A new O-cinnamoyl threonine derivative from gene adpA overexpression strain Streptomyces sp. HS-NF-1222A

Article abstract of DOI:10.1080/14786419.2019.1573234

A new O-cinnamoyl threonine derivative, O-(2-(3-methyloxiranyl) cinnamoyl) threonine (1), was isolated from the gene adpA overexpression strain Streptomyces sp. HS-NF-1222A. The structure of 1 was determined based on HRESIMS and extensive NMR analysis.

Full text of DOI:10.1080/14786419.2019.1573234

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new O-cinnamoyl threonine derivative from gene
adpA overexpression strain Streptomyces sp. HS-
NF-1222A
Huan Qi, Zheng Ma, Zheng-Lian Xue, Zhen Yu, Qing-Yu Xu, Hui Zhang, Xiao-
Ping Yu & Ji-Dong Wang
To cite this article: Huan Qi, Zheng Ma, Zheng-Lian Xue, Zhen Yu, Qing-Yu Xu, Hui Zhang,
Xiao-Ping Yu & Ji-Dong Wang (2019): A new O-cinnamoyl threonine derivative from gene
adpA overexpression strain Streptomyces sp. HS-NF-1222A, Natural Product Research, DOI:
10.1080/14786419.2019.1573234
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1573234
SHORT COMMUNICATION
A new O-cinnamoyl threonine derivative from gene adpA
overexpression strain Streptomyces sp. HS-NF-1222A
a,c
a
b
c
c
c
Huan Qi , Zheng Ma , Zheng-Lian Xue , Zhen Yu , Qing-Yu Xu , Hui Zhang ,
a
b,c
Xiao-Ping Yu and Ji-Dong Wang
a
Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine College of Life
b
Sciences, China Jiliang University, Hangzhou, China; College of Biochemical Engineering, Anhui
Polytechnic University, Wuhu, China; Zhejiang Key Laboratory of Antifungal Drugs, Zhejiang Hisun
c
Pharmaceutical Co., Ltd., Taizhou, China
ABSTRACT
ARTICLE HISTORY
A new O-cinnamoyl threonine derivative, O-(2-(3-methyloxiranyl)
cinnamoyl) threonine (1), was isolated from the gene adpA over-
expression strain Streptomyces sp. HS-NF-1222A. The structure of
Received 30 October 2018
Accepted 18 January 2019
KEYWORDS
1
was determined based on HRESIMS and extensive NMR analysis.
Streptomyces; adpA
overexpression;
O-cinnamoyl threonine
derivative; structure
elucidation
1. Introduction
Microorganisms have provided abundant sources of natural products which have been
developed as commercial products for human medicine, animal health, and plant crop
protection (Leonard and Richard 2016). Nevertheless, the growing number of
sequenced microbial genomes has revealed a remarkably large number of natural
product biosynthetic clusters for which the products are still unknown (Chiang et al.
2011). Investigators have uncovered numerous cryptic secondary metabolite biosyn-
thetic pathways not associated with the production of known metabolites, suggesting
that the majority of these gene clusters are silent or are expressed below detectable
levels (Luisa et al. 2011; Gregory 2008). To activate such gene clusters, many strategies
have been developed (Jin et al. 2006; Li et al. 2014, 2017). It has been reported that
manipulation of regulatory genes could activate the expression of silent gene clusters,
CONTACT Xiao-Ping Yu
yuxiaoping19630306@163.comJi-Dong Wang
jdwang@hisunpharm.com
Supplemental data for this article is available online at at https://doi.org/10.1080/14786419.2019.1573234
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
H. QI ET AL.
such as overexpression of global positive regulator gene adpA (Liu et al. 2013; Yasuo
et al. 1999, 2005). During the course of searching for novel microbe-derived bioactive
secondary metabolites, we obtained a hygromycin A producing strain Streptomyces sp.
HS-NF-1222. In order to tap the potential of this strain, we tried to activate the silent
gene clusters in Streptomyces sp. HS-NF-1222. As a result, while we over-expressed the
gene adpA in Streptomyces sp. HS-NF-1222 by an integrative expression vector, a
recombination strain HS-NF-1222A with an extra peak compared to parental strain in
the HPLC chromatogram of the fermentation product was obtained. Further chemical
investigation led to the isolation of a new O-cinnamoyl threonine derivative 1 corre-
sponding to the extra peak. In this paper, we describe the fermentation, isolation and
structure elucidation of 1.
2. Results and discussion
2.1. Structure determination
Compound 1 was isolated as white amorphous powder. The molecular formula of 1
þ
was determined to be C H NO on the basis of HRESIMS at m/z 306.1337 [M þ H]
16
19
5
(
calcd for C H NO , 306.1336) in combination with NMR data (Table S1), suggesting
16 20 5
eight degrees of unsaturation. The IR spectrum displayed absorption band for carbonyl
ꢀ
1
1
group (1680 cm ). The H NMR spectrum (Table S1) of 1 showed the signals of a 1,2-
disubstituted benzene ring at d 7.24 (1H, d, J = 7.7 Hz), 7.29 (1H, dd, J = 7.6, 8.0 Hz),
H
7
.31 (1H, dd, J = 7.7, 8.0 Hz), 7.64 (1H, d, J = 7.6 Hz), a trans double-bond at d 6.79
H
(
(
(
1H, d, J = 15.5 Hz), 7.95 (1H, d, J = 15.5 Hz), two aliphatic doublet methyls at d 1.25
H
3
3H, d, J = 5.8 Hz), 1.46 (3H, d, J = 4.8 Hz), four sp methine proton signals at d 2.91
H
1
3
1H, m), 3.86 (1H, s), 4.35 (1H, br s), 4.56 (1H, br s). The C NMR spectrum along with
2
DEPT135 revealed the presence of two carbonyl carbons at d 176.0, 166.8, six sp
methines at d 122.9, 124.8, 125.9, 127.7, 129.4, 136.9, two sp quaternary carbons at
d 133.5, 136.8, four sp methines at d 56.8, 58.1, 59.1, 67.9 and two methyls at d
1
subsequently accomplished by the H- H COSY, HMQC and HMBC spectra. The H- H
COSY correlation (Figure S2) of H-7/H-8 and the observed HMBC correlations (Figure
S2) from H-8 to C-1 and from H-7 to C-9 indicated the presence of a cinnamic acid
C
2
C
3
C
C
C
1
13
6.6, 18.5. The complete assignment of all the H and C NMR spectral data of 1 was
1
1
1
1
0
0
0
1
1
structure unit. The correlations of H-2 /H-3 /H-4 in the H- H COSY spectrum and the
0
0
0
0
long-range correlations from H-4 to C-2 and from H-3 to C-1 in conjunction with the
1
13
0
1
1
H and C NMR data of C-2 revealed the presence of a threonine moiety. The H- H
1
13
COSY correlations of H-10/H-11/H-12 and the H and C NMR chemical shifts of C-10
and C-11 supported a 3-methyl oxiran structural unit in 1. The connectivity of the 3-
methyl oxiran structural unit with C-2 was evidenced by the HMBC correlation from H-
0
1
0 to C-2. Finally, the connection of C-9 to C-3 via an oxygen atom was confirmed by
0
the HMBC correlation between H-3 and C-9. Considered the formula C H NO of 1,
16
19
5
the gross structure of 1 was established as shown in Figure 1. Previously, a synthetic
analogue, O-cinnamoyl threonine, was reported as a deodorant composition (Laney
1998). Difference between 1 and O-cinnamoyl threonine was that a 3-methyl oxiran
moiety was present in 1. The configuration of threonine residue in 1 was investigated
using the advanced Marfey’s method after acidic hydrolysis (Peter 1984; Soumini et al.

NATURAL PRODUCT RESEARCH
3
4
'
O
COOH
6
1
7
3'
1'
9
5
4
O
2
'
8
1
1
NH2
2
1
2
3
1
0
O
Figure 1. The chemical structure of compound 1.
2
016) and the configuration of threonine was determined as L. The configurations at
C-10 and C-11 remained unassigned.
2.2. Acaricidal activity
The larvicidal capacity of compound 1 against Tetranychus cinnabarinus reared in the
laboratory was evaluated. The results showed that compound 1 exhibited lethality
against female Tetranychus cinnabarinus with LC50 value of 48.5 mg/L.
3. Experimental
3.1. Organism and fermentation
The parental strain Streptomyces sp. HS-NF-1222 was isolated from soil sample col-
lected from the Jiujiang, Jiangxi province, China. The strain was identified as the genus
Streptomyces because its 16s rDNA sequence (accession No.: MH727816) exhibited a
high sequence similarity of 99% with that of Streptomyces endus strain NRRL 2339
(accession No.: NR_043379.1). The strain was grown and maintained on GYM agar
plate containing malt extract 1%, yeast extract 0.4%, glucose 0.4%, and agar 2.0% at
pH 7.0. The global positive regulator gene adpA was generated from Streptomyces coe-
licolor. A3(2) by PCR and harbored by an integrative expression vector (Figure S1).
Escherichia coli DH5a was used for cloning purpose, and E. coli ET12567 (pUZ8002)
was used for conjugation. Both of the E. coli strains were cultured in LB medium. After
the vector transformed into the parental strain, the recombination strains obtained
and each recombination strain was fermented by shake flask with 25 mL medium con-
sisted of malt extract 1%, yeast extract 0.4%, glucose 0.4%, soluble starch 4.0% and
ꢁ
CaCO 0.2% at pH 7.2 on a rotary shaker (250 rpm, 28 C) for 7 days. The metabolic
3
profiles of the fermentation product were assayed by HPLC. Then, the chromatogram
of a recombination strain exhibited an extra peak (Figure S3) which absent in the
chromatogram of the parental strain and the recombination strain was named
Streptomyces sp. HS-NF-1222A.
The fermentation for the Streptomyces sp. HS-NF-1222A was carried out in a 50 L
fermenter containing 30 L of production medium consisted of malt extract 1%, yeast
extract 0.4%, glucose 0.4%, soluble starch 4.0% and CaCO 0.2% at pH 7.2. The seed
3
medium consisted of glucose 0.4%, malt extract 1%, yeast extract 0.4% and CaCO3
0
.2% at pH 7.2. A total of 1 liter seed was incubated by 1 liter flasks containing 250

4
H. QI ET AL.
ꢁ
mL of the seed medium at 28 C for 24 h, shaken at 250 rpm. The fermentation was
ꢁ
carried out at 28 C for 7 days, stirred at 200 r.p.m. with the aeration rate of 1000 L of
air per hour, tank pressure control at 0.05 MPa.
3.3. Extraction and isolation
The completed fermentation broth (30 L) was separated into supernatant and mycelia
by centrifugation. The mycelia was washed with water (3 L) and subsequently
extracted with MeOH (3 L). The supernatant and the wash water were subjected to
a Diaion HP-20 resin column eluting with 95% EtOH. The MeOH extract and the EtOH
eluents were combined and evaporated in vacuo to yield the crude extract. The mix-
ture was chromatographed on a silica gel column and successively eluted with a step-
wise gradient of CHCl /MeOH (95:5-40:60, vol/vol) to give six fractions (Fr.1-Fr.6) based
3
on the TLC profiles. TLC was performed on silica-gel plates with a solvent system of
CHCl /MeOH (9:1) and the developed TLC plates were observed under a UV lamp at
3
254 nm or by heating after spraying with sulfuric acid-ethanol, 5:95 (vol/vol). The Fr.4
was subjected to a Sephadex LH-20 column eluted with CHCl /MeOH (1:1, vol/vol) and
3
detected by TLC to give three subtractions (Fr.4-1–Fr.4-3). The Fr.4-2 was further
purified by semi-preparative HPLC (Agilent 1100, Zorbax SB-C18, 5 lm, 250 ꢂ 9.4 mm
ꢀ
1
inner diameter; 1.5 mL min ; 220 nm; Agilent, Palo Alto, CA, USA) eluting with
CH CN/H O (13:87, vol/vol) to give compound 1 (t 18.6 min, 35 mg).
3
2
R
2
5
Compound 1. White amorphous powder; [a] -87 (c 0.05, EtOH); UV (EtOH) k
nm
D
max
(
1
log e): 221 (4.51), 277 (4.57) nm; IR (KBr) v : 3339, 2972, 2922, 1680, 1612, 1421,
max
ꢀ
1
1
13
218, 1085, 1048, 976, 878, 765 cm ; H and C NMR spectral data see Table S1;
þ
HRESIMS: m/z 306.1337 [M þ H] (calcd for 306.1336).
3.4. Acid hydrolysis and Marfey’s method
ꢁ
Compound 1 (1 mg) in 2 M HCl (1 mL) was heated at 100 C in a sealed vial for 2 ꢃ 3
h, after which the hydrolysate was concentrated to dryness under a stream of dry N .
2
The hydrolysate was then treated with 1 M NaHCO (1 mL) and -FDAA (10% solution
3
D
ꢁ
in acetone, 2 mL) at 40 C for 1 h, after which the reaction was neutralized with 1 M
HCl, filtered (0.45 lm PTFE) prior to HPLC analysis. An aliquot (5 lL) of the analyte
ꢁ
was injected into an Agilent Eclipse XDB-C18 column, 5 lm, 9.4 ꢂ 250 mm, 30 C,
with a 1 mL/min, 30 min linear gradient elution from 30% to 70% MeCN/H O with
2
0
.1% trifluoroacetic acid in H O. The threonine content in analytes was assessed by UV
2
(340 nm), with comparison to authentic standard.
3.5. Acaricidal activity test
The acaricidal capacity of compound 1 against Tetranychus cinnabarinus reared in the
laboratory was evaluated according to our reported paper (Huang et al. 2015).

NATURAL PRODUCT RESEARCH
5
4. Conclusion
In this study, a new O-cinnamoyl threonine derivative, O-(2-(3-methyloxiranyl) cinna-
moyl) threonine (1), was obtained from the fermentation broth of the gene adpA over-
expression strain Streptomyces sp. HS-NF-1222A. To the best of our knowledge,
compound 1 represent a new kind of natural O-cinnamoyl threonine derivatives and
the study may provide a viable option for screening novel secondary metabolites from
known antibiotic-producing streptomycetes. In addition, studies on the O-cinnamoyl
threonine derivative with diverse activities are currently underway.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China (31772213),
excellent youth fund of Zhejiang province, China (LR17C140002).
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