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

#### **2.1 Gene editing for metabolites discovery**

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

#### *2.1.1 Gene cloning*

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


#### **Table 2.**

*Different genomic engineering techniques used in Metabolomic.*

new cloning tools have been introduced, including Cas9-assisted targeting of chromosome segments (CATCH), transformation-assisted recombination (TAR), and TAR-CRISPR [22–24]. Basically, gene cloning steps include: determining the suitable heterologous host, cloning of the target BGC, transfer of the BGC into the chosen host, expression in chosen host system, and optimization of production.

**5**

*Enhancement and Identification of Microbial Secondary Metabolites*

Cas9-assisted targeting of chromosome segments (CATCH) is a cloning technique that utilizes the CRISPR-Cas9 gene editing system for direct BGCs cloning into the host. Comparing with traditional cloning tools such as PCR and restricted enzymes, CATCH is predicted to become a useful molecular tool for direct cloning of large gene clusters. Transformation-assisted recombination (TAR) has been used for cloning of large BGCs for about decades. However, the TAR approach is associated with a low cloning efficiency, which means it requires screening of hundreds of colonies to detect few positive clones [22, 24]. To address this challenge, TAR and CRISPR-Cas9 have been coupled resulting in a new approach called TAR-CRISPR [24]. By coupling TAR with CRISPR, a significant increase of the clone efficiency has been reported. Comparing with traditional TAR cloning, the advantages of TAR-CRISPR are that the positive clones could be achieved with secondary screening and lesser manpower and also it does not require a high experience of working with yeast [24]. In fact, the TAR-CRISTAR cloning will allow for the development of BGC cloning and SM production in the future.

Gene refactoring or replacement is useful not only in BGCs' activation but also for novel SMs' discovery. In fact, several silent BGCs have been refactored by replacing the BGC promoter to yield natural products such as secondary metabo-

Another new tool in gene refactoring is multiplexed CRISPR-Cas9- and transformation-associated recombination (TAR)-mediated promoter engineering method (mCRISTAR) [21]. This new tool combines the advantages of the CRISPR-Cas9 system and TAR. It is different than the TAR-CRISPR that was discussed earlier. Comparing with TAR-CRISPR, which is a yeast-based method, basically mCRISTAR uses CRISPR-Cas9 to break 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. Another gene refactoring tool that has aided in the faster cloning and refactoring of BGCs is the direct pathway cloning (DiPaC). Direct pathway cloning (DiPaC) depends on PCR amplification and in vitro DNA assembly for biosynthesis gene cluster capture and their expression. DiPaC was recently employed for the capture of biosynthesis gene cluster, which is small in size, followed by their activation and expression of novel natural products [29]. DiPaC was also able to successfully clone mid and large size of BGC [29].

A large number of researches have documented the effect of gene knockout/in on BGC expression or levels of SM production. However, conventional methods of gene editing are time-intensive, while CRISPR-Cas9-based approach allows for much faster and efficient gene editing [30]. The emergence of CRISPR-Cas9 has opened up a new era in gene editing opportunities [31]. Recently, CRISPR gene editing approach has been used to insert promoter in order to activate microor-

Nowadays, CRISPR-Cas9 is used to introduce promoter at multiple BGCs, and at the same time, resulting in the activation of BGCs followed by the production of SMs [32]. Multiplexed site-specific genome engineering (MSGE) was also used for multiple BGCs' editing [33]. MSGE has led to a significant increase in the secondary

While, gene editing approaches provide a significant platform to manipulate the genetic machinery of microbes toward the production of novel, natural secondary

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

*2.1.2 Gene refactoring*

*2.1.3 Gene insertion or deletion*

ganisms' SMs' production [32].

metabolites' production.

lites [25–28].

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

Cas9-assisted targeting of chromosome segments (CATCH) is a cloning technique that utilizes the CRISPR-Cas9 gene editing system for direct BGCs cloning into the host. Comparing with traditional cloning tools such as PCR and restricted enzymes, CATCH is predicted to become a useful molecular tool for direct cloning of large gene clusters. Transformation-assisted recombination (TAR) has been used for cloning of large BGCs for about decades. However, the TAR approach is associated with a low cloning efficiency, which means it requires screening of hundreds of colonies to detect few positive clones [22, 24]. To address this challenge, TAR and CRISPR-Cas9 have been coupled resulting in a new approach called TAR-CRISPR [24]. By coupling TAR with CRISPR, a significant increase of the clone efficiency has been reported. Comparing with traditional TAR cloning, the advantages of TAR-CRISPR are that the positive clones could be achieved with secondary screening and lesser manpower and also it does not require a high experience of working with yeast [24]. In fact, the TAR-CRISTAR cloning will allow for the development of BGC cloning and SM production in the future.

#### *2.1.2 Gene refactoring*

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

It does not require protein engineering steps, very simple to test multiple gRNA

It works by including double-strand breaks in target DNA or singlestrand DNA nicks (Cas9 nickase)

CRISPR consists of single monomeric protein and chimeric RNA

been observed

Easy and very fast procedure

It can cleave methylated DNA in human cells. This aspect is of special concern for plants as this has not been much explored

advantage of CRISPR, and several genes can be edited at same time. Only Cas9 is needed

6 Components crRNA, Cas9 proteins Zn-finger

5 Mutation rate Low mutation rate has

1 Protein

2 Mode of action

4 Structural proteins

7 Length

8 Target

9 Level of experiment

10 Methylated DNA cleavage

11 Multiplexing This is the main

*Different genomic engineering techniques used in Metabolomic.*

of target sequence (bp)

recognition efficiency

engineering steps

**CRISPR/Cas9 Zinc finger** 

3 Cloning Not Required Required Required

**nucleases (ZFNs)**

It requires complex steps to test gRNA

It can induce double-strand breaks in target DNA

ZFNs work as dimeric and only protein component is required

High mutation rate has been observed in plants

domains, nonspecific FOKI nuclease domain

20–22 18–24 24–59

High High High

Complicated procedure and need for expertise in protein engineering

Highly difficult to achieve this through ZFNs

**Transcription factorlike effector nucleases (TALENs)**

TALENs need protein engineering steps to test gRNA

Induces DSBs in target DNA

TALENs also work as dimeric and require protein component

Mutation rate is high as compared to CRISPR

Zn-finger domains, nonspecific folk nuclease domain

Relatively easy procedure

marks upon the capacity of TALENs to perform methylated DNA cleavage

Very difficult to obtain multiplexed genes by means of TALENs. Because it needs separate dimeric proteins specific for each target

Unable to do so There are many question

new cloning tools have been introduced, including Cas9-assisted targeting of chromosome segments (CATCH), transformation-assisted recombination (TAR), and TAR-CRISPR [22–24]. Basically, gene cloning steps include: determining the suitable heterologous host, cloning of the target BGC, transfer of the BGC into the chosen host, expression in chosen host system, and optimization of production.

**4**

**Table 2.**

Gene refactoring or replacement is useful not only in BGCs' activation but also for novel SMs' discovery. In fact, several silent BGCs have been refactored by replacing the BGC promoter to yield natural products such as secondary metabolites [25–28].

Another new tool in gene refactoring is multiplexed CRISPR-Cas9- and transformation-associated recombination (TAR)-mediated promoter engineering method (mCRISTAR) [21]. This new tool combines the advantages of the CRISPR-Cas9 system and TAR. It is different than the TAR-CRISPR that was discussed earlier. Comparing with TAR-CRISPR, which is a yeast-based method, basically mCRISTAR uses CRISPR-Cas9 to break 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. Another gene refactoring tool that has aided in the faster cloning and refactoring of BGCs is the direct pathway cloning (DiPaC). Direct pathway cloning (DiPaC) depends on PCR amplification and in vitro DNA assembly for biosynthesis gene cluster capture and their expression. DiPaC was recently employed for the capture of biosynthesis gene cluster, which is small in size, followed by their activation and expression of novel natural products [29]. DiPaC was also able to successfully clone mid and large size of BGC [29].
