Efficient Biosynthesis of (R)- or (S)-2-Hydroxybutyrate from l-Threonine through a Synthetic Biology Approach

Article abstract of DOI:10.1002/adsc.201600468

An efficient multi-enzyme cascade reaction for the synthesis of (R)- or (S)-2-hydroxybutyric acid [(R)- or (S)-2-HB] from l-threonine was developed by using recombinant Escherichia coli cells expressing separately or co-expressing l-threonine deaminase from Escherichia coli K-12 (ilvA), formate dehydrogenase (FDH) from Candida boidinii and l-lactate dehydrogenase (l-LDH) from Oryctolagus cuniculus or d-lactate dehydrogenase (d-LDH) from Staphylococcus epidermidis ATCC 12228. Up to 750 mM of l-threonine were completely transformed to (R)- or (S)-2-HB in optically pure form (>99% ee) with high isolated yields. This one-pot multi-enzyme transformation provides a new practical method for the synthesis of these important optically pure compounds. (Figure presented.).

Full text of DOI:10.1002/adsc.201600468

FULL PAPERS
DOI: 10.1002/adsc.201600468
Efficient Biosynthesis of (R)- or (S)-2-Hydroxybutyrate from
l-Threonine through a Synthetic Biology Approach
+
a
+a
a
a
a
Peiyuan Yao, Yunfeng Cui, Shanshan Yu, Yuncheng Du, Jinhui Feng,
a,
a,
Qiaqing Wu, * and Dunming Zhu *
a
National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport
Economic Area, Tianjin 300308, Peopleꢀs Republic of China
Fax : (+86)-22-2482-8703; phone: (+86)-22-2482-8703; e-mail: wu_qq@tib.cas.cn or zhu&lowbardm@tib.cas.cn
+
The authors contributed equally to this work.
Received: May 1, 2016; Revised: June 29, 2016; Published online: && &&, 0000
Supporting information for this article can be found under: http://dx.doi.org/10.1002/adsc.201600468.
Abstract: An efficient multi-enzyme cascade reac- 750 mM of l-threonine were completely transformed
tion for the synthesis of (R)- or (S)-2-hydroxybutyric to (R)- or (S)-2-HB in optically pure form (>99%
acid [(R)- or (S)-2-HB] from l-threonine was devel- ee) with high isolated yields. This one-pot multi-
oped by using recombinant Escherichia coli cells ex- enzyme transformation provides a new practical
pressing separately or co-expressing l-threonine de- method for the synthesis of these important optically
aminase from Escherichia coli K-12 (ilvA), formate pure compounds.
dehydrogenase (FDH) from Candida boidinii and l-
lactate dehydrogenase (l-LDH) from Oryctolagus Keywords: biotransformations; enzyme catalysis;
cuniculus or d-lactate dehydrogenase (d-LDH) from (R)-2-hydroxybutyric acid; (S)-2-hydroxybutyric
Staphylococcus epidermidis ATCC 12228. Up to acid; multi-enzyme cascade reaction
Introduction
conditions. However, the prochiral 2-ketobutyric acid
is not commercially available. Although it can be gen-
Optically active 2-hydroxy acids are widely used as erated through two major metabolic pathways such as
chiral intermediates, chiral auxiliaries and resolving the threonine degradation pathway and the citrama-
agents in the pharmaceuticals, biodegradable materi- late pathway, the highest l-threonine concentration
[
1]
À1
[
als, and the chemical industry. Among them, (R)- was only 30 gL 5be,9e]cause of the inhibition effect of 2-
and (S)-2-hydroxybutyric acids [(R)- and (S)-2-HB] ketobutyric acid.
are the key precursors for the synthesis of the anti-ep-
ileptic drugs levetiracetam and brivaracetam, the tion technology with a global production capacity of
l-Threonine was industrially produced by fermenta-
[
2]
[
10]
peroxisome
proliferator-activated
receptor about 75000 tons in 2009. Therefore, we have devel-
[
3]
a (PPARa) agonist (R)-K-13675 and the PPARg ag- oped a synthetic biology approach to synthesize enan-
onist MK-0533. They have also found wide applica- tiopure (R)- or (S)-2-HB from the readily available
tions in biodegradable materials with enhanced ther- bulk chemical l-threonine by employing l-threonine
mal and material properties. Thus, the development deaminase, lactate dehydrogenases and an NADH-re-
of new practical methods for the preparation of opti- generation system. Time-consuming and yield-reduc-
[4]
[
5]
cally pure 2-HB is in demand.
ing isolation and purification of (unstable or toxic) in-
Numerous approaches have been described previ- termediates can be avoided by implementing this bio-
[
3,6]
ously, including traditional chemical methods, enzy- catalytic cascade that has recently attracted significant
[
7]
[11]
matic kinetic resolution, and asymmetric bioreduc- attention.
[
8]
tion of 2-ketobutyric acid. Currently, the asymmetric
bioreduction of 2-ketobutyric acid by using dehydro-
genases is the method of choice due to its excellent
[
8d,e]
enantioselectivity,
high yield and mild reaction
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Results and Discussion
the cofactor NADH and its expression vector
pET21d-FDH was stored in our laboratory.
The inhibitory effects of l-threonine, 2-ketobutyric
[
14]
The multi-enzyme cascade involves dehydration of l-
threonine by l-threonine deaminase (TD) giving the acid and 2-HB on the TD, l-LDH and d-LDH activi-
prochiral 2-ketobutyric acid, which is stereoselectively ty were investigated by using the corresponding puri-
reduced either to (R)- or (S)-2-HB depending on the fied enzymes (Figure S1 in the Supporting Informa-
stereopreference of the selected lactate dehydrogen- tion). The TD activity was adversely affected by high
ase (LDH) (Scheme 1).
concentrations of 2-ketobutyric acid (Figure S2A in
the Supporting Information). The reaction rate de-
clined sharply at concentrations higher than 120 mM.
In contrast, l-threonine significantly improved the
enzyme activity even at the concentration of 200 mM
(
Figure S2B in the Supporting Information), which
[12g]
was consistent with the results in the literature.
2-
HB had a little effect on the TD activity (Figure S2C
in the Supporting Information). For l-LDH or d-
LDH, 2-ketobutyric acid was found to exert substrate
inhibition. The reaction rate declined slowly at con-
centrations higher than 160 mM and 40 mM, respec-
tively. On the contrary, l-threonine and 2-HB did not
affect l-LDH or d-LDH activity up to 200 mM (Fig-
ure S3 and Figure S4 in the Supporting Information).
Therefore, the cascade reaction was investigated to
address the inhibition of 2-ketobutyric acid on the en-
Scheme 1. One-pot multi-enzyme cascade for the synthesis
of enantiomerically pure (R)- or (S)-2-HB starting from l-
threonine.
To implement the cascade reactions shown in zymes activities.
Scheme 1, we set out to clone the genes encoding TD,
The biotransformation of l-threonine to (R)- or
l-LDH and d-LDH into expression vectors, respec- (S)-2-HB was carried out in a one-pot multi-enzyme
tively. TD from E. coli K-12 (ilvA) (NCBI reference cascade by using the whole cells separately expressing
sequence NP_418220.1) has been characterized and the TD and d-LDH or l-LDH genes. After optimiza-
used to synthesize l-2-aminobutyric acid in combina- tion of the reaction conditions, it was found that up to
[
12]
tion with l-leucine dehydrogenase or transaminase.
750 mM of l-threonine was completely converted to
The ilvA gene was amplified by PCR using the pri- (R)- or (S)-2-HB within 24 h and 36 h, respectively
mers ilvA-F and ilvA-R, and then cloned into (Figure 1). The concentration of the intermediate 2-
a pRSFduetꢂ-1 expression vector using BamH I and ketobutyric acid remained below 7 mM in the d-LDH
Hind III restriction sites, leading to pRSFduet-ilvA. system, whereas it was up to 91 mM in the l-LDH
Sequence alignment with the ilvA gene from E. coli system. This could be due to the fact that the total
K-12 showed an Ala10Thr amino acid substitution. enzyme activity of TD was about 1.8 times as much as
The plasmid pRSFduet-ilvA was transformed into E. that of l-LDH and only 16% of that of d-LDH. The
coli BL21 (DE3). The recombinant TD enzyme was reaction mixture was adjusted to pH 1–2 with 6M
À1
purified and displayed a specific activity of 6 Umg . HCl solution. After centrifugation, the supernatant
Two LDHs with complementary stereoselectivity was extracted with ethyl acetate. Removal of the sol-
were selected for the reduction of 2-ketobutyric acid. vent gave the desired products (R)- and (S)-2-HB in
l-LDH from Oryctolagus cuniculus (NCBI reference optically pure form (ee >99%) with the yields of 95%
sequence NP_001075746.1) is known to reduce 2-ke- and 97%, respectively. Thus there is no effect on the
tobutyric acid to (S)-2-HB with high enantioselectivi- stereoselectivity of d-LDH and l-LDH by using
[13]
ty, whereas d-LDH from Staphylococcus epidermi- a whole-cell system with NADH regeneration.
dis ATCC 12228 (NCBI reference sequence To further simplify the process, the co-expression of
NP_765629.1) produces (R)-2-HB using NADH as co- multiple genes in E. coli was investigated. In the first
[8d]
factor. The l-LDH and d-LDH genes were cloned step, we constructed two binary expression vectors
into the Nde I/Hind III restriction sites of pET32a pRSFduet-ilvA-l-LDH and pRSFduet-ilvA-d-LDH
(
+) and the EcoR V/Kpn I restriction sites of under the control of a strong inducible promoter (T7/
pRSFduetꢂ-1, respectively. The resulting plasmids lac) and all genes carried the same ribosome binding
were transformed into E. coli BL21 (DE3). The re- site (RBS). The PCR fragment of FDH ORF of
spective specific activities of the recombinant l-LDH pET21d-FDH was then cloned into pRSFduet-ilvA-l-
and d-LDH towards 2-ketobutyric acid were 0.8 and LDH and pRSFduet-ilvA-d-LDH after the pRSFduet
À1
1
1.5 Umg . An FDH from Candida boidinii (Uni- vectorꢀs second ORF using the One Step Cloning Kit.
ProtKB ID O13437) was used for the regeneration of The recombinant plasmids pRSFduet-ilvA-l-LDH-
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Preparative biotransformation of l-threonine to
À1
(
R)- or (S)-2-HB was carried out by using 20 gL of
wet E. coli BL21(DE3) cells harboring pRSFduet-
ilvA-d-LDH-FDH or pRSFduet-ilvA-l-LDH-FDH at
7
50 mM of substrate concentration. The reactions
were completed within 8 h and 24 h, respectively, and
R)- and (S)-2-HB were obtained in optically pure
(
form (ee >99%) with the yields of 93% and 97%, re-
spectively (Figure 3A). No or only a little 2-ketobuty-
ric acid was detected during the reactions. 12.41 g of
(
S)-2-HB or 11.02 g of (R)-2-HB were produced from
Figure 1. Time courses of the biotransformations of l-threo-
nine to (R)- or (S)-2-HB employing recombinant E. coli
cells expressing separately TD and d-LDH or l-LDH genes
[
(R)-2-HB (*), (S)-2-HB ("), 2-ketobutyric acid for l-LDH
(
~), 2-ketobutyric acid for d-LDH (
&
)]. The reaction mix-
tures contained 100 mM sodium phosphate buffer (pH 7.0)
in an initial total volume of 100 mL and 2M HCl solution
was automatically added to control the pH at 7.0. Reaction
conditions: l-threonine (750 mM), TD (0.6 g, 240 U), FDH
(
10 mL, 10 U), PLP (0.1 mM), HCO Na·2H O (1.5M),
2 2
+
NAD (0.75 mM), d-LDH (1.6 g, 1493 U) or l-LDH (1.6 g,
33 U), 308C, 200 rpm.
1
Figure 2. Expression constructs of pRSFduet-ilvA-l(d)-
LDH-FDH.
FDH and pRSFduet-ilvA-d-LDH-FDH were ob-
tained (Figure 2).
Analysis of the E. coli BL21(DE3) cells harboring
pRSFduet-ilvA-l-LDH-FDH or pRSFduet-ilvA-d-
LDH-FDH by protein gel electrophoresis of cell-free
lysates showed that all four enzymes were expressed,
but the protein productivity of FDH in the systems
was better than that of ilvA, l-LDH and d-LDH (Fig-
ure S5 in the Supporting Information). The activities Figure 3. Time courses of the biotransformations of l-threo-
nine to (R)- or (S)-2-HB employing recombinant E. coli/
pRSFduet-ilvA-l-LDH-FDH and E. coli/pRSFduet-ilvA-d-
of co-expression strains were then measured. The ac-
tivities of ilvA, l-LDH and FDH for pRSFduet-ilvA-
À1
À1
LDH-FDH cells [(R)-2-HB (*), (S)-2-HB ("), 2-ketobutyric
l-LDH-FDH were 400 mUmg , 54 mUmg
and
and
acid for l-LDH (~
), 2-ketobutyric acid for d-LDH ( )]. The
&
À1
5
2 mUmg , while those of pRSFduet-ilvA-d-LDH-
reaction mixtures contained 100 mM sodium phosphate
buffer (pH 7.0) in an initial total volume of 100 mL and 2M
HCl solution was automatically added to control the pH at
À1
À1
FDH
were
440 mUmg ,
192 mUmg
À1
6
6 mUmg , respectively. Fortunately, E. coli
BL21(DE3) cells harboring pRSFduet-ilvA-l-LDH-
7
.0. Reaction conditions: l-threonine (750 mM), PLP
+
FDH or pRSFduet-ilvA-d-LDH-FDH allowed a good [0.1 mM (A) or 0 (B)], HCO Na.2H O (1.5M), NAD
2
2
balance between the dehydration and reduction steps. (0.75 mM), 308C, 200 rpm.
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the cells grown in 1 L of the culture without optimiza- Bacterial Strains and Culture Conditions
tion of the fermentation conditions. We also examined
the biotransformation without adding external PLP
cofactor in the reaction systems. The results demon-
strated that endogenous PLP in E. coli could be
The plasmids constructed in the above section were trans-
formed into E. coli BL21(DE3) cells. Recombinant E. coli
BL21(DE3) was propagated in 1 L of Luria-Bertani medium
À1
À1
containing 50 mgmL kanamycin (100 mgmL ampicillin
enough for these cascade reactions, although the reac- for pET32a-l-LDH) at 378C. The culture was induced by
tion velocities were slower than those with addition of addition of isopropyl b-d-1-thiogalactopyranoside with
external PLP (Figure 3B).
a final concentration of 0.1 mM when the optical density
l=600 nm) reached 0.6–0.8 and then incubated for addi-
(
tional 16 h at 258C with 200 rpm (1.0 mM IPTG and 188C
for pET32a-l-LDH). After centrifugation at 6000ꢀg and
Conclusions
4
0
8C for 20 min, the recombinant cells were washed with
.9% NaCl solution to give 3.04 g of pRSFduet-ilvA-d-
In summary, we have established efficient biocatalytic
cascade reactions for the synthesis of (R)- and (S)-2-
HB from l-threonine by expressing separately or co-
LDH-FDH wet cells, 3.28 g of pRSFduet-ilvA-l-LDH-FDH
wet cells, 4.00 g of pRSFduet-ilvA wet cells, 4.29 g of
pRSFduet-l-LDH wet cells, 3.81 g of pRSFduet-d-LDH wet
expressing TD, FDH and l-LDH or d-LDH in E. coli cells, or 4.20 g of pET21d-FDH wet cells, which were cryo-
cells. The cascade reaction avoids the use of expensive preserved at À208C without loss of activity within 2 weeks.
cofactors and the inhibition of intermediates. Up to
750 mM of l-threonine were completely transformed
Protein Purification
to (R)- or (S)-2-HB in optically pure form (>99% ee)
with high isolated yields without requiring chromato-
graphic purification. These results suggest that this
cascade reaction should be a very promising approach
for the synthesis of (R)- or (S)-2-HB.
The recombinant cells were suspended in 100 mM potassium
phosphate buffer (pH 7.0) and lysed by a French press at an
operating pressure of 12000 psi. The cell debris was removed
by centrifugation at 10,000ꢀg for 30 min at 48C. The result-
ing cell-free extract was mixed with 0.025% PEI solution.
After centrifugation, the supernatant was precipitated with
4
0–60% ammonium sulfate. The resulting precipitate was
collected after centrifugation and dissolved in potassium
phosphate buffer (100 mM, pH 7.0). This purifying method
is suitable for the individual expression strain (Figure S1 in
the Supporting Information).
Experimental Section
Materials
The l-threonine, sodium 2-oxobutyrate and (R/S)-2-HB
were purchased from Aladdin Industrial Corporation,
Sigma–Aldrich Corporation and TCI (Shanghai) Develop-
ment Co., Ltd., respectively. DifcoTM LB Broth, Miller
Analytical Methods
HPLC analysis was performed on an Agilent 1200 series
HPLC system with an Acclaim 120 C18 column (4.6ꢃ
250 mm, 5.0 mm) and monitored at a wavelength of 210 nm
with a UV detector. The eluent was a mixture of phosphoric
acid solution (0.05%) and methanol (95/5, v/v), column tem-
(
Luria-Bertani) was purchased from Becton Dickinson and
Company. Ampicilin was purchased from Beijing Probe Bio-
science Co., Ltd. Isopropyl b-d-1-thiogalactopyranoside
À1
(
IPTG) was purchased from AMRESCO Inc. All other
perature was 308C and flow rate was 0.8 mLmin . Under
chemicals were purchased from Sinopharm Chemical Re-
agent Co., Ltd. The restriction enzymes and other reagents
for molecular biology were supplied by Fermentas (Germa-
ny) and TaKaRa (Japan). The GC analysis was performed
on an Agilent 7890A GC system. HPLC analysis was per-
these conditions, the retention times for standard samples of
2-HB and 2-ketobutyric acid were 11.72 and 8.29 min, re-
spectively (Figure S6 in the Supporting Information).
GC analysis was performed using a CP-ChiraSil-DEX CB
column (25 mꢃ0.25 mmꢃ0.25 mm) with helium as the carri-
er gas and a flame ionization detector. The injector and de-
1
formed on an Agilent 1200 series HPLC system. H and
1
3
C NMR spectra were recorded on a Bruker Avance III
00 MHz NMR spectrometer. High-resolution MS were re-
tector temperature were set at 2208C, and the oven temper-
À1
4
ature was controlled as follows: 508C (0 min) – 58Cmin
–
corded on a Bruker micrOTOF-QII.
1008C (2 min). The retention times for standard samples of
(R/S)-methyl 2-hydroxybutyrate which were derived from
(R/S)-2-HB were 6.87 and 7.49 min, respectively (Figure S7
in the Supporting Information).
l-Lactate dehydrogenase (l-LDH) from Oryctolagus cuni-
culus (NCBI reference sequence NP_001075746.1) and d-
lactate dehydrogenase (d-LDH) from Staphylococcus epi-
dermidis ATCC 12228 (NCBI reference sequence
NP_765629.1) were synthesized by Shanghai Xuguan Bio-
technological Development Co., Ltd. (China). l-Threonine
deaminase gene was amplified from the genome of E. coli
K12 (NCBI reference sequence NP_418220.1). Formate de-
hydrogenase gene from Candida boidinii (UniProtKB ID
O13437) was amplified from pET21d-FDH (the plasmid was
stored in our laboratory). The vector pRSFDuet-1 was used
for co-expression of these genes.
Activity Assay
The activities of LDHs and FDH were measured at 308C in
À1
À1
a 96-well plate at 340 nm (e=6.22 mM cm ) using a Spec-
tra Max M2 microplate reader (MD, USA). For LDH activi-
ty assays, the total volume of 200 mL contained 1 mM 2-ke-
tobutyric acid in 100 mM potassium phosphate buffer
(pH 7.0), 0.5 mM NADH, and an appropriate amount of the
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1
enzyme. For FDH activity assays, the reaction (200 mL) was
À4.88 (c=0.96, H O). H NMR (400 MHz, CDCl ): d=4.26
2
3
performed in 100 mM potassium phosphate buffer (pH 7.0)
(dd, J=6.7, 4.5 Hz, 1H), 1.84–1.97 (m, 1H), 1.76 (dquin, J=
14.3, 7.2, 7.2, 7.2, 7.2 Hz, 1H), 1.02 (t, J=7.5 Hz, 3H);
C NMR (100 MHz, CDCl ): d=179.59, 71.31, 27.27, 8.95;
+
containing 4 mM sodium formate, 1 mM NAD and an ap-
1
3
propriate amount of FDH. One unit of activity was defined
3
À
as the amount of enzyme that converted 1 mmol of NADH
MS (ESI): m/z=103.0405, calcd. for C H O : 103.0395
4
7
3
+
À
or NAD per minute.
[MÀ1] .
The TD activity was determined in reaction mixtures
Whole cells of E. coli BL21 (DE3) containing plasmids
pRSFduet-ilvA-d-LDH-FDH gave (R)-2-HB; yield: 7.25 g
[6c]
(
(
0.5 mL) containing 100 mM potassium phosphate buffer
pH 7.0), 20 mM PLP, 40 mM threonine and a suitable
2
5
(93%); ee=99.6%; [a] : +4.2138 (c=3.02, H O); lit.
D
2
25 1
amount of TD enzyme. The reaction mixture was incubated
[a] : +4.18 (c=1.6, H O). H NMR (400 MHz, CDCl ): d=
D
2 3
at 308C for 10 min. The reaction was terminated by adding
4.26 (dd, J=6.8, 4.6 Hz, 1H), 1.83–1.99 (m, 1H), 1.76
(dquin, J=14.3, 7.2, 7.2, 7.2, 7.2 Hz, 1H), 1.01 (t, J=7.4 Hz,
2
5 mL of 6.0M HCl and diluted with 475 mL of 5% MeOH
aqueous solution. After centrifugation at 13,000ꢃg for
min, the supernatant was collected and the concentration
1
3
3H); C NMR (100 MHz, CDCl ): d=179.55, 71.32, 27.26,
3
À
2
8.95; MS (ESI): m/z=103.0401, calcd for C H O : 103.0395
4
7
3
À
of 2-ketobutyric acid was measured by HPLC analysis as de-
scribed above. One unit of enzyme activity was defined as
the amount of enzyme that produced 1 mmol 2-ketobutyric
acid per min.
[MÀ1] .
Acknowledgements
Substrate and Product Inhibition
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 21302215) and
Youth Innovation Promotion Association of the Chinese
Academy of Sciences (Grant No. 2016166).
Initial reaction rates were measured to examine l-threonine,
2-ketobutyric acid and 2-HB inhibition in the dehydration
or reduction reaction. For the dehydration reaction, 0.802 U
TD solution, 2 mL of 1 mM PLP and 100 mM phosphate
buffer (pH 7.0) were used and the initial volume of the reac-
tion mixture was 500 mL. Reaction conditions to examine
substrate inhibition were 0–200 mM l-threonine. Product in-
hibition was measured at 40 mM l-threonine, 0–200 mM 2-
ketobutyric acid or 2-HB.
For the reduction reaction, 5.5 mU l-LDH or 14 mU d-
LDH solution, 20 mL of 5 mM NADH and 100 mM phos-
phate buffer (pH 7.0) were used and the initial volume of
the reaction mixture was 200 mL. Reaction conditions to ex-
amine substrate inhibition were 0–200 mM 2-ketobutyric
acid. l-Threonine and 2-HB inhibitions were measured at
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mids pRSFduet-ilvA-l-LDH-FDH or pRSFduet-ilvA-d-
LDH-FDH were suspended in a sodium phosphate buffer
(
100 mL, 100 mM, pH 7.0) containing the substrate l-threo-
+
nine (8.93 g, 75 mmol, 750 mM), NAD (0.75 mM),
PLP(0.1 mM) and HCO Na·2H O (1.5M). The suspension
2
2
was stirred at 308C for 24 h and 2M HCl solution was auto-
matically added to control the pH at 7.0. At the indicated
time intervals, aliquots (100 mL each) were taken, quenched
by 25 mL of 2M HCl solution and diluted with a mixture of
phosphoric acid solution (0.05%) and methanol (95/5, v/v,
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75 mL). Then the samples were monitored by HPLC analy-
sis. After this time, the reaction was acidified to pH 1 with
.0M HCl solution. The supernatant was extracted with
6
ethyl acetate (4ꢃ250 mL) after centrifugation at 6000ꢀg
and 258C for 15 min. The organic phases were combined,
dried over sodium sulfate, filtered through a pad of silica gel
and evaporated under vacuum to afford (S)-2-HB or (R)-2-
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Efficient Biosynthesis of (R)- or (S)-2-Hydroxybutyrate
from l-Threonine through a Synthetic Biology Approach
7
Adv. Synth. Catal. 2016, 358, 1 – 7
Peiyuan Yao, Yunfeng Cui, Shanshan Yu, Yuncheng Du,
Jinhui Feng, Qiaqing Wu,* Dunming Zhu*
Adv. Synth. Catal. 0000, 000, 0 – 0
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ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!