New product identification in the sterol metabolism by an industrial strain Mycobacterium neoaurum NRRL B-3805

Article abstract of DOI:10.1016/j.steroids.2018.02.001

Mycobacterium neoaurum NRRL B-3805 metabolizes sterols to produce androst-4-en-3,17-dione (AD) as the main product, and androsta-1,4-dien-3,17-dione, 9α-hydroxy androst-4-en-3,17-dione and 22-hydroxy-23,24-bisnorchol-4-en-3-one have been identified as by-products. In this study, a new by-product was isolated from the metabolites of sterols and identified as methyl 3-oxo-23,24-bisnorchol-4-en-22-oate (BNC methyl ester), which was proposed to be produced via the esterification of BNC catalyzed by an O-methyltransferase using S-adenosyl-L-methionine as the methyl group donor. These results might open a new dimension for improvement of the efficiency of microbial AD production by eliminating this by-product via genetic manipulation of the strain.

Full text of DOI:10.1016/j.steroids.2018.02.001

Accepted Manuscript
New product identification in the sterol metabolism by an industrial strain My-
cobacterium neoaurum NRRL B-3805
Xuemei Li, Xi Chen, Yu Wang, Peiyuan Yao, Rui Zhang, Jinhui Feng, Qiaqing
Wu, Dunming Zhu, Yanhe Ma
PII:
S0039-128X(18)30029-1
https://doi.org/10.1016/j.steroids.2018.02.001
STE 8228
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 June 2017
18 January 2018
1 February 2018
Please cite this article as: Li, X., Chen, X., Wang, Y., Yao, P., Zhang, R., Feng, J., Wu, Q., Zhu, D., Ma, Y., New
product identification in the sterol metabolism by an industrial strain Mycobacterium neoaurum NRRL B-3805,
Steroids (2018), doi: https://doi.org/10.1016/j.steroids.2018.02.001
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1 New product identification in the sterol metabolism by an
2 industrial strain Mycobacterium neoaurum NRRL B-3805
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4
Xuemei Li1, 2, Xi Chen1, Yu Wang1, Peiyuan Yao1, Rui Zhang1, Jinhui Feng1*,
Qiaqing Wu1, Dunming Zhu1 and Yanhe Ma1
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1National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering
Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin
300308, China
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2University of Chinese Academy of Sciences, Beijing 100049, China
*Corresponding author: Jinhui Feng, Email: feng_jh@tib.cas.cn, Phone:
86-22-24828734, Fax: 86-22-84861996
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Running title: New product of M. neoaurum NRRL B-3805
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Abstract
Mycobacterium neoaurum NRRL B-3805 metabolizes sterols to produce
3
androst-4-en-3,17-dione (AD) as the main product, and androsta-1,4-dien-3,17-dione,
9α-hydroxyandrost-4-en-3,17-dione and 22-hydroxy-23,24-bisnorchol-4-en-3-one
have been identified as by-products. In this study, a new by-product was isolated
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from
the
metabolites
of
sterols
and
identified
as
methyl
7
3-oxo-23,24-bisnorchol-4-en-22-oate (BNC methyl ester), which was proposed to
be produced via the esterification of BNC catalyzed by an O-methyltransferase using
S-adenosyl-L-methionine as the methyl group donor. These results might open a new
dimension for improvement of the efficiency of microbial AD production by
eliminating this by-product via genetic manipulation of the strain.
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Key words: sterol degradation pathway, Mycobacterial neoaurum,
androst-4-en-3,17-dione, methyl 3-oxo-23,24-bisnorchol-4-en-22-oate
2

1 1. Introduction
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3
Steroidal pharmaceuticals are widely used in clinical applications.
Androst-4-en-3,17-dione (AD), androsta-1,4-dien-3,17-dione (ADD) and 9α-hydroxy
androst-4-en-3,17-dione (9-OH-AD) are the most marketed precursors for the
production of steroidal drugs [1, 2]. Many actinobacteria can use sterols including
cholesterol or phytosterols as carbon and energy sources and convert sterols to these
steroidal precursors in a biotechnological step.
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Mycobacterium neoaurum NRRL B-3805 (hereafter B-3805), which was isolated
after two rounds of ultraviolet irradiation, has been used at industrial level to produce
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AD from various sterols with up to 39% yield [3-6].
22-hydroxy-23,24-bisnorchol-4-en-3-one (4-HBC)
However, ADD,
and
22-hydroxy-23,24-biosnorchol-1,4-dien-3-one (1,4-HBC) were produced in small
amounts, limiting the product yield and increasing the difficulty in the product
purification. The cholesterol metabolic pathway was proposed as Figure 1 [7-11].
Recently, the genome of B-3805 was sequenced (GenBank accession number
CP011022), providing essential genetic information for molecular engineering of this
industrial strain [12]. Identification of the products in the microbial transformation
of sterols provides useful information for further understanding of the microbial sterol
degradation pathway, thus offering guidance for molecular engineering of the
industrial strains to improve the process efficiency for the production of the useful
steroidal intermediates. Therefore, we attempted to characterize the products in the
transformation of cholesterol with B-3805. This would lead to further understand
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the metabolic pathway of sterols by this strain and provide more information for its
engineering to eliminate the by-products, thus enhancing the AD production
efficiency of this strain.
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1 2. Materials and methods
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2.1Materials
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Cholesterol (>95%), β-sitosterol (75%), campesterol (95%), AD, ADD, 4-HBC,
1,4-HBC, testosterone (TS) and other chemical reagents and solvents were supplied
by chemical companies with reagent grade or the highest purity available. Silica gel
and thin layer chromatography (TLC) plate were both obtained from Qingdao
Haiyang Chemical Company. The 3-oxo-23,24-bisnorchol-4-en-22-oate (BNC, V)
was prepared by the oxidation of 4-HBC in our laboratory according to the reference
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[13, 14]. The H nuclear magnetic resonance (NMR) spectra of BNC was recorded
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on Bruker AVANCE III at 400 MHz and shown in Figure S1.
11 2.2Microorganism cultivation and biotransformation
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Strain B-3805 was grown on Luria-Bertani (LB) medium at 30°C in a rotary shaker
at 200 rpm for 3 days. The LB pre-grown culture was inoculated into 18 mL of
medium SCN (inoculum volume 1:9, v/v), which contained defatted soy flour (10
g/L), corn steep powder (5 g/L), (NH4)2HPO4 (2 g/L), cholesterol (20 g/L) or BNC
(10 g/L), and soy oil (200 g/L), and the pH was adjusted to pH 7.5 by NaOH. The
resulting culture was shaken at 30°C in a rotary shaker at 200 rpm for 4-7 days. The
biotransformation products were worked-up and analyzed as described below.
19 2.3Analytical methods
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The bioconversion mixture was extracted with 3 volume of ethyl acetate, and the
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solvent was removed to give a residue, which was re-dissolved in methanol. The
resulting solution was used for TLC and HPLC analysis. When the BNC served as a
substrate, the products were extracted with 4 volume of CH2Cl2. TLC was developed
with petroleum ether and acetone (7:3, v/v) and visualized by UV light (254 nm) or by
phosphmolybdic acid. HPLC was performed on an Agilent 1200 system equipped
with a C18-column (250 mm × 4.6 mm × 5 µm) and a diode array detector (DAD) at
the wavelength of 210 nm, 230 nm and 254 nm. A mixture of methanol and water
(80:20, v/v) was used as mobile phase at flow rate of 0.8 mL/min. The retention
times of AD, 9-OH-AD, BNC, ADD, 4-HBC, 1,4-HBC and TS were 5.6 min, 4.1 min,
4.5 min, 4.7 min, 12.1 min, 8.9 min and 6.2 min, respectively. The products were
also analyzed by LC-MS with an Agilent 1200 HPLC and a Bruker micro TOF_QII
high resolution mass spectrometer (HRMS) equipped with an ESI ionization source.
Masses were recorded over a 50-1000 m/z range in positive mode.
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In order to examine the potential effect of methanol on the formation of methyl
ester, acetonitrile was also used to instead of methanol in the analytical procedure.
HPLC was performed with a mixture of acetonitrile and water (80:20, v/v) as mobile
phase. Some of the samples were re-dissolved in acetonitrile. The retention time
of BNC methyl ester was 16.1 min.
19 2.4Isolation of the new product
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To isolate the products, the biotransformation mixture was extracted with 3
volumes of ethyl acetate. After being centrifuged at 4000 × g, the supernatant was
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separated and dried under reduced pressure. Then suitable volumes of methanol
were added to extract the products and the extract was dried again under the same
conditions. After that, the product was dissolved in CH2Cl2 and applied to a silica
gel column. The column was eluted by petroleum ether and ethyl acetate (6:1, v/v)
to separate the products. Preparative reverse phase recycling high performance
liquid chromatography (HPLC, Agilent 1260) with C18-column was used for the final
purification, and was performed with the mixture of water and methanol (30:70, v/v)
as eluent at flow rate of 11 mL/min. The detecting wavelengths were set as 254 nm
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and 210 nm. The H and 13C NMR spectra were recorded at 290.15 K on a Bruker
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AVANCE III at 400 MHz. Tetramethylsilane (TMS) served as the internal standard
in CDCl3 and chemical shifts (δ) are shown as parts per million (ppm) relative to TMS
(Table 1).
7

1 3. Results and discussion
2
3.1Products of strain B-3805
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Because of the poor water-solubility of sterols, the biocompatible organic-aqueous
two-liquid phase systems were used in the biotransformation by Mycobacterium
strains [15, 16]. In this study, soy oil (200 g/L) was used as the organic phase and
the incubated medium SCN without any sterols served as the control. As shown in
Figure 2A, only a very small amount of AD, which was formed from the phytosterols
in the soy oil, was detected in the control experiment. Strain B-3805 metabolized
cholesterol to produce AD as main product (peak 3), and 9-OH-AD (peak 1), ADD
(peak 2) and 4-HBC (peak 4) as by-products (Figure 2B, C, D, and E, and Table 2).
When campesterol or β-sitosterol served as substrate, same products were found
(Figure S2, Table S1 and Table S2). These results are in accordance with earlier
report [3]. In this study, trace amount of 9-OH-AD was detected (peak 1). It is
reasonable because two kshA genes and one kshB gene is annotated as the
3-ketosteroid-9α-hydroxylase gene in the genome [12]. In addition to these
previously identified by-products, a new by-product (named compound A, peak 5)
was detected with larger peak area than 9-OH-AD. Xu et al. found a trace amounts
of TS and boldenone from the metabolism of cholesterol by derivative M. neoaurum
strain [17].
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The putative sterol degradation pathway was proposed according to the identified
intermediates and the catabolic gene cluster in Mycobacterium, Nocardia, and
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Rhodococcus [1, 7-10, 18, 19]. As shown in Figure 1, the metabolic pathway is
initiated by the cholesterol oxidase (Cho) and/or 3β-hydroxysteroid dehydrogenase
(3β-HSD), which transform 3β-ol-5-en- to 3-keto-4-en- moiety [20, 21]. This is
followed by the sequential terminal oxidations catalyzed by cytochromes P450
(CYP125 and/or CYP142), to produce the terminal alcohol and the carboxylic acid
(III) [22, 23]. Further degradation of the side-chain is proposed to employ a
β-oxidation-like mechanism, which is catalyzed by sterol-CoA ligases,
dehydrogenases, hydratases, and acyl-CoA thiolases. As such, cholesterol or
phytosterols are shortened to a C24 carboxylic acid compound (IV). Another two
β-oxidation-like cycles are proceeded to produce AD (VI) or ADD (VIII) [10]. The
transformations of AD to ADD or 9-OH-AD (VII) are catalyzed by
3-ketosteroid-∆1-dehydrogenases (KstDs) or 3-ketosteroid-9α-hydroxylase (KshAB),
respectively [24, 25]. The 9α-hydroxy-1,4-androstadiene-3,17-dione (9-OH-ADD)
can also be produced, which is unstable and the B ring will be destructed
spontaneously to afford 3-hydroxy-9,10-seco-androst-1,3,5(10)-triene-9,17-dione
(3-HSA, IX) [26]. The steroidal polycyclic ring opening can occur at the earlier
steps of the metabolic pathway by following similar mechanisms with both the
3-keto-1,4-dien moiety and 9α-hydroxy group in the molecule [1]. The compound
IX is further degraded via oxygenolytic cleavage of ring A to produce
9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (DOHNAA, X) and
2-hydroxyhexa-2,4-dienoic acid [27, 28].
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Some studies have been carried out to improve the yield and purity of the main
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product by engineering the production strains. The kstDs were inactivated for the
production of AD and 9-OH-AD [25, 29]. On the contrast, the enhanced kstD
improved the yield of ADD [25]. The genes of kshB and kstD were also knocked out
in the strain M. smegmatis mc2155 for the production of ADD (∆MSMEG_6039,
∆kshB1) and AD (∆MSMEG_6039-∆MSMEG_5941, ∆kshB1-∆kstD1) [30]. The
characterizations of KstDs and KshABs were studied in detail [24, 26, 31, 32].
There were some studies to produce other products, such as TS and 4-HBC/1,4-HBC,
by engineering strains. Fernández-Cabezón et al. cloned and overexpressed the
17-ketosteroid reductase in the strain M. smegmatis mc2155 to produce TS from
phytosterols or AD [33]. Xu et al. knocked out hsd4A in the strains of M. neoaurum
to produce 4-HBC or 1,4-HBC [17].
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In this context, genetic manipulation would reduce or eliminate the by-products to
improve the yield and purity of the main product. Identification of by-products is
essential for understanding their formation mechanisms, and thus providing useful
information for the genetic manipulation. As such, we were interested in the
isolation and characterization of the new by-product (compound A) in the metabolism
of sterols by strain B-3805.
18 3.2Isolation and identification of the new by-product
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To isolate the compound A, 480 mL culture of strain B-3805 with cholesterol as
the substrate were extracted with ethyl acetate. After the solvent was removed, the
products were separated on a silica column. The fractions with compound A were
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combined and the solvents were removed. The residue was re-dissolved in methanol,
and the compound A was purified by a preparative HPLC to give a white powder (11
mg). HR-MS analysis indicated that the compound A with m/z 358.2509 possibly
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1
had an additional methyl group in its structure compared to BNC. The H NMR
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spectra of the compound A clearly showed that it had the same main carbon skeleton
with BNC. There were two main differences of BNC with the compound A. One
is the chemical shift of proton on C21 was changed (δ 1.19, 1.18 for compound A, δ
1.25, 1.23 for BNC), but still as a duplet peak; the other was a new single peak
appeared (δ 3.67). The 13C NMR spectra of compound A showed the compound
bearing 23 carbon, which is one more carbon than BNC, but the C22 (δ 177.2) still the
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carbonyl group. By HSQC, HMBC and H NMR analyses, δ 3.64 was attached to
51.4, and showed no interaction with the main skeleton (Table 1). Therefore, the
compound A was identified as the methyl ester of BNC, and named as BNC methyl
ester.
Owen et al. have identified some intermediates in the cholesterol degradation
pathway of strain Pseudomonas sp. NCIB 10590 by infrared spectra, NMR and MS.
These
intermediates
include
acid,
chol-4-ene-3-one-24-oic
acid
(IV),
acid
chola-1,4-dien-3-one-24-oic
pregn-4-ene-3-one-20-carboxylic
(3-oxo-23,24-bisnorchol-4-ene-22-oate, V), and pregna-1,4-dien-3-one-20-carboxylic
acid [11]. The last compound was also found in the sterol degradation by
Rhodococcus rhodochrous strain RG32, a kshA deletion mutant [34]. These results
are consistent with the proposed β-oxidation-like mechanism of the side chain
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degradation. On the other hand, in the sterol metabolism by the engineering strains
of M. neoaurum, 4-HBC, 1,4-HBC, and 9,22-dihydroxy-23,24-bisnorchol-4-en-3-one
(9-OH-HBC) were observed. These compounds may be produced via reduction of
3-oxo-23,24-bisnorchol-4-en-20-carbaldehyde which formed by an retro-aldol
reaction [17]. The same reaction also ultimately produced 17-keto-steroids
including AD and ADD [7-9].
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In theory, the C22 terminal aldehyde compound can be oxidized to BNC in vivo.
However, the authors did not detect the carboxylic acid (V) [17]. Thomas et al.
found the accumulation of methyl 1β-(2’-propanoate)-3aα-H-4α-(3’-propanoic
acid)-7aβ-methylhexahydro-5-indanone (methyl ester of DOHNAA), which was
identified by HRMS and NMR. However, no more suggestion was given for the
esterification reaction [35]. In this study, the possibility for the formation of methyl
ester during the reaction work-up and analysis was excluded, because similar amounts
of BNC methyl ester was formed when acetonitrile was used instead of methanol for
the reaction work-up and analysis (Figure S3). Therefore, BNC methyl ester should
be produced during the degradation of sterols by strain B-3805.
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17 3.3Biotransformation of BNC
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BNC, which could be oxidatively converted from 4-HBC, was tested as the
substrate for the biotransformation by strain B-3805 with 100 g/L soy oil, and the
products were analyzed. Only trace amount of AD was detected from the control
experiment without BNC (Figure 3A). As shown in Figure 3B, 2 g/L BNC was
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converted to 0.46 g/L AD and 0.36 g/L BNC methyl ester after 168 h, and 1.21 g/L
BNC was recovered. Similar results were obtained when the concentration of BNC
was increased to 10 g/L, after 168 h, the concentrations of AD, BNC methyl ester and
remaining BNC were 0.75 g/L, 0.80 g/L and 8.17 g/L, respectively (Figure 3C).
These results suggested that BNC was converted to AD and BNC methyl ester by
strain B-3805, and this may be the reason that the carboxylic acid compounds (BNC
and pregna-1,4-dien-3-one-20-carboxylic acid) did not detected for strain B-3805.
It has been reported that O-methyltransferases can act on carboxyl moiety of
molecules using S-adenosyl-L-methionine (SAM) as the methyl group donor [36]. It
is possible that the esterification reaction to yield BNC methyl ester might be a methyl
transfer reaction catalyzed by an O-methyltransferase in the strain B-3805, although
the gene encoding the methyltransferase is still unknown.
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1 4. Conclusion
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In summary, in the sterols metabolism by M. neoaurum NRRL B-3805, AD was
produced as the main product. In addition to the by-products 9-OH-AD, ADD, and
4-HBC, BNC methyl ester was isolated and identified. This new by-product might
be produced by the esterification of BNC catalyzed by an O-methyltransferase using
SAM as the methyl group donor. These results would provide new opportunity for
the genetic manipulation of strain B-3805 to improve the efficiency of AD production.
In this context, further studies on the gene encoding O-methyltransferase and
characterization of the enzyme are warranted.
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Acknowledgement
This work was financially supported by Youth Innovation Promotion Association (to
Jinhui Feng) and the STS Program of Chinese Academy of Sciences
(KFJ-SW-STS-164), and National High-Tech Research & Development Program of
China (863 Program, No. 2011AA02A211).
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Phytochemistry 67 (2006) 1686-1698.
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1
2
Figure legends
Figure 1 Overview of the cholesterol degradation pathway [7-11]. Cholesterol
degradation is initiated by the oxidation of 3β-hydroxyl group, and the side-chain was
degraded by sequential terminal oxidations and then by 3 rounds of β-oxidation like
reactions to yield AD or ADD. Retro-aldol reaction maybe participate in the
degradation pathway. The ring B is destructed spontaneously via 9-OH-ADD to
form 3-HSA (IX), and then was metabolized further to produce DOHNAA (X) and
2-hydroxyhexa-2,4-dienoic acid.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Figure 2 Phenotypic analyses of cholesterol metabolism of M. neoaurum NRRL
B-3805. A, HPLC chromatogram of the products from the degradation of
cholesterol by strain B-3805. Black line, cholesterol served as substrate; red line,
control experiment. B, MS of metabolite 9-OH-AD (peak 1). C, MS of metabolite
ADD (peak 2). D, MS of metabolite AD (peak 3). E, MS of metabolite 4-HBC
(peak 4). Strain B-3805 can use cholesterol and phytosterol as substrate to
accumulate AD as main product, and 9-OH-AD, ADD, and 4-HBC, et al. as
by-products.
Figure 3 The biotransformation of BNC by strain B-3805. A, only minimum of AD
was detected from the culture of control. B, the transformation of 2 g/L BNC. C,
the transformation of 10 g/L BNC. Both AD and BNC methyl ester were detected
from the culture of strain B-3805 when BNC served as substrate.
21

1
Table 1. 1H and 13C Data of compound A.
2
3
Compound A
13 C
(δ, ppm)
35.7
1 H
(δ, ppm)
2.02, 1.72
2.42, 2.35
-
Carbon No.
1
2
34.0
3
199.6
123.8
171.3
32.9
4
5.78
5
-
6
2.39, 2.29
1.85, 1.04
1.54
7
32.0
8
35.6
9
53.7
0.98
10
11
12
13
14
15
16
17
18
19
20
21
22
23
38.6
20.9
1.55, 1.45
1.98, 1.27
-
39.4
42.5
52.8
1.64
24.2
1.65
27.0
1.73, 1.36
1.08
55.4
12.1
0.77
17.3
1.18
42.4
2.45
17.0
1.18, 1.19
-
177.2
51.3
3.67
4
22

1
Table 2. HPLC-HRMS Data of the products in the cholesterol degradation by M. neoaurum NRRL B-3805.
HPLC
Molecular
formula
Predicted
Experimental Identification of
Compound ID
(RTa, min)
molecular massb
ionic mass
303.1921
325.1756
285.1833
307.1720
287.2004
309.1822
331.2610
353.2431
product ions
[M+H]+
9α-hydroxy androst-4-en-3,17-dione
4.6
5.2
C19H26O3
C19H24O2
C19H26O2
C22H34O2
302.1882
284.1776
286.1933
330.2559
(9-OH-AD)
androsta-1,4-dien-3,17-dione
(ADD)
[M+Na]+
[M+H]+
[M+Na]+
[M+H]+
androst-4-en-3,17-dione
(AD)
6.2
[M+Na]+
[M+H]+
22-hydroxy-23,24-bisnorchol-4-en-3-one
(4-HBC)
12.7
[M+Na]+
2
3
a RT, retention time.
b The predicated molecular mass was calculated using the atom mass of 12C (12.0000), 16O (15.9949), and 1H (1.0078).
23

1
COOH
COOH
III
I
HO
O
II
O
IV
O
COOH
O
O
VI (AD)
O
OH
O
VII (9-OH-AD)
O
V (BNC)
O
O
O
H
H
O
O
O
O
H
H
OH
+
OH
O
VIII (ADD)
HO
X
OH
IX
2
3
4
5
6
7
8
9
Figure 1 Overview of the cholesterol degradation pathway [7-11]. Cholesterol
degradation is initiated by the oxidation of 3β-hydroxyl group, and the side-chain was
degraded by sequential terminal oxidations and then by 3 rounds of β-oxidation like
reactions to yield AD or ADD. Retro-aldol reaction maybe participate in the
degradation pathway. The ring B is destructed spontaneously via 9-OH-ADD to
form 3-HSA (IX), and then was metabolized further to produce DOHNAA (X) and
2-hydroxyhexa-2,4-dienoic acid.
24

1
2
Figure 2 Phenotypic analyses of cholesterol metabolism of M. neoaurum NRRL
B-3805. A, HPLC chromatogram of the products from the degradation of
cholesterol by strain B-3805. Black line, cholesterol served as substrate; red line,
control. B, MS of metabolite 9-OH-AD (peak 1). C, MS of metabolite ADD (peak
2). D, MS of metabolite AD (peak 3). E, MS of metabolite 4-HBC (peak 4).
Strain B-3805 can use cholesterol and phytosterol as substrate to accumulate AD as
3
4
5
6
7
8
main product, and 9-OH-AD, ADD, and 4-HBC, et al. as by-products.
25

100
80
60
40
20
A
0
0
5
10
15
20
100
Time (min)
80
60
40
20
0
BNC
B
AD
BNC methyl ester
0
5
10
15
20
300
280
260
Time (min)
BNC
C
240
100
AD
80
60
40
20
0
BNC methyl ester
0
5
10
15
20
Time (min)
1
2
3
4
Figure 3 The biotransformation of BNC by strain B-3805. A, only minimum of AD
was detected from the culture of control. B, the transformation of 2 g/L BNC. C,
the transformation of 10 g/L BNC. Both AD and BNC methyl ester were detected
from the culture of strain B-3805 when BNC served as substrate.
5
6
26

1
2
3
4
5
6
7
8
9
Highlights:
1. A new by-product was isolated from the degradation of sterol by the strain
Mycobacterium neoaurum NRRL B-3805.
2. It was identified as methyl 3-oxo-23,24-bisnorchol-4-en-22-oate by NMR and
HRMS.
3. The methyl ester was formed from the corresponding acid catalyzed by an
O-methyltransferase.
4. This provides new information on the microbial metabolism pathway of sterols.
10
11
27