Identification andin situremoval of an inhibitory intermediate to develop an efficient phytosterol bioconversion process using a cyclodextrin-resting cell system

Article abstract of DOI:10.1039/d1ra02774c

A classically versatile steroid intermediate, 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD), can be obtained by phytosterol (PS) bioconversion usingMycobacterium. In this study, a cyclodextrin-resting cell reaction system with a high concentration of PS (50 g L^?1) was used to produce 9α-OH-AD. However, the inhibitory effect of metabolic intermediates is a key factor limiting production efficiency. After the separation and identification of a series of metabolic intermediates, it was found that 4-ene-3-keto steroids, which are the first metabolites of sterol side-chain degradation, accumulated at the beginning of the bioprocess and had a remarkable inhibitory effect on bioconversion. The bioconversion rate was greatly improved when 5 g L^?1of macroporous adsorbent resin D101 was added to the reaction system in the initial phase. A certain amount of resin acted as a reservoir to remove the inhibitory intermediatein situand facilitated the bioconversion process, and the 9α-OH-AD space-time yield increased to 8.51 g L^?1d^?1, which was 23.15% higher than that without resin addition (6.91 g L^?1d^?1) after 72 h bioconversion. In summary, we identified an inhibitory intermediate that limits the bioconversion rate and provided a solution based on resin adsorption for improving 9α-OH-AD production efficiency in a commercial-scale process.

Full text of DOI:10.1039/d1ra02774c

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PAPER
Identification and in situ removal of an inhibitory
intermediate to develop an efficient phytosterol
bioconversion process using a cyclodextrin-resting
cell system†
Cite this: RSC Adv., 2021, 11, 24787
Da Wang, ‡ Jian Zhang,‡ Dan-Dan Cao, Xuedong Wang * and Dongzhi Wei
A classically versatile steroid intermediate, 9a-hydroxyandrost-4-ene-3,17-dione (9a-OH-AD), can be
obtained by phytosterol (PS) bioconversion using Mycobacterium. In this study, a cyclodextrin-resting
ꢀ1
cell reaction system with a high concentration of PS (50 g L ) was used to produce 9a-OH-AD.
However, the inhibitory effect of metabolic intermediates is a key factor limiting production efficiency.
After the separation and identification of a series of metabolic intermediates, it was found that 4-ene-3-
keto steroids, which are the first metabolites of sterol side-chain degradation, accumulated at the
beginning of the bioprocess and had a remarkable inhibitory effect on bioconversion. The bioconversion
ꢀ1
rate was greatly improved when 5 g L of macroporous adsorbent resin D101 was added to the reaction
system in the initial phase. A certain amount of resin acted as a reservoir to remove the inhibitory
intermediate in situ and facilitated the bioconversion process, and the 9a-OH-AD space–time yield
Received 9th April 2021
ꢀ1 ꢀ1
ꢀ1 ꢀ1
increased to 8.51 g L
d
, which was 23.15% higher than that without resin addition (6.91 g L
d )
Accepted 30th June 2021
DOI: 10.1039/d1ra02774c
rsc.li/rsc-advances
after 72 bioconversion. In summary, we identified an inhibitory intermediate that limits the
h
bioconversion rate and provided a solution based on resin adsorption for improving 9a-OH-AD
production efficiency in a commercial-scale process.
solubility of PS. Among them, the cyclodextrin-resting cell
system is an ideal method that has been successfully used for
1
. Introduction
In the past decades, researchers have focused on the microbial the production of a variety of steroid intermediates. It was
side-chain degradation of phytosterols (PS) or cholesterol to observed that not only was the expression of genes related to PS
1,2
produce steroid intermediates. 9a-Hydroxyandrost-4-ene-3,17- metabolism inuenced but the degradation of steroid inter-
10,11
dione (9a-OH-AD) is one of the intermediates that can further be mediates was also inhibited in this reaction system.
None-
chemically modied to steroid drugs, such as hydrocortisone and theless, an extended bioconversion period and a lower reaction
3
eplerenone. However, the bioavailability of PS and the cytotoxicity rate still exist, especially at high PS concentrations. The inhib-
of intermediate metabolites are the key factors that limit produc- itory effect of metabolic intermediates is a key factor limiting
4
4
tion efficiency, particularly at high substrate concentrations.
production efficiency. Thus, many resin-based in situ removal
Currently, the synthesis efficiency of steroid intermediates is methods have been used to alleviate the inhibition of inter-
enhanced by a variety of metabolic regulation methods. Genetic mediates and improve the bioconversion efficiency of many
engineering modication of the production strains is a widely steroids,
such
as
androsta-1,4-diene-3,17-dione
and
1
2–14
used method to improve bioconversion. Yao et al. reported an testosterone.
5
engineered Mycobacterium-transformed PS to 9a-OH-AD. In
In situ product removal (ISPR) is a common method used for
addition, diverse sterol-solubilizing reaction systems, including the simultaneous separation of inhibitory products during
4,6
a cyclodextrin complexing system, biocompatible ionic liquid fermentation and biotransformation. Macroporous resins are
7
system, microdispersion/emulsication system, and surfactant promising candidates for ISPR materials and have many
8,9
addition,
were established to improve the poor water advantages, such as alleviated feedback suppression, low cost,
15,16
and high phase stability.
In recent years, macroporous
resins have been successfully used to produce various metabo-
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology,
East China University of Science and Technology, Shanghai 200237, China. E-mail:
xdwang@ecust.edu.cn; Fax: +86-21-64250068; Tel: +86-21-64253715
lites, such as diepoxin z, biogenic amines, botrallin and TMC-
17–19
264.
However, the study on the inuence of adsorption resin
†
Electronic supplementary information (ESI) available: Experimental details, on the performance of Mycobacterium to produce 9a-OH-AD
Table S1, Fig. S1 and S2. See DOI: 10.1039/d1ra02774c
‡
These authors contributed equally to this work.
©
2021 The Author(s). Published by the Royal Society of Chemistry
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from PS at high substrate concentrations has not been pub- exploring the optimum time to add resin). The transformation
lished and requires further investigation. of PS was performed under non-sterile conditions with a 10 mL
ꢀ
1
In this study, 9a-OH-AD was obtained from PS through PB (20 mM, pH ¼ 8.0) system consisting of 100 g L wet cells,
ꢀ
1
ꢀ1
biotransformation using the cyclodextrin-resting cell system at 50 g L PS, and 200 g L HP-b-CD, placed on a shaker at
ꢁ
a high substrate concentration. We evaluated the effect of the a rotation speed of 200 rpm and 30 C for the reaction. All
adsorption resin on the reaction efficiency of 9a-OH-AD experiments were repeated three times.
production. The ISPR system with different resin types, times,
and amounts was measured to optimize the bioconversion 2.4 Metabolites analytical methods
process. Furthermore, we investigated the mechanism by which
the adsorption resin improved phytosterol bioconversion effi-
ciency, and we separated and identied the inhibitory interme-
diate that limits the bioconversion rate. Using the in situ removal
strategy, we were able to provide a solution for improving 9a-OH-
AD production efficiency at high substrate concentrations.
The resins were separated from the reaction system using
a single layer gauze. The samples from the reaction solution and
resins were extracted with ten volumes of ethyl acetate (before
desorption, the resin was washed twice with deionized water to
remove non-adsorbed products). Aer centrifugation, an
appropriate amount of the upper organic phase was withdrawn
and allowed to evaporate completely before detection by high-
2
. Materials and methods
performance liquid chromatography (HPLC) with Hypersil
ODS2-column. Methanol–water (80 : 20, v/v) was used as the
2
.1 Microorganisms, medium and reagents
Mycobacterium neoaurum NwIB-HK86 (devoid of 3-ketosteroid-
,2-dehydrogenase activity; 3-ketosteroid-9-hydroxylase and
ꢀ1
mobile phase at a ow rate of 1 mL min and UV detector at
2
9
54 nm was used to investigate the main steroidal metabolites,
a-OH-AD et al. For the detection of phytosterols, the mobile
1
cholesterol oxidases overexpressed) can bioconvert PS to
produce 9a-OH-AD as the main product. MYC/01 seed medium
was used as an amplication medium for microorganisms, as
previously described by Gao et al. The seeds were then inocu-
lated at 5% (v/v) into 500 mL shake asks containing 100 mL of
phase and detection wavelength were changed to acetonitrile–
isopropanol (70 : 30, v/v) and 210 nm, respectively.
4
3
. Results and discussion
ꢁ
MYC/04 broth and cultured at 30 C for 72 h on a 200 rpm 3.1 Transformation of PS by Mycobacterium neoaurum
shaker. MYC/04 were composed of (g L ) K HPO $3H O, 0.5; NwIB-HK86 under resting cell system
ꢀ1
2
4
2
4 2
MgSO $7H O, 0.5; glucose, 30; sodium citrate, 2.8; ammonium
ferric citrate, 0.05; starch, 2; corn steep powder, 3; and PS, 0.1.
The initial pH was controlled at 8.0.
The reagents described in the above medium composition
were purchased from Shanghai Titan Scientic Co., Ltd.
In our previous study, Mycobacterium neoaurum NwIB-HK86
efficiently produced the main product 9a-OH-AD from phytos-
terols in HP-b-CD-resting cell system, accompanied by more
than 20% of by-products such as androstenedione (AD), 9,22-
dihydroxy-23,24-bisnorchol-4-en-3-one (DHBC), and 9,24-
(
(
Shanghai, China) or Sinopharm Chemical Reagent Co., Ltd.
Shanghai, China). 9a-OH-AD, AD standards were obtained
20
dihydroxychol-4-en-3-one (DHC). Furthermore, the results
demonstrated that the reaction was remarkably inhibited by
from Sigma (Shanghai, China). PS and HP-b-CD were purchased
from Shanxi Sciphar Natural Products Co., Ltd. (Shangluo,
China) and Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.
(Binzhou, China), respectively. The organic solvents used in the
mobile phase, e.g., methanol, acetonitrile, isopropanol, were all
HPLC grade.
2.2 Preparation of resting cells and macroporous resins
Resting cells were prepared according to the method described
4
by Gao et al. Ten types of macroporous adsorbent resins were
employed as carriers for the in situ adsorption technique during
the production of 9a-OH-AD, and were purchased from
Shanghai Huazhen Technology Co., Ltd. (Shanghai, China). The
physical and chemical properties of the resins are listed in
Table S1.† The resins were pre-treated by soaking in ethanol at
ꢁ
3
0 C for 48 h, then washed three times with deionised water for
ꢁ
2
4 h each, followed by drying at 55 C to a constant weight.
Fig. 1 Time profiles of 9a-OH-AD production by M. neoaurum NwIB-
2
.3 Bioconversion with ISPR by addition of macroporous
HK86 transforming phytosterols under cyclodextrin-resting cells
ꢀ1
system. The reaction system consisted of 50 g L of phytosterols,
resins
ꢀ
1
ꢀ1
1
00 g L of wet biomass and 200 g L of HP-b-CD, were carried out
Precisely, weighed resin was added to the reaction solution at
in 250 mL shake flasks containing 10 mL of reaction solution for 5
the beginning of the bioconversion (except for experiments d (error bars for standard deviations, n ¼ 3).
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high levels of substrates and metabolites. As shown in Fig. 1, 9a-
3.2.2 Optimization of the concentration of D101 adsorbent
OH-AD production increased slowly during the rst 24 h. resin in bioconversion. A set of experiments was conducted with
Subsequently, the accumulated products entered a rapid growth different concentrations of resin D101, ranging from 0 to 50 g
ꢀ1
ꢀ1
stage and reached the maximum level (25.1 g L ) at 96 h. The
L , to determine the optimum resin concentration for the 9a-
PS was also depleted aer 96 h of bioconversion, following the OH-AD production process, as shown in Table 2. As the adsor-
ꢀ1
accumulation of 9a-OH-AD. Aer further incubation, 9a-OH-AD bed resin concentration increased from 0 to 5 g L , D101
entered the degradation stage, and the yield began to decrease. exhibited an increased effect on the production of 9a-OH-AD
and the bioconversion of PS. The space–time yield of 9a-OH-
ꢀ
1
ꢀ1
AD increased from 6.91 to 8.51 g L
dependent manner. And the PS was completely exhausted at
.2.1 Selection of different adsorption resins in biocon- 72 h, which was 14.7% higher than that of the control. Unex-
d
in a concentration-
3
.2 Application of ISPR in the process
3
version. To further improve the efficiency of 9a-OH-AD pectedly, PS bioconversion and 9a-OH-AD production showed
bioconversion, the ISPR method was used in the system to reverse trends as the D101 concentration continuously
ꢀ1
ꢀ1
alleviate the inhibition of metabolites. The inuence of ten increased from 5 g L to 50 g L , even though the proportion
types of adsorption resins on 9a-OH-AD production was evalu- of 9a-OH-AD adsorbed in the resin continued to increase and
ated in this study. The production of 9a-OH-AD and the reached a maximum of 20.23%, which further demonstrates
consumption of PS were remarkably inuenced by the different that 9a-OH-AD adsorption is not an essential factor in
adsorption resins (Table 1). Among them, the D101 resin increasing the reaction rate. Aer resin desorption, it was found
signicantly increased the space–time yield of 9a-OH-AD (from that the higher the dose of resin D101 added during the
ꢀ
1
ꢀ1
6
.91 to 7.76 g L
d ), leading to a 12.25% increase in the total bioconversion process, the more metabolic intermediates
ꢀ
1
yield of 9a-OH-AD (23.28 g L ) compared to the control group adsorbed in the resin, which may be the reason for lower
aer 72 h of bioconversion by Mycobacterium, which was most production rate of 9a-OH-AD. Furthermore, the high concen-
suitable for further exploration. In addition, the strains showed tration of resin in the system could restrict mass transfer and
strong bioconversion activity of PS aer the addition of D101 thus affect the space–time yield of 9a-OH-AD. These results
resin. The PS in the D101 group was consumed by 92.78% aer indicate that the addition of 5 g L of D101 is sufficient to
2 h, with a 7.48% increase compared to the control group, facilitate the production of 9a-OH-AD and that an overdose of
which could contribute to production cost savings by reducing resin inhibits the conversion of PS.
ꢀ
1
7
the reaction period. However, all the other nine resins exerted
3.2.3 Optimization of resin D101 addition time in the
negative effects on PS conversion and 9a-OH-AD production. bioconversion process. The addition time of the adsorbent resin
Consequently, D101 is the most effective resin for in situ is another key factor in maximizing resin utilization efficiency
adsorption. Furthermore, the distribution of 9a-OH-AD in the during PS bioconversion by Mycobacterium in the resting cell
reaction solution and resins was investigated. Initially, we systems. The inuence of different D101 addition times,
thought that the feasibility of using D101 as an absorbent may ranging from 0 to 60 h, on 9a-OH-AD production process was
be due to, in part, its ability to adsorb the main product, 9a-OH- investigated. As shown in Table 3, the results showed that resin
ꢀ
1
AD. However, only 2.23 g L of 9a-OH-AD converged at the D101 was optimally added at 0 h, allowing PS to be entirely
resins, accounted for the 9.58% of the total 9a-OH-AD yield, depleted, and the 9a-OH-AD space–time yield increased by
ꢀ
1
ꢀ1
suggesting that the adsorption of 9a-OH-AD may not be a key 23.15% to 8.51 g L
d
compared to the control group at 72 h.
factor in improving the reaction rate.
a
Table 1 Effects of different resins on 9a-OH-AD production, space–time yield and phytosterols consumption
9
a-OH-AD
9a-OH-AD
in resins (g L
Conversion rate
of PS (%)
Space–time
yield (g L
ꢀ1
ꢀ1
ꢀ1
ꢀ1 ꢀ1
Resin
in supernatant (g L
)
)
Total 9a-OH-AD (g L
)
d )
CK
D101
20.74 ꢂ 0.54
21.05 ꢂ 0.38
17.90 ꢂ 1.12
12.24 ꢂ 0.24
15.94 ꢂ 0.78
6.36 ꢂ 0.01
16.24 ꢂ 0.13
3.63 ꢂ 0.15
11.83 ꢂ 0.70
7.04 ꢂ 0.51
5.71 ꢂ 0.59
—
20.74 ꢂ 0.74
23.28 ꢂ 0.86
19.06 ꢂ 0.49
12.96 ꢂ 0.38
17.27 ꢂ 0.51
6.90 ꢂ 0.25
17.00 ꢂ 0.53
3.77 ꢂ 0.19
12.52 ꢂ 0.45
7.52 ꢂ 0.47
6.00 ꢂ 0.32
85.30 ꢂ 0.39
92.78 ꢂ 0.30
82.72 ꢂ 0.73
66.67 ꢂ 0.65
78.22 ꢂ 0.71
57.89 ꢂ 0.57
75.41 ꢂ 0.81
42.80 ꢂ 0.55
70.89 ꢂ 1.25
55.09 ꢂ 0.68
52.26 ꢂ 0.53
6.91 ꢂ 0.24
7.76 ꢂ 0.19
6.35 ꢂ 0.18
4.32 ꢂ 0.27
5.76 ꢂ 0.31
2.30 ꢂ 0.22
5.67 ꢂ 0.27
1.22 ꢂ 0.11
4.17 ꢂ 0.24
2.51 ꢂ 0.13
2.00 ꢂ 0.21
2.23 ꢂ 0.04
1.16 ꢂ 0.01
0.72 ꢂ 0.09
1.33 ꢂ 0.03
0.54 ꢂ 0.02
0.76 ꢂ 0.05
0.14 ꢂ 0.01
0.69 ꢂ 0.04
0.48 ꢂ 0.04
0.29 ꢂ 0.01
SP 207
HZ 820
HZ 818
HZ 806
HZ 801
HPD 826
HPD 500
HPD 300
HPD 100
a
ꢀ1
Except for the control group with no resin addition (CK), all the resins were added at a concentration of 20 g L at 0 h. — means not detected. All
ꢀ
1
ꢀ1
ꢀ1
experiments contained 50 g L of phytosterols, 100 g L of wet biomass, 200 g L of HP-b-CD, and 10 mL of reaction solution bioconverted in
50 mL shake asks for 3 d (error bars for standard deviations, n ¼ 3).
2
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a
Table 2 Effects of different concentrations of resin D101 on 9a-OH-AD production, space–time yield and phytosterol consumption
9
a-OH-AD
9a-OH-AD
in resins (g L
Conversion rate
of PS (%)
Space–time
yield (g L
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1 ꢀ1
D101 concentrations (g L
)
in supernatant (g L
)
)
Total 9a-OH-AD (g L
)
d )
0
3
5
1
2
3
4
5
20.74 ꢂ 0.54
22.02 ꢂ 0.34
24.75 ꢂ 0.43
22.45 ꢂ 0.18
21.05 ꢂ 0.38
17.96 ꢂ 0.58
15.88 ꢂ 0.17
14.63 ꢂ 0.58
—
20.74 ꢂ 0.74
22.25 ꢂ 0.51
25.53 ꢂ 0.23
23.71 ꢂ 0.50
23.28 ꢂ 0.86
20.70 ꢂ 0.38
19.20 ꢂ 0.73
18.34 ꢂ 0.49
85.30 ꢂ 0.39
89.16 ꢂ 0.34
100 ꢂ 0.00
6.91 ꢂ 0.24
7.42 ꢂ 0.10
8.51 ꢂ 0.15
7.90 ꢂ 0.44
7.76 ꢂ 0.19
6.90 ꢂ 0.15
6.40 ꢂ 0.25
6.11 ꢂ 0.17
0.23 ꢂ 0.02
0.78 ꢂ 0.18
1.26 ꢂ 0.09
2.23 ꢂ 0.04
2.74 ꢂ 0.31
3.32 ꢂ 0.29
3.71 ꢂ 0.47
0
0
0
0
0
94.68 ꢂ 0.12
92.78 ꢂ 0.30
82.98 ꢂ 0.83
79.44 ꢂ 0.15
76.28 ꢂ 0.96
a
ꢀ1
ꢀ1
ꢀ1
D101 resin was added at the beginning of the reaction. All experiments contained 50 g L phytosterols, 100 g L wet biomass, 200 g L HP-b-CD,
0 mL reaction solution bioconverted in 250 mL shake asks for 3 d (error bars for standard deviations, n ¼ 3).
1
a
Table 3 Effects of different addition times of resin D101 on 9a-OH-AD production, space–time yield and phytosterol consumption
D101 addition
times (h)
9a-OH-AD
in supernatant (g L
9a-OH-AD
in resins (g L
Conversion rate
of PS (%)
Space–time
yield (g L
ꢀ
1
ꢀ1
ꢀ1
ꢀ1 ꢀ1
)
)
Total 9a-OH-AD (g L
)
d )
CK
0
20.74 ꢂ 0.54
24.75 ꢂ 0.43
23.72 ꢂ 0.54
22.24 ꢂ 0.64
21.25 ꢂ 0.17
20.44 ꢂ 0.65
20.39 ꢂ 0.52
—
20.74 ꢂ 0.74
25.53 ꢂ 0.23
24.27 ꢂ 0.23
22.81 ꢂ 0.50
21.80 ꢂ 0.67
20.75 ꢂ 0.38
20.61 ꢂ 0.73
85.30 ꢂ 0.39
100 ꢂ 0.00
6.91 ꢂ 0.24
8.51 ꢂ 0.15
8.09 ꢂ 0.11
7.60 ꢂ 0.07
7.27 ꢂ 0.12
6.92 ꢂ 0.10
6.87 ꢂ 0.39
0.78 ꢂ 0.18
0.55 ꢂ 0.08
0.57 ꢂ 0.02
0.55 ꢂ 0.07
0.31 ꢂ 0.04
0.22 ꢂ 0.01
12
24
36
48
60
95.2 ꢂ 0.37
92.02 ꢂ 0.49
86.98 ꢂ 0.43
84.16 ꢂ 0.55
82.30 ꢂ 0.83
a
ꢀ1
ꢀ1
ꢀ1
ꢀ1
The D101 resin was added at a concentration of 5 g L . All experiments contained 50 g L phytosterols, 100 g L wet biomass, 200 g L HP-b-
CD, 10 mL reaction solution bioconverted in 250 mL shake asks for 3 d (error bars for standard deviations, n ¼ 3).
Apart from this, the PS bioconversion efficiency decreased conditions for its use were optimized. The results showed that
gradually when the addition of D101 was later than 0 h because the optimum time for the addition of D101 was at the beginning
ꢀ1
a short incubation time for the resin D101 might not necessarily of the reaction, and the most suitable concentration was 5 g L
alleviate the inhibitory effect of the toxic products. These results
.
are contrary to the conclusions of a previously published study
on the timing of resin addition for the production of ADD,
3
.3 Analysis of the mechanism of D101 in improving the
bioconversion process
3.3.1 Separation and identication of putative inhibitory
diepoxin z, botrallin, and TMC-264 under the growing cells
12,17,19
system,
mainly because premature resin addition can
adsorb nutrients and thus lead to a decrease in biomass. intermediates. The aforementioned results indicated that the
However, this situation did not exist in this resting cell system adsorption of 9a-OH-AD by D101 was not a key factor in
and early resin addition provided an excellent protective improving the space–time yield, since only 3.1% of the total 9a-
ꢀ
1
microenvironment for bioconversion by adsorbing toxic OH-AD yield was adsorbed by the resin when 5 g L D101 was
products.
added. Multiple types of metabolites produced during biocon-
In this section, macroporous adsorbent resin D101, which version were adsorbed by the resins. To better understand the
favourably produced 9a-OH-AD, was selected and the effect of resin adsorption on space–time yield, the putative
inhibitor was separated and puried by silic gel
Table 4 Proportion of phytosterol metabolites adsorbed by resin D101 after 3 d of bioconversion
ꢀ1
ꢀ1
Metabolites
a-OH-AD
AD
DHBC
DHC
In supernatant (g L
)
In resins (g L
)
Absorption ratio (%)
9
24.75 ꢂ 0.33
0.46 ꢂ 0.04
2.99 ꢂ 0.03
2.17 ꢂ 0.17
—
0.78 ꢂ 0.02
0.06 ꢂ 0.01
0.14 ꢂ 0.02
0.29 ꢂ 0.02
0.05 ꢂ 0.01
3.05 ꢂ 0.04
11.54 ꢂ 0.11
4.47 ꢂ 0.09
11.79 ꢂ 0.23
100 ꢂ 0.00
4
-Ene-3-keto steroids
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Fig. 2 Degradation pathway of M. neoaurum HK86 for sitosterol, according to ref. 5 and 21. ChoMs: cholesterol oxidase, Cyp125: steroid C27-
monooxygenase, KSH: 3-ketosteroid-9a-hydroxylase. The black dashed arrows indicate multi-step enzyme-catalyzed side chain degradation
reactions.
chromatograph, and identied using ultra-performance liquid was not detectable aer 24 h of reaction, indicating that 4-ene-3-
chromatography-tandem mass spectrometry (UPLC-MS/MS) to keto steroids only accumulated at the beginning of the bio-
determine the molecular weight and fragmentation pattern of process and did not have a clear effect in the middle and late
these compounds (Fig. S1†). As described in Table 4, these stages of bioconversion. Notably, there was no 4-ene-3-keto
compounds are all intermediates of PS bioconversion, and 4- steroids detected in the supernatant aer treatment with
ene-3-keto steroids, which were the rst intermediate of PS D101 at 0 h, and all of them were adsorbed by the resin.
conversion through an oxidation reaction catalysed by choles- Moreover, the amount of 4-ene-3-keto steroids adsorption
terol oxidases (ChoMs) (Fig. 2), showed the largest adsorption increased with an increase in the amount of resin, and when the
ꢀ
1
rate in the intermediates. Moreover, 4-ene-3-keto steroids was system was treated with 30 g L of D101, the amount of 4-ene-3-
detected only in the resin and not in the supernatant when the keto steroids adsorption reached 3 times that attained with 5 g
ꢀ
1
process was treated with resin. Other intermediates, such as AD
L
of D101. However, the excess adsorption of 4-ene-3-keto
and DHC, mostly remained in the reaction system. Therefore, steroids reduced the 9a-OH-AD yield and the conversion rate
we speculated that D101 enhanced the space–time yield of 9- of PS (Table 2). The initial reaction rate aer the addition of
ꢀ
1
OH-AD mainly by the adsorption of 4-ene-3-keto steroids and resin was evaluated (Fig. 3B). When 5 g L D101 was added at
alleviated its toxic effect on Mycobacterium cells. 0 h, the initial reaction rate was enhanced by 23.44% compared
.3.2 Effect of 4-ene-3-keto steroids on bioconversion to the control group (17.28 g L d .
ꢀ1
ꢀ1
ꢀ1 ꢀ1
3
d
), reaching 21.33 g L
process. The quantitative characterization of 4-ene-3-keto Interestingly, there were no signicant differences in the initial
steroids using the relative concentrations method (referred reaction rates at different resin concentrations. These data
Fig. S2†) is shown in Fig. 3A. In the control group, the accu- demonstrated that the accumulation of 4-ene-3-keto steroids
mulation of 4-ene-3-keto steroids was observed only at 12 h and was closely related to the production efficiency of 9a-OH-AD,
and the removal of 4-ene-3-keto steroids by resin adsorption
markedly increased the initial reaction rate of the bioprocess.
However, the excess adsorption of 4-ene-3-keto steroids by the
resin in the process was detrimental to further degradation of
ꢀ1
sterol side chains as an intermediate metabolite, and 5 g L of
D101 resin was optimum for 9a-OH-AD production in this
system.
To verify if 4-ene-3-keto steroids accumulation could have an
inhibitory effect on bioconversion, we exogenously supple-
mented 4-ene-3-keto steroids and detected its inhibitory effect
on 9a-OH-AD production. The initial reaction rate decreased
signicantly in the presence of exogenous 4-ene-3-keto steroids
Fig. 3 Quantitative characterization of 4-ene-3-keto steroids in the
absence or presence of resin D101 (A), and effect of resin adsorption of
(Fig. 4), and the higher the concentration of 4-ene-3-keto
4
-ene-3-keto steroids on initial reaction rate (B). The 4-ene-3-keto
steroids, the lower the initial reaction rate. The initial reaction
steroids content in the 12 hour sample in the absence of resin was set
ꢀ
1
ꢀ1
to 1, and the initial reaction rate indicates the consumption of PS at 1 d. rate decreased by 19.39% to 13.93 g L
d
aer adding the 4-
ꢀ
1
B, control group, no resin added; -, 4-ene-3-keto steroids in 5 g L
ene-3-keto steroids at a relative concentration of 1 (4-ene-3-keto
steroids content of the control group at 12 h was set to a relative
concentration of 1). These results suggested that the accumu-
lation of 4-ene-3-keto steroids had an inhibitory effect on the
ꢀ1
D101; :, 4-ene-3-keto steroids in 10 g L D101; A, 4-ene-3-keto
steroids in 30 g L D101. 4-Ene-3-keto steroids was not detected in
the supernatant ingredients after the addition of resin and is not
ꢀ1
ꢀ1
depicted in the figure. All experiments contained 50 g L phytosterols,
ꢀ1
ꢀ1
1
00 g L wet biomass, 200 g L HP-b-CD, 10 mL reaction solution biotransformation process, thus reducing the space–time yield
bioconverted in 250 mL shake flasks for 3 d (error bars for standard
deviations, n ¼ 3).
of 9a-OH-AD.
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2021 The Author(s). Published by the Royal Society of Chemistry
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the addition time of the resins. The results revealed that D101
resin was the most suitable adsorption carrier for this ISPR
system, and the space–time yield of 9a-OH-AD was markedly
ꢀ
1
improved when it was added at a concentration of 5 g L at the
beginning of PS bioconversion. This study indicated that the in
situ removal of the inhibitory intermediate is a potential
candidate for producing 9a-OH-AD under the cyclodextrin-
resting cell system at high substrate concentrations.
Conflicts of interest
The authors declare no conict of interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (31570079, 21706072).
Fig. 4 Effect of exogenous addition of 4-ene-3-keto steroids on initial
reaction rate. The 4-ene-3-keto steroids content in the 12 hour
sample was set to a relative concentration of 1 in the absence of resin,
and the initial reaction rate indicates the consumption of PS at 1 d.
Different amounts of 4-ene-3-keto steroids were added in reaction
solution at the beginning of bioconversion. All experiments contained
References
1
W. Y. Tong and X. Dong, Recent Pat. Biotechnol., 2009, 3, 141–
ꢀ1
ꢀ1
ꢀ1
153.
5
0 g L phytosterols, 100 g L wet biomass, 200 g L HP-b-CD,
1
0 mL reaction solution bioconverted in 250 mL shake flasks for 1
2 R. Batth, C. Nicolle, I. S. Cuciurean and H. T. Simonsen,
Plants, 2020, 9, 1144.
d (error bars for standard deviations, n ¼ 3).
3
M. V. Donova and O. V. Egorova, Appl. Microbiol. Biotechnol.,
012, 94, 1423–1447.
2
The generation of 4-ene-3-keto steroids is the initial step in
4 X. Q. Gao, J. X. Feng, Q. Hua, D. Z. Wei and X. D. Wang,
Biocatal. Biotransform., 2014, 32, 343–347.
5 K. Yao, L. Q. Xu, F. Q. Wang and D. Z. Wei, Metab. Eng., 2014,
24, 181–191.
6 L. Su, S. Xu, Y. Shen, M. Xia, X. Ren, L. Wang, Z. Shang and
M. Wang, Appl. Environ. Microbiol., 2020, 86, e00441-20.
7 J. J. Yuan, Y. X. Guan and S. J. Yao, ACS Sustainable Chem.
Eng., 2017, 5, 10702–10709.
the catabolism of PS by Mycobacterium neoaurum and has been
22
demonstrated to be the rate-limiting step in PS bioconversion.
The augmentation of cholesterol oxidase expression levels is
a commonly used method to enhance the formation of 4-ene-3-
keto steroids, but the increased response rate was in turn
inhibited by feedback from the excessive accumulation of 4-ene-
3
-keto steroids. Therefore, enhancing cholesterol oxidase
activity alone did not result in a sustained improvement in PS
bioconversion. In this study, we demonstrated that the
adsorption of 4-ene-3-keto steroids by resin accompanied with
the reaction progress was a key method to help mitigate 4-ene-3-
8 R. A. Mancilla, C. Little and A. Amoroso, Appl. Biochem.
Biotechnol., 2018, 185, 494–506.
9 L. Zhou, H. Li, Y. Xu, W. Liu, X. Zhang, J. Gong, Z. Xu and
J. Shi, Process Biochem., 2019, 87, 89–94.
keto steroids toxicity, and achieving a balance between 4-ene-3- 10 V. Y. Shtratnikova, M. I. Schelkunov, D. V. Dovbnya,
keto steroids production and consumption was a key factor
affecting PS bioconversion.
E. Y. Bragin and M. V. Donova, Appl. Microbiol. Biotechnol.,
2017, 101, 4659–4667.
1
1
1 S. M. Khomutov, G. V. Sukhodolskaya and M. V. Donova,
Biocatal. Biotransform., 2009, 25, 386–392.
2 M. A. Molchanova, V. A. Andryushina, T. S. Savinova,
T. S. Stytsenko, N. V. Rodina and N. E. Voishvillo, Russ. J.
Bioorg. Chem., 2007, 33, 354–358.
4
. Conclusions
PS bioconversion to 9a-OH-AD proceeded in a series of bio-
reactions and produced many intermediates that putatively
inhibited cell activity. Mechanistic probing revealed that the 13 W. H. Liu, C. W. Kuo, K. L. Wu, C. Y. Lee and W. Y. Hsu, J.
increased space–time yield of 9a-OH-AD was mainly achieved by Ind. Microbiol. Biotechnol., 1994, 13, 167–171.
in situ removal of 4-ene-3-keto steroids, which is the rst 14 C. Y. Lee, C. D. Chen and W. H. Liu, Appl. Microbiol.
intermediate metabolite of sterol degradation, to alleviate its Biotechnol., 1993, 38, 447–452.
feedback inhibition rather than the adsorption of the nal 15 J. T. Dafoe and A. J. Daugulis, Biotechnol. Lett., 2014, 36, 443–
product 9a-OH-AD. A method of in situ adsorption of the toxic 460.
intermediate metabolite using D101 resin as a carrier in 16 A. G. Santos, T. L. Albuquerque, B. D. Ribeiro and
a cyclodextrin-resting cell system was rst used to promote the M. A. Z. Coelho, Biotechnol. Appl. Biochem., 2020, 1–14.
production efficiency of 9a-OH-AD. We evaluated the types and 17 J. Zhao, Y. Li, T. Shan, Y. Mou and L. Zhou, World J.
amounts of resins added to the reaction system and optimized
Microbiol. Biotechnol., 2011, 27, 2753–2758.
24792 | RSC Adv., 2021, 11, 24787–24793
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
1
1
2
8 S. W. Han, Y. R. Choi and J. S. Shin, Catal. Lett., 2021, DOI: 21 J. K. Capyk, R. Kalscheuer, G. R. Stewart, J. Liu, H. Kwon,
10.1007/s10562-021-03535-6.
R. Zhao, S. Okamoto, W. R. Jacobs, L. D. Eltis and
W. W. Mohn, J. Biol. Chem., 2009, 284, 35534–35542.
22 K. Yao, F. Q. Wang, H. C. Zhang and D. Z. Wei, Metab. Eng.,
2013, 15, 75–87.
9 H. Luo, H. Liu, Y. Cao, D. Xu, Z. Mao, Y. Mou, J. Meng, D. Lai,
Y. Liu and L. Zhou, Molecules, 2014, 19, 14221–14234.
0 X. Q. Gao, J. X. Feng, X. D. Wang, Q. Hua and D. Z. Wei,
Chem. Biochem. Eng. Q., 2015, 29, 567–573.
©
2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 24787–24793 | 24793