Background

Actinomycetes, particularly those belonging to the genus Streptomyces, are a prolific source of bioactive secondary metabolites with significant applications in medicine, agriculture, and industry. Among them, Streptomyces species have garnered attention for their potential to produce novel antimicrobial, anticancer, and even plant growth-promoting compounds [1]. Microbial strains offer a sustainable and environmentally friendly alternative to synthetic agrochemicals, playing a crucial role in biocontrol and plant health enhancement [2]. Traditional methods for isolating Streptomyces from environmental sources involve selective culturing techniques using specific growth media, followed by morphological and biochemical characterization [3]. However, recent advances in molecular biology and whole genome sequencing (WGS) have revolutionized the identification and functional analysis of these microorganisms [4]. WGS enables comprehensive insights into the genetic architecture of Streptomyces, facilitating the discovery of biosynthetic gene clusters (BGCs) responsible for secondary metabolite production [5]. This genomic approach overcomes the limitations of conventional screening methods, which often fail to detect cryptic or silent gene products under standard laboratory conditions [6]. The protocol described herein focuses on the isolation, genome sequencing, and functional characterization of Streptomyces sp. VITGV100, an endophytic actinomycete strain isolated from Lycopersicon esculentum (tomato) plants [5]. Compared to traditional bioactivity-guided screening, this genomic approach enhances the efficiency of novel bioactive compound discovery by directly linking genetic information to metabolite production [7]. A key advantage of this protocol is its ability to systematically identify and analyze BGCs involved in secondary metabolite biosynthesis using tools such as antiSMASH [8] and ARTS [9]. This computational approach significantly accelerates the identification of novel antibiotics [10]. Furthermore, comparative genomic analyses using ANI and dDDH facilitate the taxonomic positioning and evolutionary relationships of the newly isolated strain, contributing to a deeper understanding of Streptomyces diversity and genetic variability [11]. Beyond antibiotic discovery, the protocol has broad applications in biotechnology and agriculture. Endophytic Streptomyces strains have been reported to enhance plant growth through phytohormone production, nitrogen fixation, and antagonism against phytopathogens [12]. In summary, this protocol represents a comprehensive strategy for isolating, characterizing, and harnessing the biosynthetic potential of endophytic Streptomyces strains.