Microbial transformation of flavonoids by isaria fumosorosea ACCC 37814

Article abstract of DOI:10.3390/molecules24061028

Glycosylation is an efficient strategy to modulate the solubility, stability, bioavailability and bioactivity of drug-like natural products. Biological methods, such as whole-cell biocatalyst, promise a simple but highly effective approach to glycosylate biologically active small molecules with remarkable regio- and stereo-selectivity. Herein, we use the entomopathogenic filamentous fungus Isaria fumosorosea ACCC 37814 to biotransform a panel of phenolic natural products, including flavonoids and anthraquinone, into their glycosides. Six new flavonoid (4-O-methyl)glucopyranosides are obtained and structurally characterized using high resolution mass and nuclear magnetic resonance spectroscopic techniques. These compounds further expand the structural diversity of flavonoid glycosides and may be used in biological study.

Full text of DOI:10.3390/molecules24061028

molecules
Article
Microbial Transformation of Flavonoids by
Isaria fumosorosea ACCC 37814
Fangmin Dou, Zhi Wang, Guiying Li and Baoqing Dun *
The National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences,
Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing 100081, China;
doufangmin@hotmail.com (F.D.); wangzhi@caas.cn (Z.W.); liguiying@caas.cn (G.L.)
* Correspondence: dunbaoqing@caas.cn; Tel.: +86-10-8210-8746
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 February 2019; Accepted: 11 March 2019; Published: 15 March 2019
Abstract: Glycosylation is an efficient strategy to modulate the solubility, stability, bioavailability
and bioactivity of drug-like natural products. Biological methods, such as whole-cell biocatalyst,
promise a simple but highly effective approach to glycosylate biologically active small
molecules with remarkable regio- and stereo-selectivity. Herein, we use the entomopathogenic
filamentous fungus Isaria fumosorosea ACCC 37814 to biotransform a panel of phenolic natural
products, including flavonoids and anthraquinone, into their glycosides. Six new flavonoid
(4-O-methyl)glucopyranosides are obtained and structurally characterized using high resolution
mass and nuclear magnetic resonance spectroscopic techniques. These compounds further expand
the structural diversity of flavonoid glycosides and may be used in biological study.
Keywords: Isaria fumosorosea; flavonoids; microbial transformation; methylglycosylation
1. Introduction
Flavonoids constitute the largest group of plant-derived polyphenol secondary metabolites which
could be found in various vegetables, fruits, nuts, grains, spices, wine and tea. The daily intake of
flavonoids can range from 50 to 800 mg, depending on the consumption of flavonoid-containing
foods and beverages [1]. Many studies have shown that flavonoids not only have beneficial effects
to human health, such as antioxidative, cardioprotective and neuroprotective properties but also
display a wide range of pharmacological activities including anticancer, anti-inflammatory, antibiotic
and so forth [2–4]. The basic flavonoid structure consists of 15 carbon atoms that arranged in a
C6-C3-C6 triple-ring system in which two aromatic rings (ring A and B) are connected by a C3
moiety (ring C). Based on the oxidation level and substitution pattern of ring C, flavonoids can be
categorized into several subclasses such as flavones, flavanones, isoflavones, flavonols, flavanonols
and flavan-3-ols (Figure 1). The structural diversity of individual flavonoids within these subclasses,
however, arises from the variations of substitution pattern on ring A and B [
flavonoids exist in the form of glycosides in which single or multiple sugar moieties are attached to
the flavonoid scaffold via O- or C-glycosidic bonds [ ]. Many clinically important natural products,
such as the antibiotic vancomycin and erythromycin and the antineoplastic doxorubicin, require the
sugar moiety to maintain their bioactivities [ ]. However, glycosylation of flavonoids has a much
1]. Most naturally occurring
2
5–7
more complicated impact on their biological activities which is not only influenced by the structural
variations of the flavonoid core and the sugar unit but also by the number of sugar moieties and
the position that the sugars are attached to [2,8,9]. Despite the fact that flavonoid aglycones usually
exhibit stronger activities than their sugar-conjugates in cell-based bioassays, the potential application
of aglycones are often limited by their insufficient solubility in organic and aqueous solvents, low
bioavailability after oral administration and rapid metabolism in vivo [10,11]. Flavonoid glycosides,
Molecules 2019, 24, 1028; doi:10.3390/molecules24061028
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1028
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on the other hand, have satisfying solubility and in many cases display improved bioavailability
when comparing to the aglycones. For instance, quercetin 3- and 40-O-
-D-glucosides are much more
efficiently absorbed than quercetin and quercetin 3-O- -D-rutinoside in the small intestine [10 12],
β
β
,
which probably due to the active transport of glucosides by Na-dependent glucose transporter 1
(SGLT1) presented in the brush-border membrane of the small intestine [13]. As glycosylation plays
such a substantial role in modulating the physicochemical, biological and metabolic properties of
flavonoids, it is important to further expand the structural diversity of flavonoid glycosides via
chemical and/or biological approaches.
Figure 1. Basic structures of major flavonoid subclasses.
Chemical glycosylation of structurally complex natural products often requires laborious
protection-deprotection procedures to achieve regio- and stereo-selectivity. By contrast, biological
methods, such as whole-cell biocatalysis, in vivo total biosynthesis and in vitro enzymatic reaction
using isolated glycosyltransferases, offer alternative approaches to realize one-pot and one-step regio-
and stereospecific glycosylations that proceed under mild conditions and avoid the use of toxic
reagents [
9
,14]. Filamentous fungi, especially species of genera Aspergillus and Beauveria, are rich
sources of whole-cell biocatalyst for the glycosylation of natural products [15
–17]. Entomopathogenic
fungi are an ecologically highly specialized group of microorganisms that infect, kill or seriously
disable insects and many other arthropods. Isaria fumosorosea is an important member of this group
of fungi which is isolated from over 40 species of arthropods and also presents in soil, water and
plants of every continent except for Antarctica [18]. I. fumosorosea alone or in combination with other
entomopathogenic fungal species have been successfully commercialized as mycopesticides in America,
Europe and Asia [11]. Species of genus Isaria produce a wide range of biologically active natural
products, such as the immunosuppressant sphingoid ISP-I [19], the antioxidative pseudo-dipeptide
hanasanagin [20], the insecticidal cyclodepsipeptides, isariins, isarolides and safelins/isaridins [21–24],
the carotane-type sesquiterpenes, trichocaranes E and F, CAF-603 and trichocarane C [25] and the novel
skeleton polyketide tenuipyrone [26]. More importantly to us, several I. fumosorosea species have shown
prominent ability in biotransforming a variety of natural products into their glycosides [11,27–29].
To further exploit the biocatalytic potential of I. fumosorosea and to expand the chemical
diversity of flavonoid glycosides, we used Isaria fumosorosea ACCC 37814 as whole-cell
biocatalyst to glycosylate eight flavonoids which cover the major subclasses of flavonoid,
such as flavanone (naringenin
apigenin ), isoflavone (formononetin
as another two phenolic compounds (emodin
methylglucopyranosides, namely 5,7-dihydroxyflavanone 40-O-
1b, 5-hydroxyflavanone 40,7-di-O- -D-(4-O-methyl)glucopyranoside 1d, 40,5,7-trihydroxyflavone
30-O- 30,5,7-trihydroxyflavone 40-O-
-D-(4-O-methyl)glucopyranoside 2a -D-(4-O-methyl)
glucopyranoside 2b, 5,7-dihydroxy-40-methoxyflavone 30-O-
-D-(4-O-methyl)glucopyranoside 3a and
40-methoxyisoflavone 7-O-
-D-(4-O-methyl)glucopyranoside 4a, along with two known compounds
40,5-dihydroxyflavanone 7-O-
-D-(4-O-methyl)glucopyranoside 1a and 5,7-dihydroxyflavanone
40-O-β-D-glucopyranoside 1c were obtained and structurally characterized.
1
and hesperetin
and genistein
and curcumin 10). As a result, six new flavonoid
-D-(4-O-methyl)glucopyranoside
6
), flavone (luteolin
2
, diosmetin
3 and
7
4
8) and flavonol (kaempferol
5), as well
9
β
β
β
,
β
β
β
β

Molecules 2019, 24, 1028
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2. Results and Discussion
2.1. Biotransformation Assay
Small-scale fermentations of I. fumosorosea ACCC 37814 in the presence of each substrate were
carried out to screen for all possible glycosylated products. The ethyl acetate extract of each
fermentation culture was analyzed by HPLC-HRMS/MS and the potential glycosylated products were
identified after examining their LC and HRMS/MS spectra. Satisfactorily, all tested compounds except
for curcumin 10 were converted by I. fumosorosea ACCC 37814 into at least one glycosylated products.
The LC peak areas (recorded at 300 nm) of all products and the unreacted substrate were used to
calculate the substrate-to-product conversion rate for each compound (Figure 2 and Supplementary
Materials, Table S1). In all cases, mono-methylglucoside was produced as the major product, while
mono-glucoside was also detected for some flavonoids such as naringenin
hesperetin . Mono-methylglucoside regioisomers were presented in extracts of naringenin
, kaempferol , genistein and emodin but only naringenin had di-methylglucosylated product
1
, kaempferol
5 and
6
1, luteolin
2
5
8
9
(Figure 2 and Supplementary Materials, Table S1). The precise position to which the glycosyl and
methyl substituents were attached, however, remained unclear at this point where NMR measurements
were required to unambiguously characterize the structure.
Figure 2. The biotransformation of phenolic substrates by I. fumosorosea ACCC 37814. (
conversion rate [%] of each tested substrate. All data represent the means SDs in three independent
experiments. Mono-Glc: mono-glucoside; Mono-MeGlcA and –B: mono-(4-O-methyl)glucoside
A) Total
±
regioisomers; Di-MeGlc: di-(4-O-methyl)glucoside. NA: not applicable (B) Chemical structures of
substrates 1–10. See Supplementary Materials, Table S1 for tabulated data.
2.2. Structure Characterization
Scale-up biotransformations of flavonoids 1, 2, 3 and 4 by I. fumosorosea ACCC 37814 were
carried out to obtain enough amount of their glycosides for NMR measurements. The fermentation
culture was extracted by ethyl acetate and the resulting crude extract was subjected to silica gel
column chromatography and semi-preparative HPLC to give the single compounds 1a–1d, 2a,

Molecules 2019, 24, 1028
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2b, 3a and 4a (Figure 3). Their structures were unambiguously characterized by mass and NMR
spectroscopic techniques.
Figure 3. Product profiles of flavonoids 1–4 biotransformed in the I. fumosorosea ACCC 37814 cultures
(reverse phase High Performance Liquid Chromatography (HPLC) traces recorded at 300 nm). HPLC
traces of each standard substrate (colored in red) were used as references.
2.3 mg of 1a, 3.2 mg of 1b, 6.5 mg of 1c and 1.5 mg of 1d were isolated from the scale-up
fermentation culture of I. fumosorosea ACCC 37814 (Scheme 1). The negative mode HRESIMS spectra
of 1a and 1b displayed [M
176 amu larger than that of naringenin
−
H]− ion peaks at m/z 447.1289 and 447.1294, respectively, which were
1
and thus indicated the presence of a methylated hexose unit
in their structures. This was further supported by H- and 13C-NMR spectra of 1a and 1b, which
were quite similar, and both showed characteristic resonances of a naringenin core and a methylated
D-glucosyl substituent (Table 1). Next, comprehensive analysis of HSQC and HMBC spectra revealed
that 1a and 1b were regioisomers in which the sugar substituent was attached to different positions.
The HMBC spectrum of 1a displayed correlations of the anomeric proton (δH 5.01, d, J = 7.8 Hz) and two
aromatic protons (H-6, δH 6.15, brs; H-8, δH 6.13, brs) with the oxygen-bearing aromatic carbon at δC
165.1 (C-7), revealing that the D-glucosyl substituent was connected to OH-7 of naringenin (Scheme 1).
1
Moreover, the large coupling constant of the anomeric proton confirmed the
glucosidic bond. Similarly, the D-glucose fragment in 1b was confirmed to be connected to C-40 of
naringenin via an O- -D-glycosidic bond because of the large coupling constant of the anomeric proton
δH 4.91, d, J = 7.7 Hz) and HMBC interaction between the anomeric proton and the oxygen-bearing
β-configuration of the
β
(
0
aromatic carbon at δC 157.4 (C-4 ). In both cases, HMBC correlations between the methoxy protons (1a
δH 3.44, s; 1b δH 3.45, s) and C-4 of the glucose moiety (1a δC 78.8; 1a: δC 79.0) confirmed that the
methylation has been taken place on the OH-4 of the glucose fragment (Scheme 1). Hence, the structures
:
:
:
of 1a and 1b were defined as 40,5-dihydroxyflavanone 7-O-
β-D-(4-O-methyl)glucopyranoside and
5,7-dihydroxyflavanone 40-O-β-D-(4-O-methyl)glucopyranoside, respectively.
The HRESIMS-determined molecular weight of 1c was 14 amu lower than those of 1a and 1b
.
Moreover, H- and 13C-NMR spectra of 1c were almost superimposable to those of 1b, except for
the absence of the methoxy signals and some chemical shift variations on C-3, C-4 and C-5 of the
glucosyl substituent (Table 1). These evidence indicated that 1c has a glucosyl substituent, instead of
a 4-O-methylated glucosyl group, attached to the 40-OH of naringenin. Comprehensive analysis of
HSQC and HMBC spectra of 1c further supported this speculation (Scheme 1) and thus the structure
of 1c was elucidated as 5,7-dihydroxyflavanone 40-O-β-D-glucopyranoside.
1

Molecules 2019, 24, 1028
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Scheme 1. The microbial transformation of naringenin
1 in the I. fumosorosea ACCC 37814 culture.
Key Heteronuclear Multiple Bond Correlations (HMBC) correlations of each product are indicated by
red arrows.
The HRESIMS determined molecular weight of 1d was 176 amu larger than those of 1a and 1b
and its 1H- and 13C-NMR spectra showed resonance signals corresponding to two methylated-glucosyl
substituents (Table 1), suggesting that 1d was the di-(4-O-methyl)glucopyranosyl substituted derivative
of naringenin. After careful examination of the 2D-NMR spectra (Scheme 1), the structure of 1d was
defined as 5-hydroxyflavanone 40,7-di-O-β-D-(4-O-methyl)glucopyranoside.
The scale-up biotransformation of luteolin
2 in the culture of I. fumosorosea ACCC 37814 resulted
in 2.8 mg of product 2a and 2.3 mg of product 2b (Scheme 2). The (
−
)-HRESIMS spectra of 2a and 2b
−
displayed [M
−
H] ions at m/z 461.1100 and 461.1097, respectively, which were consistent with that of
1
(4-O-methyl)glucosyl substituted luteolin. H- and 13C-NMR spectra of 2a and 2b showed resonance
signals corresponding to a luteolin core structure, a D-glucose unit and a methoxy functionality.
Apparently, 2a and 2b were regio-isomers which have the sugar fragment attached to different positions
of luteolin. In the case of 2a, the coupling constant of the anomeric proton (δH 4.93, d, J = 7.8 Hz)
along with the HMBC correlation between the anomeric proton and the oxygen-bearing aromatic
carbon at δC 145.7 (C-30) confirmed that the glucosyl substituent was connected to C-30 of luteolin
via a O-
between the anomeric proton (δH 4.92, d, J = 7.8 Hz) and the oxygen-bearing aromatic carbon at
δC 148.4 (C-40), confirming an O- -glucosidic linkage between the C-40 of luteolin and the anomeric
carbon of the sugar unit (Scheme 2). Based on HMBC correlation between the methoxy protons (2a
and 2b δH 3.47, s) and C-4 of the glucose moiety (2a δC 79.3; 2b δC 79.0), the methoxy groups in both
2a and 2b were confirmed to be attached to C-4 of the glucosyl substituent. Hence, the structures of
2a and 2b were established as 40,5,7-trihydroxyflavone 30-O-
-D-(4-O-methyl)glucopyranoside and
30,5,7-trihydroxyflavone 40-O-β-D-(4-O-methyl)glucopyranoside, respectively.
β-glucosidic linkage (Scheme 2). By contrast, the HMBC spectrum of 2b exhibited cross peak
β
:
:
:
β
Scheme 2. The microbial transformation of luteolin
2 in the I. fumosorosea ACCC 37814 culture. Key
HMBC correlations of each product are indicated by red arrows.

Molecules 2019, 24, 1028
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1
Table 1. H- (600 MHz) and 13C-NMR (150 MHz) Data of 1a, 1b, 1c and 1d in DMSO-d6.
1a
δ , Mult. (J in Hz)
1b
δ , Mult. (J in Hz)
1c
δ , Mult. (J in Hz)
1d
δ , Mult. (J in Hz)
No.
δC, Type
78.7, CH
42.0, CH2
δC, Type
78.0, CH
42.0, CH2
δC, Type
δC, Type
H
H
H
H
2
3
5.50, brd (12.7)
3.35, m
2.73, dt (17.4, 3.6)
5.52, brd (12.2)
3.27, m
2.73, dt (17.1, 3.2)
78.1, CH
5.52, brd (12.2)
3.27, m
2.73, dt (17.1, 3.2)
3.36, m
2.80, dd (17.1, 2.8)
42.1, CH2
4
5
6
7
8
197.2, C
162.9, C
95.4, CH
165.1, C
96.5, CH
162.8, C
103.2, C
128.6, C
128.4, CH
115.2, CH
157.8, C
115.2, CH
128.4, CH
99.1, CH
73.2, CH
76.0, CH
78.8, CH
75.6, CH
196.1, C
163.5, C
95.9, CH
166.8, C
95.0, CH
162.8, C
101.7, C
131.9, C
128.0, CH
116.1, CH
157.4, C
116.1, CH
128.0, CH
99.9, CH
73.4, CH
76.3, CH
79.0, CH
75.6, CH
195.9, C
163.5, C
96.0, CH
167.3, C
95.2, CH
162.7, C
101.6, C
197.0, C
162.9, C
95.4, CH
165.1, C
96.5, CH
162.6, C
103.3, C
131.7, C
128.1, CH
116.2, CH
157.5, C
116.2, CH
128.1, CH
99.1, CH
73.2, CH
76.0, CH
78.8, CH
75.6, CH
6.15, brs
6.13, brs
5.89, brs
5.88, brs
5.87, brs
5.86, brs
6.17, brs
6.14, brs
9
10
0
1
2
3
4
5
6
1
2
132.0, C
0
7.33, d (8.2)
6.80, d (8.2)
7.43, d (8.2)
7.06, d (8.2)
128.0, CH
116.2, CH
157.5, C
116.2, CH
128.0, CH
100.3, CH
73.2, CH
77.1, CH
69.7, CH
76.6, CH
7.43, d (8.2)
7.06, d (8.2)
7.45, d (8.2)
7.06, d (8.2)
0
0
0
6.80, d (8.2)
7.33, d (8.2)
5.01, d (7.8)
3.24, t (8.1)
3.40, m
7.06, d (8.2)
7.43, d (8.2)
4.91, d (7.7)
3.24, m
3.41, m
3.03, t (9.4)
3.38, m
7.06, d (8.2)
7.43, d (8.2)
4.88, d (7.4)
3.24, m
3.33, m
3.16, t (9.0)
3.26, m
7.06, d (8.2)
7.45, d (8.2)
5.01, d (7.8)
3.24, t (8.1)
3.40, m
0
00
00
00
3
4
5
00
3.01, t (9.3)
3.43, m
3.01, t (9.3)
3.43, m
00
3.60, brd (11.8)
3.63, dd (11.8, 4.4)
3.69, btd (11.8)
3.60, brd (11.8)
00
6
60.1, CH2
59.6, CH3
60.2, CH2
59.7, CH3
60.7, CH2
60.1, CH2
3.47, m
3.44, s
3.49, m
3.45, s
3.45, m
3.47, m
3.44, s
4.91, d (7.7)
3.24, m
3.41, m
3.03, t (9.4)
3.38, m
00
4 -OCH3
59.6, CH3
99.9, CH
73.4, CH
76.3, CH
79.0, CH
75.6, CH
000
1
2
3
4
000
000
000
000
5
3.63, dd (11.8, 4.4)
000
6
60.2, CH2
59.7, CH3
3.49, m
3.45, s
12.02, s
000
4
-OCH3
5-OH
4 -OH
12.01, s
9.62, s
12.13, s
12.15, s
0

Molecules 2019, 24, 1028
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2.0 mg of compound 3a was isolated from the scale-up fermentation culture of I. fumosorosea
ACCC 37814. The HRESIMS determined molecular weight of 3a was consistent with that of
(4-O-methyl)glucosylated derivative of diosmetin 3. Detailed analysis of 1D- and 2D-NMR spectra
further confirmed that 3a is the methylglucosylated derivative of diosmetin. HMBC interaction
between the anomeric proton (δH 5.17, d, J = 7.8 Hz) and the oxygen-bearing aromatic carton
at δC 146.5 (C-30) indicated that the glucose unit was attached to OH-30 of diosmetin (Scheme 3).
The β-configuration of the glucosidic bond was proved by the coupling constant of the anomeric
proton. HMBC correlation between the methoxy protons (δH 3.47, s) and the oxygen-bearing
sp3-carbon at δC 79.1 (C-400) confirmed that the methylation has been taken place on the 4-OH
of the glucose moiety. Thus, the structure of 3a was defined as 5,7-dihydroxy-40-methoxyflavone
30-O-β-D-(4-O-methyl)glucopyranoside.
Scheme 3. The microbial transformation of diosmetin
3 in the I. fumosorosea ACCC 37814 culture. Key
HMBC correlations of each product are indicated by red arrows.
2.0 mg of compound 4a was purified from the scale-up biotransformation of formononetin 4 in
the culture of I. fumosorosea ACCC 37814. The negative mode HRESIMS spectrum of 4a displayed a
[M
−
H]− ion at m/z 489.1422, indicating 4a was the methylglucosylated derivative of formononetin.
This speculation was supported by the presence of resonance signals corresponding to a formononetin
core, a D-glucose unit and a methoxy functional group in the H- and 13C-NMR spectra of 4a
HMBC interaction between the anomeric proton (δH 5.14, d, J = 7.8 Hz ) and the oxygen-bearing
aromatic carton at δC 161.3 (C-7) as well as the large coupling constant of this anomeric proton
.
1
indicated that the glucose fragment was attached to C-7 of formononetin via an O-β-glucosidic linkage.
The HMBC spectrum of 4a also displayed cross peak between the methoxy protons (δH 3.47, s) and
the oxygen-bearing sp3-carbon at δC 78.9 (C-400), confirming that the C-4 of the glucose moiety has a
methoxy substituent (Scheme 4). Hence, the structure of 4a was elucidated as 40-methoxyisoflavone
7-O-β-D-(4-O-methyl)glucopyranoside.
Scheme 4. The microbial transformation of formononetin 3 in the I. fumosorosea ACCC 37814 culture.
Key HMBC correlations of each product are indicated by red arrows.
Most of the naturally occurring flavonoids exist in the form of O-glycosides and flavone and
flavonol O-glycosides account for the largest two groups of flavonoid glycosides [2]. Theoretically
speaking, any free phenolic hydroxyl group on the flavonoid scaffold can be glycosylated except
for OH-5 which forms hydrogen bond with the C-4 carbonyl group and thus has relatively low
reactivity [
naturally occurring flavones, flavanones and isoflavones tend to be glycosylated at OH-7, while
flavonols and flavanols usually have sugar fragments attached to OH-3 and/or OH-7 [ ]. Therefore, it
is interesting that I. fumosorosea ACCC 37814 prefers to glycosylate the OH-30 or OH-40 on ring B of
8]. However, nature seems to favor some positions rather than others. The majority of
2

Molecules 2019, 24, 1028
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flavones such as luteolin and diosmetin. By contrast, the widely studied biocatalyst, B. bassiana, which
is also an entomopathogenic filamentous fungus and genetically close to I. fumosorosea, favors OH-7 as
methylglucosylation site [14].
The biosynthesis of natural flavonoid O-glycosides in plant cells are catalyzed by various
uridine diphosphate-dependent glycosyltransferases (UGTs). Over the past two decades, highly
efficient biosynthesis of flavonoid O-glycosides has been achieved by using genetically engineered
Escherichia coli strains that harbor UGTs from Arabidopsis thaliana, Oryza sativa, Medicago truncatula and
so forth [30–32]. Bacteria also utilize a wide range of UGTs to glycosylate their secondary metabolites.
E. coli strains that expressing heterologous bacterial UGTs were used to biosynthesize not only natural
flavonoid O-glucosides [33
–35] but also “unnatural” flavonoid glycosides which incorporate amino-
and deoxysugars in their structures [36
,37]. Despite the fact that fungi have long been used as
whole-cell biocatalyst for the glycosylation of a variety of natural products, only a few fungal UGTs
have been identified and functional characterized so far [9,14,38]. Very recently, Xie et al. identified a
glycotransferase-methyltransferase (GT-MT) biosynthetic module from B. bassiana which proficiently
synthesized (4-O-methyl)glucopyranosides of various phenolic substances, including benzendiol
lactones, anthraquinones and flavonoids, in Saccharomyces cerevisiae host [14]. Recent studies and our
present work demonstrated that I. fumosorosea is capable of methylglucosylating a number of flavonoid
scaffolds and prefers the free aromatic hydroxyl group on ring B as glycosylation site [11,27–29].
These results indicate that I. fumosorosea is a promising source to exploit novel fungal GT-MT module
that exhibit different chemo- and regio-selectivity than those from B. bassiana.
Many studies have proved that glycosylation significantly improves the aqueous solubility of
natural products such as flavonoids, while methylation on one of the hydroxyl groups of the sugar
moiety does not significantly alter the solubility [5,14]. However, the biological and pharmacological
significance of the 4-O-methylation of the sugar moiety is less clear. Flavonoids and their glycosides
undergo intense metabolism by both intestine epithelium and gut microbiota after they are taken into
human body, which eventually diminishes the beneficial effects of flavonoids observed in vitro [12].
A recent study has shown that attaching methyl group to the OH-4 of glucose moiety can substantially
avert the rapid de-glucosylation process of benzendiol lactone glucosides in both yeast and mammalian
cell models [14] and thus may increase the in vivo stability and bioavailability of these drug-like
compounds. Consequently, the effect of methylglucosylation of flavonoids would be an interesting
topic to further explore.
3. Conclusions
The current study describes the microbial transformation of natural phenolics using the
entomopathogenic filamentous fungus I. fumosorosea ACCC 37814. Nine out of ten substrates
were converted to their glycosides in the culture of I. fumosorosea ACCC 37814 after four days
of biotransformation. Mono-methylglucosides are the major products for all substrates but
di- methylglucosylated and mono-glucosylated products were also detected in several cases.
Four substrates representing different sub-classes of flavonoid were selected for scale-up fermentation.
As a result, six new flavonoid glycosides and two known compounds were isolated from the
scale-up cultures and their structures were unambiguously characterized. The results indicated
that I. fumosorosea ACCC 37814 prefers free aromatic hydroxyl groups on ring B of flavonoid scaffold
as glycosylation site and could be developed as regio-selective biocatalyst for the methylglucosylation
of phenolic natural products.
4. Materials and Methods
4.1. General Experimental Orocedures
All substrates were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai,
China). Samples were routinely analyzed on an Agilent 1260 Infinity II HPLC instrument (Santa Clara,

Molecules 2019, 24, 1028
9 of 13
CA, USA). The HPLC was equipped with an Agilent Eclipse Plus C18 RRHD column (1.8
µm,
2.1 mm × 50 mm) and eluted with a linear gradient of 10–95% of acetonitrile-water for 20 min, 95%
acetonitrile-water for 10 min and drop down to 10% in 5 min, then 10% acetonitrile-water for 5 min
at a flow rate of 0.5 mL/min. 1H-, 13C-, HSQC- and HMBC-NMR spectra were recorded on an
Agilent 600 DD2 spectrometer. Chemical shift values (δ) are given in parts per million (ppm) and the
coupling constants (J values) are in Hz. Chemical shifts were referenced to the residual solvent peaks
of DMSO-d6. HPLC-HRESIMS spectra were acquired on an Agilent 1290 Infinity II HPLC coupled with
an Agilent QTOF 6530 instrument operated in negative ion mode using capillary and cone voltages of
3.6 kV and 40–150 V, respectively. For accurate mass measurements the instrument was calibrated each
time using a standard calibration mix (Agilent) in the range of m/z 150–1900.
4.2. Culture and Biotransformation Procedures
The fungus Isaria fumosorosea ACCC 37814, acquired from the Agricultural Culture Collection
of China (ACCC), was maintained on potato-dextrose agar (PDA, Difco, Sparks, MD, USA) at 26 ◦C
for 7 days. Spores were collected after 10 days of growth by flooding the plates with sterile water
(0.1% Triton-X100). For small scale biotransformation experiment, the collected spores with 106/mL
concentration were inoculated into a 100 mL Erlenmeyer flask containing 50 mL potato-dextrose
◦
broth (PDB, Difco, Sparks, MD, USA) medium. The flask was placed in a rotary shaker at 25 C
and shook at 220 rpm for three days. After that, 0.5 mg of substrates (10 uL DMSO solution of
substrate at the concentration of 50 mg/mL) was added and the flask was maintained under the
same conditions for an additional four days. Two controls were incubated under the same conditions:
(1) I. fumosorosea ACCC 37814 fermentation broth with the addition of substrate-free DMSO solution
and (2) microorganism-free PDB medium with the addition of DMSO solution containing the substrate.
For large scale fermentation, a total of 3 L fermentation culture and 75 mg of substrates were used
(2.5 mg/flask) in the 250 mL flasks, each containing 100 mL of PDB and incubated under the same
conditions as described for the small scale experiment.
4.3. Extraction, Isolation and Purification of the Products
The scale-up fermentation cultures were combined and extracted with an equal volume of ethyl
acetate for three times. The extracts were pooled and dried in vacuo to give the crude extracts I
(481.6 mg/L), II (844.0 mg/L), III (868.1 mg/L) and IV (868.1 mg/L) for substrates 1–4, respectively.
Each crude extract was first subjected to silica gel column chromatography and eluted with a
gradient of dichloromethane/methanol (100:0, 98:2, 95:5, 93:7 and 90:10, v/v) to give five fractions
(Fr. A–E). These fractions were analyzed by LC-MS instrument (Agilent Technologies, Santa Clara,
CA, USA) to detect potential products. Fractions containing the target compounds were subsequently
purified by semi-preparative HPLC on an Agilent Eclipse XDB-C18 reversed-phase column (5 µm,
9.4 mm × 250 mm) using an Agilent 1260 Infinity II instrument. Specifically, Fr. D of extract I was
isolated using acetonitrile-water (25:75, v/v) as eluent at the flow rate of 2 mL/min to give compound
1a (2.3 mg, tR = 23.2 min), compound 1b (3.2 mg, tR = 27.3 min), compound 1c (6.5 mg, tR = 18.5 min)
and compound 1d (1.5 mg, tR = 13.4 min); Fr. D of extract II was isolated using acetonitrile-water
(30:70, v/v) as eluent at the flow rate of 2 mL/min to give compound 2a (2.8 mg, tR = 16.2 min) and
compound 2a (2.3 mg, tR = 14.7 min). Fr. D of extract III was isolated using acetonitrile-water (30:70,
v/v) as eluent at the flow rate of 2 mL/min to give compound 3a (2.0 mg, tR = 17.5 min). Fr. E of
extract IV was isolated using acetonitrile-water (50:50, v/v) as eluent at the flow rate of 2 mL/min to
give compound 4a (4.0 mg, tR = 8.5 min).

Molecules 2019, 24, 1028
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1
Table 2. H- (600 MHz) and 13C-NMR (150 MHz) Data of 2a, 2b, 3a and 4a in DMSO-d6.
2a
2b 3a
4a
No.
δC, Type
δ , Mult. (J in Hz)
H
δC, Type
δ , Mult. (J in Hz)
δC, Type
δ , Mult. (J in Hz)
H
δC, Type
δ , Mult. (J in Hz)
H
H
2
3
4
5
6
7
8
9
10
163.4, C
103.0, CH
181.7, C
161.4, C
98.9, CH
164.3, C
94.1, CH
157.3, C
103.6, C
121.1, C
114.4, C
145.7, C
151.4, C
116.6, CH
122.0, CH
101.5, CH
73.5, CH
75.8, CH
79.3, CH
75.7, CH
163.1, C
104.0, CH
181.7, C
161.4, C
98.9, CH
164.3, C
94.0, CH
157.3, C
103.8, C
124.7, C
113.6, C
146.9, C
148.4, C
115.8, CH
118.5, CH
100.7, CH
73.4, CH
75.8, CH
79.0, CH
75.6, CH
163.0, C
103.7, CH
181.7, C
161.4, C
99.1, CH
no show
94.2, CH
157.4, C
103.4, C
122.9, C
112.8, C
146.5, C
152.1, C
112.4, C
121.0, CH
99.4, CH
73.4, CH
76.6, CH
79.1, CH
75.6, CH
153.6, CH
123.4, C
174.6, C
127.0, CH
115.6, CH
161.3, C
103.3, CH
157.0, C
8.44, s
6.79, s
6.82, s
6.89, s
8.06, d (8.8)
7.15, dd (8.8, 1.8)
6.18, brs
6.49, brs
6.20, brs
6.50, brs
6.17, brs
6.49, brs
7.24, d (1.8)
118.5, C
124.0, C
0
1
2
3
4
5
6
1
2
0
7.74, d (2.2)
7.50, brs
7.70, d (2.0)
130.1, CH
115.6, CH
159.0, C
115.6, CH
130.1, CH
99.6, CH
73.3, CH
76.2, CH
78.9, CH
75.7, CH
7.53, d (8.4)
7.00, d (8.4)
0
0
0
6.95, d (8.5)
7.64, dd (8.5, 2.2)
4.93, d (7.8)
3.34, m
7.22, d (8.5)
7.51, brd (9.0)
4.92, d (7.8)
3.34, m
3.52, m
3.06, t (9.3)
3.46, m
7.16, d (8.6)
7.64, dd (8.6, 2.0)
5.17, d (7.8)
3.30, m
7.00, d (8.4)
7.53, d (8.4)
5.14, d (7.8)
3.30, m
3.46, m
3.06, t (9.1)
3.52, m
0
00
00
00
3
4
5
3.52, m
3.04, t (9.2)
3.46, m
3.46, m
3.05, t (9.3)
3.52, m
00
00
3.70, brs (11.4)
3.66, brs (11.4)
3.64, brs (11.0)
3.66, brs (11.0)
00
6
60.4, CH2
60.2, CH2
60.2, CH2
60.2, CH2
3.56, m
3.52, m
3.52, m
3.52, m
0
4 -OCH3
55.8, CH3
59.7, CH3
3.86, s
3.47, s
55.2, CH3
59.7, CH3
3.79, s
3.47, s
00
4 -OCH3
59.7, CH3
3.47, s
59.7, CH3
3.47, s
5-OH
12.96, s
12.90, s
12.90, s

Molecules 2019, 24, 1028
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40,5-Dihydroxyflavanone 7-O-
β
-D-(4-O-methyl)glucopyranoside (1a): 1H- and 13C-NMR data see
Table 1; (−)-HRESIMS m/z 447.1289 [M − H]− (calcd. for C22H23O10, 447.1291).
5,7-Dihydroxyflavanone 40-O- -D-(4-O-methyl)glucopyranoside (1b): 1H- and 13C-NMR data see
β
Table 1; (−)-HRESIMS m/z 447.1294 [M − H]− (calcd. for C22H23O10, 447.1291).
5,7-Dihydroxyflavanone 40-O- -D-glucopyranoside (1c): 1H- and 13C-NMR data see Table 1;
β
(−)-HRESIMS m/z 433.1122 [M − H]− (calcd. for C21H21O10, 433.1135).
5-Hydroxyflavanone 40,7-di-O- -D-(4-O-methyl)glucopyranoside (1d): H- and 13C-NMR data see
β
1
Table 1; (−)-HRESIMS m/z 623.1950 [M − H]− (calcd. for C29H35O15, 623.1976).
40,5,7-Trihydroxyflavone 30-O- -D-(4-O-methyl)glucopyranoside (2a): 1H- and 13C-NMR data see
β
Table 2; (−)-HRESIMS m/z 461.1100 [M − H]− (calcd. for C22H21O11, 461.1084).
30,5,7-Trihydroxyflavone 40-O-
β
-D-(4-O-methyl)glucopyranoside (2b): H- and 13C-NMR data see
1
Table 2; (−)-HRESIMS m/z 461.1097 [M − H]− (calcd. for C22H21O11, 461.1084).
5,7-Dihydroxy-40-methoxyflavone-30-O- -D-(4-O-methyl)glucopyranoside (3a): H- and 13C-NMR
β
1
data see Table 2; (−)-HRESIMS m/z 475.1261 [M − H]− (calcd. for C23H23O11, 475.1240).
40-methoxyisoflavone 7-O-
β
-D-(4-O-methyl)glucopyranoside (4a): H- and 13C-NMR data see Table 2;
1
(−)-HRESIMS m/z 489.1422 [M − H]− (calcd. for C24H25O11, 489.1397).
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Resources, B.D.; conceptualization, Z.W. and B.D.; formal analysis, Z.W.; investigation, F.D.
and Z.W.; methodology, F.D. and Z.W.; supervision, G.L. and B.D.; validation, F.D. and Z.W.; visualization, F.D. and
G.L.; writing—original draft, F.D. and Z.W.; writing—review & editing, Z.W. and B.D.; funding acquisition, B.D.
Funding: This research was funded by the Agricultural Science and Technology Innovation Program
(CAAS-ASTIP-2017-ICS) and Special Fund for Agroscientific Research in the Public Interest of China (201503135).
Acknowledgments: We thank Jingang Gu of Agricultural Culture Collection of China for generously providing
the fungus Isaria fumosorosea ACCC 37814. We also thank the Research Facility Center of the Biotechnology
Institute for providing analytical instruments and software.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
Pietta, P.-G. Flavonoids as antioxidants. J. Nat. Prod. 2000, 63, 1035–1042. [CrossRef] [PubMed]
Veitch, N.C.; Grayer, R.J. Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 2011, 28,
1626–1695. [CrossRef] [PubMed]
3.
4.
Xiao, J. Dietary flavonoid aglycones and their glycosides: Which show better biological significance? Crit. Rev.
Food Sci. Nutr. 2017, 57, 1874–1905. [CrossRef] [PubMed]
Yang, B.; Liu, H.L.; Yang, J.L.; Gupta, V.K.; Jiang, Y.M. New insights on bioactivities and biosynthesis of
flavonoid glycosides. Trends Food Sci. Technol. 2018, 79, 116–124. [CrossRef]
5.
6.
Chen, X. Fermenting next generation glycosylated therapeutics. ACS Chem. Biol. 2011, 6, 14–17. [CrossRef]
Losey, H.C.; Peczuh, M.W.; Chen, Z.; Eggert, U.S.; Dong, S.D.; Pelczer, I.; Kahne, D.; Walsh, C.T. Tandem action
of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: Novel glycopeptides.
Biochemistry 2001, 40, 4745–4755. [CrossRef] [PubMed]
7.
8.
9.
Fujita, H.; Ogawa, K.; Tone, H.; Iguchi, H.; Shomura, T.; Murata, S. Pharmacokinetics of doxorubicin,
(2R)-4 -O-tetrahydropyranyl-adriamycin and aclarubicin. Jpn. J. Antibiot. 1986, 39, 1321–1336. [PubMed]
0
Hofer, B. Recent developments in the enzymatic O-glycosylation of flavonoids. Appl. Microbiol. Biotechnol.
2016, 100, 4269–4281. [CrossRef]
Feng, J.; Zhang, P.; Cui, Y.; Li, K.; Qiao, X.; Zhang, Y.-T.; Li, S.-M.; Cox, R.J.; Wu, B.; Ye, M.; et al. Regio-
and stereospecific O-glycosylation of phenolic compounds catalyzed by a fungal glycosyltransferase from
Mucor hiemalis. Adv. Synth. Catal. 2017, 359, 995–1006. [CrossRef]

Molecules 2019, 24, 1028
12 of 13
10. Brglez Mojzer, E.; Knez Hrncic, M.; Skerget, M.; Knez, Z.; Bren, U. Polyphenols: Extraction methods,
antioxidative action, bioavailability and anticarcinogenic effects. Molecules 2016, 21, 901. [CrossRef]
11. Dymarska, M.; Grzeszczuk, J.; Urbaniak, M.; Janeczko, T.; Plaskowska, E.; Stepien, L.; Kostrzewa-Suslow, E.
Glycosylation of 6-methylflavone by the strain Isaria fumosorosea KCH J2. PLoS ONE 2017, 12, e0184885.
[CrossRef]
12. Hollman, P.C.; Bijsman, M.N.; van Gameren, Y.; Cnossen, E.P.; de Vries, J.H.; Katan, M.B. The sugar moiety
is a major determinant of the absorption of dietary flavonoid glycosides in man. Free Radical Res. 1999, 31,
569–573. [CrossRef]
13. Wang, W.; Sun, C.; Mao, L.; Ma, P.; Liu, F.; Yang, J.; Gao, Y. The biological activities, chemical stability,
metabolism and delivery systems of quercetin: A review. Trends Food Sci. Technol. 2016, 56, 21–38. [CrossRef]
14. Xie, L.; Zhang, L.; Wang, C.; Wang, X.; Xu, Y.-M.; Yu, H.; Wu, P.; Li, S.; Han, L.; Gunatilaka, A.A.L.; et al.
Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial
biosynthesis. Proc. Natl. Acad. Sci. USA 2018, 115, E4980–E4989. [CrossRef]
0
15. Zhan, J.; Gunatilaka, A.A.L. Selective 4 -O-methylglycosylation of the pentahydroxyflavonoid quercetin by
Beauveria bassiana ATCC 7159. Biocatal. Biotransform. 2006, 24, 396–399. [CrossRef]
0
16. Zeng, J.; Yang, N.; Li, X.M.; Shami, P.J.; Zhan, J. 4 -O-methylglycosylation of curcumin by Beauveria bassiana.
Nat. Prod. Commun. 2010, 5, 77–80.
17. Sordon, S.; Popłonski, J.; Tronina, T.; Huszcza, E. Microbial glycosylation of daidzein, genistein and biochanin
A: Two new glucosides of biochanin A. Molecules 2017, 22, 81. [CrossRef]
18. Kozlowska, E.; Dymarska, M.; Kostrzewa-Suslow, E.; Janeczko, T. Isaria fumosorosea KCH J2
entomopathogenic strain as an effective biocatalyst for steroid compound transformations. Molecules
2017, 22, 1511. [CrossRef]
19. Fujita, T.; Inoue, K.; Yamamoto, S.; Ikumoto, T.; Sasaki, S.; Toyama, R.; Chiba, K.; Hoshino, Y.; Okumoto, T.
Fungal metabolites. Part 11. A potent immunosuppressive activity found in Isaria sinclairii metabolite.
J. Antibiot. 1994, 47, 208–215. [CrossRef]
20. Sakakura, A.; Suzuki, K.; Katsuzaki, H.; Komiya, T.; Imamura, T.; Aizono, Y.; Imai, K. Hanasanagin: A
new antioxidative pseudo-di-peptide, 3,4-diguanidinobutanoyl-dopa, from the mushroom, Isaria japonica.
Tetrahedron Lett. 2005, 46, 9057–9059. [CrossRef]
21. Vining, L.C.; Taber, W.A. Isariin, a new depsipeptide from Isaria cretacea. Can. J. Chem. 1962, 40, 1579–1584.
[CrossRef]
22. Sabareesh, V.; Ranganayaki, R.S.; Raghothama, S.; Bopanna, M.P.; Balaram, H.; Srinivasan, M.C.; Balaram, P.
Identification and characterization of a library of microheterogeneous cyclohexadepsipeptides from the
fungus Isaria. J. Nat. Prod. 2007, 70, 715–729. [CrossRef] [PubMed]
23. Briggs, L.H.; Fergus, B.J.; Shannon, J.S. Chemistry of fungi-IV: Cyclodepsipeptides from a new species of
Isaria. Tetrahedron 1966, 22, 269–278. [CrossRef]
24. Baute, R.; Deffieux, G.; Merlet, D.; Baute, M.A.; Neveu, A. New insecticidal cyclodepsipeptides from the
fungus Isaria felina. I. Production, isolation and insecticidal properties of isariins b, c and d. J. Antibiot. 1981
34, 1261–1265. [CrossRef] [PubMed]
,
25. Zhang, J.; Liu, S.-S.; Yuan, W.-Y.; Wei, J.-J.; Zhao, Y.-X.; Luo, D.-Q. Carotane-type sesquiterpenes from cultures
of the insect pathogenic fungus Isaria fumosorosea. J. Asian Nat. Prod. Res. 2017, 19, 1–7. [CrossRef]
26. Asai, T.; Chung, Y.M.; Sakurai, H.; Ozeki, T.; Chang, F.R.; Yamashita, K.; Oshima, Y. Tenuipyrone, a novel
skeletal polyketide from the entomopathogenic fungus, Isaria tenuipes, cultivated in the presence of epigenetic
modifiers. Org. Lett. 2012, 14, 513–515. [CrossRef] [PubMed]
27. Dymarska, M.; Janeczko, T.; Kostrzewa-Suslow, E. Glycosylation of methoxylated flavonoids in the cultures
of Isaria fumosorosea KCH J2. Molecules 2018, 23, 2578. [CrossRef]
28. Dymarska, M.; Janeczko, T.; Kostrzewa-Suslow, E. Glycosylation of 3-hydroxyflavone, 3-methoxyflavone,
quercetin and baicalein in fungal cultures of the genus Isaria. Molecules 2018, 23, 2477. [CrossRef] [PubMed]
29. Dymarska, M.; Janeczko, T.; Kostrzewa-Suslow, E. Biotransformations of flavones and an isoflavone
(daidzein) in cultures of entomopathogenic filamentous fungi. Molecules 2018, 23, 1356. [CrossRef]
30. Willits, M.G.; Giovanni, M.; Prata, R.T.; Kramer, C.M.; De, L.V.; Steffens, J.C.; Graser, G. Bio-fermentation of
modified flavonoids: An example of in vivo diversification of secondary metabolites. Phytochemistry 2004, 5,
31–41. [CrossRef]

Molecules 2019, 24, 1028
13 of 13
31. Kim, J.H.; Shin, K.H.; Ko, J.H.; AHn, J.-H. Glucosylation of flavonols by Escherichia coli expressing
glucosyltransferase from rice (Oryza sativa). J. Biosci. Bioeng. 2006, 102, 135–137. [CrossRef]
32. He, X.-Z.; Li, W.-S.; Blount, J.W.; Dixon, R.A. Regioselective synthesis of plant (iso)flavone glycosides in
Escherichia coli. Appl. Microbiol. Biotechnol. 2008, 90, 253–260. [CrossRef]
33. Ko, J.H.; Kim, B.G.; Ahn, J.-H. Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus.
FEMS Microbiol. Lett. 2006, 258, 263–268.
34. Kim, J.H.; Kim, B.G.; Kim, J.A.; Park, Y.; Lee, Y.J.; Lim, Y.; Ahn, J.-H. Glucosylation of flavonoids with E. coli
expressing glycosyltransferase from Xanthomonas campestris. J. Microbiol. Biotechnol. 2007, 17, 539–542.
35. Ahn, B.C.; Kim, B.G.; Jeon, Y.M.; Lee, E.J.; Lim, Y.; Ahn, J.-H. Formation of flavone di-O-glucosides using a
glycosyltransferase from Bacillus cereus. J. Microbiol. Biotechnol. 2009, 19, 387–390. [CrossRef]
36. Yoon, J.-A.; Kim, B.-G.; Lee, W.J.; Lim, Y.; Chong, Y.; Ahn, J.-H. Production of a novel quercetin glycoside
through metabolic engineering of Escherichia coli. Appl. Microbiol. Biotechnol. 2012, 78, 4256–4262. [CrossRef]
37. Kim, B.G.; Yang, S.M.; Kim, S.Y.; Cha, M.N.; Ahn, J.-H. Biosynthesis and production of glycosylated
flavonoids in Escherichia coli: Current state and perspectives. Appl. Microbiol. Biotechnol. 2015, 99, 2979–2988.
[CrossRef]
38. Xie, K.; Dou, X.; Chen, R.; Chen, D.; Fang, C.; Xiao, Z.; Dai, J. Two novel fungal phenolic UDP
glycosyltransferases from Absidia coerulea and Rhizopus japonicus. Appl. Environ. Microbiol. 2017, 83, e03103-16.
[CrossRef]
Sample Availability: Samples of the compounds 1–10, 1a–1d, 2a, 2b, 3a and 4a are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).