biological asymmetric synthesis
method becomes the preferred strategy for the production of
chiral amines because of its theoretical conversion of up to
100%.16–19 Therefore, the synthesis of chiral amines by biolog-
ical asymmetric synthesis has been of increasing interest.
u-Transaminases (u-TAs) are pyridoxal phosphate (PLP)
dependent enzymes that could be used for the synthesis of
chiral amines.20–23 Since the transamination reaction catalysed
by u-TA is a reversible reaction, there are two methods to make
Frontier Science Center for Synthetic Biology, Key Laboratory of Systems
Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin
University, Tianjin, 300350, P. R. China. E-mail: hsong@tju.edu.cn; Tel:
+86-18722024233
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra06140a
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Table 1 Plasmids used in this study
Plasmids
Characteristics
Source
p2A4
AmpR
Our lab
p2A4T7
p2A4T7-ArR-uTA
p2A4tet
p2A4tet-LDH
p2A4Lac
p2A4Lac-Ybiw
p2A4Lac-pB
p2A4Lac-pD
p2A4Lac-tdcE
p3C5
p2A4 derivate, PT7
ArR-uTA gene in p2A4T7
p2A4 derivate, Ptet
LDH gene in p2A4tet-LDH
p2A4 derivate, PLac
Ybiw gene in p2A4Lac
pB gene in p2A4Lac
pD gene in p2A4Lac
tdcE gene p2A4Lac
CmR
This work
This work
This work
This work
Our lab
This work
This work
This work
This work
Our lab
p3C5BAD
p3C5BAD-GDH
p3C5 derivate, PBAD
GDH gene in p3C5BAD
This work
This work
were avoided in the optimal sequence. The gdh gene (NCBI Gene
ID: 938377) originated from Bacillus subtilis was obtained in the
same way as the arR-uTA gene. The arR-uTA gene and gdh gene
were in vitro synthesized. The ybiw (NCBI Gene ID: 945444), pB
(NCBI Gene ID: 945514), pD (NCBI Gene ID: 948454), tdcE
(NCBI Gene ID: 947623) and ldh (NCBI GenBank: APQ21713.1)
were amplied from the genomic DNA of the E. coli MG1655
through PCR with primers. The gene of green uorescent
protein (GFP) was from our research lab. Tables S1 and S2† list
all primers utilized and synthesized gene sequences in this
research, respectively.
ArR-uTA, Ybiw, PB, PD, TdcE, LDH and GFP were con-
structed in the p2A4 vector containing pBR322 origin. GDH and
GFP were constructed in the p3C5 vector containing p15A
origin. All genes were inserted into vectors by biobrick. All the
plasmids utilized in this research are shown in Table 1. E. coli
BL21 (DE3) was utilized for gene expression. Fig. 2A shows
schematic of plasmids used in this research.
Fig. 1 Asymmetric amination of ketones using u-transaminase. (A)
Pyruvate is removed by the TdcE/FDH/LDH system. Ketones:
substrates for transamination reaction; amine: products for trans-
amination reaction; R1 and R2: methyl, propyl, cyclohexyl, ethylphenyl;
u-TA: u-transaminase; PLP: pyridoxal phosphate; LDH: lactate
dehydrogenase; TdcE: formate acetyltransferase; FDH: formate
dehydrogenase. (B) Substrates for transamination reaction in this
study. (C) Products for transamination reaction in this study.
(Fig. 1A). The substrates and products for the transamination
reaction in this study were shown in Fig. 1B and C, respectively.
Secondly, we optimized the reaction conditions, including D-
alanine, DMSO, and PLP with different concentration of 2-
pentanone (as a model substrate) to increase the conversion of
ketones to amines. Thirdly, the conversions of 2-pentanone, 4-
phenyl-2-butanone and cyclohexanone by using the ArR-uTA/
TdcE/FDH/LDH system were 84.5%, 98.2% and 79.3%, respec-
tively. Besides, the pyruvate to formate pathway (i.e. TdcE/FDH)
dominates the by-product pyruvate removal in the trans-
amination reaction. This work demonstrated that the ArR-uTA/
TdcE/FDH/LDH system could effectively convert ketones to
amines.
2.2 Gene expression in E. coli
The transformed strains were cultured in LB liquid medium
containing appropriate antibiotics (100 mg ampicillin per L or
34 mg chloramphenicol per L), and cultivation for overnight at
ꢀ
37 C and 220 rpm. The seed cultures were diluted 1 : 100 in
fresh Terric Broth medium containing 1% (w/v) glucose and
appropriate antibiotics, and they were incubated at 37 ꢀC. When
the optical density at 600 nm (OD600) reached approximately
0.6, the temperature was decreased to 25 ꢀC with shaking at
200 rpm. Then, a nal concentration of 1 mM isopropyl-b-D-
thiogalactopyranoside (IPTG), 10 mM aTc or 1 mM L-arabinose
ꢀ
was added, and cells were cultured at 25 C for 16 h.
2. Materials and methods
2.3 Representative example for amination
2.1 Gene synthesis and plasmid construction
Cells containing ArR-uTA, TdcE, LDH or GDH were collected by
FDH is from Candida boidinii (Sigma). (R)-Selective u-trans- centrifugation at 6000 rpm for 10 min at 4 ꢀC, respectively.
aminase derived from Arthrobacter sp. (NCBI GenBank: Then, cells were suspended in PBS buffer (100 mM, pH 7.5) for
BAK39753.1) was identied from NCBI. Its amino acid the standardization of samples (OD600 ¼ 120) and lysed through
sequences were optimized in the E. coli strain BL21 (DE3) in sonication over ice for 30 min with 5 s pulses at 10 s intervals.
JCAT, and the restriction sites of NdeI, SpeI, HindIII and XbaI Cells lysates were regarded as crude products. All reactions were
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Fig. 2 SDS analysis and the analysis of four formate acetyltransferases. (A) Schematic diagram of plasmids used in this study. Four promoters: T7,
lac, tet and bad; the gene of (R)-selective u-transaminase from Arthrobacter sp.: arR-uTA; four formate acetyltransferase genes: ybiW, pflB, pflD
and tdcE; the gene of lactate dehydrogenase (LDH): ldh; the gene of glucose dehydrogenase (GDH): gdh. (B) SDS-PAGE analysis of purified
formate acetyltransferases. M, protein marker; lane 1, YbiW; lane 2, PflB; lane 3, TdcE; lane 4, PflD. (C) SDS-PAGE analysis. M, protein marker; lane
1, the blank control; lane 2, ArR-uTA; lane 3, LDH; lane 4, GDH. (D) Mechanism of measurement of formate acetyltransferases activity. FDH,
formate dehydrogenase. (E) Specific activity of the four formate acetyltransferases. IPTG was added to a final concentration of 1 mM. Results are
represented as mean ꢃ SD of three replicates.
ꢀ
carried out at 30 C and 180 rpm for 24 h in PBS buffer (1 mL, the mixture, and Na2SO4 was used to dry the organic phases.
100 mM, pH 7.5, 1.5 mM PLP, 1 mM NAD+). The ArR-uTA/LDH/ The conversion of ketones to amines was measured by gas
GDH system: 500 mL crude product of ArR-uTA, 120 mL crude chromatograph (GC).
product of LDH, 120 mL crude product of GDH, D-alanine
(250 mM, 22.3 mg), ketone (25 mM), glucose (150 mM) and 30%
(v/v) DMSO were added. The ArR-uTA/TdcE/FDH/LDH system:
500 mL crude product of ArR-uTA, 120 mL crude product of LDH,
2.4 GC method for conversion and optical purity
determination
120 mL crude product of TdcE, FDH (10 U), D-alanine (250 mM,
22.3 mg), ketone (25 mM), CoA (0.1 mM) and 30% (v/v) DMSO
were added. Aqueous NaOH (200 mL, 10 N) was added to stop
the reaction. Ethyl acetate (500 mL, twice) was utilized to extract
The conversions were analysed by GC-MS on TG-5MS column
(30 m ꢁ 0.32 mm ꢁ 0.25 mm; Shimadzu). The GC program
parameters included the following: injector 250 ꢀC; constant
ow 1.8 mL minꢂ1; temperature program 40 ꢀC/hold 2 min; 80
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ꢀC/rate 5 ꢀC per min/hold 2 min; 250 ꢀC/rate 20 ꢀC per min/hold
10 min. GC analyses and mass spectrometry analyses in this
study are shown in Table S3 and Fig. S1,† respectively. The
external standard method was utilized in gas chromatography
experiments and the standard curves of three substrates are
shown in Fig. S2.†
The optical purity of reaction products were determined by
GC on CP-Chirasil-Dex CB column (25 m ꢁ 0.32 mm ꢁ 0.25 mm;
Agilent) aer derivatization. Derivative method and analytical
method are from Mutti et al.33 The chiral analyses of products
are shown in Table S4.†
2.6 Purication of formate acetyltransferase
Collecting of cells was performed via centrifugation at 6000 rpm
for 10 min at 4 ꢀC and suspended in phosphate buffer (100 mM,
pH 7.5). Cell lysed through sonication over ice for 30 min with
5 s pulses at 10 s intervals. The soluble fraction of cell lysates
was separated by centrifugation (10 000 rpm for 20 min at 4 ꢀC).
The formate acetyltransferase were carried out by Ni-Sepharose
purication (Thermo Fisher Scientic). The puried proteins
were utilized for SDS-PAGE analysis and measurement of
enzyme activity.
2.5 SDS-PAGE
2.7 Measurement of formate acetyltransferases activity
Collecting of cells was performed via centrifugation at 6000 rpm
and lysed through sonication on ice. The soluble fractions were The enzyme activities of the four formate acetyltransferases
extracted from supernatant at 10 000 rpm for 10 min at 4 C. were determined by the double enzymatic method,34 based on
Protein samples were analysed on 10% acrylamide gels. the following reactions:
ꢀ
Fig. 3 Optimization of the condition of transamination reaction. (A–D) Effect of varied D-alanine, DMSO, substrate and PLP concentrations on
the asymmetric amination of 2-pentanone catalyzed by the ArR-uTA/TdcE/FDH/LDH system, respectively. (E) Effect of different CoA
concentrations on the asymmetric amination of 2-pentanone catalysed by the ArR-uTA/TdcE/FDH/LDH system. Conditions: 2-pentanone (25
mM), 30% (v/v) DMSO, D-alanine (250 mM), PBS buffer (pH 7.5, 100 mM), PLP (1.5 mM), NAD+ (1 mM), FDH (10 U), ArR-uTA (500 mL), TdcE (120 mL),
LDH (120 mL), shaking at 30 ꢀC and 180 rpm for 24 h. Results are represented as mean ꢃ SD of three replicates.
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Pyruvate þ CoA formate acetyltransferase formate þ acetyl-CoA formate
ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ!ƒ
lyase, is an enzyme to transform pyruvate into formate. There
are four formate acetyltransferases derived from E. coli MG1655
(i.e., Ybiw, PB, PD and TdcE) catalysing the reaction from
pyruvate to formate. In this study, we constructed plasmids for
these four genes (Fig. 2A) and conrmed their soluble expres-
sion (Fig. 2B). The catalytic activity of four formate acetyl-
transferases were compared through the formation of formate.
Considering the reversible reaction between pyruvate and
formate, FDH was introduced to decompose formate and
produce NADH (Fig. 2D). Thus, we tested these four enzymes
þNADþ formate dehydrogenase CO þ H O þ NADH
ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ!ƒ
2
2
The reactions were carried out in total volume of 1 mL, with
the compositions of sodium pyruvate (20 mM), CoA (0.1 mM),
NAD+ (1 mM), FDH (1 U), PBS buffer (100 mM, pH 7.5) and
puried formate acetyltransferase (0.69 mg mLꢂ1). The mixture
ꢀ
was shaken at 30 C for 10 s, and the concentration of NADH
activities by determining the concentration of NADH at OD340
.
was measured as OD340. The curves of OD340 with time are
We demonstrated that the overexpression of TdcE enabled the
highest specic activity (299.3 U mgꢂ1 puried protein) in
comparison to the other three formate acetyltransferases
(Fig. 2E). Therefore, TdcE was selected as the enzyme to catalyse
the reaction from pyruvate to formate for subsequent experi-
ments. Furthermore, TdcE/FDH was regarded as the formate
pathway to remove pyruvate. Considering the recycle of co-
factor NADH, LDH was introduced to recover NAD+. In
shown in Fig. S3.†
3. Results and discussion
3.1 Overexpression and analysis of formate
acetyltransferases
Pyruvate can be catalysed to produce lactate and formate in E.
coli. Formate acetyltransferase, also known as pyruvate formate
Fig. 4 Asymmetric amination of ketones using ArR-uTA. (A) Effect of different systems (the ArR-uTA/LDH/GDH system, and the ArR-uTA/TdcE/
FDH/LDH system) on the asymmetric amination of ketones. A/L/G, the ArR-uTA/LDH/GDH system; A/T/F/L, the ArR-uTA/TdcE/FDH/LDH
system. ArR-uTA/LDH/GDH system, conditions: substrate (25 mM), 30% (v/v) DMSO, D-alanine (250 mM), glucose (150 mM), PBS buffer (pH 7.5,
100 mM), PLP (1.5 mM), NAD+ (1 mM), ArR-uTA (500 mL), LDH (120 mL), GDH (120 mL), shaking at 30 ꢀC and 180 rpm for 24 h; ArR-uTA/TdcE/FDH/
LDH system, conditions: substrate (25 mM), 30% (v/v) DMSO, D-alanine (250 mM), PBS buffer (pH 7.5, 100 mM), PLP (1.5 mM), NAD+ (1 mM), CoA
(0.1 mM), FDH (10 U), ArR-uTA (500 mL), TdcE (120 mL), LDH (120 mL), shaking at 30 ꢀC and 180 rpm for 24 h. (B) The conversion of 2-pentanone to
pentan-2-amine at different reaction times. A/L/G, the ArR-uTA/LDH/GDH system; A/T/F/L, the ArR-uTA/TdcE/FDH/LDH system. ArR-uTA/
LDH/GDH system, conditions: substrate (25 mM), 30% (v/v) DMSO, D-alanine (250 mM), glucose (150 mM), PBS buffer (pH 7.5, 100 mM), PLP (1.5
mM), NAD+ (1 mM), ArR-uTA (500 mL), GDH (120 mL), LDH (120 mL); ArR-uTA/TdcE/FDH/LDH system, conditions: substrate (25 mM), 30% (v/v)
DMSO, D-alanine (250 mM), PBS buffer (pH 7.5, 100 mM), PLP (1.5 mM), NAD+ (1 mM), CoA (0.1 mM), FDH (10 U), ArR-uTA (500 mL), TdcE (120 mL),
LDH (120 mL). (C) Effect of two pyruvate removal pathways (the LDH pathway, pyruvate to lactate; the TdcE/FDH pathway, pyruvate to formate)
on the asymmetric amination of ketones. A/L, the ArR-uTA/LDH system; A/T/F, the ArR-uTA/TdcE/FDH system. ArR-uTA/LDH system,
conditions: substrate (25 mM), 30% (v/v) DMSO, D-alanine (250 mM), PBS buffer (pH 7.5, 100 mM), PLP (1.5 mM), NADH (1 mM), CoA (0.1 mM),
ArR-uTA (500 mL), LDH (120 mL), shaking at 30 ꢀC and 180 rpm for 24 h; ArR-uTA/TdcE/FDH system, conditions: substrate (25 mM), 30% (v/v)
DMSO, D-alanine (250 mM), PBS buffer (pH 7.5, 100 mM), PLP (1.5 mM), NAD+ (1 mM), CoA (0.1 mM), FDH (10 U), ArR-uTA (500 mL), TdcE (120 mL),
shaking at 30 ꢀC and 180 rpm for 24 h. Results are represented as mean ꢃ SD of three replicates. **p < 0.01; ***p < 0.001.
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addition, LDH was regarded as the lactate pathway to remove conversion of 2-pentanone to pentan-2-amine reached 84.5%.
pyruvate. Thus, a novel multi-enzyme system, i.e., the u-TA/ Thus, subsequent experiments were performed at 250 mM D-
TdcE/FDH/LDH system, was established for transamination alanine, 30% (v/v) DMSO, 25 mM substrate and 1.5 mM PLP.
reaction (Fig. 1A).
In order to determine the expression level of promoters, we
constructed four inducible promoters (i.e., PT7, Plac, Ptet, Pbad) to
drive the expression of gfp gene, respectively (Fig. S4†). The
expression levels of TdcE, LDH and GDH should be similar,
thus avoiding the effect of enzyme dosages on the experimental
3.3 Analysis the asymmetric amination of the ArR-uTA/
TdcE/FDH/LDH system
Three substrates (i.e., 2-pentanone, 4-phenyl-2-butanone and
cyclohexanone) were examined to compare the conversion of
ketones to amines obtained with both the ArR-uTA/TdcE/FDH/
LDH system and the ArR-uTA/LDH/GDH system. By using the
ArR-uTA/TdcE/FDH/LDH system, the conversion of 2-penta-
none to pentan-2-amine was increased from 75.1% (by the ArR-
uTA/LDH/GDH system) to 84.5%, the conversion of 4-phenyl-2-
butanone to 4-phenylbutan-2-amine was increased from 74.9%
to 98.2%, and the conversion of cyclohexanone to cyclohexyl-
amine was increased from 55.8% to 79.3%, respectively
(Fig. 4A). Besides, the conversion of 2-pentanone to pentan-2-
amine and the conversion of 4-phenyl-2-butanone to 4-
phenylbutan-2-amine with the ArR-uTA/LDH/GDH system re-
results. The results showed that the GFP/OD600 values of Plac
,
Ptet and Pbad were all about 20 000, when the inducer concen-
trations were 1 mM IPTG, 10 mM aTc and 1 mM L-arabinose,
respectively. Therefore, 1 mM IPTG, 10 mM aTc and 1 mM L-
arabinose were selected for further studies. The soluble frac-
tions of cells containing ArR-uTA, LDH or GDH were analysed
by SDS-PAGE (Fig. 2C).
3.2 Optimization of the condition of transamination
reaction
Reaction condition is a key factor in the transamination reac- ported in the literature were only 67% and 82%, respectively.33
tion. We investigated the effects of D-alanine, DMSO, substrate The enantiomeric excess of reaction products was shown in
concentration and PLP on the conversion of ketones to amines. Table 2. It is remarkable that the ArR-uTA/TdcE/FDH/LDH
2-Pentanone was used as a model substrate in the optimization system could efficiently synthesize chiral amines. Moreover,
of reaction conditions.
the ArR-uTA/TdcE/FDH/LDH system does not need to be
The conversion was improved with the concentration of D- provided with co-substrate (i.e., glucose).
alanine increasing from 50 mM to 250 mM; however, there was
2-Pentanone was used as a model substrate to compare the
a slight increase in conversion when the concentration of D- conversion of these two systems at different reaction times. The
alanine was more than 250 mM (Fig. 3A). The conversion could conversion with the ArR-uTA/TdcE/FDH/LDH system reached
increase up to 60.2% at 250 mM D-alanine. Thus, 250 mM D- the maximum value within 4 to 8 h, while the conversion with
alanine was selected for further experiments. When the DMSO the ArR-uTA/LDH/GDH system reached the maximum value
concentration increased from 0 to 30% (v/v), the conversion was within 8 to 12 h (Fig. 4B). Compared with the ArR-uTA/LDH/
also enhanced. While the conversion decreases when the DMSO GDH system, the ArR-uTA/TdcE/FDH/LDH system led to
concentration was over 30% (v/v). Optimization of the DMSO a shorter reaction time and a higher conversion. The ArR-uTA/
concentration led to 70.6% conversion at 30% (v/v) DMSO TdcE/FDH/LDH system has two enzymes, TdcE and LDH, to
(Fig. 3B). It is noted that the conversion was decreased with the remove the by-product pyruvate, thereby shiing the equilib-
concentration of substrate increases at 30% (v/v) DMSO and rium toward amine synthesis. Furthermore, FDH and LDH in
250 mM D-alanine concentration (Fig. 3C). The conversion the ArR-uTA/TdcE/FDH/LDH system could not only recycle the
reached 79.3% at 25 mM substrate concentration. The conver- redox cofactor (i.e., NAD+ and NADH), but also promote the
sion of ketone to amine was increased when more PLP was removal of pyruvate by decomposing formate.
added, while the concentration of PLP has minor positive
De-acetylation of pyruvate to formate catalysed by TdcE was
impact on the conversion at the PLP concentration above accompanied by the conversion of CoA to acetyl CoA. Thus,
1.5 mM (Fig. 3D). Upon optimization of reaction conditions, the regeneration of CoA from acetyl CoA would be a crucial step for
Table 2 Effect the ArR-uTA/LDH/GDH system and the ArR-uTA/TdcE/FDH/LDH system on the asymmetric amination of ketones
A/L/G systema
A/T/F/L systemb
Conversion [%]
c
c
Entry
Substrate
Conversion [%]
eeamine [%]
eeamine [%]
1
2
3
2-Pentanone
4-Phenyl-2-butanone
Cyclohexanone
75.1
74.9
55.8
>99(R)
>99(R)
n.a.d
84.5
98.2
79.3
>99(R)
>99(R)
n.a.d
a
A/L/G, the ArR-uTA/LDH/GDH system. Conditions: substrate (25 mM), 30% (v/v) DMSO, D-alanine (250 mM), glucose (150 mM), PBS buffer (pH 7.5,
100 mM), PLP (1.5 mM), NAD+ (1 mM), ArR-uTA (500 mL), LDH (120 mL), GDH (120 mL), shaking at 30 ꢀC and 180 rpm for 24 h. b A/T/F/L, the ArR-
uTA/TdcE/FDH/LDH system. Conditions: substrate (25 mM), 30% (v/v) DMSO, D-alanine (250 mM), PBS buffer (pH 7.5, 100 mM), PLP (1.5 mM),
c
NAD+ (1 mM), CoA (0.1 mM), FDH (10 U), ArR-uTA (500 mL), TdcE (120 mL), LDH (120 mL), shaking at 30 ꢀC and 180 rpm for 24 h. Optical
d
purity of reaction products was determined by GC. n.a.: not applicable.
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the feasibility of ArR-uTA/TdcE/FDH/LDH cascade. Acetyl-CoA enzymes production cost in our system compared with the re-
reacted with oxaloacetate to give CoA and citrate in the tricar- ported system. In addition, the conversion with our system
boxylic acid cycle. The crude enzyme preparations contained reached maximum within 4 to 8 h, while the conversion with the
GltA (citrate synthase) that catalysed this reaction, thereby reported system reached maximum within 8 to 12 h. Compared
regenerating CoA from acetyl-CoA. We also performed experi- with the reported system, our system led to a shorter reaction
ments on varied concentrations of CoA in the ArR-uTA/TdcE/ time and ꢄ20% increase in the conversion. Thus, our system
FDH/LDH system, in which 2-pentanone was used as a model has advantages in the production cost.
substrate, as shown in Fig. 3E. The results showed that the
conversion of 2-pentanone to pentan-2-amine barely increased
when the concentration of CoA was more than 0.1 mM. It
Conflicts of interest
proved that 0.1 mM CoA was sufficient, and the regeneration of There are no conicts of interest to declare.
CoA from acetyl CoA was feasible in the ArR-uTA/TdcE/FDH/
LDH system.
Acknowledgements
In order to investigate which of the two pyruvate removal
pathways dominates, three substrates were tested to compare We sincerely acknowledge the nancial support from the
the conversion of ketones to amines obtained with the ArR-uTA/ National Natural Science Foundation of China (NSFC
LDH system and the ArR-uTA/TdcE/FDH system. 1 mM NADH 21621004).
was added to the ArR-uTA/LDH system, and 1 mM NAD+ was
added to the ArR-uTA/TdcE/FDH system. Under the ArR-uTA/
TdcE/FDH system, the conversions of 2-pentanone, 4-phenyl-
Notes and references
2-butanone and cyclohexanone were 76.3%, 89.6% and 62.7%,
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LDH system were 49.9%, 71.5% and 47.4%, respectively
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amines than that of the ArR-uTA/LDH system. Furthermore, it
illustrated that the formate pathway (i.e. TdcE/FDH) dominates
the by-product pyruvate removal in the transamination
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