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Current Pharmaceutical Analysis, 2020, 16, 000-000 1

RESEARCH ARTICLE

Metabolite Fingerprinting of Novel Streptomyces UK-238 from the Hima-

layan Forest

Nidhi Srivastava1 and Indira P. Sarethy1,*

1
Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector-62, Noida-201309, India

Abstract: Aims: Characterization of antimicrobial metabolites of novel Streptomyces sp. UK-238.

ARTICLE HISTORY
Received: September 03, 2019
Revised: November 11, 2019
Accepted: January 20, 2020
DOI:
10.2174/1573412916666200206160836
Keywords: Multidrug resistant, natural products, forest, Streptomyces, antimicrobial, metabolite fingerprinting.
Background: Novel antimicrobial drug discovery is urgently needed due to emerging multi antimicro-
bial drug resistance among pathogens. Since many years, natural products have provided the basic
skeletons for many therapeutic compounds including antibiotics. Bioprospection of un/under explored
habitats and focussing on selective isolation of actinobacteria as a major reservoir of bio and chemodi-
versity has yielded good results.
Objective: The main objectives of the study were the identification of UK-238 by 16S rDNA sequenc-
ing and antimicrobial metabolite fingerprinting of culture extracts.
Method: In the present study, a promising isolate, UK-238, has been screened for antimicrobial activity
and metabolite fingerprinting from the Himalayan Thano Reserve forest. It was identified by 16S
rDNA sequencing. Ethyl acetate extract was partially purified by column chromatography. The pooled
active fractions were fingerprinted by GC-MS and compounds were tentatively identified by collated
data analysis based on Similarity Index, observed Retention Index from Databases and calculated Re-
tention Index.
Results: UK-238 was identified as Streptomyces sp. with 98.4% similarity to S. niveiscabiei. It exhibit-
ed broad-spectrum antibacterial and antifungal activity. The GC-MS analysis of active fractions of
ethyl acetate extract showed the presence of eighteen novel compounds, some with antimicrobial activ-
ity, belonging to four major categories- alcohols, alkaloids, esters and peptide.
Conclusion: The study confirms that bioprospection of underexplored habitats can elaborate novel bio
and chemodiversity.

1. INTRODUCTION isolation of novel actinomycetes has been found to be suc-

Multidrug-resistant pathogens threaten global health and
have become a cause for concern due to the lack of effective
antimicrobial drugs. New drug discovery is urgently needed
to combat such pathogens. For many years, bioactive com-
pounds from natural sources have served to fulfill the need
as therapeutic agents due to their vast structural diversity.
Microorganisms, especially actinomycetes, have been major
reservoirs of natural products [1], with more than five thou-
sand compounds reported from them, accounting for 90% of
commercial antibiotics [2]. The search for new bioactive
compounds from known sources may lead to the rediscovery
of known compounds [3]. The combined approach of bio-
prospection of underexplored habitats, focusing on selective
*Address correspondence to this author at Department of Biotechnology,
Jaypee Institute of Information Technology, A-10, Sector-62, Noida-
201309, India; Tel: +91-120-2594202; Fax: +91-120-2400986;
E-mail: indirap.sarethy@jiit.ac.in, indira.sarethy@gmail.com
cessful in elaborating new compounds.
Actinobacteria from un/underexplored habitats harbour
diverse novel antimicrobial compounds as also substantiated
by other studies [4-6]. Forests comprise one such largely
unexplored areas. Many studies based on novel actinobacte-
rial diversity have been reported from various forests, for
instance, Streptomyces colonosanans MUSC 93JT [7], S.
monashensis [8] from mangrove forest of Malaysia and S.
gilvifuscus sp. from Pyeongchang-gun forest, Korea [9].
The Himalayas in India are a well-documented biodiver-
sity hot spot. Many studies have been reported for novel ac-
tinobacterial diversity and antimicrobial compounds [4, 10,
11] from various regions of the Himalayas. This is the first
report where the subtropical Thano Reserve forest, an unex-
plored component of the Indian Himalayas has been investi-
gated during a screening program for actinobacteria. A
promising Streptomyces isolate (UK-238) was obtained

1573-4129/20 $65.00+.00 © 2020 Bentham Science Publishers

2 Current Pharmaceutical Analysis, 2020, Vol. 16, No. 00 Srivastava and Sarethy

exhibiting broad-spectrum antibacterial and antifungal activi- 3. RESULTS AND DISCUSSION

ty in preliminary screening assays. The study was further
The isolate UK-238 was obtained on GYE media and
expanded to include the following objectives (i) identifica-
routinely maintained on BM. UK-238 showed broad spec-
tion of UK-238 by 16S rDNA sequencing (ii) metabolite
trum antibacterial and antifungal activity, showing zones of
fingerprinting of culture extracts.
inhibition against all the target organisms (Table. 1). 16S
2. MATERIALS AND METHODS rDNA sequencing of UK-238 showed 98.4% similarity to S.
niveiscabiei with 20 nucleotide differences in 1339 sites
Isolate UK-238 was isolated from the Thano Reserve (Fig. 1). The aerial and substrate mycelia of isolate UK-238
forest (30.12 °N; 78.10 °E) as colonies on Glucose Yeast were light brown and dark brown respectively with crystal-
Extract Agar media supplemented with 20 µg ml-1 Rifampic- line texture. The isolate secreted light brown diffusible as
in. It was found to be Gram-positive filamentous actinobac- well as light brown melanin pigments. Studies have shown
teria and was routinely maintained on Bennett’s medium that the closely related S. niveiscabiei was obtained from
[12] at 30 °C. UK-238 was checked for antimicrobial activity raised corky lesions on potato (from Korea) with white aerial
against Gram positive [Bacillus subtilis (MTCC-121), Mi- mycelium and with no diffusible or melanin pigment produc-
crococcus luteus (MTCC-106), Staphylococcus epidermidis tion [19]. Low similarity percentage of the 16S rDNA se-
(MTCC-435) and Brevibacterium linens (MTCC-268)] and quence (98.4%) as against 98.6% permitted variation for
Gram negative bacteria [Escherichia coli (MTCC-1679) and species delineation [20], in association with phenotypic dif-
Pseudomonas fluorescens (MTCC-2421)] and a fungus ferences in colony suggests that UK-238 is a novel taxon in
[Saccharomyces cereviseae (MTCC-1512)] using agar plugs the Streptomyces lineage.
of the isolate [13].
The bacterial DNA genome of isolate UK-238 was ex-
tracted [14]. Agarose gel electrophoresis and spectrophotom-
eter (NanoDrop1000-Thermo-Scientific) was used to check
the quality of DNA. To amplify the 16S rDNA sequence,
universal primers 8F (5′-AGA GTT TGA TCC TGG CTC
AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT TC-
3′) were used with the PCR conditions as per Ibeyaima et al.
2017 [6]. Exosap-IT was used to purify PCR product. The
same primers (8F and 1492R) were used to sequence the
amplified pure product (Applied Biosystems 3130 Genetic
Analyzer). By aligning sequences and deleting overlaps,
consensus sequence was obtained and quality-checked using
DECIPHER. Subsequently, the EzTaxon server (http://eztax
on-e.ezbio cloud.net) was used to align with sequences of
representative type strains [15]. Jukes and Cantor (1969)
method was used to compute evolutionary distances and the
bootstrap consensus tree inferred from 1000 replicates was
evaluated [16]. The phylogenetic tree was used to construct
using MEGA 6.0 software based on the neighbor-joining tree
algorithm [17]. The nearly complete 16S rDNA consensus
sequences were deposited in the GenBank database with the
following accession number: MH605520.
Based on the low similarity percentage of isolate UK-238 Fig. (1). Neighbour-joining phylogenetic tree based on 16S rDNA
with nearest matching strains (98.4%) and presuming taxo- sequence showing relationship between isolates UK-238 with relat-
nomic novelty, it was further selected for metabolite profil- ed type strains of Streptomyces sp. Bootstrap values at the nodes
ing. UK-238 was grown in 200 ml Bennett’s broth for seven indicate collated values based on 1000 resampled datasets; only
days at 250 rpm centrifugal force at 30 °C. The culture was values above >50% are given. Bar, 0.005 indicates substitutions per
then centrifuged, and the supernatant extracted with equal nucleotide.
volumes of ethyl acetate, butanol, hexane, dichloromethane
and petroleum ether in separate experiments [4]. The aque-
ous and organic solvent extracts were checked for antimicro- The EA extract exhibited maximum zones of inhibition
bial activity against same panel of target organisms [13]. The against all target organisms (Table 1) and hence it was used
ethyl acetate extract exhibited the best antimicrobial activity for further metabolite fingerprinting studies after partial puri-
and hence was subsequently purified by column chromatog- fication by column chromatography.
raphy. The fractions that showed antimicrobial activity The GC-MS fingerprint of pooled fractions exhibiting an-
against all the target organisms were pooled and analysed by timicrobial activity showed a total of eighteen metabolites
GC-MS (QP2010Ultra-SHIMADZU) following the condi- belonging to four categories - alcohols, alkaloids, esters and
tions described in Srivastava et al. (2019) [4]. The GC-MS peptide (Table 2). The obtained compounds were tentatively
data was analysed as per Babushok et al. (2011) [18]. identified as per the NIST11, WILEY8 libraries, PubChem
Metabolite Fingerprinting of Novel Streptomyces UK-238 Current Pharmaceutical Analysis, 2020, Vol. 16, No. 00 3

Table 1. Antimicrobial activity of agar plugs/aqueous and organic solvent extracts of UK-238.

Zones of inhibition

(cm)
Agar plugs/aqueous/
organic solvent extracts Mean ± SD

B. subtilis S. epidermidis B. linens M. luteus P. fluorescens E. coli S. cereviseae

Agar plugs 2.4±0.12 2.2±0.04 2.0±0.06 3.0±0.12 2.8±0.04 1.76±0.04 3.1±0.16

Aqueous 0.9±0.04 1.3±0.08 0.8±0.12 1.1 ± 0.16 1.2 ± 0.96 0.7±0.08 1.0±0.08

Ethyl acetate 1.6±0.08 1.7±0.12 1.2±0.08 2.6±0.12 2.45 ± 0.12 1.3±0.04 2.6±0.06

Butanol 1.2±0.04 0 0 1.4±0.08 0.9±0.08 0 1.2±0.04

Hexane 0 0 0 0 0 0 0

Dichloromethane 0 0 0 0 0 0 0

Petroleum ether 0 0 0 0 0 0 0

Table 2. Compounds/metabolites and their characteristics, obtained by GC-MS after column chromatography from ethyl acetate
extract of UK-238.

MS data
Similarity Chemical Ab- Retention Calculated
(Fragment information) Retention index
Chemical stracts Service indices from retention indi-
Name of compounds Time (RT) (CAS) databases ces
groups (m/z) SI
min
(%) no. (RI*) (RI$)
Mass peak Base Peak

Eucalyptol 60 43.05 6.223 96 470-82-6 1001-1243 887.8362

Fenchol 56 81.10 7.797 95 1632-73-1 ND 934.5193

Alcohols
2,3,4,5-Tetramethylcyclopent-2-en-1-
72 125.10 10.847 75 82061-20-9 ND 1014.299
ol

n-Pentadecanol 97 83.10 16.633 88 629-76-5 1765-2287 1141.092

2-Heptadecanol Acetate 98 43.05 19.89 92 56599-51-0 ND 1209.563

Benzene,1 methyl-4-(1-methylethyl) 53 119.15 6.073 87 99-87-6 1001-1288 881.8145

Cyclohexene-1-menthol, alpha, alpha,

57 59.05 9.057 91 98-55-5 1198,1209 968.7398
4-trimethyl
Alkaloids
Pyrrolo[1,2-a]Pyrazine-1,4-dione,
65 83.10 16.57 67 19179-12-5 1795 1139.769
hexahydro-

Pyrrolo[1,2-a]Pyrazine-1,4-dione,
87 70.10 17.053 83 5654-86-4 1908, 2870 1149.916
hexahydro-3-(2-methylpropyl

2,5-Piperazinedione 3, 6-bis(2-
141 170.15 20.553 75 14474-78-3 ND 1223.559
methylpropyl)
(Table 2) contd…

 
4 Current Pharmaceutical Analysis, 2020, Vol. 16, No. 00 Srivastava and Sarethy

MS data
Similarity Chemical Ab- Retention Calculated
(Fragment information) Retention index
Chemical stracts Service indices from retention indi-
Name of compounds Time (RT) (CAS) databases ces
groups (m/z) SI
min
(%) no. (RI*) (RI$)
Mass peak Base Peak

Eicosenoic acid, methyl ester 105 43 14.793 76 2390-09-2 2278-2309 1102.437

Methyl tetradecanoate 106 74.05 15.88 94 124-10-7 1705-2021 1125.273

1,2-Benzenedicarboxylic acid, butyl

130 149.10 18.357 85 84-78-6 2317 1177.311
octyl ester

Hexadecanoic acid ethyl ester 119 88.05 18.677 90 628-97-7 1968-2274 1184.034
Ester
Heptadecanoic acid ethyl ester 128 88.10 22.46 89 56599-51-0 ND 1263.817

Hexadecanoic acid, 2-hydroxy-1-

126 43.05 24.033 92 23470-00-0 2498, 2519 1297.023
(hydroxymethyl) ethyl ester

Dodecanoic acid, 1,2,3-propanetriyl

186 57.10 27.41 84 538-24-9 ND 1370.81
ester

Peptides dl-Alanyl-1-leucine 75 44.05 16.203 90 1638-60-4 ND 1132.059

*Abbreviation: ND; Not Determined.

Fig. (2). Total Ion Chromatogram of the metabolites obtained from ethyl acetate extract of UK-238. (A higher resolution / colour version of
this figure is available in the electronic copy of the article).
Metabolite Fingerprinting of Novel Streptomyces UK-238
and ChemSpider databases. Total Ion Chromatogram of the
metabolites has been shown in Fig. (2).
A standard approach to deduce the identity of the com-
pounds obtained by GC-MS has been described by Babushok
et al. (2011) using the Similarity index (SI), and reported
Kovat’s Retention Index (RI*) from databases with the ob-
served Retention Index (RI$) in cases where reference com-
pounds are not available [18]. Data analysis was started by
analyzing the SI first. Maximum SI of 96% was obtained for
Eucalyptol. The RI* of Eucalyptol form databases (1001-
1243) varied from that of RI$ (887.8362). Similarly, SI of
95% and RI$ of 934.5193 was of Fenchol and could not be
matched with RI* as it is not mentioned in the databases. SI
of 94% was obtained for Methyl Tetradecanoate with RI*
1705-2021 and RI$ 1125.273. The compounds Hexadecanoic
acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester, and 2-
Heptadecanol acetate showed SI of 92%. For Hexadecanoic
acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester, RI* of 2498
and 2519, and RI$ of 1297.023 were obtained while for 2-
Heptadecanol acetate, RI$ was 1209.563. However, its RI*
has not been reported. Similarly, SI of 91% was obtained for
Cyclohexene-1-Menthol, Alpha, Alpha, 4-Trimethyl with
RI* 1198 and 1209, and RI$ 968.7398. The compounds Hex-
adecanoic acid ethyl ester and dl-Alanyl-1-leucine showed SI
of 90%. For Hexadecanoic acid ethyl ester, RI* of 1968-
2274 and RI$ of 1184.034 were obtained. For dl-Alanyl-1-
leucine RI$ of 1132.059 was obtained but RI$ has not been
reported. Other compounds showed lower SI values between
75%-89% with widely varying RI* and RI$. Overall, the low
SI (75% to 96%) and wide variation in the RI* and RI$ val-
ues affirm the novelty of all the compounds. Hence, we can
conclude that the obtained compounds are different from
those documented in the databases and are novel.
Amongst the obtained compounds (as identified by the
databases), many have been reported for antimicrobial activi-
ty. For instance, Eucalyptol from Eucalyptus intertexta and
E. largiflorens [21], Fenchol from Zanthoxylum alatum [22],
Pyrrolo[1,2-a]Pyrazine-1,4-dione, hexahydro- from Bacillus
tequilensis [23], Pyrrolo[1,2-a]Pyrazine-1,4-dione, hexahy-
dro-3-(2-methylpropyl) from Streptomyces sp. VITMK1
[24], Benzene,1 methyl-4-(1-methylethyl) from plant ex-
tracts [25], Cyclohexene-1-menthol, alpha, alpha, 4-trimethyl
(α-terpineol) from Cinnamomum longepaniculatum leaf es-
sential oil [26]. However, other compounds have not been
directly reported for any antimicrobial activity. From the
obtained compounds, some have been reported from plant
and microbial sources. For Instance, 2,3,4,5-
Tetramethylcyclopent-2-en-1-ol has been reported from
Tasmannia lanceolata [27], 1,2-Benzenedicarboxylic acid,
butyl octyl ester from essential oils of Hydnora africana
[28], Hexadecanoic acid ethyl ester and Dodecanoic acid,
1,2,3-Propanetriyl ester from Pistia stratiotes [29].
CONCLUSION
Molecular and morphological data support the taxonomic
novelty of UK-238. Eighteen compounds, some with antimi-
crobial activity, were tentatively identified by GC-MS analy-
sis of ethyl acetate extract of UK-238. However, since the
compounds obtained in the present study do not match those
of the databases, they are presumed novel. We can conclude
Current Pharmaceutical Analysis, 2020, Vol. 16, No. 00 5
that understudied habitats can elaborate novel chemo and
biodiversity. A combination of analysing a novel taxonomic
isolate with the production of compounds can result in novel
bioactive compounds.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
Not applicable.
FUNDING
None.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
The authors are thankful to the Jaypee Institute of Infor-
mation Technology, Noida, for providing the necessary facil-
ities. Nidhi Srivastava thanks the Indian Council of Medical
Research, Government of India, for providing ICMR fellow-
ship [3/1/3JRF-2013/HRD-136 (30690)]. We acknowledge,
Advanced Institute of Research Facility (AIRF), Jawaharlal
Nehru University, New Delhi, for GC-MS analyses.
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