Enhanced production of optical (: S)-acetoin by a recombinant Escherichia coli whole-cell biocatalyst with NADH regeneration

Article abstract of DOI:10.1039/c8ra06260a

Acetoin is an important platform chemical with a variety of applications in foods, cosmetics, chemical synthesis, and especially in the asymmetric synthesis of optically active pharmaceuticals. It is also a useful breath biomarker for early lung cancer diagnosis. In order to enhance production of optical (S)-acetoin and facilitate this building block for a series of chiral pharmaceuticals derivatives, we have developed a systematic approach using in situ-NADH regeneration systems and promising diacetyl reductase. Under optimal conditions, we have obtained 52.9 g L^-1 of (S)-acetoin with an enantiomeric purity of 99.5% and a productivity of 6.2 g (L h)^-1. The results reported in this study demonstrated that the production of (S)-acetoin could be effectively improved through the engineering of cofactor regeneration with promising diacetyl reductase. The systematic approach developed in this study could also be applied to synthesize other optically active α-hydroxy ketones, which may provide valuable benefits for the study of drug development.

Full text of DOI:10.1039/c8ra06260a

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Enhanced production of optical (S)-acetoin by
a recombinant Escherichia coli whole-cell
biocatalyst with NADH regeneration†
Cite this: RSC Adv., 2018, 8, 30512
ab
b
Jian-Xiu Li,
Neng-Zhong Xie
Yan-Yan Huang,b Xian-Rui Chen,b Qi-Shi Du,
Jian-Zong Meng,a
b
ab
*
*
and Ri-Bo Huang
Acetoin is an important platform chemical with a variety of applications in foods, cosmetics, chemical
synthesis, and especially in the asymmetric synthesis of optically active pharmaceuticals. It is also
a useful breath biomarker for early lung cancer diagnosis. In order to enhance production of optical (S)-
acetoin and facilitate this building block for a series of chiral pharmaceuticals derivatives, we have
developed a systematic approach using in situ-NADH regeneration systems and promising diacetyl
reductase. Under optimal conditions, we have obtained 52.9 g Lꢀ1 of (S)-acetoin with an enantiomeric
purity of 99.5% and a productivity of 6.2 g (L h)ꢀ1. The results reported in this study demonstrated that
the production of (S)-acetoin could be effectively improved through the engineering of cofactor
regeneration with promising diacetyl reductase. The systematic approach developed in this study could
also be applied to synthesize other optically active a-hydroxy ketones, which may provide valuable
benefits for the study of drug development.
Received 24th July 2018
Accepted 21st August 2018
DOI: 10.1039/c8ra06260a
rsc.li/rsc-advances
antibiotics to synthesize optically active drugs, which could
enhance drug targeting properties and reduce side effects.12,13
1. Introduction
Native microorganisms generally produce acetoin as
a mixture of two stereoisomers, in which (R)-acetoin is
predominant and (S)-acetoin exists as a minor by-product,
leading to very low yield of (S)-acetoin and high cost of chiral
separation.14–16 Liu et al. developed an engineered Lactococcus
lactis to produce (S)-acetoin from glucose.17 However, the
nonenzymatic oxidative decarboxylation of a-acetolactate to
diacetyl occurred spontaneously and inefficiently, the engi-
neered strain could only produce 5.8 g Lꢀ1 of (S)-acetoin with
a productivity of 0.19 g (L h)ꢀ1. Several attempts to produce (S)-
acetoin by using puried enzymes have been reported previ-
ously. According to Loschonsky et al., cyclohexane-1,2-dione
hydrolase formed (S)-acetoin with a 95% enantiomeric excess
from 23 mM substrate by homocoupling of acetaldehyde.18 In
2013, Gao et al. developed a two-enzymes coupling system, in
which NADPH-dependent carbonyl reductase reduced diacetyl
to produce (S)-acetoin, and glucose dehydrogenase was used to
regenerate NADPH. Under optimal conditions, 12.2 g Lꢀ1 of (S)-
acetoin (enantiomeric purity: 96.9%) with a productivity of 9.8 g
(L h)ꢀ1 was produced.19
Enantiomeric a-hydroxy ketones are important pharmaceutical
intermediates, widely applied in antifungal agents, antitumor
antibiotics, antidepressants, farnesyl transferase inhibitors and
selective inhibitors of amyloid-b protein production.1–3 Acetoin
(3-hydroxy-2-butanone), the smallest natural chiral a-hydroxy
ketone, is a popular avor additive, preservative, plant growth
promoter and breath biomarker of early lung cancer, and is
identied as one of the top 30 potential building blocks.4–7 This
four-carbon compound has one chiral center in the molecule
and therefore exists as two stereoisomers, (R)-acetoin and (S)-
acetoin.8,9 With FEMA no. 2008, acetoin is a substance generally
recognized as safe (visit https://ntp.niehs.nih.gov/ for toxicity
and related information). Acetoin and its isomers are widely
applied in foods, cosmetics, chemical synthesis, especially in
asymmetric synthesis of optically active pharmaceuticals and
liquid crystals.10,11 Its derivate, 4-chloro-4,5-dimethyl-1,3-
dioxolan-2-one, has been extensively utilized in modifying
aState Key Laboratory for Conservation and Utilization of Subtropical
Agro-Bioresources, Life Science and Biotechnology College, Guangxi University, 100
Daxue Road, Nanning, 530004, China. E-mail: xienengzhong@gxas.cn; guruace@
163.com
bState Key Laboratory of No-Food Biomass and Enzyme Technology, National
Engineering Research Center for No-Food Biorenery, Guangxi Key Laboratory of
Biorenery, Guangxi Academy of Sciences, 98 Daling Road, Nanning, 530007, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra06260a
Recently, recombinant whole-cells have emerged as bio-
catalysts for (S)-acetoin production. He et al.20 described a whole-
cell biocatalytic process with a high yield by recombinant E. coli
cells co-expressing (2R,3R)-2,3-butanediol dehydrogenase (R,R-
BDH), NADH oxidase and hemoglobin. The recombinant strain
produced 72.4 g Lꢀ1 (S)-acetoin from a mixtures of 2,3-butanediol
isomers. However, the enantiomeric purity of the product was
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only 94.7% for the generation of (R)-acetoin. Since (S)-acetoin can
be used to provide chiral groups in high-value pharmaceuticals or
for liquid crystals, high optical purity (i.e., over 98.0%) is partic-
ularly required. Gao et al. developed a whole-cell biocatalytic
process for (S)-acetoin production by over-expressing diacetyl
reductase (DAR) in E. coli.12 When diacetyl and glucose were used
as substrate and co-substrate respectively, the recombinant
whole-cells could produce 39.4 g Lꢀ1 of (S)-acetoin in 20 h. Since
diacetyl is a prochiral compound that can be produced by
chemical synthesis or microbial fermentation, it is a preferred
substrate for optical (S)-acetoin.12,19,21,22 However, two problems
still need to be solved. First, due to the high concentration of
intracellular DAR, the endogenous cofactor regeneration system
of E. coli is typically not sufficiently efficient,23,24 limiting the titer
and productivity of (S)-acetoin; second, organic acids (e.g. acetic
acid and lactic acid) produced during glucose metabolism make
product recovery difficult and increase glucose consumption.25
In this study, an efficient and economical process was
developed for optical (S)-acetoin production from diacetyl. As
shown in Fig. 1, two in situ-NADH regeneration systems were
introduced into E. coli and determined their effectiveness in
(S)-acetoin production. DAR genes from several species were
codon-optimized and over-expressed to screen for a promising
DAR with high stereoselectivity, specic activity and stability.
Finally, a series of whole-cell biocatalytic reactions were per-
formed to achieve high (S)-acetoin production.
2. Materials and methods
2.1 Chemicals
(2S,3S)-2,3-Butanediol (99.0%) and meso-2,3-butanediol (98.0%)
were obtained from ACROS (Geel, Belgium). (2R,3R)-2,3-Buta-
nediol (97.0%), acetoin (99.0%), NADH, and NAD+ were ob-
tained from Sigma (St Louis, USA). Diacetyl (99.0%) was
obtained from Aladdin (Shanghai, China). PCR primers were
synthesized by Sangon (Shanghai, China) and DNA sequencing
was performed by Genscript (Nanjing, China). Yeast extract and
peptone were obtained from Oxoid (Hampshire, UK).
2.2 Bacterial strains and vectors
The strains and vectors used in the present study are listed in
Table 1. E. coli DH5a and E. coli BL21(DE3) were used for gene
cloning and protein expressing, respectively. The pET-28a(+) and
pETDuet-1 were used for gene expression. Luria–Bertani medium
(10 g Lꢀ1 peptone, 5 g Lꢀ1 yeast extract, and 10 g Lꢀ1 NaCl at pH
7.0) was used for E. coli cultivation and solidied with 1.5% agar,
if necessary. Kanamycin and ampicillin were used at concentra-
tions of 30 mg mLꢀ1 and 100 mg mLꢀ1, respectively.
2.3 Synthesis of DAR genes and construction of co-
expression systems
DNA manipulations were carried out using standard proto-
cols.26 The codons for the DAR genes, Kpdar from K. pneumoniae
Fig. 1 Scheme for (S)-acetoin production from diacetyl using recombinant whole-cell biocatalysts with NADH regeneration systems. dar, the
gene encoding diacetyl reductase (DAR); gdh, the gene encoding glucose dehydrogenase (GDH); fdh, the gene encoding formate dehydro-
genase (FDH); Ap: the gene encoding ampicillin resistance.
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Table 1 Strains and plasmids used in this study
of the mixture was reacted in 100 mM phosphate buffer (pH 7.5)
at 32 C and 200 rpm for 2 h. (S)-Acetoin production by whole-
ꢁ
Source or
reference
cells co-expressing fdh with DAR genes was carried out using
15.0 g Lꢀ1 of diacetyl and 12.0 g Lꢀ1 of formate (only account
molecular mass of formate ion). For (S)-acetoin production by
whole-cells co-expressing Kpdar with gdh, 15.0 g Lꢀ1 of diacetyl
and 30.0 g Lꢀ1 of glucose were required.
Strain and plasmid
Genotype and properties
Strain
E.coli DH5a
supE44OlacU169 (480 lacZOM15)
hsdR17 recA1 endA1
Novagen
Novagen
gyrA96 thi-1 relA1
E.coli BL21(DE3)
Fꢀ ompT hsdSB (rBꢀmBꢀ)
gal (l c I 857 ind1 Sam7
nin5 lacUV5 T7gene1) dcm (DE3)
2.5 Optimization of the reaction conditions
To achieve a high conversion efficiency, theꢁpH (5.5, 6.0, 6.5, ꢁ7.0,
ꢁ
ꢁ
ꢁ
7.5, or 8.0), temperature (28 C, 32 C, 36 C, 40 C, or 45 C),
molar ratio of formate to diacetyl (0.6, 0.8, 1.0, 1.2, 1.6, or 2.0),
and shaking speed (75 rpm, 100 rpm, 125 rpm, 150 rpm, or 175
rpm) were investigated in turn. An initial bioconversion was
Plasmid
pET-28a(+)
pETDuet-1
pET-28a(+)-Kpdar
pETDuet-fdh
Overexpression vector; Kanr
Overexpression vector; Ampr
Kpdar in pET-28a(+)
Novagen
Novagen
This work
This work
This work
This work
This work
This work
This work
This work
This work
fdh in pETDuet-1
ꢁ
reacted in 100 mM phosphate buffer at 32 C and 200 rpm for
pETDuet-Kpdar
pETDuet-Kpdar-gdh
pETDuet-Kpdar-fdh
pETDuet-Ppdar-fdh
pETDuet-ard II-fdh
pETDuet-adh-fdh
pETDuet-adr-fdh
Kpdar in pETDuet-1
2 h. The concentrationsꢀ1of cell, diacetyl and formate in the
Kpdar and gdh in pETDuet-1
Kpdar and fdh in pETDuet-1
Ppdar and fdh in pETDuet-1
ard II and fdh in pETDuet-1
adh and fdh in pETDuet-1
adr and fdh in pETDuet-1
reaction was 5.5 g(DCW) L , 15 g Lꢀ1 and 12 g Lꢀ1, respectively.
2.6 Fed-batch biotransformation
Fed-batch biotransformation was carried out in a 10 mL mixture
at 40 ꢁC and 125 rpm. The initial concentrations of diacetyl and
whole-cell biocatalyst were 15.0 g Lꢀ1 and 5.5 g(DCW) Lꢀ1
,
CICC 10011 (GenBank accession no. HM113375.1), Ppdar from
P. polymyxa DSM 365 (GenBank accession no. KT717931), ard II
from K. pneumoniae 342 (GenBank accession no. CP000964.1),
adh from Mycobacterium sp. B-009 (GenBank accession no.
AB905197.1), and adr from R. erythropolis WZ010 (GenBank
accession no. KC508606.1), were optimized and synthesized by
Genscript (Nanjing, China), and then inserted into pETDuet-1
between the Nco I and Sac I sites to yield expression vectors.
The formate dehydrogenase (FDH) gene fdh from S. cerevisiae
(NCBI reference sequence NM_001183808.1), and Glucose
dehydrogenase (GDH) gene gdh from B. subtilis (GenBank
accession no. CP019663.1), were also synthesized with codon
optimization. The fdh and gdh fragments were digested with
Nde I and Kpn I, respectively. Then the fragments were ligated
into the expression vectors that had been digested with the
same restriction endonucleases. The co-expression plasmids
were designated pETDuet-Kpdar-gdh, pETDuet-Kpdar-fdh, pET-
Duet-Ppdar-fdh, pETDuet-ard II-fdh, pETDuet-adh-fdh, and
pETDuet-adr-fdh, respectively.
respectively. The ratio of formate to diacetyl was 1.2, and the pH
was 6.5. During bioconversion, diacetyl was fed at intervals to
maintain its concentration at 5.0–15.0 g Lꢀ1, and pH was
adjusted to 6.5 by formic acid. At the same time, small amounts
of whole cell biocatalyst were added at the interval time, until
the whole cell concentration reached to 11.0 g(DCW) Lꢀ1
.
2.7 Purication of KpDAR
E. coli BL21(DE3) carrying plasmid pET-28a (+)-Kpdar was
cultivated at 37 ꢁC and 200 rpm to OD600 of 0.6, and then
ꢁ
0.4 mM IPTG was added to the culture at 20 C for 12 h. The
culture was centrifuged at 6000 rpm, and the cell pellet was
resuspended in buffer (pH 7.4; 20 mM potassium phosphate,
500 mM sodium chloride, 20 mM imidazole, 1 mM DTT,
0.1 mM PMSF, and 10% glycerol). Cells were disrupted by
sonication in an ice bath and centrifuged at 12 000 rpm for
30 min at 4 ꢁC. The Kpdar was puried by HisTrap HP column
(GE Healthcare) and detected by SDS-PAGE.
2.8 Assays for DARs, GDH and FDH activities
2.4 Biocatalyst preparation and bioconversion conditions
Crude proteins were extracted from recombinant E. coli using
ꢁ
The recombinant E. coli strains were cultivated at 37 C and BugBuster Master Mix reagent (Novagen). Enzyme activities
180 rpm to OD600 of 0.6. According to preliminary experi- were determined by monitoring the change in optical density at
ments, recombinant genes expression was induced with 340 nm corresponding to the reduction of NAD+ or oxidation of
0.2 mM IPTG at 25 ꢁC for 12 h. The cells were harvested by NADH (3340 ¼ 6.22/mM cm) using a UV/visible spectrophotom-
centrifugation at 6000 rpm and then washed twice with 0.85% eter (DU800, Beckman). One unit of DAR activity was dened as
NaCl. The cell pellet was resuspended in 100 mM phosphate the amount of enzyme that consumed 1 mmol of NADH per min.
buffer (pH 7.5) at a concentration of 20 g(DCW) Lꢀ1 and main- The reaction mixture contained 5 mM diacetyl and 0.2 mM
ꢁ
tained at 4 C. The dry cell weight (DCW) of E. coli was deter- NADH in 100 mM phosphate buffer (pH 6.5). One unit of GDH
mined by converting the OD600 value with a coefficient of 0.275 or FDH activity was dened as the amount of enzyme that
g(DCW) per (LOD600).27
produced 1 mmol of NADH per min. The reaction mixture con-
A typical bioconversion reaction was carried out with 5.5 tained 5 mM substrate (glucose or formate) and 0.2 mM NAD+ in
ꢀ1
g
L
whole-cell biocatalyst in 50 mL asks. A total of 10 mL 100 mM phosphate buffer (pH 6.5). The reaction solution for the
(DCW)
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KpDAR reduction assay contained 5 mM of ketone substrate and co-expressed with DAR gene in E. coli BL21(DE3). To obtain
and 0.2 mM of NADH in 100 mM phosphate buffer and for a promising DAR, ve DAR genes from several species were
KpDAR oxidation contained 5 mM of alcohol substrate and codon-optimized (see ESI Table S1†) and co-expressed with fdh or
0.2 mM of NAD+ in 100 mM phosphate buffer. Protein gdh to compare for (S)-acetoin synthesis. Recombinant DARs were
concentrations were determined via the Bradford method using detected by SDS-PAGE (see ESI Fig. S1†).
bovine serum albumin as a standard.
Batch biotransformation was conducted to evaluate the
performance of two NADH regeneration systems with formate or
glucose as co-substrate. As shown in Table 2, the conversion rate
was low, obtained by E. coli BL21/pETDuet-Kpdar with single
expression of KpDAR, and the nal titer of (S)-acetoin was only
0.8 g Lꢀ1 with an enantiomeric purity of 89.3% and productivity
of 0.4 g (L h)ꢀ1. The crude extract enzyme of E. coli BL21/pET-
Duet-Kpdar-gdh showed a KpDAR activity of 33.4 U mgꢀ1 and
a GDH activity of 16.3 U mgꢀ1. When introducing the GDH/
glucose system, the titer of (S)-acetoin increased to 4.3 g Lꢀ1
with an enantiomeric purity of 94.4% and productivity of 2.2 g (L
h)ꢀ1 by E. coli BL21/pETDuet-Kpdar-gdh. Introduction of the
FDH/formate system resulted in a higher (S)-acetoin titer,
productivity and enantiomeric purity. 6.7 g Lꢀ1 of (S)-acetoin was
obtained with an enantiomeric purity of 99.6% and productivity
of 3.4 g (L h)ꢀ1 by E. coli BL21/pETDuet-Kpdar-fdh. Crude extract
of this recombinant strain showed a KpDAR activity of 34.9 U
mgꢀ1 and a FDH activity of 0.45 U mgꢀ1, while E. coli BL21/
2.9 Analytical methods
The concentrations of diacetyl, acetoin, and 2,3-butanediol in
the supernatant were determined using a gas chromatography
(GC) system (Agilent 7890A) equipped with a polar column
(Phenomenex ZB-WAX plus, 0.32 mm ꢂ 0.25 mm ꢂ 30 m). The
injector and detector temperatures were both 250 ꢁC. The
column oven temperature was maintained at 100 ꢁC for 1 min,
then increased to 180 ꢁC at a rate of 20 ꢁC minꢀ1, nally
ꢁ
maintained at 180 C for 3 min. (R)-Acetoin and (S)-acetoin in
the supernatant were extracted using ethyl acetate and then
measured by the GC system (Agilent 7890A) equipped with
a chiral column (Agilent Cyclosil-B, 0.32 mm ꢂ 0.25 mm ꢂ 30
m). The temperatures of injector and detector were both 240 ꢁC.
The column oven temperature was maintained at 100 ꢁC for
1 min and increased to 120 ꢁC at a rate of 10 ꢁC minꢀ1. Then, it
ꢀ1
ꢁ
ꢁ
was increased to 130 C at a rate of 6 C min and nally to
230 ꢁC at a rate of 20 ꢁC minꢀ1. The enantiomeric purity of (S)-
acetoin was dened as [S]/([S] + [R]) ꢂ 100%, where [R] and [S]
represented the concentrations of (R)-acetoin and (S)-acetoin,
respectively. Formate and other organic acids concentration
were analyzed using high-performance liquid chromatography
(HPLC) system (Dionex UltiMate 3000) equipped with a Carbo-
mix H-NP column (Sepax, 7.8 ꢂ 300 mm). The analysis was
conducted at 55 ꢁC with a mobile phase of 2.5 mM H2SO4 at
pETDuet-Kpdar showed a KpDAR activity of 79.0 U mgꢀ1
.
Acetoin biosynthesis initiates with the condensation of two
pyruvate molecules to yield one a-acetolactate, then yield (R)-
acetoin by decarboxylases or diacetyl upon spontaneous decar-
boxylation.24,34,35 Diacetyl can be further converted into (R)-ace-
toin or (S)-acetoin by DAR or BDH. Liang et al. reported that E.
coli has an endogenous acetoin/2,3-butanediol pathway and
glucose can be metabolized to acetoin by diacetyl-dependent
endogenous system.24 In our study, the enantiomeric purity of
(S)-acetoin produced by E. coli BL21/pETDuet-Kpdar-gdh with
glucose as co-substrate was low (94.4%), indicating that (R)-
acetoin was produced by the intrinstic acetoin/2,3-butanediol
pathway of E. coli. While in FDH/formate system, only a trace
amount of (R)-acetoin was detected for the absence of glucose,
and the enantiomeric purity of (S)-acetoin can reach to 99.6% by
E. coli BL21/pETDuet-Kpdar-fdh.
a ow rate of 0.6 mL minꢀ1
.
3. Results and discussion
3.1 Construction of different whole-cell biocatalysts by
cofactor engineering
Redox balance plays an important role for cell growth and cellular
metabolism.28 When heterogeneous oxidoreductases are overex-
pressed, the endogenous cofactor regeneration system is typically
not sufficiently efficient.23 GDH and FDH are widely used for in
situ-cofactor regeneration.25,29–31 To investigate their effect on (S)-
acetoin production, gdh gene from Bacillus subtilis32 and fdh gene
from Saccharomyces cerevisiae33 were synthesized aer codon
optimization (see ESI Table S1†) by GenScript (Nanjing, China)
Low-molecular-weight formate has similar price with
glucose. Regeneration of 1 mol of NADH requires only 45 g of
formate comparing 172 g of glucose, indicating that the
reducing equivalent capacity of formate is approximately four-
fold higher than that of glucose.30 In addition, glucose can be
metabolized to acetic acid, lactic acid by E. coli, further
Table 2 The products of batch bioconversion with different DARs and cofactor regeneration systems
Strain
(S)-Acetoin (g Lꢀ1
)
Productivity (g (L h)ꢀ1
)
Enantiomeric purity (%)
E. coli BL21/pETDuet-Kpdar
E. coli BL21/pETDuet-Kpdar-gdh
E. coli BL21/pETDuet-Kpdar-fdh
E. coli BL21/pETDuet-Ppdar-fdh
E. coli BL21/pETDuet-ard II-fdh
E. coli BL21/pETDuet-adh-fdh
E. coli BL21/pETDuet-adr-fdh
0.8 ꢃ 0.01
4.3 ꢃ 0.12
6.7 ꢃ 0.08
3.2 ꢃ 0.04
1.2 ꢃ 0.02
1.8 ꢃ 0.03
5.5 ꢃ 0.15
0.4
2.2
3.4
1.6
0.6
0.4
2.8
89.3 ꢃ 0.97
94.4 ꢃ 0.79
99.6 ꢃ 0.08
95.9 ꢃ 0.77
54.5 ꢃ 0.07
90.2 ꢃ 0.09
98.5 ꢃ 0.19
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increasing glucose consumption.25 FDH catalyses the conver- specic activity of puried KpDAR towards diacetyl was up to
sion of formate to CO2 with no organic acid products, reducing 2887.6 U mgꢀ1. meso-2,3-Butanediol was the best substrate in
the cost associated with the co-substrate and downstream pro- the alcohol oxidation reactions, while very low activity was
cessing.30,31,36 Considering the higher enantiomeric purity and observed with (R)/(S)-acetoin, (2S,3S)-2,3-butanediol and
titter, lower cost of the co-substrate, and easier purication, the (2R,3R)-2,3-butanediol.
FDH/formate system would signicantly reduce total cost of (S)-
acetoin production.
3.3 Optimization of the reaction conditions
When co-expressing fdh with other DAR genes including
Ppdar, ard II, adr, or adh, all resulting biocatalysts produced (S)-
acetoin, but the titer and enantiomeric purity of (S)-acetoin
varied widely (Table 2). Because E. coli BL21/pETDuet-Kpdar-fdh
showed the best performance in terms of both titer and enan-
tiomeric purity among all recombinant strains, this strain
together with KpDAR was used for subsequent analysis.
The reaction conditions for (S)-acetoin production were opti-
mized to increase the efficiency of the whole-cell biocatalysis.
Because reaction pH and temperature are two important
parameters that strongly inuence enzyme activities and
cellular maintenance, the effects of pH and temperature on
whole-cell biocatalysis were rst investigated. As shown in
Fig. 2a, the titer (9.19 g Lꢀ1) and yield (59.9%) of (S)-acetoin was
high at pH 6.5. Under more acidic or alkaline conditions, the
3.2 Purication and characterization of KpDAR
yield of the transformation decreased. As shown in Fig. 2b, the
ꢁ
KpDAR was puried by Ni-affinity chromatography and optimal temperature was found at 40 C, and the yield of (S)-
conrmed by SDS-PAGE. The puried KpDAR showed single acetoin increased to 67.8%.
bands about 28 kDa on the SDS-PAGE (see ESI Fig. S2†).
The substrate specicities of KpDAR indicated that KpDAR
Oxidation and reduction reactions were conducted to investi- not only showed a high catalytic efficiency toward diacetyl, but
gate the substrate specicities of KpDAR (see ESI Table S2†). also exhibited acetoin reductase activity (see ESI Table S2 and
KpDAR had clear activities towards diacetyl, (R)/(S)-acetoin and Fig. S3†). Both reductions of diacetyl and (S)-acetoin are
meso-2,3-butanediol with NADH/NAD+ as the cofactor. Diacetyl accomplished with stoichiometric amounts of NADH
was the best substrate in the ketone reduction reactions. The consumption. Through manipulating the level of NADH/NAD+,
Fig. 2 Optimization of biocatalysis conditions. (a) pH; (b) temperature; (c) ratio of formate to diacetyl; (d) shaking speed. (C) Yield of (S)-acetoin.
Error bars indicate standard deviations (n ¼ 3).
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Lꢀ1 of (S)-acetoin was accumulated from 15.0 g Lꢀ1 of diacetyl
with a productivity of 6.6 g (L h)ꢀ1 (Fig. 2d). The yield of (S)-
acetoin was 86.0% and the enantiomeric purity was 99.6%
(Fig. 3a and b).
3.4 Fed-batch biotransformation
Diacetyl is toxic to recombinant E. coli,12,32 and therefore fed-
batch processes should be performed to avoid substrate inhi-
bition and achieve high product concentration.37 In this study,
fed-batch biotransformation was conducted with an initial
diacetyl concentration of 15.0 g Lꢀ1, while feeding small
amounts of diacetyl and formate (ratio of formate to diacetyl
approximately 1.2) at 1 h intervals. Aer 8.5 h of reaction, 52.9 g
Lꢀ1 of (S)-acetoin was accumulated from 81.4 g Lꢀ1 of diacetyl
with an enantiomeric purity of 99.5% (Fig. 3a and c) and
productivity of 6.2 g (L h)ꢀ1 (Fig. 4). The titer and productivity
were new records on high optical (S)-acetoin (enantiomeric
purity $ 98.0%) production to our knowledge. In addition,
15.6 g Lꢀ1 of (2S,3S)-2,3-butanediol was obtained (Fig. 4). It
should be pointed out that this optical active compound is also
a building block in the chiral synthesis,20 and could be sepa-
rated from (S)-acetoin by distillation for their different boiling
points. No meso-2,3-butanediol, (2R,3R)-2,3-butanediol or
organic acids were detected.
According to Liu et al., (S)-acetoin was effectively produced
by biocatalytic resolution of the mixture of meso-2,3-butanediol
and (2S,3S)-2,3-butanediol by the resting cells of B. subtilis 168,
in which meso-2,3-butanediol was converted to (S)-acetoin.38
However, the enantiomeric purity of the product (S)-acetoin was
low (96.2%) for the generation of (R)-acetoin, which may be due
to the complex metabolic network of B. subtilis. Gao et al. re-
ported that whole-cells of recombinant E. coli over-expressing
DAR could be used to produced relatively high concentration
of (S)-acetoin (39.4 g Lꢀ1) from diacetyl.12 However, the enan-
tioselective reduction of one diacetyl molecule to form (S)-ace-
toin consumes one NADH/NADPH molecule as an electron
Fig. 3 Chiral-column GC analyses of products produced by whole-
cell of E. coli BL21/pETDuet-Kpdar-fdh. (a) Standard samples of pure
stereoisomers; (b) sample from batch bioconversion by E. coli BL21/
pETDuet-Kpdar-fdh; (c) sample from fed-batch bioconversion by E.
coli BL21/pETDuet-Kpdar-fdh. Samples were extracted using ethyl
acetate and isoamyl alcohol was used as the internal standard.
production of (S)-acetoin can be improved.25 Thus, to enhance
(S)-acetoin production, the concentration of formate should be
optimized. As shown in Fig. 2c, when the molar ratio of formate
to diacetyl was low, increasing the concentration of formate
effectively increased the efficiency of (S)-acetoin production.
When the molar ratio was 1.2, (S)-acetoin production reached
its maximum level (12.96 g Lꢀ1) with a yield of 84.4%. Further
increasing formate concentration to provide more NADH
resulted in accumulation of (2S,3S)-2,3-butanediol, thus
decreased the nal yield of (S)-acetoin.
Oxygen supply can signicantly affect the ratio of NADH/
NAD+. Different oxygen supplies were investigated by varying
the shaking speeds, and the optimal shaking speed was
125 rpm, as shown in Fig. 2d. Under optimal conditions, 13.2 g
Fig. 4 Production of optical (S)-acetoin by E. coli BL21/pETDuet-
Kpdar-fdh in fed-batch bioconversion. (:) (S)-Acetoin, (-) (2S,3S)-
2,3-butanediol, (;) diacetyl, (A) formate. Error bars indicate standard
deviations (n ¼ 3).
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donor, while generating NAD+/NADP+.19,39 Once the DAR level is
no longer limiting, the NADH/NADPH levels become limiting.
Thus, it is particularly important to construct an efficient
cofactor regeneration system to further increase the
productivity.
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In the present study, DARs from several species were codon-
optimized and compared for (S)-acetoin synthesis, and two
typical in situ-cofactor regeneration systems were introduced
into recombinant E. coli. In batch biotransformation, co-
expressing KpDAR with FDH resulted in 8.4-fold increase in
titer, with enantiomeric purity of 99.6%. Compared with the
results achieved previously using glucose as a co-substrate,12 co-
expressing KpDAR with FDH improved the (S)-acetoin concen-
tration from 39.4 g Lꢀ1 to 52.9 g Lꢀ1 and productivity from 2.0 g
(L h)ꢀ1 to 6.2 g (L h)ꢀ1 in fed-batch biotransformation, indi-
cating that the NADH level of E. coli was maintained appropri-
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In this study, we developed an efficient and economical process
for high-value chiral (S)-acetoin production from diacetyl. Two
in situ-NADH regeneration systems were applied to improve the
production of (S)-acetoin, and ve DARs from several species
were also compared for (S)-acetoin synthesis. Under the optimal
conditions, 52.9 g Lꢀ1 of (S)-acetoin was produced with an
enantiomeric purity of 99.5% and a productivity of 6.2 g (L h)ꢀ1
;
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the titer and productivity were new records on high optical (S)-
acetoin (enantiomeric purity $ 98.0%) production to our
knowledge. The systematic approach developed in this study
could also be applied to synthesize other optically active a-
hydroxy ketones.
¨
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Conflicts of interest
There are no conicts to declare.
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China [grant numbers 21466007, 31460296 and
31360207]; and the Guangxi Science and Technology Develop-
ment Project [grant number 14125008-2-22]. We appreciate the
professional comments and the constructive suggestions of the
anonymous reviewers and the editor in improving the
manuscript.
¨
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