**1. Introduction**

The term secondary metabolites (SMs) was first mentioned in 1891 by A. Kossel. Microbial secondary metabolites have attracted the scientific world's attention, since the discovery of penicillin in 1940s. After that, the identification and characterization of SMs have reached the highest level between 1940s and 1960s, and this period is called as "the golden era of SMs discovery" [1, 2]. A lot of compounds have been characterized and reported during the golden era and are still utilized till now. Unfortunately, the discovery of approved novel chemical scaffolds of secondary metabolites has significantly decreased after the golden era [1]. The possible explanation of the decreasing in the SMs' identification might be due to the following: (1) using the biosynthetic modules that are used for SMs' production

by many bacteria, (2) focusing on some specific group of microorganisms such as actinobacteria, resulting in the isolation and identification of known compounds, and (3) almost 1% of the microbial community can be cultured in the laboratory [2] due to the difficulty in identifying their optimal medium compositions, resulting in the majority of SMs not being identified.

Microbial secondary metabolites play significant roles in our life. Because of SMs' unusual chemical structures, the microbial secondary metabolites show a variety of biological activities such as antimicrobial agents, antitumor agents, enzyme inhibitor, immunosuppressive agents, antiparasitic agents, herbicides, anthelmintic and food industry, etc. For instance, one of the huge successes in human medicine is the discovery of immunosuppression, such as cyclosporine A, which plays a significant role in establishing the organ transplant field.

All biochemical reactions carried out by organisms are called as metabolism and all products resulting from metabolism are called as metabolites. As a result of metabolism reactions, organisms produce primary and secondary metabolites. Primary metabolites are found in all living cells that are able to divide, while secondary metabolites are present only incidentally and do not affect the organism's life immediately. Nowadays, over 2,140,000 secondary metabolites (SMs) have been identified based on their vast diversity in function, structure, and biosynthesis (**Table 1**) [3]. The major sources of SMs are plants (about 80%) and microorganisms. Among microorganisms, bacteria, especially actinobacteria, and fungi have been reported to produce the majority of SMs that have been identified till now [4].

Microbial secondary metabolites (SMs) such as antibiotics, alkaloids, toxins, pigments, growth hormones, antitumor agents, and others are low molecular mass products that are produced by microorganisms, usually during the late growth phase. In fact, microbial secondary metabolites are not essential for the growth and development of microorganisms that produce them but are associated with some other functions such as competition, interactions, defense, and others [3, 5].

The development and advances of omics-based techniques such as genomics, metabolomics, proteomics, and trascriptomics have revealed that microorganisms have the potential to produce more secondary metabolites than were originally expected [6, 7]. These products are often coded by clustered genes present on the chromosomal DNA and rarely on plasmid DNA. In fact, most of these new SMs


**3**

*2.1.1 Gene cloning*

*Enhancement and Identification of Microbial Secondary Metabolites*

have been predicted only by using bioinformatics analysis, which analyzes the putative SMs gene clusters in a sequenced genome. This is because, all of the new revealed SMs are not produced naturally under the lab conditions, or even though they are produced, this in very low amount that the traditional detection techniques are unable to detect them [8, 9]. Metabolomics approach aims to discover and characterize secondary metabolites in natural or engineered biosystems, and it can measure as many low molecular weight compounds as possible. Metabolomicsbased technologies such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been identified as significant analytical methods to detect SMs produced under specific conditions [10]. The present chapter provides an overview of present-day metabolomic and genetic engineering approaches for secondary

Biosynthesis gene clusters (BGCs) are the genes associated with the biosynthesis of secondary metabolites. These BGCs include all genetic information necessary for SMs' biosynthesis, assembly, modification, and regulation of their export and transport [11]. Microorganisms' genome contains variety of cryptic or silent genes that are responsible for the production of secondary metabolites but are not expressed under laboratory conditions. It has been reported that most BGCs remain silent and cannot be fully expressed under standard laboratory conditions. These silent BGCs are potentially significant in the discovery of novel SMs [11–14].

Due to the development of genomic and bioinformatic field, we are able to access extensive sequencing data and genetic information and enable genome mining of relevant BGCs with the potential for valuable SM production [15]. Therefore, biosynthetic biology and genetic engineering tools are now utilized for identification of novel BGCs. In fact, genetic engineering is now widely used and moving beyond traditional tools, which has opened a new era in the detection of novel secondary metabolites [16]. Genetic engineering for the production of SMs can be carried out in heterologous as well as homologous host. In fact, gene manipulation in heterologous host enables the activation of biosynthesis gene clusters (BGCs) obtained from unculturable organisms, whereas gene manipulation in homologous host allows the retention of all natural factors essential for the production of secondary metabolites [17]. While there is no single approach that will work for all genes of interest, a variety of techniques have been developed to induce the expression of these genes. In fact, several genome techniques have emerged and are utilized in the metabolomic production field, including transcriptional activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9) [18, 19]. Each genome engineering technology has its own advantages and disadvantages (**Table 2**). For instance, ZFNs and TALENs have been successfully utilized in various microbes but still have limitation which includes the difficulty to engineer them [20]. Recently, CRISPR-Cas9 has been reported to be a significant and promising genome editing technology in the

Direct cloning of the entire BGCs into the heterologous host is the most general

and widely used approach for the activation of silent BGCs. Nowadays, many

*DOI: http://dx.doi.org/10.5772/intechopen.93489*

metabolites' enhancement and identification [11].

**2.1 Gene editing for metabolites discovery**

discovery and production of SMs [17, 16, 21].

**2. Genomic for screening and enhancement of SMs**

#### **Table 1.**

*Approximate number of identified natural metabolites.*

*Enhancement and Identification of Microbial Secondary Metabolites DOI: http://dx.doi.org/10.5772/intechopen.93489*

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

the majority of SMs not being identified.

role in establishing the organ transplant field.

the majority of SMs that have been identified till now [4].

by many bacteria, (2) focusing on some specific group of microorganisms such as actinobacteria, resulting in the isolation and identification of known compounds, and (3) almost 1% of the microbial community can be cultured in the laboratory [2] due to the difficulty in identifying their optimal medium compositions, resulting in

Microbial secondary metabolites play significant roles in our life. Because of SMs' unusual chemical structures, the microbial secondary metabolites show a variety of biological activities such as antimicrobial agents, antitumor agents, enzyme inhibitor, immunosuppressive agents, antiparasitic agents, herbicides, anthelmintic and food industry, etc. For instance, one of the huge successes in human medicine is the discovery of immunosuppression, such as cyclosporine A, which plays a significant

All biochemical reactions carried out by organisms are called as metabolism and all products resulting from metabolism are called as metabolites. As a result of metabolism reactions, organisms produce primary and secondary metabolites. Primary metabolites are found in all living cells that are able to divide, while secondary metabolites are present only incidentally and do not affect the organism's life immediately. Nowadays, over 2,140,000 secondary metabolites (SMs) have been identified based on their vast diversity in function, structure, and biosynthesis (**Table 1**) [3]. The major sources of SMs are plants (about 80%) and microorganisms. Among microorganisms, bacteria, especially actinobacteria, and fungi have been reported to produce

Microbial secondary metabolites (SMs) such as antibiotics, alkaloids, toxins, pigments, growth hormones, antitumor agents, and others are low molecular mass products that are produced by microorganisms, usually during the late growth phase. In fact, microbial secondary metabolites are not essential for the growth and development of microorganisms that produce them but are associated with some other functions such as competition, interactions, defense, and others [3, 5].

The development and advances of omics-based techniques such as genomics, metabolomics, proteomics, and trascriptomics have revealed that microorganisms have the potential to produce more secondary metabolites than were originally expected [6, 7]. These products are often coded by clustered genes present on the chromosomal DNA and rarely on plasmid DNA. In fact, most of these new SMs

**Source All known compounds Bioactive** Plant kingdom 600,000–700,000 150,000–200,000 Microbes Over 50,000 22,000–23,000 Higher plants 500,000–600,000 ~100,000 Animal kingdom 300,000–400,000 50,000–100,000 Protozoa Several hundreds 100–200 Vertebrates 200,000–250,000 50,000–70,000 Marine animals 20,000–25,000 7000–8000 Invertebrates ~100,000 NA Algae, lichens 3000–5000 1500–2000 Insects, worms 8000–10,000 800–1000

**2**

**Table 1.**

*NA—data not available. Source: Bérdy [4].*

*Approximate number of identified natural metabolites.*

have been predicted only by using bioinformatics analysis, which analyzes the putative SMs gene clusters in a sequenced genome. This is because, all of the new revealed SMs are not produced naturally under the lab conditions, or even though they are produced, this in very low amount that the traditional detection techniques are unable to detect them [8, 9]. Metabolomics approach aims to discover and characterize secondary metabolites in natural or engineered biosystems, and it can measure as many low molecular weight compounds as possible. Metabolomicsbased technologies such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been identified as significant analytical methods to detect SMs produced under specific conditions [10]. The present chapter provides an overview of present-day metabolomic and genetic engineering approaches for secondary metabolites' enhancement and identification [11].
