Microbial transformation of diosgenin to diosgenone by Wickerhamomyces anomalus JQ-1 obtained from Naxi traditional Jiu Qu

Article abstract of DOI:10.1016/j.bioorg.2019.103508

Diosgenone [(20S,22R,25R)-spirost-4-en-3-one, C₂₇H₄₀O₃] has been considered as a potential therapeutic alternative remedy for malaria. An efficient and economical approach of microbial transformation with diosgenin to diosgenone by the yeast strain Wickerhamomyces anomalus JQ-1 from Naxi traditional Jiu Qu was developed in this study. Chromatographic analysis confirmed that 85% of 0.1 mM diosgenin was transformed to diosgenone within 72 h. This research demonstrates that diosgenin could be converted to diosgenone through two-step pathway including 3β-hydroxyl oxidation and double bond isomerization rather than through one-step pathway, which prompted a further inference that the oxidation activity in W. anomalus JQ-1 has the same function with the Oppenauer-type oxidation which can convert diosgenin into diosgenone. Gaining specific functional strains from traditional fermented products will be a potential direction and ethnobotanical researches could provide helps with discovery and utilization of microbial resources.

Full text of DOI:10.1016/j.bioorg.2019.103508

Journal Pre-proofs
Microbial transformation of diosgenin to diosgenone by Wickerhamomyces
anomalus JQ-1 obtained from Naxi traditional Jiu Qu
Liangqun Li, Yanqiang Zhao, Bayi Lang, Yuying Xie, Manfred Shaowu
Meng, Yun He, Lixin Yang, Aizhong Liu
PII:
S0045-2068(19)31148-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103508
YBIOO 103508
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Bioorganic Chemistry
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12 December 2019
14 December 2019
Please cite this article as: L. Li, Y. Zhao, B. Lang, Y. Xie, M. Shaowu Meng, Y. He, L. Yang, A. Liu, Microbial
transformation of diosgenin to diosgenone by Wickerhamomyces anomalus JQ-1 obtained from Naxi traditional
Jiu Qu, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103508
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Microbial transformation of diosgenin to diosgenone by Wickerhamomyces
anomalus JQ-1 obtained from Naxi traditional Jiu Qu
Liangqun Li a, 1, Yanqiang Zhao b, 1, Bayi Lang c,1, Yuying Xie a, Manfred Shaowu Meng d, Yun
He e, Lixin Yang a, c, *, Aizhong Liu a, c, f,
*
a
Key Laboratory of Economic Plants and Biotechnology, Yunnan Key Laboratory for Wild
Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming
650201, China
b
c
d
e
f
College of Forestry and Vocational Technology in Yunnan, Kunming 650224, Yunnan, China
Center for Biodiversity and Indigenous Knowledge, Kunming 650034, Yunnan, China
Duke Global Health Institute, Duke University, NC 27708, USA
Lijiang Yunxin Green Biological Development Co., Ltd., Lijiang 674119, Yunnan, China
Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains
of China, Ministry of Education, Southwest Forestry University, Kunming 650224, China
Keywords: Diosgenone; Oppenauer oxidation; Wickerhamomyces anomalus; Biotranformation
Abstract:
Diosgenone [(20S,22R,25R)-spirost-4-en-3-one, C27H40O3] has been considered as a
potential therapeutic alternative remedy for malaria. An efficient and economical
approach of microbial transformation with diosgenin to diosgenone by the yeast strain
Wickerhamomyces anomalus JQ-1 from Naxi traditional Jiu Qu was developed in this
study. Chromatographic analysis confirmed that 85% of 0.1 mM diosgenin was
transformed to diosgenone within 72 hours. This research demonstrates that diosgenin
could be converted to diosgenone through two-step pathway including 3β-hydroxyl
oxidation and double bond isomerization rather than through one-step pathway, which
prompted a further inference that the oxidation activity in W. anomalus JQ-1 has the
same function with the Oppenauer-type oxidation which can convert diosgenin into
diosgenone. Gaining specific functional strains from traditional fermented products will
be a potential direction and ethnobotanical researches could provide helps with
discovery and utilization of microbial resources.
* Corresponding authors at: Key Laboratory of Economic Plants and Biotechnology, Yunnan Key Laboratory
for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China (L.
Yang). Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China,
Ministry of Education, Southwest Forestry University, Kunming 650224, China (A. Liu).
E-mail addresses: rattan@mail.kib.ac.cn (L. Yang), liuaizhong@mail.kib.ac.cn (A. Liu).
1 These authors contributed equally to this work
1

1. Introduction
Active compound diosgenone, namely, 20(S),22(R),25(R)-spirost-4-en-3-one, is a
natural steroid having six rings (A/B/C/D/E/F) with 27 carbons which exists only in a
few species of plants [1,2]. This steroid has been considered as a potential alternative
for malaria treatment [3,4]. Initially, this finding come from the traditional usage of
Solanun nudum by indigenous people for treating anti-malarial at Tumaco (Narino,
Colombia) [5]. Further, diosgenone was reported as active compound to inhibit IFN-γ
production [6]. Thus, diosgenone and its derivatives have provided new sources for the
new drug development of neuroprotective and apoptosis inducing agents [7,8].
Generally, diosgenone and its analogues were obtained from extracts of plant tissues,
but the content of diosgenone is usually low in plant tissues. Furthermore, with the
development and utilization of medicines, wild plants containing diosgenone have been
over-harvested. Therefore, it is unsustainable that obtaining active compound
diosgenone by extraction of plant tissues [1]. Besides, obtaining of active steroidal
compounds by experimentally structural modification or chemical synthesis is often
high energy and cost consumption [9]. In comparison to traditional phytoextraction and
chemical synthesis, biotransformation of steroids is one of the energy-saving and
environmentally friendly methods, which is normally used as an approach to modify
chemical substances with multi-enzymatic reactions, especially in the cases of common
hydroxylation, oxidation, reduction, hydrolysis, epimerization, isomerization, and
rearrangement [10].
Recent development of biotransformation technology can provide a new insight
for generating the active steroidal ingredients [11]. Microbial transformation of
diosgenin has been extensively employed for developing steroidal drugs [12-15].
Steroidal bioconversion of abiraterone to D4A drives the development of anti-tumour
drugs in prostate cancer treatment [16]. The premise of this technology is to find
specific microbial resources (strains) with high biotransformation efficiency. So far, a
few researches have demonstrated that the obtaining of diosgenone or diosgenin by
microbial transformation [17-23]. An actinomycete strain Streptomyces virginiae IBL-
14 was isolated from soil, which can transform diosgenin to isonuatigenone
2

accompanied by the formation of diosgenone [17,18]. Due to the lack of microbial strain
resources, the production and utilization of diosgenone by microbial transformation
have been restricted. On another hand, diverse microbial resources have been identified
from traditional microbial fermented foods [24-27]. In particular, traditional Chinese
Jiu Qu (that can produce ethanol by microbial fermentation) is one of the most
important microbial resources. Herein, this study intends to isolate microbial strains
that can convert diosgenin to diosgenone from traditional Chinese Jiu Qu.
The Naxi, indigenous ethnic group living in Lijiang area (Northwest Yunnan
Province of Southwest China), have accumulated abundant and distinctive traditional
knowledge on microbial fermentation [28]. Naxi people have developed rich
ethnobotanical knowledge on producing diverse Jiu Qu by fermentation using different
plants, resulting in diverse microbial resources for making their traditional wine and
fermented foods. For instance, they mixed and ground the chopped fresh aerial part of
Gentiana cephalantha Franch. ex Hemsl and bran with cold water for fermentation. The
wet bran and plant tissues were pressed into a pie with 7-8 cm in diameter. The Naxi
Jiu Qu pies were ready after they were dried under natural conditions about 15 days.
The making process of Naxi Jiu Qu is as shown in Fig. 1. Often, the quality of Naxi Jiu
Qu (containing different microbial species) is variable due to different plants used. The
Jiu Qu contains diverse microbial species that provide useful resources for discovery of
functional microbial species or strains.
Fig. 1. Here
The yeast strain Wickerhamomyces anomalus JQ-1 from Naxi Jiu Qu was isolated
and explored its capacity to transform diosgenin to diosgenone through Oppenauer-type
oxidation (3β-hydroxyl oxidation, subsequently double bond isomerization) pathway in
this study, which provides an efficient approach for producing active compound
diosgenone by microbial transformation.
2. Results and discussion
2.1. Isolation and identification of the strains from Naxi traditional Jiu Qu
According to the characteristics of yeast and mold strains growing on the medium,
3

the colonies were selected after preliminary culture, and then those colonies were
separated by streaking plate method. After three times purification, two types of yeast
colonies were isolated: one kind of colony is milky white, smooth, flat, opaque, creamy,
named JQ-1; the other colony is white and rounded, named JQ-2. The two yeasts from
Lijiang traditional Jiu Qu were suspected and inoculated into medium of yeast culture,
growing at 30℃.To determine species of two strains, we obtained the ITS fragment
sequences. Specifically, we performed PCR reaction using conserved primers (Tab.
S1-S2 of the Supplementary materials). The resulting PCR products were sequenced
(Beijing Qingke Xinye Biotechnology Co., Ltd.) and the obtained sequences were used
as query to perform BLASTN against public database NCBI. The results showed that
the ITS fragment from JQ-1 strain shared 95% similarity with that of Wickerhamomyce
anomalus, and the ITS sequence from JQ-2 shared 97% similarity with that of
Rhizomucor variabilis, respectively (Fig. S1-S2). Furthermore, phylogenetic analysis
(based on the neighbor-joining criteria) confirmed that JQ-1 strain was clustered with
W. anomalus, and JQ-2 stain was clustered with Mucor circinelloides (Fig.2). Therefore,
we named the JQ-1 strain as “Wickerhamomyce anomalus JQ-1” and the JQ-2 as
“Rhizomucor variabilis JQ-2”.
Fig. 2 here
2.2. Biotransformation of diosgenin by Wickerhamomyces anomalus JQ-1
To test whether W. anomalus JQ-1 and R. variabilis JQ-2 were able to transform
diosgenin in yeast culture, we initially performed a preliminary screening experiment,
scattered the steroidal substrate diosgenin (16.6 mg) in 400 mL liquid medium for
culturing W. anomalus JQ-1 and R. variabilis JQ-2, respectively. After incubated for 5
days, mycelium and fermentation broth were collected and extracted (crude extract) by
adding ethyl acetate. Thin layer chromatography (TLC) was used to detect the organic
phase, these results showed that W. anomalus JQ-1 could transform diosgenin to one
new product, whereas R. variabilis JQ-2 could not transform (Fig. 3A). Further,
experiments on transforming diosgenin using W. anomalus JQ-1 at the preparative scale
4

(414 mg of diosgenin was scattered in 10 L liquid medium) was proceeded within 25
Erlenmeyer Flasks. The samples were successively detected by TLC and high
performance liquid chromatography (HPLC) after 0,12,24,36,48,60 and 72 hours (Fig.
3B-3C). To be clarified, TLC analysis was the complement of HPLC analysis because
the absorption wavelength of product III is 211 nm, while that of product I and substrate
diosgenin (II) is 273 nm (Fig. 3C). Taking bioconversion time as the horizontal
coordinate and relative content of I, II and III as the vertical coordinate draw the line
graph (Fig. 3D). During 12 hours to 36 hours after substrate added, the relative content
of substrate diosgenin (II) was gradually decreased. The content of intermediate III
increased from 12 hours, peaked at 36 hours, and then gradually decreased to 0 during
the next 36 hours. The content of product I increased gradually from 12 hours to 72
hours, with the fastest growth rate from 36 hours to 48 hours. Thus, it can be seen that
substrate diosgenin (II) have started to be converted into products I and III within 24
hours, and substrate diosgenin (II) and intermediate III have been completely
converted into final product I within 72 hours. Previous researches exhibited that it is
deficient in the microbial resources for the conversion of diosgenin and the products of
biotransformation are complex and low [19-22]. Clearly, this experiment exhibited W.
anomalus JQ-1 was able to convert substrate diosgenin (II) into products I and III,
suggesting W. anomalus JQ-1 might be useful for the bioconversion of steroidal
productions in industry.
Fig. 3 here
2.3. Structure elucidation of the isolated metabolites
To purify products I and III, the preparative scale microbial transformation was
extinguished by adding ethyl acetate after the bioconversion proceeded for 60 hours
and ultrasonic extraction was then performed. The culture was filtered and the filtrate
was extracted three times with the same volume of ethyl acetate (EtOAc) each time.
The crude extract was evaporated to dryness and the resulting mixture of brown gum
was obtained (2.79 g). The compounds I and III were separated from the crude extract
5

by multiple column chromatography and the weights were 273 mg and 79 mg,
respectively.
In order to elucidate chemical structure of the product I and III, X-ray, IR, HRMS,
and NMR were used to clarify the structure configuration, functional groups, molecular
formula and the types and relative numbers of H and C atoms, respectively.
Identification of the original data for the product I and III were placed in the
supplementary Data section.
Product I was obtained as colorless needle (ethanol), melting point:185-187°C.
The molecular formula of product I was assigned as C27H40O3 by the HRMS ions at
+
m/z 435.2874 [M+Na] (calcd for C27H40O3Na, 435.2874). The IR spectrum showed
the characteristic absorptions attributable to ketone carbonyl group (3436 cm-1) and a
double bond (1676 cm-1). The 1H NMR data displayed resonances for four methyls (δH
0.72, 0.75, 0.89,1.10). The 13C and dept NMR spectrum gave a new peak at δ 199.8 and
171.4, indicating an unsaturated ketene group. The structure of product I was tentatively
defined as (25R)-spirost-4-en-3-one via the 2D NMR data analysis (Fig. S7-S11).
Single-crystal X-ray diffraction analysis with Cu Kα radiation revealed that CH3-10,
CH3-13, CH3-20, CH3-25, C-22 and H-14 were disordered in the crystal structure
(CCDC Number: 1939859. Fig. S14 and Tab. S3), indicating that two dominant
interconverting conformations existed in ethanol. The resulting Flack parameter and the
calculated Hooft parameter could be used to define the (20S,22R,25R) absolute
configuration of diosgenone (the structural formula of the compound was shown in Fig.
4). The 1H and 13C NMR data of product I was in fitting agreement with those reported
in literature [17].
Metabolic intermediate III was isolated as a white, amorphous powder with a
molecular formula of 435.2870 (calcd for C27H40O3Na, 435.2870) based on the analysis
of its HRMS ion at m/z 435.2870 [M+Na]+. The NMR data (Fig. S3 and S4) of product
III were closely similar to those of diosgenin (II), except for a ketone carbonyl group
(δC 210.0, C-3) in compound III, replacing the methine (δH 3.53, H2-2; δ C 71.6, C-2)
in product III. Thus, product III is the 3-hydroxyl group oxidated derivative of
diosgenin (II). The structure of compound III was defined as (25R)-spirost-4-en-3-one
6

(the structural formula of the compound was shown in Fig. 4).
After structure elucidation of two pure metabolites, to investigate the
transformation rate of diosgenone, HPLC was used and the analysis exhibited that 16.6
mg (0.1mM) diosgenin was transformed to 14 mg diosgenone within 72 hours. So far,
there are few reports on the microbial transformation of diosgenin to diosgenone, which
was just a by-product [17]. However, diosgenone transformed by W. anomalus JQ-1
strain was the main product with a high conversion rate of 85%.
2.4. Determination of plausible metabolic pathways
In order to understand the metabolic pathway of diosgenin in the biotransformation
by W. anomalus JQ-1, time course of the production of steroid metabolites was
investigated by HPLC analysis (Fig. 3C-3D). Diosgenin was converted to diosgenone
(I) and spirost-5-en-3-one (III) within 24 hours of incubation by the yeast. The reaction
mixture contained a larger percentage of both diosgenone (I) and spirost-5-en-3-one
(III) after 48 hours of incubation; however, after 72 hours, the metabolite III was
nonexistent and the amount of diosgenone (I) had considerably increased in the medium.
It can thus be inferred that spirost-5-en-3-one (III) is intermediate metabolite in the
production of diosgenone (I). The two process of biotransformation were included that
the 3-position hydroxyl group of the substrate diosgenin (II) was oxidized to yield
intermediate product spirost-5-en-3-one (III) and double bond isomerization of
compound III was yielded diosgenone (I), which is indicated the presence of biological
Oppenauer-type oxidation activity efficacy (3β-hydroxyl oxidation and double bond
isomerization in succession) in the yeast strain.
Plausible Oppenauer-type oxidation pathway of diosgenin by W. anomalus JQ is
as follows (Fig. 4): In the initial step of the molecular mechanism, the secondary alcohol
substrate diosgenin (II) coordinates with the microbial receptors to generate a complex,
which in the next step, the oxidant acetone goes on to the alkoxide intermediate. The
acetone coordinates with the organism which can activate it for the hydride transfer
from the intermediate. The organism-catalyzed hydride shift from the secondary
alcohol to the carbonyl group of acetone occurs over a six-membered transition state
intermediate. Finally, the designated ketone of interest is generated after the hydride
7

transfer [30]. This research demonstrates that diosgenin (II) could be converted to
diosgenone (I) through a two-step pathway including 3β-hydroxyl oxidation and double
bond isomerization instead of the one-step pathway reported in the former literature
[17]. It could further prompt that the oxidation activity in the yeast strain has the same
function with the Oppenauer-type oxidation which can convert diosgenin into
diosgenone.
Fig. 4 here
3. Conclusions
Based on an ethnobotanical method we isolated and identified a new yeast strain
W. anomalus JQ-1 from Naxi traditional Jiu Qu pies. Using bioconversion experiments
we found that W. anomalus JQ-1 was able to convert diosgenin to diosgenone with a
relatively high rate (85% within 72 hours). The potential Oppenauer-type oxidation
pathway in the bioconversion of diosgenone was determined during fermentation of W.
anomalus JQ. This study provides a new bioresource for potential utilization to produce
the active compound diosgenone by bioconversion methods in industry. In particular,
this study demonstrates a potential direction toward identification and utilization of
microbial resources by ethnobotanical researches.
4. Experimental Part
4.1. Reagent materials
The substrate diosgenin in 95% purity was purchased from Shanghai Yuanye
Biotechnology Co., Ltd and was recrystallized in methanol before use. Analytic
solvents were of chromatographic grade, and other reagents and solvents were of
analytical grade. Ethyl acetate and acetone were of the industrial purity and distilled
prior to use. Column chromatography was carried out on silica gel (200-300 mesh,
Qingdao Marine Chemical Factory, Qingdao, P.R. China), Thin-layer chromatography
(TLC) was performed on silica gel (Qingdao Marine Chemical Factory, Qingdao,
China).
4.2. Microbial material
8

Naxi Jiu Qu pies were collected from Ludian village (one of Naxi communities),
Yulong Naxi Autonomous County, Lijiang City, Northwest Yunnan, China (100.25°E,
26.86°N) in April 2018, where local people have used Naxi Jiu Qu pies for making
traditional wine for thousand years. They were made from the mixture of Gentiana
cephalantha Franch. ex Hemsl and bran with cold water by fermentation. The dried
pies were sampled for analysis in this study.
4.3. Isolation and identification of the yeast strains
Dried Jiu Qu pies were grinded into powders, and 10 g Jiu Qu powders was
weighed and poured into a beaker containing 100 mL dd H2O and 20 glass beads. After
well-shook 10-1 Jiu Qu suspension. The suspension was diluted into gradient bacterial
suspensions of 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 by multiple dilution.
Each of 0.1mL suspension of 10-4, 10-5 and 10-6 gradient bacteria was inoculated
on the YMG medium (yeast extract 20 g /L, peptone 40 g/L, glucose 40 g/L, agar 20
g/L, (NH4)2SO4 1 g/L, KH2PO4 1 g/L, MgSO4·7H2O 1 g/L, distilled water to 1000 mL).
with 0.1 mL plate coating method. Each dilution concentration was inoculated on 3
plates, which were incubated at 18℃, 25℃ and 30℃ for 1-3 days respectively.
According to the colony characteristics of yeast and mold, the colonies with
corresponding characteristics were selected after preliminary culture, and then those
colonies were separated by streaking plate method. The suspected grayish white or
milky white colony was taken and inoculated in the medium. The yeast colony was
selected to be inoculated in the class culture medium and cultured at 30℃ for
observation. The suspected white flocculent or dense colonies were selected and
inoculated in the medium. These colonies were also cultured at 30℃.
After three times purification, the suspected yeasts JQ-1 and JQ-2 were inoculated
into yeast medium and cultured at 30℃. After observation, these strains were kept
refrigerated at 4℃ for preservation. The samples were deposited at the Kunming
Institute of Botany.
To determine the origin of these two strains, ITS-based sequencing was performed.
Specifically, yeast DNA was isolated from collected colonies using yeast DNA
extraction kit and diluted to 50 ng/μL as described by José Manuel [31]. Then, ITS1
9

region from two DNA sample extracts were amplified by PCR using high specificity
primers (Tab. S1). The resulting PCR fragments were sequenced and used as query
sequence to perform BLASTN against public database NCBI as described [32].
Sequences with best-blast hits were further selected to perform multiple sequence
alignment and phylogenetic analysis. The multiple sequence alignment was performed
using clustal W [33], and aligned results were visualized by BioEdit version 7.2.5 [34].
The neighbor-joining (NJ) tree was constructed using MEGAX [29] with the “pairwise
deletion” option and “Poisson correction” model. Bootstrap support was estimated from
1000 replicates to evaluate the reliability of internal branches.
4.4. Biotransformation conditions and product extraction
All biotransformation experiments were performed in liquid YMG medium.
Preliminary screening scale biotransformation of diosgenin was carried out in 1000
mL Erlenmeyer flasks containing 400 mL of liquid medium. The flasks were placed on
a rotary shaker (180 rpm, 28°C). The substrate (diosgenin) gave a concentration of 8
mg mL-1 by acetone. After 48 hours of pre-culture, 16.6 mg of diosgenin was added
into each flask. The incubation was allowed to continue for 5 days. Culture controls
consisted of fermentation blanks with microorganisms grown under identical non-
substrate conditions. Substrate controls were composed of sterile medium with
substrate, and they were incubated without microorganisms.
Preparative scale biotransformation of diosgenin by W. anomalus JQ-1 was
conducted in a 1 L Erlenmeyer Flask. The substrate (16.6 mg) dissolved in acetone (2
mL) was added to pre-cultured medium (400 mL) and was incubated for an additional
3 days. Finally, 414 mg diosgenin was used for the preparative experiment.
The culture was filtered and the filtrate was extracted three times with the same
volume of EtOAc each time. The extract was evaporated to dryness on a rotary
evaporator under reduced pressure (0.02 MPa) at 45°C. The resulting product (2.79 g)
was obtained, which had a characteristic of brown gum.
4.5. Product purification and structure identification
Final residue of the dried extract was dissolved in CH2Cl2 and subjected to be
isolated by silica gel column chromatography (200-300 mesh, 2.5 cm by 40 cm with 50
10

g of silica gel). The elution system was cyclohexane/ethyl acetate (8:2 v: v). The
purified metabolites were analyzed and structurally identified by 1 HNMR, 13 C NMR,
2D NMR, HRMS, IR and X-rays.
1 H, 13 C and 2D nuclear magnetic resonance (NMR) spectra were recorded using
Bruker DRX-600 NMR spectrometers with TMS as an internal standard. HRESIMS
and HRESIMS were recorded on Finnigan LCQ-Advantage mass spectrometer and VG
Auto-Spec-3000 mass spectrometer. Optical rotations were measured on a Jasco DIP-
370 digital polarimeter. Melting points were recorded on a Fisher-Johns melting
apparatus and were uncorrected. The IR spectra were measured on a Perkin-Elmer
983G infrared spectrometer with KBr pellets. Samples from diosgenin and its
metabolites were analyzed by 1100 high-performance liquid chromatography apparatus
(Agilent Technologies, USA) with an Agilent ZORBAX SB-C18 column (5μm,
4.6×250 mm), DAD detector, and a quaternary pump system.
4.6. Time course experiment and HPLC analysis
A time course study was performed to investigate the metabolic pathways used in
the biotransformation of diosgenin. After adding the substrate, sampling and extraction
were conducted on all experiments at 0, 12, 24, 36, 48, 60 and 72 hours. Each of the six
fermented broths was each extracted with ethyl acetate, then analyzed by TLC. Samples
from diosgenin and its metabolites in experiments at the 24 hours period were analyzed
by 1100 high-performance liquid chromatography apparatus. The operating conditions
were as follows: column temperature was 40°C, mobile phase was methanol/water
(8.5:1.5), and flow rate of mobile phase was 1 mL/min. Analytes were detected with
UV simultaneously at 211 and 273 nm.
Acknowledgements
We thank Dr. Xiaojun Pu, Ms Yuying Xie, Dr. Jiangmiao Hu, Dr. Renqiang Mei,
Sudi Wang and Connor Mauche for draft suggestions. We acknowledge the Department
of Instrumental Analysis of Kunming Institute of Botany for measuring the optical
rotations, UV, NMR and mass spectra.
This study was funded by Natural Science Foundation of China (No. 31670340),
11

Basic Research Foundation of Yunnan Province (No. 2017FD043), Yunnan Post-
doctoral Fund (No. Y835481261), Project on Creative Research Group by Science &
Technology of Yunnan Department (No. 2018HC009), Dr. Plant R&D Center (No.
Y65J8322C1), and Paris polyphylla Project of Yunxin Co., Ltd (No. Y75A6812C1).
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Fig. 1 The traditional making process of Naxi Jiu Qu
15

Fig.2 A phylogenetic analysis confirms the identification of W. anomalus JQ-1 and R.
variabilis JQ-2 strains (The bootstrap values less than 50% are not shown)
Fig. 3 Time course of biotransformation. (A)TLC analysis of W. anomalus JQ-1 and R. variabilis
JQ-2 ;(B) TLC study on substrate and products (C) HPLC profile where substrate (II) was added
to the 48-hrs-old culture for 24 hrs; (D) The line chart of relative content and bioconversion time
by W. anomalus JQ-1. (Lanes 2-7 indicate yeast transformation at 12,24,36,48,60 and 72 hrs,
respectively).
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O
O
O
oxi
iso
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
HO
O
O
O
H
II
diosgenin
I (m/z:412)
diosgenone
III (m/z:412)
spirost-5-en-3-one
H
O
Six menbered chair-like transition state
Fig. 4 Plausible Oppenauer-type oxidation pathway of II to I by W. anomalus JQ-1. The structure
illustrated in brackets is a postulated intermediate. The oxi denotes steroid oxidase and the iso
denotes double bond isomerase
17

Declaration of Competing Interest
The authors claim that the researchers in this study have no conflict of interest.
18