Metabolomics and Genetic Engineering for Secondary Metabolites Discovery

*Ahmed M. Shuikan, Wael N. Hozzein, Rakan M. Alshuwaykan and Ibrahim A. Arif*

### **Abstract**

Since 1940s, microbial secondary metabolites (SMs) have attracted the attention of the scientific community. As a result, intensive researches have been conducted in order to discover and identify novel microbial secondary metabolites. Since, the discovery of novel secondary metabolites has been decreasing significantly due to many factors such as 1) unculturable microbes 2) traditional detection techniques 3) not all SMs expressed in the lab. As a result, searching for new techniques which can overcome the previous challenges was one of the most priority objectives. Therefore, the development of omics-based techniques such as genomics and metabolomic have revealed the potential of discovering novel SMs which were coded in the microorganisms' DNA but not expressed in the lab or might be produced in undetectable amount by detecting the biosynthesis gene clusters (BGCs) that are associated with the biosynthesis of secondary metabolites. Nowadays, the integration of metabolomics and gene editing techniques such as CRISPR-Cas9 provide a successful platform for the detection and identification of known and unknown secondary metabolites also to increase secondary metabolites production.

**Keywords:** metabolomics, genetic engineering, secondary metabolites identification, genomic, CRISPR-Cas9, production of secondary metabolites, microorganisms, gene editing

### **1. Introduction**

Since the discovery of penicillin in the 1940s, microbial secondary metabolites (SMs) have attracted the attention of scientists all over the world. In fact, penicillin discovery has been shown to be a promising solution for many kinds of infections. As a result, the scientific world starts to search for other products that are produced by microbes that can be utilized for treating a different disease or can be useful for any aspect of our life. Therefore, the period between the 1940s – 1960s The "golden period of SM discovery" [1, 2] is referred to as "the golden era of SM discovery." During the golden era, several SMs were discovered, characterized, and reported, and they are still used today. Unfortunately, after the golden era, the development of authorized novel chemical

scaffolds of secondary metabolites has declined dramatically [1] the decrease in the microbial secondary metabolites detection and identification could be due to 1) almost 99% of the microbial community unculturable [2], due to the difficulty to identify their optimal medium compositions, which means that the majority of SMs are definitely unidentified, 2) the scientists have been focused on specific groups of microorganisms such as *Actinobacteria* which resulting to the identification of known compounds and do not develop a new methodology for screening in other microorganisms.

All biochemical reactions carried out by organisms is called metabolism and all products resulting from metabolism is called metabolites. In fact, there are two kinds of metabolites resulting from the biochemical reactions that are called primary and secondary metabolites. The difference between primary and secondary is that primary metabolites are found in all living cells able to divide while secondary metabolites are present only incidentally and are not affect the organism's life immediately. Microbial SMs is low molecular mass products with an unusual chemical structure that are produced by microorganisms usually during the late growth phase and are not essential for the growth and development of the microbe but are associated with some other functions such as competition, interactions, defense, and others [3, 4]. In fact, SMs have shown a variety of biological activities that can be utilized in different aspects such as antitumor agents, immunosuppressive agents, antimicrobial agents, antiparasitic agents, anthelmintic, and food industry etc. An example for the importance of SMs in our life is the discovery of immunosuppression such as cyclosporine A, which plays a significant role in establishing the organ transplant field.

Nowadays, Over 2 million SMs have been found based on their vast diversity in structure, function, and biosynthesis (**Table 1**). Plants (about 80%) and microbes (approximately 20%) are the primary sources of secondary metabolites discovered [3]. Actinobacteria and fungi have been found to create the bulk of SMs discovered to date [5]. Nowadays, omics-based techniques such as genomics, metabolomics, proteomics, and transcriptomics have overcome the problem of


#### **Table 1.**

*Approximate number of identified natural metabolites.*

#### *Metabolomics and Genetic Engineering for Secondary Metabolites Discovery DOI: http://dx.doi.org/10.5772/intechopen.102838*

identification of unculturable microbes and have revealed that microorganisms have the potential to produce more secondary metabolites than were originally expected [6, 7]. By conducting omics techniques scientists were able to detect SMs that are coded by clustered genes present on chromosomal DNA directly without doing microbial culturing.

Due to the development of the genomic and bioinformatic field, scientists are now able to access extensive genetic information and enable genome mining of relevant Biosynthesis gene cluster (BGCs) with the potential for valuable SM production [8]. therefore, genetic engineering has now become widely used and moving beyond traditional tools which open a new era in the detection of novel secondary metabolites [9]. In fact, by using bioinformatic analysis that analyzes the putative secondary metabolites genes cluster in the sequenced genome, scientists were able to predict new SMs that were not identified by using traditional techniques because all new revealed SMs are not produced naturally under the lab conditions or even though produced but in very low amount that the traditional techniques were unable to identify them [10, 11]. Metabolomics aims to characterize and identify SMs in natural and engineered biosystems.

Metabolomics based techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) is accurate that can measure as low molecular weight compounds as possible. In fact, mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been reported as significant analytical techniques to detect secondary metabolites under specific conditions [12]. This chapter provides an overview of metabolomics and genetic engineering techniques especially the CRISPR-Cas9 technique for the discovery and production enhancement of microbial secondary metabolites.

### **2. Genetic engineering for SMs detection**

The genes associated with the biosynthesis of secondary metabolites is named biosynthesis gene cluster (BGCs). In fact, BGCs include all genetic information required for secondary metabolites regulation, assembly, modification, and biosynthesis [13]. As mentioned previously, not all microorganisms can be cultured in the laboratory resulting in not all SMs can be expressed by using traditional techniques (culturing and detection) also a lot of microbes contains silent or cryptic genes in their genome that are responsible for the production of secondary metabolites. In fact, these silent BGCs have potentially significant in the discovery of novel secondary metabolites [13–16].

Nowadays, instead of traditional detection techniques, genetic engineering tools are utilized for the identification of novel biosynthesis gene cluster BGCs [9]. However, genetic engineering can be used in both heterologous and homologous hosts. While gene manipulation in a homologous host allow the retention of factors necessary for the production of SMs, also gene manipulation in a heterologous host enable activation of BGCs obtained from unculturable microorganism [17].

In fact, a variety of genetic engineering techniques have been developed in order to induce the expression of all genes of interest. Therefore, in metabolomic production field, several genome techniques have been utilized in order to detect and enhance secondary metabolites production such as clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9), zinc finger nucleases (ZFNs), and transcriptional


#### **Table 2.**

*Comparing different genomic engineering techniques used in metabolomics.*

activator-like effector nucleases (TALENs) [18, 19]. While each technique has its advantages and disadvantages (**Table 2**), CRISPR-Cas9 has been reported to be the most promising and significant technique that can be used in the discovery and enhancement of SMs production [9, 17, 20, 21].

#### **2.1 Gene insertion/deleting**

Gene insertion or deletion is useful not only in biosynthesis gene clusters activation but also for novel SMs discovery [22]. In fact, several silent biosynthesis gene clusters have been refactored by replacing the biosynthesis gene clusters promoter to yield natural products such as secondary metabolites [23–26].

Nowadays, the promising technique has been developed in the genetic engineering field that is multiplexed CRISPR-Cas9 and transformation-associated recombination (TAR)-mediated promoter engineering method (mCRISTAR) [21, 27–30]. mCRISTAR actually combined the advantages of TAR technique and CRISPR-Cas9 technique. Basically, mCRISTAR mode of action is that CRISPR-Cas9 breaks the double-stranded in the promoter region of the biosynthesis gene cluster (BGCs), then the fragments produced are reassembled by TAR with synthetic gene-cluster specific promoter cassettes [21].

#### **2.2 Gene cloning**

Basically, gene cloning consists of some steps include 1) determining the suitable heterologous host 2) cloning the target gene, 3) transferring the gene into the suitable host, 4) expression of the gene in the suitable host system, 5) optimization of production [31].

However, many new and useful cloning techniques have been introduced such as transformation assisted recombination (TRA), Cas9-assisted targeting of chromosome segments (CATCH), and TAR-CRISPR [20, 32, 33]. CATCH is a cloning tool that uses the CRISPR-Cas9 system for direct BGCs cloning into the host. However, compared to PCR and restriction enzyme cloning techniques, CATCH is appeared to be more useful for direct cloning of large genes clusters. Whether, TAR technique has been utilized for about a decade in the cloning of large BGCs, but the TAR technique is associated with low cloning efficiency [20, 33]. To address this challenge TAR and CRISPR-Cas9 have been coupled resulting in a new approach called TAR-CRISPR [33]. Therefore, TAR-CRISPR is different than mCRISTAR as discussed earlier. It is yeast-based method, while mCRISTAR uses CRISPR-Cas9 to breaks the double-stranded in the promoter region of the BGC, and the fragments produced are reassembled by TAR with synthetic gene-cluster specific promoter cassettes. As a result, by coupling CRISPR with TAR significant increase of clone efficiency has been reported [33]. In fact, TAR-CRISTAR cloning will allow for the development of BGC cloning and SM production in the future.

While gene-editing techniques play a significant role in the detection and production of microbial secondary metabolites, metabolomics is also important in the identification and characterization of secondary metabolites produced by native or genetically modified microorganisms.
