Molecular and biotechnological aspects of secondary metabolites in actinobacteria

Journal Pre-proof Molecular and Biotechnological Aspects of Secondary Metabolites in Actinobacteria Richa Salwan, Vivek Sharma

PII:

S0944-5013(19)31098-5

DOI:

https://doi.org/10.1016/j.micres.2019.126374

Reference:

MICRES 126374

To appear in:

Microbiological Research

Received Date:

28 September 2019

Revised Date:

10 November 2019

Accepted Date:

11 November 2019

Please cite this article as: Salwan R, Sharma V, Molecular and Biotechnological Aspects of Secondary Metabolites in Actinobacteria, Microbiological Research (2019), doi: https://doi.org/10.1016/j.micres.2019.126374

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Molecular and Biotechnological Aspects of Secondary Metabolites in Actinobacteria

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Salwan Richa1* and Sharma Vivek2*

*Corresponding author: 1

Richa Salwan, College of Horticulture and Forestry, Department of Social Science And Basic Sciences Neri, Hamirpur Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan (H P), 177001 Email ID: [email protected] 2

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Vivek Sharma, University Centre for Research and Development, Chandigarh University, 140413 Email ID: [email protected]

Highlights of review article 

Actinobacteria taxonomy based on whole genome

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Mining actinobacteria for bioactive metabolites and their biosynthetic pathways



Genomic annotations for identification of cryptic genes/gene clusters

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Precision based genome editing and artificial operons for novel compounds and enhanced

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production for different applications

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Abstract

The ability to produce plethora of secondary metabolites and enzymes for pharmaceutical,

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agricultural and biotechnological applications make actinobacteria one of the most explored microbes among prokaryotes. The secondary metabolites and lytic enzymes of actinobacteria are

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known for their role in various physiological, cellular and biological processes including environmental sensing, mineral acquisition and recycling, and establishing social communication. In addition, the basic scaffold of secondary metabolties derived from actinobacteria is a source of

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inspiration for chemists. Recent development in “gene to metabolites” and “metabolites to gene” based omics technologies have played major role in revealing the prevalence of silent gene clusters

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in the genome of actinobacteria. Moreover, the development in precision-based genome editing tools and use of artificial gene operon for pathway engineering have emerged as a key player in activation of these silent/cryptic gene clusters for novel metabolites at large scale which were previously found to be poorly expressed and difficult to characterize in lab conditions. The access to diverse uncharacterized biosynthetic gene clusters of different types and the leverage of modern

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gene editing tools for modulated expression of the operons would contribute to novel product discovery and product diversification compared to traditional way of mining metabolites. Here, in review article, we have discussed the taxonomic status, genomic potential of actinobacteria for various secondary metabolites and role of genetic engineering to explore these microbes for human welfare.

Keywords: Metabolites; Genome mining; Gene cluster; Gene editing

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Introduction Actinobacteria represents the largest group of prokaryotes. It includes Gram positive bacteria

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which exhibit variable morphological growth characteristics (Bhatti et al., 2017). They are aerobic or anaerobic, filamentous, spore-forming bacteria, found in aquatic as well as terrestrial habitats,

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are well known for giving characteristic earthy odour in soil, due to the production of organic product known as geosmin. Actinobacteria are promising candidates for the production of secondary metabolites which are explored as antibiotics and immunosuppressants in medicine. In

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agriculture, the ability of actinobacteria to produce organic acids, fix atmospheric nitrogen and decompose organic matter is of vital importance (Bhatti et al., 2017). Actinobacteria belonging to

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the genus Glycomyces and Streptomyces have been reported to inhibit the growth of penicillin resistant Staphylococcus aureus (Golinska et al., 2015). The secondary metabolites of actinobacteria constitute about two-thirds of all naturally derived bioactive products with

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pharmaceutical importance (Tiwari & Gupta, 2012). Among 23,000 bioactive metabolites from microorganisms, about 10,000 metabolites are reported from actinobacteria. Among actinobacteria, Streptomyces alone contributes approximately 7600 compounds with ability to suppress multidrug-resistant pathogens and other pharmaceutical properties (Berdy, 2012). Besides their importance in the medical field, actinobacteria have significant impact on

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agricultural productivity and mineral recycling in ecosystem by decomposing plant biomass and chelating minerals in soil. Actinobacteria alongwith other soil inhabitants organisms like pathogens and commensals, is known to constitute about 20% of the soil microbial flora in spring, >30% in autumn and only 13% in winter (Barka et al., 2016). The endophytic association of actinobacteria is found to promote plant growth under abiotic and biotic stresses, due to their plant growth regulators production, nitrogen fixation and solubilisation of minerals. (Bouizgarne and Ben Aouamar, 2014). Even free-living members of actinobacteria such as Frankia was the first

actinobacteria reported for nitrogen fixation in angiosperms (Verma et al., 2009). Actinobacteria are also known for their role as biocontrol agents and plant probiotics potential because of their plant root colonization and in situ antibiotic production (Kunoh, 2002; Cao et al., 2004; Sharma and Salwan, 2018). Moreover, bioactive compounds from actinobacteria have been reported from crops like maize, tomato and banana plant which are responsible for suppressing growth of plantpathogens (de Araujo et al., 2000; Cao et al., 2005; Goudjal et al., 2014; Trujillo et al., 2015). These bacteria are known for the production of siderophores and phosphorus solubilization, to

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overcome the iron and phosphorus limited conditions, respectively (Kandel et al., 2017). So far, siderophores such as trihydroxamate,desferrioxamine coelichelin, heterobactin and griseobactin have been reported from Streptomyces, Rhodococcus and Nocardia (Meiwes et al. 1990; Imbert et

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al. 1995; Carrano et al. 2001; Yamanaka et al. 2005; Lautru et al. 2005; Mukai et al. 2009; Patzer

multi-drug resistant bacterial strains.

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Historical and Taxonomy of Actinobacteria

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and Braun 2010). Moreover, siderophores are being explored for developing antibiotics against

Actinobacteria originated about 270 million years ago in oxygen deficient environment were obligate anaerobes, non-filamentous and non-spore formers with simple morphological features

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(Chandra and Chater, 2014; Lewin et al., 2016). Members of actinobacteria including Streptomyces have been characterized and classified based on their sporulation patterns,

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morphological and physiological features and pigmentation (Sousa and Olivares, 2016). Micromonospora belonging to Micromonosporaceae was reported as filamentous, aerobic, spore forming and pigment producing organisms (Trujillo et al., 2014a). Actinobacteria have complex mycelial morphology including presence or absence of a straight or aerial mycelium, production of pigments that impart color to the mycelium and sporulation pattern. Actinobacteria can be

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coccus shaped including Micrococcus, coccobacilli including Arthrobacter, fragmenting hypha including Nocardia spp. and branched mycelia including Streptomyces spp. (Ventura et al., 2007). The variations in morphological features have been reported as elongated filaments without true mycelium in Rhodococci, to substrate hyphae fragmented into flagellated motile elements in Oerskovia, filamentous growth at apex without lateral wall extension and no mycelial growth in corynebacteria (Barka et al., 2016). The characteristic patterns of spores and pigments have been reported as characteristic features in the taxonomy of Actinobacteria (Barka et al., 2016). Actinobacteria produce either as single cell spores, or in chains, inside vesicles known as sporangia

which are either flagellated or non-flagellated. They produce pigments which are commonly known as melanoid polymers and are similar to the humic substances present in soil (Manivasagan et al., 2013; Barka et al., 2016). These pigments are not required for the growth of bacteria but have significant contribution for better survival (Barka et al., 2016). Now days, the identification based on molecular tools including availability of 16S rRNA sequence and biochemical parameters has facilitated their accurate taxonomic and phylogenetic relationships (Chandra and Chater, 2014; Sousa & Olivares, 2016). Cohn first reported actinobacterial species Strepthrotrix foersteri in 1875

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which exhibit both bacterial and fungal features in morphology (Sousa & Olivares, 2016). The classification of actinobacteria has changed over time with the availability of knowledge and existing molecular tools. Based on 16S rRNA sequencing and phylogenetic

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relationship, actinobacteria comprises six classes, six orders, and fourteen suborders (Gao and Gupta, 2012). The class actinobacteria is the largest among 43 families and 16 orders (Zhi et al.,

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2009), while the classes Acidimicrobiia, Coriobacteriia, Nitriliruptoria, Rubrobacteria, and Thermoleophilia contain 10 families only (Barka et al., 2016). Besides the use of 16S rRNA, other

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molecular markers including rpoB, atpD, gyrB, recA, trpB and ssgB are also used to distinguish the closely related genera (Ventura et al., 2007; Girard et al., 2013; Barka et al., 2016). Moreover,

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the genome of actinobacteria is large, compared to other bacteria which varied from 1-12 Mb in size with average genome size of 5 Mb. Limited number of plasmids also have been reported in the genome. Te genome of actinobacteria is characterized by the presence of high G+C content of

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>70% reported in Streptomyces and Pseudonocardia, and <50% in Gardnerella vaginalis and Tropheryma whipplei (Ahmed et al., 2012; Lewin et al., 2016). Comparative study of the whole genome sequences taken from NCBI public domain database revealed conserved features and their true relationships among actinobacterial strains (Fig. 1). Here, the phylogenetic relationship based on whole genome sequences using Composition Vector (CV) Tree version 3.0 provide alignment

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free but closely related to 16S rRNA phylogeny for taxonomic classification (Qi et al., 2004; Verma et al., 2013). The phylogenetic relationship of actinobacteria has also been reported based on single gene like 16S rRNA, 23S rRNA genes, and conserved genes, and overlapping genes (Verma et al.,

2013). Keeping in view, the multiple beneficial roles of actinobacteria in ecosystem, present review article disucss the underlying molecular mechanisms starting from early colonization to imparting beneficial effect to the associated host plant. Attachment and entry to the host plant surface

The establishment of microbes in plant rhizosphere is one of the important factors which determine the success of beneficial microbes in the field before imparting benefits to the associated host plant in the form of pathogen suppression to boost plant health. The early chemical acquaintance between microbes and host plant roots is essential for successful root colonization through adherence to the plant root surface. The host plant exudates, in the rhizosphere serve as chemoattractants. These exudates are complex of different compounds and their composition vary at significant level between plants (Bais, et al., 2006; Haichar, et al., 2014). The molecules such as

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plant exopolysaccharides (EPSs), flavonoids, amino acids, aromatic compounds and organic acids are known to facilitate and mimic the conducive environment for quorum-sensing (Ling et al.,

fold and modify the surrounding soil (Beauregard, 2015).

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2011; Hassan & Mathesius, 2012). These exudates can prime microbial populations upto 10-1000

Other molecules of associated host plant such as strigolactones are known to induce

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repetitive colonization whereas microbial exudates trigger activation of the SYM pathway involved in signal transduction and induction of early symbiosis. The early colonization of

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actinobacteria in roots induces the assembly of cytoplasm as pre-penetration apparatus (PPA) near epidermal cell and outer underlying cortical cell (Benizri et al. 2001). These molecules are also

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known to affect the modulation of transcription, translation and then determine the outcome of interactions between two partners. The plant and microbial derived mucilage in association with organic and mineral matter influence the community structure in plant rhizosphere (Walker et al.

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2003). After adherence, the bacterial cells get access to the root cells by the natural openings like wounds and mechanical injuries.

The process of root colonization and attachment over plant surface has been widely studied in root nodulating bacteria such as Rhizobia and phytopathogens (Rodríguez-Navarro et al., 2007). On the other hand, limited studies are available on root colonization by endophytes. Endophytes

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are perceived to enter the host plant through root hairs, stomata, wounds and hydathodes in shoots to access the plant tissues (Hardoim, et al., 2015; Kandel et al., 2017). Other bacterial structures like flagella, fimbriae and exopolysaccharides may facilitate root adherence to the plant surface (Kandel et al., 2017). Moreover, lipopolysaccharides are also considered as an important structure for root attachment and colonization. Studies on root colonization of maize by H. seropedicae have been reported (Balsanelli et al., 2010; Kandel et al., 2017). Endophytes also secrete enzymes like cellulases, pectinases and xylanases which degrades plant cell wall components and facilitate the

entry between root cells (Kandel et al., 2017) (Fig. 2). Further studies have demonstrated that Nacetyl glucosamine, a sugar residue of LPS binds with lectins secreted by roots of the maize plant during colonization (Balsanelli et al., 2013). Various species of actinobacteria Microbispora, Microbacterium, Micrococcus, Micromonospora,

Nocardia,

Rhodococcus,

Streptomyces,

Streptosporangium

and

Streptoverticillium have been reported as endophytes as well as from root surface of the plants (de Araújo et al., 2000; Kim et al., 2012). The metagenomic studies emphasizing on microbial

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communities of agricultural crops have shown predominance of actinobacteria among other microbes. In similar study, actinobacteria have contributed around 15% of the microbial community in Nipponbare and Kasalath varieties of rice (Okubo et al., 2014). Studies have also

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reported Pseudonocardia followed by Nocardiopsis, Micromonospora and Streptosporangium from medicinal plants (Trujillo et al., 2015). Both culture-dependent and independent approaches

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have also revealed diversity of Amnibacterium, Actinostreptospora, Catenuloplanes, Pseudokineococcus and Quadrisphaera from the plant Maytenus austroyunnanensis which has not

Quorum sensing in actinobacteria

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been reported as endophytes (Qin et al., 2012).

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The continuous increase in world population, expanding needs of agricultural produce and growing resistance against antibiotics by the pathogens, demands efforts for developing alternate

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approaches to effectively combat plant stresses which can efficiently increase the productivity. Quorum sensing (QS) coordinate the modulation of genes responsible for a phenotypic shift like non-virulent to virulent or planktonic to biofilm (Rutherford and Bassler 2012). QS is essential part of cell-cell to communication in a population where bacteria invade, defend, develop resistance against antibiotics and build populations through coordinated gene expression.

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Therefore, approaches engineered to impede quorum sensing are attractive tools to modulate the microbial processes or traits which can be used for human welfare (LaSarre & Federle, 2013). The signaling processes in quorum-sensing (QS) or quorum-sensing inhibition (QSI) are facilitated by different signals which differ from bacteria to bacteria. Through quorum sensing, the bacteria maintain their own population density via autoinducers (Danhorn & Fuqua, 2007; Beauregard, 2015). Different autoinducers in bacteria like the first class autoinducer-1 (AI-1) includes acylated homoserine lactones (AHLs), restricted to Gram-negative bacteria, whereas the second class includes peptide signals, restricted to Gram-positive bacteria and third class includes autoinducer-

2 (AI-2) are reported from different types of bacteria. Another class involving quinolone, diffusible signaling molecules and autoinducer-3 (AI-3) has also been reported (LaSarre & Federle, 2013). In general, AHLs are synthesized by LuxI of AHL synthases family using S-adenosyl methionine (SAM) as substrates and acylated carrier protein (acyl-ACP) with few exceptions. AHLs are composed of a homo-serine lactone unit which is attached to an acyl chain of variable carbon atoms length. These AHL molecules also differ in their oxidation state at C-3 carbon and saturation of acyl chain ( Rutherford & Bassler, 2012; LaSarre & Federle, 2013). Even though actinobacteria is one of the abundant groups in the bacterial kingdom but only

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25 genera have been reported for quorum sensing regulation. The γ-butyrolactone (GBL) system is well established in actinobacteria and has structural similarity to the AHL in Gram negative

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bacteria. GBL is a one-component system where the communicating molecule which is a sensing protein acts as response regulator which induces expression of the target genes (Takano, 2006).

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GBL-based system is well characterized in Streptomyces sp., but still the communication is scarcely explored (Takano, 2006). The signaling molecules such as SCB1/SCB2.SCB3, MMF1,

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MMF2, MMF3, MMF4, MMF5, C4 homoserine lactone, A-factor, P1-factor interact with the cognate receptor scb1 (Fig. 3a). Alternatively, the antibiotics such as actinorhodin and

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undecylprodigiosin, interact with pseudo-receptors (JadP2/scbR2) and activate a series of events which lead to morphogenetic changes and secondary metabolites production (Fig. 3b). Genomic studies have revealed presence of one LuxR domain among 991 proteins of 53 different

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actinobacterial species with variation in distribution pattern ranging from single sequence in Mycobacterium leprae and over 50 sequences in Streptomyces spp (Santos et al., 2012). The domain architecture of LuxR is characterized by the presence of helix turn helix domain at the Cterminus composed of N-terminal module which binds their specific QS signal, and C- terminal region that binds DNA and modulates gene expression (Santos et al., 2012).

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The first step of pathogenicity and quorum sensing involving biofilm formation is known

to regulate virulence or benficial outcome of an interaction. The antiquorum sensing nowadays is emerging as new strategy in managing bacterial diseases through the prevention of biofilm formation in pathogens (Miao et al., 2017). Quorum quenching molecules inhibit expression of virulence factors through the production of enzymes which are capable of breaking down signaling molecules and quorum sensing peptides, involved in the biosynthesis and activation of quorum sensing receptors (Rajput et al, 2015; Betancur et al., 2017). Inhibition of the QS system,

could assist in the termination of the bacterial resistance without killing the bacteria (Hentzer and Givskov, 2003). In particular, the fsr QS system of Enterococcus faecalis and agr QS system of Staphylococcus aureus have been studied (Cook and Federle 2014; Singh and Nakayama 2015). In the agr system, an extracellular build-up of 6 to 8- thiolactone AIP activates, AgrCA, a twocomponent system which results in the upregulation of virulence genes such as toxic shock syndrome toxin-1 and α-hemolysin (Novick 2003; Singh and Ray, 2014). In the fsr system, an 11residue lactone AIP activates FsrCA regulatory system which controls the transcription of two

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extracellular proteases involved in pathogenicity (Nakayama et al., 2001). The emergence of multidrug-resistant bacterial strains, demands alternative approaches to target these virulent

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strains, hence targeting QS systems is an attractive target (Scutera et al., 2014). Metabolites of Actinobacteria

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Actinobacteria representing Streptomyces, Arthrobacter, Actinomyces, Corynebacterium, Micrococcus, Frankia, Micromonospora and several other members are one of the most

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economical and biotechnologically relevant microbial communities. Secondary metabolites of actinobacteria are known for diverse biological activities (Manvisagan et al., 2014). Approximately, 23,000 antibiotics have been discovered from different microbes and the

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contribution (~10,000) of actinobacteria is unchallenged in microbial community. Among actinobacteria, the genus Streptomyces constitute a major source of bioactive molecules and each

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strain is estimated to produce 10–20 secondary metabolites (Lam, 2006). Among members of actinobacteria, Streptomyces is known to produces a plethora of bioactive metabolites such as antibiotics, volatile compounds, siderophores and several others (Table 1). These metabolites are known for their antimicrobial, antitumor, and anti-inflammatory have been reviewed for their diverse biological activities by Manivasagan et al., 2014. A comprehensive list of different type of

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metabolites, their source and their biological activity obtained from different actinobacteria can be retrieved from http://www.bio.nite.go.jp/pks/. Antibiotics

Antibiotics are the chemical substances which inhibit the growth of other organisms. These antibiotics produced by the bacteria as well as filamentous fungi but actinomycetes especially the genus Streptomyces are considered as a promising source (Tishkov, 2001). The history of antibiotics discovery started with streptothricin in 1942 from Streptomyces and in 1944, antibiotic

streptomycin was discovered from the same genus (Fig. 4). Different antibiotics are classified based on the mode of action, structure, administration route and spectrum of biological activity (Calderon and Sabundayo, 2007). In general, the molecular structures of antibiotics include βlactams, aminoglycosides, tetracyclines, macrolides, glycopeptides, quinolones, oxazolidinones and sulphonamides (Adzitey, 2015). These antibiotics are produced as secondary metabolites by different microbes (Hamaki et al., 2005). Presently, around 80% of the antibiotics of microbial orgin discovered so far are derived from actinomycetes (Emerson et al., 2012). Moreover,

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Streptomyces and other species of actinobacteria constitute 45.6% and 16% of these antimicrobials products (Velho-Pereira and Kamat, 2011). These antibiotics are in existence since forty years ago and has been modified since then by the structural changes to combat clinical resistance

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(Genilloud, 2017). The antibiotics are synthesized from the precursors of primary metabolism such as glycolysis, fatty acid and protein catabolism. Besides that, non-proteinogenic amino acids and

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other precursors such as malonyl Co-A, ribostomycin, d-glucopyranose 6-phosphate and terpenoids (Fig. 5) and capuramycin-type nucleoside based antibiotics class composed of three

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components uridine-5’-carboxamide, unsaturated hexuronic acid and an aminocaprolactam are also used as common precursors for the biosynthesis of different types of antibiotics (Cai et al.,

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2015).

The type of precursors used in the biosynthesis, depends upon the type of antibiotics. For example, glycopeptide based antibiotics biosynthesis (GPAs) such as vancomycin reported from

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Streptomyces orientalis previously known as Amycolatopsis orientalis, is derived from both proteinogenic and non-proteinogenic amino acids. Here, the building unit involves both proteinogenic amino acids such as Asn, Ala, Glu, Leu and Tyr and and non-proteinogenic amino acids

such

as

4-hydroxyphenylglycine

(hpg),

β-hydroxytyrosine

(bht),

and

3,

5-

dihydroxyphenylglycine (dpg). Its operon includes a cluster of genes has been reported in the

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genome for the biosynthesis of vancomycin (Fig. 9). The biosynthesis of Bht in vancomycin-which is glycopeptide antibiotics varies from teicoplanin-type. Initially, discovered in novobiocin and nikkomycin producers, the enzymes for β-hydroxylation of tyrosine have been reported from vancomycin-type producing strains and reviewed comprehensively (Yim et al., 2014). In addition, the other enzymes involved in GPA processing such as glycosyltransferases, halogenases, acyltranserases, sulfotransferases and methyltransferases, alters the amino acids residues specifically on the heptapeptide backbones. The biosynthesis of teicoplanin A2 belonging to class

lipoglycopeptide antibiotic (LGPA) was introduced for clinical use in the mid-1980s. It was isolated from Actinoplanes teichomyceticus and then reported for its superior activity against enterococci and other organisms which was comparable in spectrum to vancomycin (Solecka et al., 2012). Further processing such as acylation, glycosylation and other modification of basic scaffolds, modifies the biological as well as chemical properties such as solubility, antimicrobial activity (Yim et al., 2014). The biosynthesis of antibiotics such as GPA having heptapeptide backbone is synthesized by non-ribosomal peptide synthetases (NRPS) complex. The NRPS

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domain is known to vary with GPAs and have diverse ORFs module arrangements. The NRPS assembly line contains multimodular enzyme machinery which assembles the precursor amino acids in a specific manner. This NRPS assembly line module encodes various domains involved

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in the condensation, adenylation, peptidyl carrier protein, thiolation, epimerization and thioesterase domains. These domains are essential for the biosynthesis of basic scaffold and

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tailoring of skeleton from the NRPS assembly. Despite the several success stories on discovery and the advancements in the production of antibiotics, diseases still remain the major cause of loss

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to human kind and agricultural productivity. The biosynthesis of antibiotics is energy consuming process and required considerable efforts from the organism since most of these antibiotics are

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either derived from amino acids, sugar derivatives and other primary metabolites. In general, the biosynthesis of antibiotics is an energy consuming process and involves the utilization of NADPH (Fig. 5). The biosynthesis of streptomycin which was discovered in 1944,

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requires three precursors; Myo-inositol, dTDP-β-L-rhamnose and d-glucopyranose 6-phosphate. The first two precursors after processing produces O-1, 4-α-L-dihydrostreptosyl-streptidine 6phosphate which combines with CDP-N-methyl-L-glucosamine derived from third precursor dglucopyranose 6-phosphate. Both of these products give rise to Streptomycin 6-phosphate (Fig. 5). The cephalosporin-C discovered in 1945 from S. clavuligenes. The biosynthetic pathway involves

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three amino acids L-cysteine, L-valine and L-2-aminoadipate which in a series of steps are converted into cephalosporin and its derivatives. The biosynthesis of antibiotic neomycin discovered in 1949 from S. fradiaeinvolves only single precursor d-glucopyranose 6-phosphate derived from glucose metabolism is similar to streptomycin. In a series of events, using glutamate, H2O and UDP-NAG, the primary precursor is converted to neomycin-c which is then converted into neomycin (Fig. 5). Other antibiotics like nystatin, jadomycin, tetracyclin and its derivatives

use different number of acetyl co-A/malonyl co-A and NADPH whch ultimately leads to the formation of these antibiotics (Fig. 5). The targets of different antibiotics involves molecular, biochemical, and structural changes on multiple cellular sites including DNA replication, transcription and translation and cell wall synthesis inhibition (Emerson et al., 2012). Antibiotics from microbial sources are also source of synthetic and semi-synthetic drugs. For example, retapamulin was prepared by the chemical modification of terpenoid based pleuromutilin reported from fungi in 1950 (Genilloud, 2017).

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Similarly, another narrow spectrum antibiotic known as fidaxomicin effective against Gram positive bacteria was reported in 1975 from Actinoplanes deccanensis but in 2011 same antibiotic derived from tiacumicin has been reported for the treatment of colitis caused by C. difficile

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(Genilloud, 2017). Such modified antibiotics including semi-synthetic glycopeptides are already available in the market whereas other antibiotics modified from dalvabancin, oritavancin,

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telavancin and vancomycin are permitted for clinical trials.

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Siderophores and their biosynthetic pathways in actinobacteria

Iron predominantly present as ferric ions in aerobic environment is essential element for organisms. The low solubility of ferric in water at neutral pH, makes it one of the limited minerals.

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The acquisition of iron is a major challenge across saprophytic and pathogenic microorganisms (Barona-Gómez, et al., 2004). Therefore, to sequester iron from surrounding environment,

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organisms have siderophore-mediated iron uptake/import system (Matzanke, 2005). Siderophores are high-affinity metal or in particular iron chelators. Siderophores produced by both lower and higher organisms are low-molecular-weight, water-soluble, extracellular fluorescent compounds of 200–2,000 Da, generally produced under low iron conditions and are involved in facilitating iron uptake in soil (Schalk et al., 2011).

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Siderophores with different chemical structural compositions, constitutes a family of about 500 compounds. The competition between microbes or between microbes and hosts for Fe3+ has led to evolution of structural diversity in the structure of siderophores and their corresponding receptors (Challis and Hopwood, 2003). Metals other than iron are also known to induce the production of siderophores (Wang et al., 2014). In medical field, siderophores derived drugs such as “Desferal” are used to medicate iron or aluminum overdose arising due to multi blood transfusions or genetic disorders. The siderophore based antibiotics such as sideromycins are attractive target for developing shuttle transport system in multiple-drug-resistant infections and can strongly increase

the efficiency of antibiotics and lower the minimal inhibitory concentration (MIC) of antibiotic to 100-fold lower (Braun et al. 2009; Wang et al., 2014). Siderophores either contain bidentate oxygen ligands such as hydroxamates/ (desferrioxamine E) (Fig. 6), or catecholates (petrobactin) (Fig. 7a), or hydroxycarboxylates (achromobactin) (Miethke and Marahiel, 2007) and nitrogen heterocycles like yersiniabactin, pyochelin utilize phenolates or carboxylates or a combination of additional ligands for Fe3+ acquisition. In some cases, these molecules deploy oxygen and nitrogen heterocycle ligands for

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ferric acquisition (Miethke and Marahiel, 2007). Two types of siderophores mediated shuttle (a) and displacement model (b) for iron acquisition mechanisms has been proposed (Fig. 7b). The ferric–siderophore complex interacts with specific receptors on cell surface, scavenged Fe3+ via an

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energy dependent system known as membrane-associated ATP-dependent transport which triggers the iron transport into the microbial cell via membrane-spanning pores (Miethke and Marahiel,

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2007). Once excreted siderophore scavenge Fe3+ from the surrounding, the resulting Fe3+ siderophore complex is then readsorbed by the cell via a membrane-associated ATP-dependent

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transport system. The transporters system is known to exhibits high substrate specificity. Diverse pathways for the acquisitions of Fe3+ through siderophore complexes and reduction to Fe2+ for

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storage and utilization have been studied (Barona-Gómez et al., 2004). Once inside the cell, Fe3+ is reduced to F2+ and is used for different biological activities whereas siderophores are secreted outside (Fig.7b) (Inomata et al., 2007).

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The siderophores nocardimicins A- F of Nocardia sp. TP-A0674 origin are found to have muscarinic M3 receptor inhibiting activity and thus have potential to treat respiratory, gastrointestinal and urinary disorders (Ikeda et al., 2005). In the rhizosphere, siderophores from bacteria are known for their biological role in managing soil borne plant pathogens. The siderophores of endophytic Streptomyces are involved in the antagonism to Fusarium oxysporium

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f. sp. cubense and promotes the plant growth (Cao et al., 2005). The Streptomyces lydicus WYEC108 in roots can influence pea root nodulation. Siderophores are also well known for their activity against Gram-positive bacteria and fungi (Sontag et al. 2006). To enhance the uptake of iron, siderophores form complex with Fe3+ in soil and then interact with receptors on cell membrane (Raymond and Dertz, 2004). Actinobacteria are one of the most prolific producer of siderophores (Bhatti et al., 2017). Among actinobacteria, approximately 44 percent are found positive on chrome azurol sulphate

(CAS) plate assay (Nakouti et al., 2012) and several species of Streptomyces are reported for desferrioxamine G, B, and E types of siderophores (Challis and Hopwood 2003). Mesylate salt of desferrioxamine produced by S. pilosus belonging to type B, marketed as Desferal is recommended for treating iron intoxication. Besides this, a number of siderophores such as heterobactin from Nocardia and Rhodococcus (Lee et al., 2012), tsukubachelin B from Streptomyces sp. TM-74 (Kodani et al. 2012), Streptomyces sp. GW9/1258 (Sontag et al., 2006), nocardamine from Citricoccus sp. KMM3890 erythrobactin from Saccharopolyspora erythraea SGT2 (Oliveira et

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al., 2006) have been reported. S. coelicolor is found to produce E & G type of desferrioxamine, coelichelin and coelibactin type of siderophores (Challis and Ravel, 2000). The nocardamine siderophore from Citricoccus sp. KMM3890 is known for its colony formation inhibition effects

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in PRMI-7951, SK- Mel-5, T-47D and SK-Mel-228 tumor cell lines as well as weak antimicrobial activity against Gram-positive bacteria (Kalinovskaya et al., 2011). Siderophores of nocardimicins

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A-F obtained from Nocardia sp. TP-A0674 are found promising in treating respiratory, gastrointestinal and urinary disorders (Ikeda et al. 2005). A mixture of fifteen trihydroxamate and

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amphiphilic siderophores have been reported from S. coelicolor M1455 strain (Sidebottom et al., 2013).

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Biosynthetic pathways of siderophores in actinobacteria

Based on the structures, siderophores are broadly categorised into catechol and hydroxamic acids

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(Schalk et al., 2011). Other siderophores like α-hydroxycarboxylates and thiazolines/oxazolines are also reported from actinobacteria (Challis and Hopwood, 2003). The siderophores are biosynthesized by both non-ribosomal peptide synthetase (NRPS) also known as NRPS-dependent pathway which contains multi-enzyme family. The second class representing non-peptide platforms, contains NRPS-independent pathway. The nonribosomal peptide synthetase (NRPS)

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suit has been studied and the biosynthetic pathways for different types of siderophores have been explored. The biosynthetic platform of hydroxamic acid-based siderophores contains building blocks of alternating dicarboxylic acid and diamine which are joined together by amide bonds. An assay based on ATP–pyrophophate exchange system can be used to monitor the specificity of adenylation domains within multienzymes synthetase. For NRPS-independent pathways, an alternative assay traps the activated carboxyl group from hydroxylamine and then form hydroxamic acid which can form ferric complex and then can be detected using spectrophotometer. The first assay is not suitable for NRPS-independent siderophore (NIS) synthetase system which

may be because pyrophosphate is not released from enzyme-carboxylic acid complex (Kadi & Challis, 2009). NRPS-dependent Pathways Understanding the role of cryptic pathways involved in natural products biosynthesis is a bottle neck in exploiting the biosynthetic diversity of actinomycetes (Genilloud, 2017). The biosynthesis of siderophores is complex and uses a family of novel synthetase enzymes (Baroma-Gomez et al., 2006). Here, in NRPS dependent pathways nitrogen heterocycle-based siderophores are assembled

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from building blocks of amino and/or aryl acid Felnagle et al., 2008). The distinct modular structure and presence of multiple catalytic domains for recognition, activation and modification

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of amino acids within single or few polypeptide chains in NRPS collectively function as an assembly line for the synthesis of complex product. The minimal basic chain-initiation NRPS

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module is composed of adenylation (A) domain, specifically catalyzes adenylation of its carboxyl group and thiolation (T) or peptidyl carrier protein (PCP) domain uses thiol of phosphopantetheine

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arm which is post-translationally installed for capturing the activated carboxyl group of the adenylate. In some cases, TE domain is substituted by alternate domain reductase (R) domain. The last condensation (C) domain is involved in the acylation of the thioester. In some modules, C

NRPS-independent Pathway

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domain is susbtituted by a heterocyclization (Cy) domain (Kadi & Challis, 2009).

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Limited studies for NRPS-independent pathways reported α-hydroxycarboxylates and hydroxamate-based siderophores (Challis, 2005). In recent studies, several siderophores are known to assemble via NRPS-independent pathway involving similar enzymes to those involved in aerobactin biosynthesis (Challis, 2005). The aerobactin, a metabolic product of Yersinia, Salmonella, Aerobacter, Escherichia and several other bacteria as a pathogenic factor is

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synthesized using NRPS-independent pathway (Challis, 2005). The biosynthetic pathway contained gene clusters iucABCD with the iutA gene that encodes the outer membrane receptor ferric-aerobactin and is assembled from a molecule of citric acid, two L-lysine, acetyl-CoA and oxygen (Challis, 2005). The presence of one or several genes encoding a homolog of IucA/ IucC has been recognized as a hallmark of NRPS-independent biosynthetic gene clusters. Hybrid NRPS/NIS Pathway

An example of Hybrid NRPS/NIS Pathway was originally isolated from Marinobacter hydrocarbonoclasticus. The biosynthetic assembly of petrobactin unusually contained two catecholate ligands and one a-hydroxycarboxylate ligand for ferric-iron chelation (Barry & Challis, 2009). Desferrioxamines is a well-known member of non-peptide hydroxamate siderophores. The biosynthesis of desferrioxamine B in S. pilosus, involves an operon containing a set of four genes (desA-D) (Barona-Gomez et al., 2004). Initially, the decarboxylation of Llysine via PLP-dependent amino acid decarboxylases (desA), leads to the formation of cadaverine.

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The cadaverine is then hydroxylated via FAD-dependent amine monooxygenses (desB) to give Nhydroxycadaverine (Fig. 6). An inverted repeat sequence upstream of operon acts as iron (II)dependent repressor. However, the formation of desferrioxamine from N-hydroxycadaverine is

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still unclear. The relaxed CoA- dependent acyl transferases mediated acylation either with acetyl CoA or succinyl CoA (desC) is assumed to catalyse the acylation of N-hydroxycadaverine to give

(DesD) gives rise to

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hydroxamic acid which via nucleotide triphosphate dependent oligomerization and cyclization macrocycle (Barona-Gomez

et al., 2004; Barry & Challis, 2009).

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Desferrioxamine B is known to for its antioxidant activity either by donating hydrogen atom or an electron from hydroxamate (Shimon et al., 1998). Similar to the desferrioxamine, the operon for

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aerobactin contain gene iucABCD genes for iron uptake chelate alongwith iutA gene for iron uptake transport which are encoded by plasmid pSMN1 (Waters and Crosa, 1988) and pColV-K30 (Warner et al., 1981). It was first identified siderophore and contains dihydroxamate of N-Acetyl-

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N-hydroxy-L-lysine and citrate. Although the biosynthetic pathway for aerobactin was identified originally from Klebsiella aerogenes but most of knowledge has been gained from Escherichia coli K-12 strain.

Petrobactin belonging to class of catechol type siderophores contains bis-catecholate, alpha-hydroxy acid siderophore. The initial biosynthetic pathways of petrobactin was proposed in

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2007 and it involves six genes namely asbAF. The precursor dihydroxybenzoate adenylate is derived from shikimate pathway from D-erythrose 4-phosphate whereas other two are readily available as primary metabolites. The second precursor sperimidine is derived from S-adenosylL-methionine and third moiety of citrate is derived from TCA cycle. Spermidine and citrate are ligated into N-citryl-spermidine by the asbA-encoded ligase. The three precursors for petrobactin includes dihydroxybenzoate adenylate, spermidine and citrate which combine pre-activated by adenylation to dihydroxybenzoyl-aryl-carrier protein by acyl and aryl carrier proteins and ligated

by 3,4-dihydroxybenzoate[aryl-carrier protein ligase encoded by asbC gene. In subsequent steps, two molecules of 3,4-dihydroxybenzoyl-aryl-carrier protein, one N-citryl-spermidine molecule, and another spermidine are ligated by asbB encoded ligase and asbE-encoded petrobactin synthase through two path and ultimately leads to the formation of petrobactin. The Fe3+ forms complex with petrobactin in presence of natural sunlight which results in the decarboxylation of siderophore ligand and reduction of Fe3+ to Fe2+. Bioactive volatile metabolites

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Production of volatile organic compounds (VOCs) from actinobacteria has been explored ( Schöller et al., 2002) but Streptomyces is well known for their ability to secrete a variety of

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secondary metabolites. Over 1400 VOCs have been detected from Streptomyces (Jones and Elliot, 2017). These volatile metabolites can diffuse in air and soil through pores and finds application as

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house contamination, in interspecies communication as infochemicals or semi-ochemicals (Herrmann 2010), plant pathogen suppression, biofilm formation and several other areas (Jones &

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Elliot, 2017). These VOCs also play important role as indicators during differentiation, mycelium formation and sporulation (Schöller et al., 2002). The VOCs are usually carbon based compounds, hydrophobic in nature, have low boiling point and polarity and vaporizes at 0.01 kPa (Hung et al.,

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2012). They have diverse structural compositionsand composed of hydrocarbons such as alkane, alkene, alcohol, amines, thiols, and terpenes (Lemfack et al., 2014). According to the literature,

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actinobacteria produce off-odor, musty and aroma compounds known as geosmin and 2methylisoborneol which are well characterized. Geosmin has also been reported for reducing the alcohols and esters contributed by fermenting yeast (Du et al., 2015). Comparatively, less attention has been paid on the characterization and physiological roles of other VOCs produced by actinobacteria (Scholler et al., 2002). A total of 120 diffusible VOCs produced by Streptomyces

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spp. have been reported from which 53 compounds have been identified as terpenoids (Scholler et al., 2002). Similarly, S. albidoflavus have also been reported for the production of alcohols and ketones, sesquiterpenes, ammonia, methylamine, diethylamine and ethylamine (Claeson & Sunesson 2005). VOCs such as trimethylamine makes the surrounding environment alkaline and promotes exploration of nearby Streptomyces cultures (Jones & Elliot, 2017). Biosynthesis of VOCs

Although various VOCs are known from actinobacteria but their biosynthesis has not been reported so far. These VOCs are hydrocarbons which are usually produced from carbohydrates, proteins and lipid metabolism (Audrain et al., 2015). Some studies have reported engineering of these compounds from fatty acids by condensation of their polar head groups (Sukovich et al., 2010), release of hydroxyl group from aldehyde to alcohol and elongation-decarboxylation reactions (Brown and Shanks 2012). Similarly, fatty acids undergo α and β-oxidation and release long-chain aliphatic alcohols (Hamilton-Kemp et al., 2005), whereas under anaerobic conditions,

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2, 3-butanediol like short- chain alcohols are produced (Whiteson et al., 2014) and branched chain alcohols methyl-butanol are produced from amino acids via Ehrlich pathway (Ramos et al., 2000; Xiao and Xu 2007). Ketones like 3-hydroxy-2-butanone and 2, 3-butanedione are produced under

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anaerobic conditions from pyruvate (Ryu et al., 2003). Actinbacteria produce α-ketoglutarate, citrate, lactate, malate, oxalate, pyruvate and succinate but phosphate solubilization via organic

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acid production is poorly reported. Moreover, actinobacteria except Streptomyces lack pyrroloquinoline quinone-dependent glucose dehydrogenase pathway to form gluconic acid and

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2-ketogluconic acids from glucose (Jog et al., 2012; Bhatti et al., 2017). Besides the production of organic acids, actinobacteria are also known for the release of sulfur

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compounds including dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide and methyl thioacetate by degrading methionine and by oxidizing methanethiol (Scholler et al., 2002). The formation of these compounds involves removal of 3-dimethylsulfoniopropionate, produced from

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L-methionine in marine algae and higher plants (Stefels, 2000). Other important secondary metabolites include terpenoids, formed from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as basic building blocks. Terpenoids are synthesized by Mevalonic acid (MVA) pathway in fungi and animals and Methylerythritol Phosphate Pathway (MEP) in algae and bacteria. Both MVA and MEP pathways have been reported in both plants and bacteria

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(Grawert et al., 2011). Actinobacteria have been reported for the release of monoterpenes, sesquiterpenes and their derivatives (Citron et al., 2012). Majority of the actinobacteria are known for their ability to produce geosmin except Haliangium ochraceum, Kribbella flavida, Catenulispora acidiphila and Stackebrandtia nassauensis. The biosynthesis of geosmin includes cyclization of farnesyl pyrophosphate to germacradienol, germacrene D, octalin, and geosmin. The first report on isolation of enzyme germacradienol geosmin synthase catalyzing synthesis of geosmin has been reported from Streptomycces coelicolor. Another important compound

albaflavenone exclusively reported in Streptomyces was first reported from Streptomyces albidoflavus (Audrain et al., 2015). Inorganic volatile compounds from Actinobacteria Inorganic volatile compounds like ammonia, nitric oxide, hydrogen sulfide and hydrogen cyanide have also been reported from actinobacteria. These inorganic compounds are produced by the breakdown of amino acids such as H2S production from the degradation of cysteins, NO from Larginine (Mattila and Thomas, 2014), NH3 from peptides, L-aspartate like amino acids, aspartate

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conversion to fumarate in the presence of aspartate ammonia lyase (Bernier et al., 2011). Actinobacteria are also reported for the production of phytohormones like Indole-3-acetic acid

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(IAA) which helps in regulating cell division, elongation and differentiation and increased root hair formation to enhance absorption of nutrients from soil. According to literature, Streptomyces

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spp have been reported for the enhanced synthesis of IAA in the presence of tryptophan. Other species including S. violaceus and S. exfolitus catabolize indole-3- acetamide (IAM), indole-3-

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lactic acid (ILA), indole-3-ethanol (IEt) and indole-3-acetaldehyde (IAAld) into IAA by different pathways. Actinomycetes are known as durable organisms for soil applications because of high cell loading capability, high retention of cell viability, production of antimicrobial products.

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Actinomycetes are well known for nitrogen fixation and form associations with non-leguminous plants to supply fixed nitrogen to the plants. Approximately 15% of the fixed nitrogen provided to

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the plants is due to mutual relationships of actinobacteria with the host plants. Such actinobacteria includes members of family Frankia which provides fixed nitrogen to the plants (Bhatti et al., 2017).

Cell-wall degrading enzymes

Actinobacteria are among the important candidates involved in the decomposition of plant biomass

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as nutritional symbionts as well as releasing natural products as defensive agents (Lewin et al., 2016). Actinobacteria are free living and play vital role in various ecological processes like carbon cycling. Streptomyces is one of the most widely studied genus among actinobacteria is a potent producer of various lytic enzymes which contribute to the antagonistic properties against plant pathogens. Actinobacteria have the ability to degrade plant biomass as they contain a reservoir of lytic enzymes (Berlemont and Martiny, 2013; Lewin et al., 2016; Salwan and Sharma, 2018). Many Streptomyces produce extracellular lytic enzymes which are also responsible for the

inhibition of fungal growth and other pathogens (Chater et al. 2010). Lytic enzymes like chitinases and glucanases have been reported as biocontrol agents as anthracnose disease of pepper was suppresses in the presence of S. cavourensis SY224 (Lee et al. 2012). Similar studies on glucanases obtained from Streptomyces spp. have reported suppression of pathogens causing infectious diseases in agricultural crops (El-Tarabily et al., 2009). Chitinases are also known for their biocontrol potential as reported for S. violaceusniger XL-2 which suppresses the wood-rotting fungi (Shekhar et al. 2006). Chitinase production is extensively distributed among species of

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Streptomyces corroborating its colonization in habitats like insects, compost, and soil (Kim et al. 2012; Jog et al. 2014). Similarly, proteases of S. phaeopurpureus has been reported for their biocontrol role against Colletotrichum coccodes (Palaniyandi et al., 2013), and Streptomyces sp.

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A6 against Fusarium udum (Singh and Chhatpar, 2011). Streptomyces also produce exo and endocellulases which inhibitsphytopathogens like oomycetes containing cellulose in their cell walls

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(van Bruggen and Semenov, 2000).

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Antimicrobial peptides

Actinobacteria are known for their ability to secrete diversity of bioactive metabolites having antimicrobial activity. Among these bioactive metabolites, bacteriocins represent a significant

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resource of drugs to overcome the antibiotic resistance acquired by the pathogens. Bacteriocins produced by bacteria are ribosomally synthesized and have antimicrobial properties. These are

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small in size, cationic or amphipathic and have antiagonistic effect non-related bacteria, protozoans, yeasts and fungi and viruses (Richard et al., 2015). Besides antimicrobial effect, bacteriocins also have anti-inflammatory, anti-allergic, antitumour and antinociceptive activities (Gomes et al., 2017). Bacteriocins are categorized into Class I, Class II and Class III based on their size and post translational modification. Class I bacteriocins are ribosomally synthesized, have

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size smaller than 10 kDa and are modified by post translational modifications. Class II bacteriocins are also smaller in size but do not undergo post translational modification whereas class III bacteriocins have size larger than 10 kDa but do not undergo post translational modification. Actinobacteria produce all types of bacteriocins but their production is very costly. So, recombinant antimicrobial peptides are being produced as an alternative to chemical synthesis. Streptomyces lividans has been proved as an effective heterologous host for the production of antimicrobial peptide over E. coli.

Genomic organization of actinobacteria The genomes of actinobacteria are circular or linear and contain plasmids. The first genome sequence of actinobacteria has been reported for Mycobacterium tuberculosis H37Rv and more than 20 species of actinobacteria have been sequenced (Ventura et al., 2007). The actinobacterial taxa

including

Actinomyces,

Amycolatopsis,

Actinoplanes,

Streptoverticillium,

and

Micromonospora has shown linear genome with size ranging from 7.7 Mb to 9.7 Mb and also contains large plasmids (Ventura et al., 2007). These genomes have been explored for gene

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clusters involved in production of secondary metabolites similar to the clusters reported in genome of S. coelicolor (Tiwari and Gupta, 2012). Another species including Salinispora tropica and S.

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arenicola have reported for the presence of genes encoding hydroxamate-type and phenolate-type siderophore (Wang et al., 2014). Ishikawa, 2008 reported that majority of the gene clusters

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involved in secondary metabolism have remained unexplored from actinobacteria. Therefore, genomic tools have gained more attention and proved as promising tools for exploring cryptic

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pathways responsible for antibiotic biosynthesis. Genome mining for cryptic antibiotic pathways

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Although decades back, mining of microbes and other organisms gave access to different metabolites. The identification of key biosynthetic enzymes was done manually either using nBLAST, pBLAST or PSI-BLAST or alignments of amino acid sequences in genome data. The

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clusters of genes were then explored on both side of hit sequence. Using MultiGeneBlast, the genome mining was further improved for analyzing operons containing cluster of genes. At present, this technique is only limited only to the identification of certain which are otherwise difficult to cover with automated tools (Weber & Kim, 2016). The developments in genome sequencing techniques and computation tools have brought a shift in paradigms and led to the

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discovery of automated genome mining techniques. The two variants of genome sequencing i.e. Pan-genome mining and genome mining were discovered. While the pan genome mining approach focuses on multiple strains from a specific species the genome mining targets only a specific strain of species. The former represents a global view of gene repertoire in a species across the strains and are of vital importance for studying the genetic diversity and their environmental adaptation. For example, the pan-genome analysis of a marine actinomycete of Salinispora representing 75 strains have shown the distributions, evolution of biosynthetic pathways and also provided insights

of chemical diversity among strains. On other side, the complete genome sequencing of several actinomycetes has shown an abundance of gene clusters which is far more than the metabolites characterized so far (Niu, 2018). The computational analysis of gene clusters involved in biosynthesis are based on “Metabolites to Genes” in comparison to “Genes to Metabolites” based approach. This retro approach enumerate the enzymatic reactions or biochemical transformations which generate a chemical moiety from precursor molecules and then identifies the potential enzyme responsible

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for catalyzing the proposed transformations (Khater et al., 2016). The computational tools in reconstruction of biosynthetic pathways have been reviewed by Khater et al., 2016 and Weber & Kim, 2016. The automated genome consortium databases such as such as antiSMASH,

IMG-ABC/

Integrated

Microbial

Genomes:

Atlas

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ClusterMine360, https://magarveylab.ca/, MIBiG, Orphan assembly line polyketide synthases, Biosynthetic

Gene

Clusters

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(http://www.secondarymetabolites.org/databases/) have identified PKS and NRPS clusters for biosynthetic products. These projects are helpful to assist researchers in linking several thousands

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of bioactive metabolites to their biosynthetic gene clusters. These databases, allow the researchers to deposit and retrieve the biosynthetic gene clusters data and also permit users to develop

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comparative and comprehensive analysis tools (Table 2). A comparative overview of actinobacteria which inlcudes Streptomyces albus J1074, Streptomyces griseoflavus Tu4000, Streptomyces bingchenggensis BCW-1, Micromonospora sp. ATCC 39149, Frankia sp., CcI3,

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Thermomonospora chromogena strain DSM 43794, Kineococcus radiotolerans SRS 30216, Kribbella catacumbae DSM 19601, Conexibacter woesei DSM 14684, Acidimicrobium ferrooxidans DSM 10331, Salinispora pacifica DSM 45543 is described in table 2. In general, high G+C content (~70%) and vast variations in the total genes are present in the gneome of actinobacteria (Table 2). Besides, gene clusters and their domain organizations, these databases

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also provide information of compound families of secondary metabolites. For example, the ClusterMine360 provides information of 290 gene clusters which are responsible for over 200 nonribosomal and polyketides biosynthesis. In additions, Integrated Microbial Genomes-Atlas of Biosynthetic gene Clusters like database (IMG-ABC) also provides genomic locus for gene clusters of metabolites. The other databases such as MIBiG, NP. Searcher, ClustScan, NRPSpredictor, antiSMASH integrated with tools like ClusterFinder, Prediction Informatics for Secondary Metabolomes (PRISM) permits to identification of gene clusters such as NRPS, PKS

or PKS-NRPS which are responsible for encoding novel class metabolites. These databases have helped in the identification of thio template domains, specific adenylation, acyltransferase and domains catalyzing tailoring reactions (Fig. 8). In comparison to this, Pep2Path faciltiatied the identification of the chemical structure of non-ribosomal peptides by matching mass spectra based on amino acids to their gene clusters. The gene cluster provides valuable informations about the arrangement of operon in a cluster like erythromycinencodes enzymes for L-mycarose and Ddesosamine biosynthesis (Oliynyk et al., 2007;). On the other side, the gene clusters for glycopeptide based antibiotics is known to contain

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enzymes for nonproteinogenic amino acids 4-hydroxyphenylglycine, β-hydroxytyrosine, and 3, 5dihydroxyphenylglycine for scaffolds assembly (Kahne et al., 2005). Genomics informations

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played vital role in the identification of cryptic/silentor orphan gene clusters. The impact of genome projects can be analysed by the facts that now actinomycetes are being considered as a

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powerhouse of antibiotics, contain a number of gene clusters and the most them are cryptic (Zazopoulos et al., 2003). Presently, more than 90% of these cryptic genes clusters involved in the

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biosynthesis of various metabolites have been explored in actinomycetes (Ishikawa, 2008). Zazopoulos et al. (2003) reported that majority of the clusters of gene in Streptomyces are silent in

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nature under normal environmental conditions. Genome mining has identified several hundereds of different molecules coelichelin, stambomycins, salinilactam, or streptocollin among actinomycetes (Zazopoulos et al., 2003). The genomics tool facilitates the mining of rare

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actinomycetes which are considered a reservoir for novel antibiotics. Although, these gene clusters are phenotypically silent, but can be expressed using nonstandard fermentation or gene/genome editing or a combination of both (Ishikawa, 2008; Tiwari & Gupta, 2012). These informations will of paramount importance in the area of synthetic biology for developing active gene clusters through genome editing and synthetic functional operon which are functional under lab conditions.

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Gene/genome editing for cryptic pathways Modern next generation genome sequencing has revealed the secondary metabolites biosynthetic pathways in the form of silent/cryptic gene clusters in actinobacteria. Therefore, this gene pool is promising source for the identification of novel compounds discovery and their related biosynthetic pathways, and enhanced production of various metabolites. The refactoring/rewiring the cognate biochemical pathways in the heterologous or natural host, genetic manipulation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPER)/cas or using synthetic

biology approaches is an attractive but challenging task. The heterologous studies may demand expression of one or complete operon or several genes either individually and in combination (Lazarus et al., 2014). In general, a heterologous study involves the identification of desired gene cluster from genomic databases followed by construct development for expression in suitable heterologous host and then characterization of related metabolite. The heterologous host based expression for different metabolites has been done (Ongley et al., 2013; Gomez-Escribano and Bibb, 2014; He et al., 2018).

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The genetic manipulation of silent/ cryptic gene clusters in Streptomyces is time consuming and costly. Both the disruption methods based on single crossover or double cross over integration have their own limitations (Cobb et al., 2015). Therefore, introduction of double-crossover

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integrants has been achieved in Streptococcus pyogenes in a single step using double-strand break (DSB) at the target site of genome via CRISPR/Cas tool box (Cobb et al., 2015). The CRISPR/Cas

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system which is known to act as a part of adaptive immunity in bacteria and archaea, where spacer sequence of already exposed bacteriophages, facilitate the Cas proteins in recognizing and cleaving

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the exogenous DNA specifically (Barrangou et al., 2007; Horvath and Barrangou, 2010). The adaptive immunity uses naive and primed adaptation. In general, the CRISPR/Cas consists of the

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nuclease Cas9, short RNAs target the site specificity through spacers which acts as recognition elements and, trans-activates short RNA which facilitates crRNA processing and recruitment to Cas9. In the naive stage, spacer is acquired from new foreign element whereas in primed stage,

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spacers are acquired from the invading cognate genetic elements (Qiu, et al., 2016). To harness the CRISPR/Cas system for genome editing, two variants of pCRISPomyces (pCRISPomyces1containing tracrRNA and CRISPR array cassettes and pCRISPomyces-2 with sgRNA expression array cassette and cas9) expression system has been developed for cognate biosynthetic pathways in three different Streptomyces species. Compared to other genome editing tool, the engineered

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CRISPR/Cas system has been found to work with unprecedented modularity (Cobb et al., 2015). Beside that multiple genome editing has been applied using pCRISPomyces system and multiple spacer sgRNA cassettes for a 31 kb biosynthetic gene cluster for undecylprodigiosin in Streptomyces lividans (Zhang et al., 2017). Till then, the CRISPR/Cas9 system in Streptomyces has been applied for different applications. The pKCcas9dO based CRISPR/Cas9 system in S. coelicolor M145 strain has been successfully used for one gene/ cluster of gene deletion and point mutation for converting Lys88 to to Glu in rpsL in S. coelicolor (Huang et al., 2015). Another

precision genome editing with 100% efficiency has been developed for reversible modulation of actinorhodin production in S. colicolor using a catalytically inactive Cas9 nuclease was able to target the promoter region or open reading frame of actIORF1 (Tao et al., 2018). The recent development is genome editing tools using synthetic biology have changed our approach of refactoring the cryptic gene clusters for enhanced production of target metabolites. The “Artificial Gene Operon assembly System” (AGOS) using synthetic biology and a derepressed promoter system for in Streptomyces is already has been developed. AGOS which is a

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plug-and-play system, uses a set of plasmids for entry where artificial gene operons are assembled by Red/ET-catalysed recombination. The biosynthesis of novobiocin has been successfully done using AGOS synthetic biology approach, and established for different precursors of novobiocin

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and then LC-MS and LC-MS/MS (Basitta et al., 2017). However, assembly of artificial operon is not a straightforward and it demands efforts. Based on the assembly line platform, two type of

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mechanisms have been proposed. The first restriction- ligation-based approach, deploys BioBrickTM, BglBrick, Master and Golden Gate tools. The second sequence homology-based

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tools, deploy assembly which is a sequence and ligation independent by using circular polymerase extension. Former approach uses cut and paste mechanism for the consecutive assembly of DNA

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fragments including transcription regulator elements, followed by coding sequences and terminators but it relies on the presence of internal restriction sites and leaves scar-sequence in joined DNA fragments in the assembly line (Basitta et al., 2017).

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Applications of Acitnobacteria metabolites

Like PGPRs, actinomycetes have also been reported for mineral acquisition, phytohormone production, inducing immunity of the plants in response to pathogen attack. Actinobacteria pose direct and indirect effects on plant growth promotion which includes mineral uptake, nitrogen

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fixation, siderophore synthesis and antibiotic production (Fig. 9). Studies on Micromonospora belonging to the class actinomycetes have been reported for plant growth promotion (MartinezHidalgo et al., 2014; Trujillo et al., 2014b). Another species of actinomycetes have beneficial effect for plant growth and antagonistic effect for plant pathogens (Bulgarelli et al., 2013). Actinomycetes secrete antibiotics and other bioactive molecules for preventing the pathogens to invade and establish in host plant. Several species of actinobacteria lives in association with the plants and are known to have nitrogen fixing capability. Studies have reported Corynebacterium sp. AN1 and Pseudonocardia dioxanivorans CB1190 as alternative nitrogenous fertilizers

(Mahendra S & Alvarez-Cohen, 2005; Giri and Pati, 2004). Although Streptomyces is reported as an effective root colonizer and can be used as a biocontrol agent, but no report is available for its use as plant growth promoting bacteria (Barka et al., 2016). Actinobacteria produce a variety of bioactive compounds and inhibit the growth of pathogenic microbes by secreting lytic enzymes, siderophores, antibiosis, nutrient competition, nitrous oxide production, and quorum quenching (Table 1; Fig. 9). Streptomyces griseorubiginouse has been reported for providing protection to the plant from Fusarium

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oxysporum f. sp. cubenese causing Fusarium wilt of banana (Getha et al., 2005). Several species of Streptomyces have been reported for cell wall degradation of phytopathogens by secreting lytic enzymes like chitinases (Xiao et al., 2002; Errakhi et al., 2007). Two bioformulations like

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actinovate has been commercialized which protects the plants against foliar and soil borne diseases.

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Members of actinobacteria such asStreptomyces species have emerged as a promising candidate, due to their biocontrol attributes against a number of plant pathogens. These organisms

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can be used as insecticides, herbicides, antifungal, and biocontrol as well as plant growth promotion. Approximately 60% of the insecticides and herbicides have been reported from

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different species of Streptomyces (Vurukonda et al., 2018). Macrotetrolides are example of insecticides, are commercialized from Streptomyces aureus and effective against mites, insects, coccidia, and helminthes (Barka et al., 2016). These macrotetrolides are composed of tetranactin,

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dinactin and trinactin. Tetranactin is an antibiotic having structure identical to cyclosporine and used as an emulsion for fruits and tea mites. Similarly, S. avermitilis is known for the production of ivermectin which is used as an antiparasitic agent (Barka et al., 2016). Different species of Streptomyces produce metabolites which may have antibacterial and antifungal activity. Streptomyces kasugaensis produce kasugamycin which act as inhibitor in the biosynthesis of

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proteins. This antibiotic has been reported for the management of fungal and bacterial diseases in crops. Similarly, polyoxins B and D produced from S. cacaoi var. asoensis having fungicidal effect on Rhizoctonia solani and other causative agents in crops. Enzymes and metabolites of Streptomyces Actinomycetes produce a variety of enzymes including protease, amylase, cellulose, lipase, pectinase, chitinase and xylanase with wide range of applications in medicine, bioremediation, food products and agriculture (Salwan and Sharma, 2018). Other enzymes like dextranase,

peroxidases, nitrile hydratase, laccases, alginate lyase and cutinase are also reported from actinomycetes (Mukhtar et al., 2017). Different species of Streptomyces are known for the production of cellulases (Kar and Ray, 2018), dextranases (Purushe et al., 2012), xylanases (Wei et al., 2019). Some species of Streptomyces are even used as alternative hosts for expressing proteins with high GC content and minimizing the inclusion bodies formation. These properties impart considerable potential as heterologous host in comparison to Escherichia coli. Apart from enzymes, actinomycetes have immense importance in secondary metabolism as they have well

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developed pathways for the biosynthesis of secondary metabolites, signaling molecules, antibiotics and antimicrobial peptides which protects the organism from harmful conditions and facilitate interactions with surroundings. Bioactive metabolites including mildiomycin, kasugamycin,

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hydroxamic acids, polyketides and chitinolytic enzymes are reported from different Streptomyces

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and other actinomycetes (Table 1). Conclusion

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Streptmyces are known to produce diverse array of natural products such as biologically active enzymes and secondary metabolites. These biomolecules are routinely used in human and therapeutics industries. Moreover, different metabolites are also known to act as signaling

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molecules for mediating microbe-microbe or host microbes interactions. The advent in ‘omics technologies, have led to a paradigm shift in natural product research. Previously, the only

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approach to identify new compounds was limited to grow the potential microorganism’s different varied conditions. Then using analytical and chemical methods were used to find access to compounds. The revolution in genomics approaches nowadays is emerging as a complementary tool for the identification of novel molecules. The recent developments in alternative approaches such as chemistry, genetics, next generation automated genome, pan-genome sequencing and

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metagenomics approaches have already played key role in mining the metabolites, their gene clusters of different secondary metabolites understanding and exploring the regulation of complex biosynthetic from different microbes (Palazzotto & Weber, 2018). This paradigm shift, led to development of different computational tools across the globe which were helpful to the researchers and played vital role in accelerating the product discovery (Weber & Kim, 2016). The “Metabologenomics” which combine genome sequencing followed by automated gene cluster annotation with metabolomics study using mass spectrometry are emerging as a new player in this field. The studies based on genes to metabolites or metabolites to genes, led to the

identification of biologically active compounds which were previously missing (Palazzotto & Weber, 2018). Moreover, the evolution and emergence of big genomics data is now gaining attention to harness the biosynthetic pathways of uncultured actinomycetes which are previsouly either under explored or explore the genome of cultured actinomycete for embedded biosynthetic pathways. To explore these microorganisms, integrated genomics- driven strategies are required which can connect the biosynthetic pathways to chemical entities for novel product discovery. The integration of genes to secondary metabolites and secondary metabolite to genes based on “Genes

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to Metabolites” using forward approach or “retro-biosynthetic approach” will be helpful in the identification of new secondary metabolites and simultaneously redesigning known biosynthetic pathways for the enhance production of novel compounds.

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The evolution in automated next generation of genomic approaches have provided big data repository for large number of PKS and NRPS clusters. Despite our success in decoding the

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mystery of these biosynthetic gene clusters using computational and genomic, the prediction of the core scaffold structure of a compound is still incomplete even for some of the well

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characterized secondary metabolites, due to either absence or gap in the implantation of biochemical knowledge. Further, the biochemical data require to train good models is very limiting

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in many cases to develop efficient machine learning-based tools followed by inaccurate prediction of gene cluster borders further need consistent efforts. Although it is in initial phase, the major challenges for metabolic engineering of these gene clusters using modern precision gene/genome

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editing, and artificial operon-based techniques will help in boosting the expression of different metabolites for human welfare. Conflict of Interests

The authors declared no conflict of interests.

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Acknowledgements

The authors are thankful to Chandigarh University for providing necessary infrastructure and SEED Division, Department of Science and Technology, GOI for providing financial benefits (SP/YO/125/2017) and (SEED-TIASN-023-2018) during the completion of this work. References

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staurosporine derivatives from the Streptomyces sp. NB-A13. Bioorganic Chemistry. 82, 33-

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Legends to Figures Fig. 1: Phylogenetic tree showing relationship of organisms among different families of actinobacteria based on whole genome alignment-free method. The tree was prepared by CVTree using composition vector approach. Fig.2: Symbiotic relation of plant beneficial actinobacteria. During early root colonization, the chemical acquaintance through root exudates such as flavonoid, amino acids, aromatic compounds

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and organic acids of associated hoist plant act as chemo-attractant and help in priming microbial populations in rhizosphere. Bacteria either colonize the host plant surface through the interaction of liposaccharides (LPS) or N-acetylglucosamine (NAG) present on their surface which interact

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with lactones or strigolactone or colonize internally through wounds on roots surface of host plants. For endophytic association, actinobacteria often produces cellulases, pectinases and xylanases.

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The using quorum sensing mostly γ-butyrolactone (GBL) system or rarely through N-Acyl homoserine lactone (AHL) for social communication, these microbes induces expression

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responsible for morphogenesis and secondary metabolites production. These metabolites are then directly or indirectly facilitate the plant growth and health.

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Fig. 3: Over view of γ-butyrolactone (GBL) system of actinobacteria. Here, signaling molecules (a) such as SCB1-SCB3, MMF1-MMF5, C4 homoserine lactone, A-factor, P1-factor interact with the cognate receptor (scb1) activate a series of events which lead to morphogenetic changes and

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secondary metabolite production. Alternatively, antibiotics such as actinorhodin and undecylprodigiosin after interaction with pseudo-receptors (JadP2/scbR2) can activate the same pathway (b).

Fig. 4: Brief history and structures of key antibiotics derived from actinobacteria. Fig. 5: Biosynthetic pathways of nystatin, lincomyacin, jadomycin, neomycin, streptomycin and

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tertracycline in actinobacteria.

Fig. 6: Biosynthesis of hydroxamates/desferrioxamine E (blue colour); and aerobactin siderophore (green colour). Both siderophores are derived from a basic precursor L-lysine. Fig. 7 (a): Biosynthesis of petrobactin (catecholate type). Details are mentioned in text. (b) Fesiderophore complex is imported into the cytoplasm SBP, permease, and ATPase systems during siderophore-shuttle and displacement mechanism without the reduction of iron. SBPs can bind to Fe3+-siderophores and apo-siderophores. Then SBP imports Fe3+- siderophore complex using

siderophore-shuttle and displacement mechanism. In shuttle exchange, SBPs exchange Fe3+ from Fe-siderophore complex to the initial bound apo-siderophore followed iron uptake. In displacement system, initially bound the apo-siderophore to the SBP is replaced by Fe3+ siderophore and followed by uptake. Fig. 8A: Pan genome analysis of Streptomyces albus J1074, Streptomyces griseoflavus Tu4000, Streptomyces bingchenggensis BCW-1, Micromonospora sp. ATCC 39149, Frankia sp., CcI3, Thermomonospora chromogena strain DSM 43794, Kineococcus radiotolerans SRS 30216,

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Kribbella catacumbae DSM 19601, Conexibacter woesei DSM 14684, Acidimicrobium ferrooxidans DSM 10331, Salinispora pacifica DSM 45543 B; Comparative overview of polyketides

in

the

genome

of

Streptomyces

sp

and

Frankia

sp

depicted

using

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https://magarveylab.ca/ database. KR- Ketoreductase, T-Thiolation, KT-ketosynthase, DHDhehydrotase in cluster 7 for polyketide containing ORF 1 and ORF2. C; Type-I modular

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biosynthetic cluster of genes for nystatin in Streptomyces noursei ATCC11455, D-type II PKS

of vancomycin in Amycolatopsis orientalis.

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oxytetracyline in Streptomyces rimosus, E-Hybrid NRPS PKS type III biosynthetic gene cluster

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Fig. 9: overview of applications of actinobacteria employed in human welfare.

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Acidimicrobium ferrooxidans DSM10331 Acidimicrobineae Rubrobacter xylanophilus DSM9941 Rubrobacterineae Conexibacter woesei DSM14684 Conexibacterineae Bifidobacterium bifidum BGN4 Bifidobacterium bifidum S17 Bifidobacterium bifidum PRL2010 Bifidobacterineae Bifidobacterium adolescentis ATCC15703 Bifidobacterium animalis ATCC25527 Bifidobacterium asteroides PRL2011 Propionibacterium acnes ATCC11828 Propionibacterium acnes TypeI A2 P acn17 Propionibacterineae Propionibacterium acnes C1 Propionibacterium_acnes 266 Propionibacterium acidipropionici ATCC4875 Arcanobacterium haemolyticum DSM20595 Salinispora tropica CNB440 Micromonosporineae Stackebrandtia nassauensis DSM44728 Glycomycineae Streptomyces davawensis JCM4913 Streptomyces venezuelae ATCC10712 Streptomyces fulvissimus DSM40593 Streptomycineae Streptomyces pratensis ATCC33331 Streptomyces bingchenggensis BCW1 Streptomyces rapamycinicus NRRL5491 Catenulisporineae Catenulispora acidiphila DSM 44928 Streptosporangium roseum DSM43021 Streptosporangineae Thermomonospora curvata DSM43183 Kribbella flavida DSM17836 Propionibacterineae Frankia sp. CcI3 Frankineae Pseudonocardia dioxanivorans CB1190 Corynebacterineae Actinosynnema mirum DSM43827 Mycobacterium tuberculosis H37Rv Arthrobacter phenanthrenivorans Sphe3 Arthrobacter chlorophenolicus A6 Arthrobacter aurescens TC1 Micrococcineae Arthrobacter arilaitensis Re117 Clavibacter michiganensis NCPPB382 Clavibacter michiganensis NCPPB2581 Kineococcus radiotolerans SRS30216 Kineosporineae Tropheryma whipplei uid57705 Tropheryma whipplei TW0827 Cryptobacterium curtum DSM15641 Coriobacterineae

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Enzymatic, wound etc.

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Population build up

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N-acetylglucosamine (NAG)

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Bacteria

Lectins

strigolactone

host plant

Endophytes or rootScolonization through biofilm formation

γ-butyrolactone (GBL) or acylated homoserine lactones (AHL) activation

Direct plant benefits

Plant pathogen suppression

a. Competition • Space and nutrients b. Pathogen suppression • Antibiotics production S • Cell wall degrading enzymes • Antimicrobial peptide production • Quorum sensing inhibition (QSI) through quorum sensing peptides and lytic enzymes

Liposaccharides ( LPS)

autoinducers

Plant

a. Plant growth promotion • Growth regulators b. Mineral acquisitions • Siderophores growth promotion • Phytases • Organic acids Plant immunity

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Catecholate pathway

apo-siderophore

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Chorismate

apo-(aryl-carrier protein) Protocatechuate

3,4-dihydroxybenzoyl holo-(aryl-carrier protein)

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Spermidine

SBP

SBP

Fe3+

Citrate

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Holo-(aryl-carrier protein)

Fe3+

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TCA cycle

Fe3+

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Fe3+ Fe3+

SBP Fe3+

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N-citryl-spermidine

N-N-citryl-bis spermidine

3,4-dihydroxybenzoyl-N-citryl-spermidine

3,4-dihydroxybenzoyl-N-citryl-spermidine

petrobactin

Fig. 7

(b)

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Table 1: Selected examples of metabolites obtained from different actinomycetes and their role in biotechnological and agricultural sectors Role Isolate Metabolite Application Reference Antibiotics Streptomyces sp. SD85 sceliphrolactam medicine Srivastava et al., 2018 Streptomyces sp. NB-A13 staurosporine antifungal and hypotensive Zhou et al., 2019 activity, anticancer agents Streptomyces sp. ZZ406 anticancer drugs inhibiting the proliferation Chen et al., 2018 of glioma cells Ivermectin antiparasitic agents Omura and Crump, S. avermitilis 2014 Planosporicin antibacterial Sherwood et al., 2013 Planomonospora alba activity against Gram +ve Actinocarbasin activity against Igarashi et al., 2011 Actinoplanes ferrugineus MRSA Plant growth Corynebacterium sp. substitute for Plant growth promotion Giri and Pati, 2004 promotion nitrogenous fertilizer Nitrogen fixation/Biocontrol Mahendra and Pseudonocardia dioxanivorans agent Alvarez-Cohen, 2005 Streptomyces sp. Plant growth promotion/ ; Barka et al., 2016 Biocontrol agent Micromonospora sp. plant growth promotion/ Martinez-Hidalgo et Biocontrol agent al., 2014; Trujillo et al., 2015 Streptomyces sp. Strain MBCN152-1 biocontrol potential against Hassan et al., 2017 Alternaria brassicicola S. lydicus WYEC 108 Actinovate soluble biofungicide Sousa and Olivares, 2016 Enzymes Streptomyces sp. H1 CMCase, xylanase, degradation of the Wei et al., 2019 Micromonospora sp. G7 lignin peroxidase, recalcitrant lignocellulose Saccharomonospora sp. T9 55

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manganese peroxidase, laccase endo-β-1,4glucanase from

S. griseus

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S. sporocinereus OsiSh-2

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Streptomyces sp. MBRL 10

hydroxamate type of siderophore

Micromonospora sp. Streptomyces spp.

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Signaling molecules VOCs

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Thermobifida fusca

Siderophore

Baeyer-Villiger monooxygenases (BVMOs) AA10 family LPMO chitin-active LPMO/Cellulase GH18 chitinase, albeit with low activity siderophore

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S. leeuwenhoekii C34

Thermobifida fusca

Streptomyces sp. MBRL 10

enhanced hydrolysis of hemicellulose into monosaccharides biocatalyst

Cecchini et al., 2018

chitin degradation

Nakagawa et al., 2015

degrade cellulosic biomass

Gaber et al., 2016; Moser et al., 2008 Gaber et al., 2016

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Streptomyces sp. G12

Vocs-alcohols, esters, acids, alkanes, ketones and alkenes VOCs

Degradation of polymeric substrates like colloidal chitin and chitosan antagonistic activity towards Magnaporthe oryzae antagonism against fungal pathogens Induction of jasmonateregulated defenses Plant disease suppression and direct antifungal activity, Modulate antibiotic production in other bacteria, protection against Rhizoctonia solani

Gran-Scheuch et al., 2018

Zeng et al., 2018 Tamreihao et al., 2018 Martínez-Hidalgo et al., 2015 Venkataraman et al. 2014; Cordovez et al. 2015)

Tamreihao et al., 2018

56

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Table 2 Genomic annotation of actinobacteria for genes/gene clusters involved in biosynthesis of bioactive metabolites

*Acylaseacylase I

6.6

G+ C content (%) Protein-coding genes

73.1 5738

Metallo-β-lactamase superfamily α/β-Hydrolase superfamily Amidohydrolase family

Cyt P450 monooxygenase

Chitinases/GH18 Glucanases Serine Proteases Amylases Polyketides Non ribosomal peptides Type II polyketide Polyketide+non-ribosomal Unknown thiotemplated Pentangular polyphenol Enediyne

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*Cell wall degrading enzymes

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Short-chain dehydrogenase *Oxidoreductase

**Gene clusters for biosynthesis of secondary metabolites

21 2 5 8 0 8 4 8 8 15 7 2 12 0 0 0

Streptomyces bingchenggensis BCW-1 NC_016582

Micromonospora sp. ATCC 39149

Frankia sp. CcI3

ACES01000001

MBLM01000001

11.9

6.5 72.3

5.4 70.1

9,262

5891

5021

32

19 4

6 3

4

4

10

3

0

0

8 10 11 12 10 7 0 0 5 0 0

1 3 3 3 5 3 2 0 0 0 0

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Genome size (Mb)

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ABYC01000001

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*Lactonases

Genome features Genome accession

Streptomyces griseoflavus Tu4000 ACFA0100000 1 7

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Streptomyces albus J1074

71.7 6520

26 3

12 4 12 5 31 0 8 6 11 11 3 8 0 0 1 0

0 9 12 15 11 43 10 0 0 8 1 0

57

Contd./-

1

24

119

0

of

Biosynthetic gene clusters

1

*Cell wall degrading enzymes

6.3 72.7 5963

2.1 68.3 2161

Salinispora pacifica DSM 45543 AQZB0100000 1 5.4 69.6 5085

5.6

24

40

24

5

14

13

20

20

2

4

4

7

8

24

2

7

23

32

22

1

6

71.6 5063

10 5

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Acidimicrobiu m ferrooxidans DSM 10331 NC_013124

4.9 74 4671

Short-chain dehydrogenase Cyt P450 monooxygenase

11

Chitinases/GH18 Glucanases Serine Proteases Amylases Polyketides Non ribosomal peptides Type II polyketide Polyketide+nonribosomal Unknown thiotemplated cluster Pentangular polyphenol

2 2 5 5 4 1 0

0 2 1 4 9 0 0 0

1 11 1 14 7 3 3 0

1 4 2 6 3 1 2 0

0 3 0 3 2 1 0 0

0 5 4 9 5 -

1

0

2

0

0

-

2 2

1 0

0 0

1 0

1 0

-

Jo

**Gene clusters for biosynthesis of bioactive metabolites

Amidohydrolase family

Conexibacte r woesei DSM 14684 NC_013739

lP

*Acylaseacylase I *Oxidoreductase

Kineococcus radiotolerans SRS 30216 NC_009664

ur na

*Lactonases

Genome size (Mb) G+ C content (%) Protein-coding genes Metallo-β-lactamase superfamily α/β-Hydrolase superfamily

Thermomonospora chromogena strain DSM 43794 FNKK01000001

-p

Genome features Genome accession

Kribbella catacumbae DSM 19601 AQUZ0100000 1 7.6 70.6 7124

re

Table 2 Genomic annotation of selected actinobacteria for genes or gene clusters

1

58

ro

of

Enediyne 1 0 0 0 0 Biosynthetic gene clusters 0 46 3 57 17 49 *Annotations for genes including Metallo-β-lactamase superfamily, α/β-Hydrolase superfamily, Phosphotriesteraselike lactonase (PLL); amidohydrolase, Short-chain dehydrogenase, Cytochrome P450 monooxygenase, Chitinases/GH18, Glucanases, Serine Proteases and Amylases was done by submitting the genomes in RAST server.

Jo

ur na

lP

re

-p

**Predictions for gene clusters for the biosynthesis of bioactive metabolites were done by submitting whole genomes in PRISM server.

59