Biosynthesis of 1,4-butanediol from erythritol using whole-cell catalysis

Article abstract of DOI:10.1080/10242422.2018.1465414

1,4-Butanediol (BDO) biosynthesis from renewable resources is of increasing interest because of global energy and environmental problems. We have previously demonstrated the production of BDO from erythritol by whole-cell catalysis. Here, the effects of several variables on BDO production were investigated, including cell density, temperature, substrate concentration and pH. It was found that the maximum BDO production was obtained at cell density (OD₆₀₀) of 30. Low temperature and weak alkaline environment were beneficial for the biotransformation. Regarding substrate concentration, 80?g/L of erythritol was found to be optimum for the bioconversion. Under the optimal conditions, the highest concentration of BDO reached 34.5?mg/L, resulting in 5.8-fold increment after optimization. These results will provide useful guidance for enhancing the bioconversion of erythritol to BDO.

Full text of DOI:10.1080/10242422.2018.1465414

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Biosynthesis of 1,4-butanediol from erythritol
using whole-cell catalysis
Lu Dai, Cui Tai, Yaling Shen, Yali Guo & Fei Tao
To cite this article: Lu Dai, Cui Tai, Yaling Shen, Yali Guo & Fei Tao (2018): Biosynthesis of 1,4-
butanediol from erythritol using whole-cell catalysis, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2018.1465414
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BIOCATALYSIS AND BIOTRANSFORMATION, 2018
https://doi.org/10.1080/10242422.2018.1465414
RESEARCH ARTICLE
Biosynthesis of 1,4-butanediol from erythritol using whole-cell catalysis
a,b
b
a
a,b
b
Lu Dai , Cui Tai , Yaling Shen , Yali Guo and Fei Tao
a
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People’s Republic
b
of China; State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, People’s Republic of China
ABSTRACT
ARTICLE HISTORY
1
,4-Butanediol (BDO) biosynthesis from renewable resources is of increasing interest because of
Received 26 January 2018
Revised 4 April 2018
Accepted 10 April 2018
global energy and environmental problems. We have previously demonstrated the production of
BDO from erythritol by whole-cell catalysis. Here, the effects of several variables on BDO produc-
tion were investigated, including cell density, temperature, substrate concentration and pH. It
was found that the maximum BDO production was obtained at cell density (OD600) of 30. Low
temperature and weak alkaline environment were beneficial for the biotransformation.
Regarding substrate concentration, 80 g/L of erythritol was found to be optimum for the biocon-
version. Under the optimal conditions, the highest concentration of BDO reached 34.5 mg/L,
resulting in 5.8-fold increment after optimization. These results will provide useful guidance for
enhancing the bioconversion of erythritol to BDO.
KEYWORDS
1
,4-Butanediol; erythritol;
whole-cell biocatalysis
1
. Introduction
based on fermentation, mostly using osmophilic
fungi (van der Woude et al. 2016). The highest yield
of erythritol from glucose is 61%, and a 56% yield
can be obtained from glycerol (Amada et al. 2012).
Considering the structures of erythritol and BDO are
highly similar, it is attractive to explore an enzyme
capable of reducing the number of hydroxyl groups,
so as to convert erythritol to BDO.
1,4-Butanediol (BDO) is an important commodity chem-
ical which is widely used in the production of plastics,
polyesters and spandex fibers (Zeng and Sabra 2011;
Forte et al. 2016). Its annual demand is approximate
one million metric tons (Liu and Lu 2015). However,
BDO cannot be produced naturally in any known micro-
organism. It is only produced from petroleum-based
feedstocks such as acetylene, propylene, and butadiene
Glycerol dehydratase (GDHt, EC 4.2.1.30) catalyzes
the coenzyme B -dependent conversion of glycerol to
1
2
(
Yang et al. 2015). Given the growing concerns over the
3
-hydroxypropionaldehyde, which is further reduced
to 1,3-propanediol by 1,3-propanediol dehydrogenase
Daniel et al. 1995). In our previous study, a mutant of
environment and volatile fossil energy costs, it becomes
an urgent need to develop a more sustainable process
for BDO production from renewable feedstocks. In
(
GDHt has been obtained by site-directed mutagenesis.
Coupling with alcohol dehydrogenase, the mutant can
individually catalyze reduction of 1,2-butanediol, 1,2,4-
butanetriol (BT), erythritol, 1,2-pentanediol, 1,2,5-penta-
netriol and 1,2,6-hexanetriol to produce 1-butanol, 1,4-
butanediol, 1,4-butanediol, 1-pentanol, 1,5-pentanediol
and 1,6-hexanediol. These products are important
chemicals with extensive applications in modern
industry. For example, the 1-alkanols have been
regarded as substitutes for diesel fuel and gasoline;
the 1,n-alkanediols have been widely used for the
production of polymers, polyesters and polyurethane
resins. As most of these alcohols are non-natural
compounds, the promiscuous functions of the GDHt
2
011, biosynthesis of BDO via engineered Escherichia
coli was first achieved by Yim et al. (2011). This pathway
requires six steps to convert TCA-cycle intermediate
succinate into BDO, and involves the overexpression of
six enzymes. Recently, other pathways for BDO biosyn-
thesis have been reported (Liu and Lu 2015; Tai et al.
2
016; Wang et al. 2017). These pathways convert
lignocellulose-derived sugar into BDO via six steps and
require five enzymes.
Erythritol, a four-carbon polyol, is a naturally occur-
ring substance which is widely distributed in nature
(
Moon et al. 2010). Its chemical synthesis is inefficient
and industrial production of this polyol is therefore
CONTACT Fei Tao
Republic of China; Yaling Shen
taofei@sjtu.edu.cn
State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, People’s
State Key Laboratory of Bioreactor Engineering, East China University of Science and
ylshen@ecust.edu.cn
Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
L. DAI ET AL.
mutant can be applied to extend natural cell metabol-
ism for renewable production of these chemicals (Dai
et al. 2017). We have previously demonstrated the
recombinant strain with overexpression of GDHt can
catalyze conversion of erythritol to BDO using whole-
cell catalysis. This is a novel pathway which contains
only four steps and requires only one heterologous
gene. Comparing to previous reports, the correspond-
ing gene regulation and genetic manipulation are
simpler. Besides, as engineering a long heterologous
pathway will bring significant metabolic burdens on
cells, the BDO pathway from erythritol could com-
mendably avoid this. Based on these advantages, the
novel route is promising and viable. However, the titer
of BDO is still unsatisfactory, leading us to investigate
the effects of different factors on BDO production
from erythritol.
2.3. Effects of cell density, temperature, substrate
and pH on BDO production
Experiments were performed with 20 mL of reaction
mixture in a 100-mL Erlenmeyer flask. To investigate
the effect of cell density on BDO production, cell pre-
cipitates prepared before were suspended in 50 mM
potassium phosphate buffer (pH 8.0). The final optical
density (OD600) of resting cells was set as 10, 20, 30
and 40. With addition of 60 g/L erythritol and 15 mM
coenzyme B , the suspensions were incubated at
1
2
ꢀ
3
0 C and 200 rpm for 20 h. Optimal bioconversion
temperature was determined over a range of tempera-
tures under the optimal cell density. Cells were sus-
pended in 50 mM potassium phosphate buffer (pH
8.0), followed by the supplement of 60 g/L erythritol
and 15 mM coenzyme B . The mixtures were individu-
1
2
ꢀ
ally incubated at 16, 25, 30, 37 and 45 C for 20 h.
To optimize the substrate concentration, the reactions
were conducted based on the optimal cell density and
temperature. Erythritol concentrations from 40 to
In this study, the effects of cell density, temperature,
substrate concentration, and pH on BDO production
were investigated. It was found that the maximum con-
centration of BDO was obtained at cell density (OD600)
100 g/L were evaluated. The effect of initial pH on
ꢀ
of 30, 16 C and pH 8.0 by using 80 g/L erythritol as
BDO production was studied under the optimal cell
density, temperature and erythritol concentration;
substrate. These results offer useful guidance for
enhancing the biotransformation of erythritol to BDO.
50 mM potassium phosphate buffer with different pH
(5.0, 6.0, 7.0, 8.0 and 9.0) was used as the reaction
buffer. For each experiment, 1 mL of reaction mixture
was collected for GC-MS/MS analysis.
2
. Materials and methods
2.1. Bacterial strains, chemicals and
culture conditions
E. coli BL21 (DE3) with overexpression of the GDHt
mutant was constructed in the previous study, which
was designated as strain EGDHt. Erythritol (99%), BT
2.4. Analytical methods
BDO and BT were derivatized by N,O-Bis(trimethylsilyl)
trifluoroacetamide (BSTFA) and quantified by GC-MS/MS.
Samples were centrifuged at 12,000 rpm for 5 min;
(
(
98%) and BDO (99%) were purchased from Aladdin
Shanghai, China). Isopropyl b-D-thiogalactoside (IPTG)
800 lL of the supernatant was mixed with supersatu-
and kanamycin were obtained from Sangon Biotech
Shanghai, China). Other chemicals were of analyt-
ical grade.
rated NaCl followed by the addition of 800 lL aceto-
nitrile. The organic phase was collected and dried
completely by using a rotary evaporator. Then, 20 lL of
(
1
mM cyclohexanol (dissolved in dimethylformamide)
and 50 lL BSTFA were subsequently added, and the
mixture was incubated at 70 C for 30 min. The derivat-
2
.2. Preparation of resting cells for bioconversion
ꢀ
of erythritol to BDO
ized samples were injected into triple quadrupole GC-
MS/MS system (Thermo & TSQ 8000) equipped with a
HP-5 MS column (0.25mm, 0.25 mm  30 m), and
quantified in SIM mode. The oven program was as fol-
For whole-cell catalysis, resting cells were prepared
with strain EGDHt. The recombinant E. coli was inocu-
lated in Luria-Bertani (LB) medium and incubated at
ꢀ
ꢀ ꢀ ꢀ
3
1
2
7 C with 200 rpm agitation. When OD
reached 0.6,
00
lows: 80 C for 1.5min, raised to 140 C at 10 C/min,
6
ꢀ
ꢀ
mM IPTG was added and the culture was induced at
ꢀ
held for 3 min, increased to 300 C at 30 C/min, held
for 5 min. Selected ion monitoring was performed by
monitoring m/z 75, 129 and 157 for cyclohexanol, m/z
101, 116 and 177 for BDO, m/z 103, 129 and 219
for BT.
0 C for 12 h. Subsequently, cells were harvested by
centrifugation (5000 rpm, 15 min) and washed twice
with PBS buffer (pH 7.4). The precipitates were stored
at 4 C and used for the following biotransformation.
ꢀ

BIOCATALYSIS AND BIOTRANSFORMATION
3
3
. Results and discussion
3.1. Effect of cell density on BDO production
The BDO pathway from erythritol by whole-cell cataly-
sis has been validated in our previous study, which
only involves four steps and overexpression of one
heterogeneous gene. In this route, erythritol is first
converted to 3,4-dihydroxy-butyraldehyde by the
GDHt mutant; 3,4-dihydroxy-butyraldehyde is then
converted to BT through alcohol dehydrogenase; fol-
lowed by another dehydration reaction, BT is con-
verted to 4-hydroxy-butyraldehyde by the GDHt
mutant; and 4-hydroxy-butyraldehyde is finally
reduced by alcohol dehydrogenase to produce BDO
Figure 1. BDO production from erythritol by whole-cell cataly-
sis. The ellipse indicates a cell of the engineered E. coli BL21
(DE3). GDHt: glycerol dehydratase; 3,4-DHBA: 3,4-dihydroxy-
butyraldehyde; BT: butanetriol; 4-HBA: 4-hydroxy-butyralde-
hyde; BDO: 1,4-butanediol.
(
Figure 1). However, the titer of BDO is not
satisfactory, leading to the investigation of effects of
several variables on BDO biosynthesis. As shown in
Figure 2, BDO concentration was optimal at cell dens-
ity (OD600) of 30 and decreased when the optical dens-
ity reached 40. The highest BDO titer obtained was
about 6 mg/L. BT is the precursor of BDO. The conver-
sion from erythritol to BDO requires two rounds of
dehydration-hydrogenation, while BT is formed via one
dehydration-hydrogenation of erythritol (Figure 1). In
the reaction culture, significant amount of BT was
accumulated and the concentration of BT increased
with the rise in cell density.
Figure 2. Effect of cell density on BDO and BT production by
recombinant E. coli.
3.2. Effect of temperature on BDO production
To evaluate the optimum temperature for whole-cell
biocatalysis, the cultures of EGDHt were incubated at
ꢀ
1
6–45 C. It was found that BDO production was max-
ꢀ
imum at 16 C with a titer of 19.9 mg/L, and decreased
remarkably with increasing temperature (Figure 3).
These results indicate that low temperature is more
beneficial for BDO production. We speculate this may
be attributed to the stability of NAD(P)H at low tem-
perature (Rover et al. 1998). As shown in Figure 1, the
transformation of erythritol to BDO is coupled with the
NAD(P)H consumption. For every mole of BDO to be
formed, two moles of NAD(P)H are required which are
all derived from intracellular reducing equivalents.
Thus, the stability of NAD(P)H is crucial for BDO produc-
tion. It has been previously reported that low tempera-
ture is advantageous for the stability of NAD(P)H, which
may thereby lead to the maximum production of BDO
Figure 3. Effect of temperature on BDO and BT production by
recombinant E. coli.
less effect on reaction rates of the first round of dehy-
dration-hydrogenation and has a greater effect on that
of the second round of dehydration-hydrogenation.
(
Wu et al. 1986). Besides, significant amount of BT was
3
.3. Effect of substrate concentration on
accumulated in the reaction mixtures, and the highest
concentration was obtained at 16 C with a titer of
BDO production
ꢀ
1
18.5 mg/L (Figure 3). It is notable that the relative con-
Varying concentrations of erythritol were used to eluci-
date the best concentration for maximum BDO pro-
duction. As depicted in Figure 4, BDO concentration
tent of BT is different at high and low temperature. We
speculate this is probably because the temperature has

4
L. DAI ET AL.
increased first, and then decreased with the rising
concentration of erythritol; 32.5 mg/L of BDO was
achieved by feeding 80 g/L erythritol, which was the
maximum BDO concentration. The titer of BT rose with
the rising erythritol level during the low concentration
range, and reduced during the high concentrate
range. The highest concentration of BT was also
obtained with 80 g/L erythritol as the substrate.
the state of organism. On the contrary, a weak alkaline
environment may be favourable for the performance
of cells, thus resulting in the higher production of
BDO. Similarly, the maximum BT production was also
obtained at pH 8.0. A significant loss of BT concentra-
tion was achieved with an increasing pH as well as a
decreasing pH.
4
. Conclusions
3
.4. Effect of initial pH on BDO production
This paper reported the effects of cell density,
temperature, substrate concentration, and pH on BDO
production. The highest concentration of BDO was
obtained at cell density (OD600) of 30. Other results
included that high temperature and acidic pH were
disadvantageous for BDO production. The maximum
BDO production was achieved with 80 g/L erythritol
as substrate. Under the optimal conditions, about
In order to evaluate the effect of initial pH on BDO
production, the whole-cell biocatalysis was conducted
at different pH (5.0–9.0). As shown in Figure 5, reaction
with pH 8.0 was more productive in BDO production.
Both an increase and decrease in pH caused the
dramatic loss of BDO concentration and detection of
BDO was not available when pH was lower than 7.0.
The highest concentration of BDO obtained at pH 8.0
was 34.5 mg/L, which was 5.8-fold higher than that at
cell density of 30. Optimum ranges of external pH are
crucial for cell growth and survival (Beales 2004).
During the whole-cell catalysis, acidic conditions may
exhibit physical stress on cells, which thereby affects
3
6
5 mg/L of BDO was produced, which was almost
-fold higher than the unoptimized conditions.
Besides, it was found that temperature and pH exerted
significant impacts on the transformation, indicating
that further optimization should give priority to these
two factors. This will provide useful guidance for
biotransformation of erythritol to BDO. During the bio-
conversion, significant amount of BT was accumulated
in the reaction mixture, indicating the catalytic effi-
ciencies of GDHt towards BT may be one of the limit-
ing steps. Thus, further efforts include engineering the
dehydratases by combination of structural biology
techniques and computational approaches. Screening
for more active homologues from other species might
also be a promising strategy.
Disclosure statement
No potential conflict of interest was reported by the authors.
Figure 4. Effect of substrate concentration on BDO and BT
production by recombinant E. coli.
Funding
This work was supported by the Science and Technology
Commission of Shanghai Municipality [17JC1404800], and
the grants from the National Natural Science Foundation of
China [31570101].
References
Amada Y, Watanabe H, Hirai Y, Kajikawa Y, Nakagawa Y,
Tomishige K. 2012. Production of biobutanediols by the
hydrogenolysis of erythritol. ChemSusChem. 5:1991–1999.
Beales N. 2004. Adaptation of microorganisms to cold tem-
peratures, weak acid preservatives, low pH, and osmotic
stress: a review. Comp Rev Food Sci Food Safety. 3:1–20.
Dai L, Tao F, Tang H, Guo Y, Shen Y, Xu P. 2017. Directing
enzyme devolution for biosynthesis of alkanols and
Figure 5. Effect of initial pH on BDO and BT production by
recombinant E. coli.

BIOCATALYSIS AND BIOTRANSFORMATION
5
1
,n-alkanediols from natural polyhydroxy compounds.
van der Woude AD, Gallego RP, Vreugdenhil A, Veetil VP,
Chroumpi T, Hellingwerf KJ. 2016. Genetic engineering of
Synechocystis PCC6803 for the photoautotrophic produc-
tion of the sweetener erythritol. Microb Cell Fact. 15:60.
Wang J, Jain R, Shen X, Sun X, Cheng M, Liao JC, Yuan Q,
Yan Y. 2017. Rational engineering of diol dehydratase ena-
bles 1,4-butanediol biosynthesis from xylose. Metab Eng.
40:148–156.
Metab Eng. 44:70–80.
Daniel R, Boenigk R, Gottschalk G. 1995. Purification of 1,3-
propanediol dehydrogenase from Citrobacter freundii and
cloning, sequencing, and overexpression of the correspond-
ing gene in Escherichia coli. J Bacteriol. 177:2151–2156.
Forte A, Zucaro A, Basosi R, Fierro A. 2016. LCA of 1,4-butanediol
produced via direct fermentation of sugars from wheat straw
feedstock within a territorial biorefinery. Materials. 9:563.
Liu H, Lu T. 2015. Autonomous production of 1,4-butanediol
Wu JT, Wu LH, Knight JA. 1986. Stability of NADPH: effect of
various factors on the kinetics of degradation. Clin Chem.
32:314–319.
via
a de novo biosynthesis pathway in engineered
Escherichia coli. Metab Eng. 29:135–141.
Yang Y, Chi YT, Toh HH, Li Z. 2015. Evolving P450pyr mono-
oxygenase for highly regioselective terminal hydroxylation
of n-butanol to 1,4-butanediol. Chem Commun (Camb).
51:914–917.
Moon H-J, Jeya M, Kim I-W, Lee J-K. 2010. Biotechnological
production of erythritol and its applications. Appl
Microbiol Biotechnol. 86:1017–1025.
Rover L, Fernandes JCB, Neto GO, Kubota LT, Katekawa E,
Serrano SHP. 1998. Study of NADH stability using ultravio-
let–visible spectrophotometric analysis and factorial
design. Anal Biochem. 260:50–55.
Tai Y-S, Xiong M, Jambunathan P, Wang J, Wang J, Stapleton C,
Zhang K. 2016. Engineering nonphosphorylative metabol-
ism to generate lignocellulose-derived products. Nat
Chem Biol. 12:247–253.
Yim H, Haselbeck R, Niu W, Pujol-Baxley C, Burgard A, Boldt J,
Khandurina J, Trawick JD, Osterhout RE, Stephen R,
et al. 2011. Metabolic engineering of Escherichia coli for
direct production of 1,4-butanediol. Nat Chem Biol.
7:445–452.
Zeng A-P, Sabra W. 2011. Microbial production of diols as plat-
form chemicals: recent progresses. Curr Opin Biotechnol.
22:749–757.