Genome based analysis for the bioactive potential of streptomyces spectabilis strain yim 121038

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

FACULTY OF BIOTECHNOLOGY
------------------------------------------------

GRADUATION THESIS
TITLE:
“GENOME-BASED ANALYSIS FOR THE BIOACTIVE
POTENTIAL OF STREPTOMYCES SPECTABILIS STRAIN
YIM 121038”

Hanoi, 3/2021


VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

FACULTY OF BIOTECHNOLOGY
------------------------------------------------

GRADUATION THESIS
TITLE:

“GENOME-BASED ANALYSIS FOR THE BIOACTIVE
POTENTIAL OF STREPTOMYCES SPECTABILIS STRAIN
YIM 121038”

Student’s name

: Vu Thi Thuy Tien

ID

: 610780

Class

: K61-CNSHE

Supervisors

: Dinh Truong Son, Ph.D.

Hanoi, 3/ 2021


COMMITMENT
I assure that the whole research process was performed by myself under the
scientific guidance of Ph.D. Dinh Truong Son.
I assure that all research contents, results and information in my thesis is
completely honest and unpublished.
Hanoi, February 24th 2021
Student

Vu Thi Thuy Tien

i


ACKNOWLEDGEMENTS
First and foremost, I would like to express deeply my gratitude to my supervisors
Ph.D. Dinh Truong Son who have supported me complete my thesis with their

patience and knowledge. Moreover, their guidance helped me in the period of writing
of this thesis.
I would like to thank all my teachers in Faculty of Biotechnology, especially
Department of Plant Biotechnology. They provided useful scientific knowledge as
well as technique for me. This is one of the most important things to help me to do
successful my thesis.
I would like to thank Department of Plant Biotechnology where supported
financial for my thesis.
I thank my lab-mate for her meaning contribution. I will appreciate the time
when we were working together.
Last but not least, I would specifically like to thank my family, especially my
parents who have loved me, cared for me, trusted me and given the best study
condition.
Hanoi, February 24th 2021
Student

Vu Thi Thuy Tien

ii


CONTENTS
COMMITMENT .............................................................................................................. i
ACKNOWLEDGEMENTS ............................................................................................ii
CONTENTS .................................................................................................................. iii
ABBREVIATION ........................................................................................................... v
LIST OF TABLES ......................................................................................................... vi
LIST OF FIGURES .......................................................................................................vii
ABSTRACT ................................................................................................................... ix
PART I. INTRODUCTION ............................................................................................ 1

1.1. Preface ...................................................................................................................... 1
1.2. Purposes and requirements ....................................................................................... 2
1.2.1. Purposes ................................................................................................................. 2
1.2.2. Requirements ......................................................................................................... 2
PART II. LITERATURE OVERVIEW .......................................................................... 3
2.1. Introduction of Streptomyces .................................................................................... 3
2.1.1. Taxonomy .............................................................................................................. 3
2.1.2. Characteristics of Streptomyces ............................................................................. 4
2.1.3. Secondary Metabolites of Streptomyces ................................................................ 8
2.1.4. Antibiotics produced by Streptomyces .................................................................. 9
2.2. Genome mining of Streptomyces............................................................................ 11
2.2.1. Genome mining ................................................................................................... 11
2.2.2. Role of Bioinformatics in Natural Product Discovery by Genome Mining ........ 12
2.2.3. Genome mining in Streptomyces ......................................................................... 12
PART III. MATERIALS AND METHODS ................................................................. 15
3.1. Materials ................................................................................................................. 15
3.2. Research equipment................................................................................................ 15
3.3. Study contents and study methods ......................................................................... 15
3.3.1. Study contents ..................................................................................................... 15
3.3.2. Study methods ..................................................................................................... 16
PART IV. RESULTS AND DISCUSSION .................................................................. 22
4.1. General genomic features Streptomyces sp. YIM 121038 ..................................... 22
iii


4.2. Phylogeny of Streptomyces sp. YIM 121038 ......................................................... 29
4.3. Biosynthetic gene clusters for secondary metabolites of Streptomyces sp. YIM
121038 ........................................................................................................................... 30
4.4. Genome annotation, functional analysis of Streptomyces sp. YIM 121038 .......... 37
4.5. Pathway of secondary metabolite biosynthesis of Zorbamycin in Streptomyces sp.

YIM 121038 (Antitumor potential of strain YIM 121038) ........................................... 50
PART V. CONCLUSION AND SUGGESTION ......................................................... 56
1. Conclusion ................................................................................................................. 56
2. Suggestion ................................................................................................................. 56
REFERENCES .............................................................................................................. 57
APPENDIX ................................................................................................................... 65

iv


ABBREVIATION

antiSMASH

Antibiotics & Secondary Metabolite Analysis Shell

ARTS

Antibiotics Resistant Target Seeker

BGCs

Biosynthetic gene clusters

CAZyme

Cacbohydrate active enzyme

COG

Clusters of Orthologous Groups

GBDP

Genome BLAST Distance Phylogeny

GGDC

Genome-to-Genome Distance Calculator

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

NRPSs

Non-ribosomal peptide synthetases

PKSs

Polyketide synthases

smBGCs

Biosynthetic gene clusters of secondary metabolites

TYGS

The Type (Strain) Genome

ZBM

Zorbamycin

v


LIST OF TABLES
Table 2.1. List of some antibiotics produced by Streptomyces sp. ............................... 10
Table 2.2. Current genome mining studies on Streptomyces species............................ 13
Table 3.1. List of 15 related strains of Streptomyces ................................................... 17
Table 4.1. Genome features of Streptomyces sp. YIM 121038 provided by NCBI,
EZBioCloud ................................................................................................. 22
Table 4.2. Number of genes of COG functional category of Streptomyces sp. YIM
121038 .......................................................................................................... 26
Table 4.3. List of Streptomyces sp. YIM 121038 and other species ............................. 29
Table 4.4. List of putative secondary metabolite producing biosynthetic clusters as
predicted by antiSMASH version 5.2.0 ....................................................... 33
Table 4.5. Species distribution of Streptomyces sp. YIM 121038 ................................ 39
Table 4.6. Top- hit species distribution of Streptomyces sp. YIM 121038 ................... 41
Table 4.7. Gene ontology categories after Blast2GO analysis ...................................... 45
Table 4.8. Deduced functions of gens in the ZBM biosynthetic gene cluster and
comparison genes in the ZBM biosynthetic gene cluster of S. Flavoviridis
to Streptomyces sp. YIM 121038. ................................................................ 55

vi

LIST OF FIGURES
Figure 3.1. General schematic diagram ......................................................................... 15
Figure 3.2. Schematic representation of the Blast2GO application .............................. 20
Figure 4.1. Features of the genome of Streptomyces sp. YIM 121038 provided by
EZBioCloud ................................................................................................. 23
Figure 4.2. Features of the genome of Streptomyces sp. YIM 121038 provided by
ARTS............................................................................................................ 23
Figure 4.3. Features of the genome of Streptomyces sp. YIM 121038 provided by
GeneMark ..................................................................................................... 24
Figure 4.4. Circular map of the Streptomyces sp. YIM 121038 .................................... 25
Figure 4.5. Color codes for gene functions presented in Table 4.2 ............................... 28
Figure 4.6. Relationship of Streptomyces sp. YIM 121038 with 15 related
Streptomyces species .................................................................................... 30
Figure 4.7. Secondary metabolite producing biosynthetic clusters as predicted by
antiSMASH version 5.2.0 ............................................................................ 31
Figure 4.8. antiSMASH biosynthetic gene clusters and their predicted core structure
for a. Lanthipeptide b. NRPS c. T1PKS-NRPS d. T3PKS-NRPS e. NRPSbacteriocin clusters from Streptomyces sp. YIM 121038 ............................ 32
Figure 4.9. Blast2GO functional annotation results overview of Streptomyces sp. YIM
121038 .......................................................................................................... 37
Figure 4.10. Number of sequences with length of Streptomyces sp. YIM 121038 ....... 38
Figure 4.11. Species distribution chart of Streptomyces sp. YIM 121038 after blastx to
NCBI nr ........................................................................................................ 38
Figure 4.12. Top- Hit species distribution chart of Streptomyces sp. YIM 121038 after
blastx to NCBI nr ......................................................................................... 40
Figure 4.13. E-value distribution for sequences of Streptomyces sp. YIM 121038 ...... 42
Figure 4.14. InterProScan Results Chart ....................................................................... 42
Figure 4.15. Mapping database sourses of YIM 121038 after mapping to B2G database .. 43
Figure 4.16. GO level distribution chart for Streptomyces sp. YIM 121038 ................ 44

Figure 4.17. Distribution of Blast2GO gene ontology (GO) categories ....................... 46

vii


Figure 4.18. Biological process combined graph of annotation of Streptomyces sp.
YIM 121038 ................................................................................................. 47
Figure 4.19. Molecular function combined graph of annotation of Streptomyces sp.
YIM 121038 ................................................................................................. 48
Figure 4.20. Cellular component combined graph of annotation of Streptomyces sp.
YIM 121038 ................................................................................................. 50
Figure 4.21. Cluster 31 in antiSMASH ......................................................................... 51
Figure 4.22. Zorbamycin biosynthetic gene cluster from Streptomyces Flavoviridis .. 51
Figure 4.23. a. A linear model for the ZBM hybrid NRPS-PKS templated assembly of
the ZBM aglycone from nine amino acids and one acetate. b. Proposed
pathway for ZBM biosynthesis. ................................................................... 53

viii


ABSTRACT
Streptomyces bacteria are recognized as an important source for antibiotics with
broad applications in human medicine and animal health. In this study, a genomebased approach Streptomyces sp. YIM 121038, a representative streptomycete, was
employed to examine the biosynthetic as well as potential compounds. A high quality
draft genome (11.09 Mb) of Streptomyces sp. YIM 121038 was obtained with a G + C
content of 72.4% and 9353 genes. Although the genome was released, the detailed
investigation analysis of this Streptomyces sp. YIM 121038 strain has never been done
which motivated us to pursue this thesis. The genome analysis of this strain revealed
39 putative biosynthetic gene clusters (BGCs) for secondary metabolites, which
showed similarity with those for antibacterial, anticancer, antifungal, antiparasitic, or

antivirus compounds. The structural diversity of secondary metabolisms predicted for
Streptomyces sp. YIM 121038 includes PKS, NRPS, PKS-NRPS hybrids, a
lanthipeptide, terpenes and siderophores. Studies of some of these clusters resulted in
the characterization of novel compounds and of previously known compounds but
never characterized in this Streptomyces species. However, the low levels of similarity
with known BGCs for most cases suggested novelty of the metabolites from those
predicted gene clusters. More importantly, we report the cluster analysis of YIM
121038 which reveals antitumor biosynthetic pathway for zorbamycin. This study
illustrates the power of genomic analyses to gain insight into the evolution of
antibiotic- producing microorganisms.

ix


PART I. INTRODUCTION
1.1. Preface
Streptomyces species are filamentous Gram-positive bacteria, high G+C content
found in the soil, water environments and a member of the largest genus of
Actinobacteria. Streptomycetes are filamentous that are prolific sources of secondary
metabolites with industrial implications. Many bioactive secondary metabolite
compounds found in Streptomycetes to date are antibiotic, antitumor, or
immunosuppressive activities (Braña et al., 2015).
Recently, increasing administration of antibiotics has led to a growing number of
resistant pathogenic bacterial strains. The problem of antimicrobial resistance increase
and its high effect on human health, there is an vital need of looking for new natural
products that could resolve this issue (Tracanna et al., 2017, Durand et al., 2019).
Therefore, genomic science has been used to determine potential drug targets and to
discover new gene clusters for natural product biosynthesis. The development of the
genome sequencing technologies has been employed in the search for novel
metabolites as well as new antibiotics. These bioactive compounds are synthesized by

biosynthetic gene clusters (BGCs) that consist of genes localized close to each other in
bacterial genomes (Naughton et al., 2017). Based on their products, BGCs are in
general categorized as non-ribosomal peptide synthetases (NRPSs), polyketide
synthases (PKSs), post-translationally modified peptides (RiPPs), and those for
saccharides, terpenoids, lanthipeptides and others (Malik et al., 2020). It was proposed
that Streptomyces might generate as many as 100,000 metabolites of antimicrobials, of
which only a small percentage was reported (Watve et al., 2001). Recognizing the
concern that the use of antibiotics currently used which become inefficient against
multiple pathogens due to the increase in the number of antimicrobial resistant
microbes, it is therefore important to look for novel strains of Streptomyces to help fill
the important need for new antibiotics (Stulberg et al., 2016).
Traditionally, the discovery of secondary metabolites has been focused on a
bioassay-guided approach involving microorganism cultivation, chemical extraction of
the generated metabolites, and final elucidation of the structure (Naughton et al.,
2017). Within the past, this approach facilitated the invention of many valuable
1


chemicals, but nowadays it leads too often to the rediscovery of known metabolites,
resulting in a dramatic reduction quantity of new molecules identified (Finking and
Marahiel, 2004). Therefore, in the last number of years, both analytical and
bioinformatic based approaches have been optimized to minimize the likelihood of
rediscovery of the same products. Hence, this detects novel biosynthetic products
quickly and easily. One of the most important factors that lead to the success of the
discovery of new secondary metabolites is genome sequence analysis, subsequent
characterization of the BGCs and related pathways for biosynthetic products. This
approach facilitates the identification of genomic entities likely responsible to produce
these new molecules.
Streptomyces sp. YIM 121038 is a potential actinomycete, isolated from soil
taken from rainy forest in Xishuangbanna, China. The whole genome of Streptomyces

sp. YIM 121038 was released, however, the detailed investigation analysis of this
Streptomyces sp. YIM 121038 strain has never been done which motivated us to
pursue this thesis “Genome-based analysis for the bioactive potential of
Streptomyces sp. YIM 121038, isolated from soil in China”.
1.2. Purposes and requirements
1.2.1. Purposes
- Analyze the whole genome of Streptomyces sp. YIM 121038 for the bioactive
potential of YIM 121038.
1.2.2. Requirements
- Determine general genomic features Streptomyces sp. YIM 121038.
- Determine the taxonomic position for raleative strains Streptomyces.
- Analyze biosynthetic gen cluster for secondary matabolites of Streptomyces sp.
YIM 121038.
- GO annotation analysis and functional annotation analysis of Streptomyces sp.
YIM 121038.
- Analyze pathway of secondary metabolite create zorbamycin of Streptomyces
sp. YIM 121038.

2


PART II. LITERATURE OVERVIEW
2.1. Introduction of Streptomyces
Streptomyces is the largest genus of Actinobacteria and the type genus of the
family Streptomycetaceae which comprise gram-positive bacteria with high G+ C
content in their DNA. Actinobacteria appeared about 2.3 billion years ago at the time
of the first oxygenation of the Earth’s atmosphere. They consist of not solely
complicated, mycelial, sporulating organisms, such as Streptomyces, however
additionally simple cocci (e.g., bacteria genus luteus), rods (e.g., Brevibacterium), and
organisms of intermediate complexness (e.g., Nocardia) (Wink et al., 2017). There are

more than 500 known species of Streptomyces. They found predominantly in soil and
decaying vegetation, most Streptomycetes produce spores. It develops as fully
mycelial organisms and reproduces by the formation of immotile spores at the tips of
the aerial hypha.
The Streptomyces is well known for its huge attribute related to the production of
secondary metabolites such as antifungals, antivirals, anti-hypertensives, antitumoral,
immunosuppressants, and especially antibiotics(Procópio et al., 2012).
2.1.1. Taxonomy
Streptomyces is the type of genus of the family Streptomycetaceae which was
proposed by Waksman and Henrici (1943) and currently more than 5000 species with
the number increasing every year. Streptomycetaceae family are in Actinobacteria
phylum and Actinomycetales order within the class Actinobacteria and the genus
Streptomyces is the sole member of this family (Anderson and Wellington, 2001).
Scientific classification of Streptomyces:
Kingdom: Bacteria
Phylum: Actinobacteria
Class: Actinomycetes
Order: Actinomycetales
Family: Streptomycetaceae
Genus: Streptomyces
(Waksman and Henrici, 1943)
Strain: Streptomyces sp. YIM 121038
3


The systematics of the genus Streptomyces have a long evolutionary history with
numerous different approaches early based on descriptions of species about
morphological procedures such as the color of aerial and substrate mycelia and spore
chain morphology along with rudimentary physiological features (Labeda et al.,
2012).In the 1940s, the discovery of antibiotics produced by Streptomyces led to

extensive approaches for novel bioactive compounds. Consequently, this led to an
extreme overclassification of the genus to patent for new species (Anderson and
Wellington, 2001). The invention of new natural products was described as new
species and patented(Anderson and Wellington, 2001). The result is the naming of
approximately 3000 species of genus Streptomyces by the year 1970, based on the
basis of small differences in morphological and cultural properties, with many of these
new names found only in the patent literature (Trejo, 1970).
In 1964, the International actinomycete Project (ISP) was initiated to introduce
commonplace criteria for the determination of species described by (Shirling and
Gottlieb, 1968a, Shirling and Gottlieb, 1968b, Shirling and Gottlieb, 1969, Shirling
and Gottlieb, 1972) so as to decrease the quantity of poorly described synonymous
species. The major disadvantage of these depictions involved morphology (i.e., spore
chain morphology, spore surface ornamentation, color of spores, substrate mycelium,
soluble pigments, and production of melanin pigment), in expansion to some
physiological properties, which were basically confined to utilization tests of different
carbon sources (Kämpfer, 2006). The ISP study was primarily focused on a limited
number of features with a heavy emphasis on morphology and pigmentation which not
provide an applicable identification scheme. In addition to the traditional methods of
using a numerical taxonomic approach based on morphological observations,
chemotaxonomic and molecular methods are now used together to enhance our
understanding of species relationship within Streptomyces genus. These consist of cell
wall composition, phage typing, protein profiling, DNA-DNA hybridization, RAPDPCR assays, ELISA and comparison of 16S rRNA and 23S rRNA sequences.
2.1.2. Characteristics of Streptomyces
The Streptomycetes are characterized as gram-positive aerobic bacteria of
complex form that present in various environments of soil including composts, water,
4


and

plants

(Hasani

et

al.,

2014).

Streptomyces

species

are

nonmotile,

chemoorganotrophic, and its shape resembles filamentous fungi but not acid-alcohol
fast. They have genomes with high GC content 69-78% (Sharma, 1999).
There are 2 types of mycelia being developed by actinomyces substrate
mycelium (vegetative mycelium) and aerial mycelium. Many types contain only
substrate mycelia, but many have only aerial mycelia. The aerial mycelium forms
chains from three to many spores at maturity and few species bear short chains of
spores on the substrate mycelium(Kämpfer, 2006).The hyphae differ greatly in length,
a few are long with limited branching, and others are short and much-branched. The
vegetative mycelium does not shape cross-walls; it does not break up into rod shape
and coccus like bodies. It reproduces by means of seven conidia formed in straight or
spirally coiled chains, or by means of bits of mycelium. Conidia are formed in huge
number as to give the colonies on artificial media a powdery appearance(Frederick,

1962). Vegetative hyphae (0.5-2.0 um in diameter) produce an extensively branched
mycelium that rarely fragments. Sclerotia, pycnidialsporangia-, and synnemata-like
structures may be formed by some species. It forms discrete and lichenoid, leathery or
butyrous colonies. Initially colonies are relatively smooth surfaced but later they
develop a weft of aerial mycelium that may appear floccose, granular, powdery, or
velvety. Produce a wide variety of pigments responsible for the colour of the
vegetative and aerial mycelia.(Kämpfer, 2006).
The steptomyces grow in pH is 6.5-8.0 (Kontro et al., 2005). The optimum
temperature for growth of most streptomycetes is in the range of 25-35˚C, although
some species can grow at temperatures within the psychrophilic and thermophilic
range. Streptomyces use a wide range of organic compounds as sole sources of carbon
for energy(Rafieenia, 2013). Generally, Streptomyces reduce nitrates to nitrites and
degrade adenine, esculin, casein, gelatin, hypoxanthine, starch, and L-tyrosine (Slim et
al., 2011). Streptomyces species can be distinguished by cell wall type to other
actinomycetes which is characterized as Type I sensu (Lechevalier and Lechevalier,
1970). The cell wall peptidoglycan contains major amounts of L-diaminopimelic acid
(L-DAP) and glycine and the lack of characteristic sugars (Uchida and Seino, 1997).
They lack mycolic acids, contain significant quantities of saturated, iso- and anteiso5


fatty acids, have either hexa- or octahydrogenated menaquinones with nine isoprene
units as the main isoprenology, and have complicated polar lipid patterns that usually
contain diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, and
mannoside phosphatidyl inositol (Cummins and Harris, 1958).
 The life cycle of Streptomycetes
In addition to fungal-like in cell structure, streptomycetes also resemble fungi in
their complex life cycle that includes sporulation and programmed cell death processes
(Yagüe et al., 2013). Streptomycetes grow as branching hyphal filaments to shape a
mat of fungus-like mycelium, from which emerge aerial branches that hold chains of
spores (Chater, 2016). This motivates the existing spores to be in an inactive state and

undergo germination to form germ tubes (Hasani et al., 2014). Germ tubes grow by tip
extension give rise to a network of filaments. The formation of branched filaments that
grow into and across on agar surface consisting of a multinucleated hyphae network
known as the "substrate mycelium"(Yagüe et al., 2012). During the vegetative growth
stage of streptomycete development, DNA replication happens without cellular
division, making the already specified filamentous structure. When the colony
continues to grow, the mycelium in the center starts to differentiate which extends
away from the substrate into the air. Differentiation leads to the formation of a new
cell type is called " aerial hyphae" (Hasani et al., 2014). Then, to become a chain of
uninucleoid compartments, the aerial hyphae undergo major septation. Finally, these
compartments differentiate to form chains of spores (Chater, 1993). Secondary
metabolism also takes place in this place with the production of secondary metabolites
including antibiotics (Urem et al., 2016). Cross-wall formation in the substrate hyphae
is infrequent, in contrast to the synchronous formation of multiple sporulation septa in
the aerial hyphae (Dyson, 2009). Both substrate and aerial mycelia are multinucleated
(Yagüe et al., 2012).
The Robinow HCl-Giemsa method of nuclear staining has been studied to
described life cycle of Streptomyces sp. comprise: (1) initial nuclear division phase ;
(2) primary mycelium ; (3) secondary mycelium (including aerial); (4) the formation
of spores (Mcgregor, 1954).
 Streptomycetes habitats
6


Streptomycetes are freeliving, saprohytes found widely distributed in soil, water
and other natural environments (L.ShantikumarSingh et al., 2006). The secondary
metabolite called geosmin (literally 'earth smell') formed by Streptomyces does not
have antibiotic activity, but gives the soil its distinctive smell, and provides an
indicator of how widespread these bacteria are in the soil. Geosmin is not known for
its adaptive significance, but its development is a well-preserved characteristic of

Streptomyces spp. It probably has an important function (Hopwood, 2007).
Streptomyces are well adapted to life within the soil where they grow as a substrate
mycelium manufactured from multiple hyphae that grow by tip extension and branch
through the soil looking for nutrients (Seipke et al., 2012). Distribution of
Streptomycetes in water and soil are affected by many factors such as temperature, pH,
food stress, salinity, soil texture and climate. Streptomyces are found spread widely in
habitats such as territories, deserts, highlands, insects, marine, invertebrates, and
marine sediments. Some of the Streptomyces habitats will be mentioned below:
Hay

and

other

organic material: Many Streptomycetes are indigenous

microorganisms isolated from the soil and have the ability to eliminate and use
different organic compounds to grow successfully(Rahmansyah et al., 2012). In
biogeochemical cycles, Streptomycetes are able to degrade cellulose, lignocellulose,
chitin, and various organic compounds(Horn et al., 2012).
Fresh water and marine habitats: Actinomycetes including Streptomyces is
distributed in clean water systems, lakes, drainage systems after heavy rainfall
(Rowbotham and Cross, 1977). The isolation of Streptomyces from marine
environments has been an abundant area of research in the past decade. They exist in
marine environments such as sediments, seawater, marine invertebrates, particularly
sponges (Selvakumar et al., 2010). Sea-derived actinomycetes have recently been
reported as a source of new antibiotics and anticancer agents (Baskaran et al., 2011).
Plants: Streptomyces spp. show life starting from benign saprophytes to beneficial
plant endosymbionts to plant pathogens. Plant pathogens in the genus Streptomyces are
rare, including the well-studied examples Streptomyces turgidi-scabies, Streptomyces

acidiscabies, Streptomyces scabies and Streptomyces ipomoeae (Seipke et al., 2012).
These pathogens have a large host range and can infect plants such as tomatoes but on
7


economically important tuber crops such as potatoes which are mainly known for their
capacity to cause necrotic scab-like lesions (Loria et al., 2003).
Animal and humans: Streptomycetes are uncommon pathogens, but
Streptomyces somaliensis and Streptomyces sudanensis can cause infections in
humans, such as mycetoma (Quintana et al., 2008). Members of the genus
Streptomyces were identified withinside the guts of numerous arthropod species, such
as termites, millipedes, beetles, wooden lice and earthworms. Streptomyces can be a
considerable population of invertebrates’ guts and probably lead to the degradation of
polymeric carbohydrates or to antimicrobial protection (Seipke et al., 2012).
2.1.3. Secondary Metabolites of Streptomyces
Streptomyces species has important role in the production of various bioactive
secondary metabolites. They ability to produce bioactive secondary metabolites such
as antifungals, antivirals, antibacterial, antitumoral, anti-hypertensives, and mainly
antibiotics and immunosuppressives (Procópio et al., 2012). It has been found that
metabolites can be broadly divided into four classes: (1) regulatory activities in
compounds, these include consideration of growth factors, morphogenic agents and
siderophores, and plants promoting rhizobia; (2) antagonistic agents, these include
antiprotozoans, antibacterials, antifungals, as well as antivirals; (3) agrobiologicals,
these include insecticides, pesticides, and herbicides; and (4) pharmacological
agents,

these include neurological agents, immunomodulators, antitumorals, and

enzyme inhibitors(Tarkka and Hampp, 2008). Streptomyces griseus and Streptomyces
coelicolor are used for industrial production of Streptomycin and novel antibiotics

such as dihydrogranticin respectively(Hasani et al., 2014). Doxorubicin and
daunorubicin such as anticancer agents are secondary metabolites produced by
Streptomyces capoamus (Mukhtar, 2012). The production of secondary metabolites
commonly coincides or slightly precedes with the development of aerial hyphae in
surface grown cultures (Bibb, 2005).
To date, approximately 17% of biologically active secondary metabolites (nearly
7600 out of 43,000) are known

from Streptomycetes (Bérdy, 2005). Soil

Streptomycetes are the major source of bioactive secondary metabolites, but a large
variety of structurally unique and biologically active secondary metabolites have
8


recently been isolated from marine actinomycetes, including those from the genus
Streptomyces.
Known secondary metabolites of Streptomyces are synthesized by six pathways
of various biosynthesis: the peptide pathway, the polyketide synthase (PKS) pathway,
the nonribosomal polypeptide synthase (NRPS) pathway, the hybrid (nonribosomal
polyketide synthetic) pathway, the shikimate pathway, the β-lactam synthetic pathway,
and the carbohydrate pathway (Hamdi and Mohamed, 2018).
It is becoming essential in biomedicine to find new secondary metabolites, and
the only way would be to restart natural strain screening. The latest Streptomyces
discoveries will be very important in designing new experimental methods to optimize
the selection process and decrease the incidence of false negatives (Yagüe et al.,
2012).
2.1.4. Antibiotics produced by Streptomyces
Streptomyces species are an crucial supply of medicines, in particular antibiotics.
Antibiotics are of the most important secondary metabolites produced by

microorganisms and specifically inhibit the growth of other bacteria or fungi or
destroy them. Streptomyces is the largest antibiotic-producing of medical and
industrial significance among microorganisms (Rafieenia, 2013).
From the late 1940s to the 1960s, also referred to as the golden age of antibiotics
discovery, many antibiotics were isolated from diverse Streptomyces species and
entered clinical use. For about two decades, the number of antimicrobial compounds
of Streptomyces recorded rose almost exponentially per year which increased steadily
to reach a peak in the 1970s but later decreased substantially in the late 1988s and
1990s. Louis Pasteur was one of the founders of modern antibiotics knowledge, in the
19th century. He discovered that other microorganisms are capable of destroying other
microorganisms (Tulchinsky and Varavikova, 2014). In 1929, Alexander Fleming
discovered the first antibiotic with "Penicillin", a molecule produced by certain molds
that kills or stops the growth of certain kinds of bacteria. Approximately 75% of all
antibiotics are derived from these filamentous prokaryotes, and 75% are produced by a
single genus, Streptomyces (Demain and Fang, 2000). The history of antibiotics of
antibiotics derived from Streptomyces began with the discovery of Streptothricin in
9


1942, and the discovery of Streptomycin in 1943, and then scientists intensified
the search for other antibiotics within the genus (Clardy et al., 2006). Nowadays,
80% of the antibiotics come from the genus Streptomyces, the most important being
actinomycetes (Procópio et al., 2012). They produce over two-thirds of the clinically
useful antibiotics of natural origin (e.g., neomycin and chloramphenicol) (Baskaran et
al., 2015). According to Nikaido, antibiotics are produced o worldwide on an
estimated scale of approximately 100,000 tons per year which are used in agriculture,
food, and health (Nikaido, 2009). Some of the antibiotics were showed on Table 2.1.
Table 2.1. List of some antibiotics produced by Streptomyces sp.
Streptomyces sp.

Antibiotic

Streptomyces sp.

Antibiotic

S. griseus

Streptomycin

S.kanamyceticus

Kanamycin

S. clavuligerus

Cephalosporins

S.avermitilis

Avermicin

S. venezuelae

Chloramphenicol

S. ribosidificus

Ribostamycin

S.nodosus

Amphotricin B

S. roseosporus

Daptomycin

S. aureofaciens

Tetracycline

S. platensis

Platensimycin

S. noursei

Nystatin

S.alboniger

Puromycin

S. vinaceus

Viomycin

S. fradiae

Neomycin

S. capreolus
S. pristinaespiralis

Fosfomycin
Virginiamycin

S. garyphalus

S. virginiae

Cycloserine

S. orchidaccus

S. lincolnensis

Lincomycin

S.tenebrarius

Tobramycin

S. orientalis

Vancomycin

S.rimosus

Oxytetracyclin

S. niveus

Noviobiocin

S.ambofaciens

Spiramycin

Despite the success of the discovery and development of antibiotics, infectious
diseases still appear to be the second cause of death in the world because of microbial
resistance. About 17 million deaths are caused by bacterial infections annually, mostly
affecting children and elderly people (Procópio et al., 2012). Their use has affected
bacterial species, causing resistance to antibiotics for example methicillin-resistant
Staphylococcus aureus (MRSA) and Gram-negative pathogens, such as Klebsiella
10


pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii (Fischbach and
Walsh, 2009). This resistance can be due to genetic modifications, such as mutation or
horizontal transfer of resistance genes, which most frequently occur in organisms with
different taxonomies (Aminov, 2009, Martinez et al., 2009). In the other hand,
microorganisms growing in a biofilm are involved with chronic and recurrent human
infections and are more resistant to antimicrobial agents (Hassan et al., 2011).
Therefore, many researchers have become interested in discovering new antibiotics or
explore new agents to inactivate enzymes that cause resistance.
2.2. Genome mining of Streptomyces
2.2.1. Genome mining
The traditional method of discovering new bioactive natural products from

microbial sources is certainly time-consuming and laborious, with the unfavorable
property of having high rates of metabolite rediscovery (Zerikly and Challis, 2009).
Therefore, the vast number of publicly accessible microbial complete genomes is
being utilized to mine for novel natural discoveries. Genome mining as a powerful
new tool for novel natural product discovery (Zerikly and Challis, 2009).
Genome mining involves the identification of previously uncharacterized natural
product biosynthetic gene clusters within the genomes of sequenced organisms,
sequence analysis of the enzymes encoded by these gene clusters, and the
experimental identification of the products of the gene clusters (Corre and Challis,
2010). It has already resulted in the identification of many novel natural product
biosynthetic gene clusters in the genomes of sequenced microbes. In some cases, these
gene clusters have been experimentally associated with the production of known
metabolites. However, in many cases, sequence analyses of the gene clusters predicted
that they direct the production of new metabolites or known metabolites not previously
identified as products of the organism in question.
In recent years, however, genome sequencing has revealed that microorganisms
still represent an important source for novel natural products by disclosing a so far
hidden secondary metabolome (Aigle et al., 2014).

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2.2.2. Role of Bioinformatics in Natural Product Discovery by Genome Mining
Genome mining relies entirely on computer technology and software for
bioinformatics. To date, a significant amount of data, represented by DNA sequences
and their annotations, has been stored in publicly accessible databases. Indeed, the
complete genome of a single prokaryote is represented in a unique sequence of four
nucleotides (A, T, C and G) with a minimum of approximately 500 000 bp (for
symbiotic organisms) and a maximum of 10 000 000 bp (for symbiotic organisms) (for
saprophytic microbes). These instruments allow for comparative analyses that not only

promote the inference of functional relationships between genes and proteins, but also
help the prediction of the specificity of the substrate, stereospecificity, and other
catalytic properties of biosynthetic enzymes. In the following pages, the use of
comparative sequence analyses in both these cases is discussed (Corre and Challis,
2010).
Based on these genome sequencing projects, genome mining, one of the
bioinformatics-based approaches to natural product discovery, has been developed and
applied to the discovery of chemical structures of new unidentified molecules (Chen et
al., 2016).

Whole genome sequencing offers knowledge-rich data that can

dramatically contribute to and guide the discovery of natural products in
microorganisms. Indeed, genome mining has been positioned as a simple
bioinformatics method in the field of natural products (Undabarrena et al., 2017).
2.2.3. Genome mining in Streptomyces
Streptomyces are a broad and useful resource of secondary metabolites which are
bioactive and complex, many of which have major clinical applications(Lee et al.,
2020a). Genome mining has become a powerful method to discover the
biotechnological potential of Streptomyces organisms, where the chemical core
structure of the molecules can be identified and even predicted by biosynthetic gene
clusters (BGCs) (Weber et al., 2015). Streptomyces have linear chromosomes (Chen et
al., 2002), unlike other bacteria, and their genome sizes are among the highest in the
bacterial world (Weber et al., 2003), ranging from 6.2 Mb for Streptomyces cattleya
NRRL 8057 (Barbe et al., 2011) to 12.7 Mb for Streptomyces rapamycinicus NRRL
5491(Baranasic et al., 2013), considering whole sequenced genomes to date(Kim et
12


al., 2015). Up to 5 percent of their genomes are dedicated to secondary metabolite

synthesis (Ikeda et al., 2003). The capacity to generate a wide range of bioactive
molecules is focused on the fact that they contain the largest number of BGCs, such as
polyketide synthases (PKS) and synthetases of non-ribosomal peptides (NRPS), or
even combinations of PKS-NRPS hybrids (Challis, 2008).
Table 2.2. Current genome mining studies on Streptomyces species
Streptomyces species

Metabolites/potentials

Reference

Streptomyces exfoliatus
UC5319 and
Streptomyces arenae Tü
469

Cytochrome P450s, CYP-161C3,
and CYP161C2, responsible for the
inal step in the biosynthesis of the
sesquiterpenoid antibiotic
pentalenolactone

(Zhu et al., 2011)

Streptomyces sp. MA37

Fluorinase beneicial in luorination
biotechnology

(Deng et al., 2014)

Streptomyces collinus Tü
365

PKS, NRPS, PKS-NRPS hybrids,
lanthipeptide, terpenes, and
siderophores

(Iftime et al.,
2016)

Streptomyces
wadayamensis A23

Antibiotic biosynthetic pathways

(Angolini et al.,
2016)

Streptomyces sp. TPA0356

Polyketide synthases and
nonribosomal peptide synthetases

(Komaki et al.,
2015)

Streptomyces
marokkonensis M10

PKS, NRPS, PKS-NRPS hybrids, a
lanthipeptide, and terpenes

(Chen et al., 2016)

Streptomyces sp. strain
CFMR 7

Two latex-clearing protein (lcp)
genes

(Nanthini et al.,
2015)

Streptomyces sp. Tü 6176 Nataxazole biosynthesis pathway

(Cano-Prieto et al.,
2015)

Streptomyces
ambofaciens
ATCC 23877

PKS, NRPS, PKS-NRPS hybrids,
and terpenes

(Aigle et al., 2014)

Streptomyces citricolor

Germacradien-4-ol and (−)-epi-αbisabolol synthases which reveal
terpene diversity

(Nakano et al.,
2011)

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Table 2.2 demonstrates recent genome mining attempts within the genus
Streptomyces in which the metabolites found are not generally experimentally
confirmed, but rather illustrate the significance of the approach in discovering new
metabolites.
Secondary metabolites are derived from multi-enzyme complexes encoded by
biosynthetic gene clusters of secondary metabolites (smBGCs). In general, smBGCs
contain complete pathways that promote their product's precursor biosynthesis,
assembly, alteration, resistance, and regulation(Lee et al., 2020a). The number of
completely sequenced genomes of Streptomyces has increased exponentially with
recent developments in DNA sequencing technology (Harrison and Studholme, 2014,
Lee et al., 2020b). Several bioinformatics tools have been developed, including
antiSMASH, ARTS, Blast2GO to identify smBGCs within the genome and pathways
of them.
This mini review focuses on the approaches to genome mining for smBGC
detection from genome data from Streptomyces and their usefulness in the discovery of
novel bioactive compounds (Lee et al., 2020a).
 Genome-based analysis the bioactive potential of Streptomyces sp. YIM 121038
In this work, we report a genome which based study on the bioactive potential of
Streptomyces sp. YIM 121038. The strain is a Gram-positive, non-motile and aerobic
actinobacterium from soil that forms largely branched substrate and aerial mycelia.
With a focus to identify genomic features related to the secondary metabolite

production, efforts were made to explore the enrichment of enzymes specific to this
streptomycete as compared to some well-known Streptomyces strains for which
genome data are available. The comparative genomic analysis reveals that strain YIM
121038 has a collection of genes encoding enzymes necessary for secondary
metabolites. Comparative analysis of the biosynthetic gene clusters and pathways
related antitumor antibiotic: Zorbamycin.

14