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
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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
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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
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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
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ꢀ
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.
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