Section 3 CRISPR Technology

## CRISPR Technology: Emerging Tools of Genome Editing and Protein Detection

*Rita Lakkakul and Pradip Hirapure*

#### **Abstract**

CRISPR technology has seen rapid development in applications ranging from genomic and epigenetic changes to protein identification throughout the last decade. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPRassociated (Cas) protein systems have transformed the ability to edit, control the genomic nucleic acid and non-nucleic acid target such as detection of proteins. CRISPR/Cas systems are RNA-guided endonucleases exhibiting distinct cleavage activities deployed in the development of analytical techniques. Apart from genome editing technology, CRISPR/Cas has also been incorporated in amplified detection of proteins, transcriptional modulation, cancer biomarkers, and rapid detection of POC (point of care) diagnostics for various diseases such as Covid-19. Current protein detection methods incorporate sophisticated instrumentation and extensive sensing procedures with less reliable, quantitative, and sensitive detection of proteins. The precision and sensitivity brought in by CRISPR-dependent detection of proteins will ensure the elimination of current impediments. CRISPR-based amplification strategies have been used for accurate estimation of proteins including aptamer-based assay, femtomolar detection of proteins in living cells, immunoassays, and isothermal proximal assay for high throughput. The chapter will provide a comprehensive summary of key developments in emerging tools of genome editing and protein detection deploying CRISPR technology, and its future perspectives will be discussed.

**Keywords:** CRISPR/Cas, genome editing, protein detection, CRISPR technology, anti-CRISPR

#### **1. Introduction**

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein modules are found to be a part of adaptive antivirus defense systems in archaea and bacteria and mediate immunity by a three-stage process called adaption, processing of the primary transcript, and interference. These systems incorporate fragments of foreign DNA (known as spacers) into CRISPR cassettes, then transcribe the CRISPR arrays including the spacers, and process them to make a guide crRNA or the clustered regularly interspaced short

palindromic repeats ribonucleic acid (CRISP RNA) which is employed to specifically target and cleave the genome of the cognate virus or plasmid. Earlier classic methods such as zinc finger motif, meganucleases, and transcription activator-like effector nucleases were deployed for genome editing but due to its prerequisite for different fusion proteins, the technology raised hurdles in its applicability. The characteristic feature of single guide RNA of CRISPR to regulate Cas protein to target specific gene sequence is highly advantageous to overcome the barriers posing from classic methods. Proteins cas1 and cas2 genes are found to be the core and active part of the information processing subsystems of the three distinct types of CRISPR/Cas

#### **Figure 1.**

*Simplified model of the immunity mechanisms of class 1 and class 2 CRISPR-Cas systems. The CRISPR-Cas systems are composed of a cas operon (blue arrows) and a CRISPR array that comprises identical repeat sequences (black rectangles) that are interspersed by phage-derived spacers (colored rectangles). Upon phage infection, a sequence of the invading DNA (protospacer) is incorporated into the CRISPR array by the Cas1-Cas2 complex. The CRISPR array is then transcribed into a long precursor CRISPR RNA (pre-crRNA), which is further processed by Cas6 in type I and III systems (processing in type I-C CRISPR-Cas systems by Cas5d). In type II CRISPR-Cas systems, crRNA maturation requires tracrRNA, RNase III and Cas9, whereas in type V-A systems Cpf1 alone is sufficient for crRNA maturation. In the interference state of type I systems, Cascade is guided by crRNA to bind the foreign DNA in a sequence-specific manner and subsequently recruits Cas3 that degrades the displaced strand through its 3*′*–5*′ *exonucleolytic activity. Type III-A and type III-B CRISPR-Cas systems employ Csm and Cmr complexes, respectively, for cleavage of DNA (red triangles) and its transcripts (black triangles). A ribonucleoprotein complex consisting of Cas9 and a tracrRNA: crRNA duplex targets and cleaves invading DNA in type II CRISPR-Cas systems. The crRNA-guided effector protein Cpf1 is responsible for target degradation in type V systems. Red triangles represent the cleavage sites of the interference machinery (Courtesy: Ref. [4]).*

*CRISPR Technology: Emerging Tools of Genome Editing and Protein Detection DOI: http://dx.doi.org/10.5772/intechopen.102516*


#### **Table 1.**

*Different types of CRISPR/Cas based on signature protein, effector, and cleavage product [3].*

systems [1]. Due to the current problems with the vast diversity and complexity of the architecture of CRISPR/Cas systems, the classification is still challenging. Based on the presence of three signature genes, the classification is as follows:

#### **1.1 Type I CRISPR/Cas systems**

Typical type I loci contain the signature cas3 gene, which codes for helicase and DNase activities within a single large protein. The detailed sequence and structural comparisons have led to the recognition of many of these proteins in the RAMP superfamily including Cas5 and Cas6 families. Type I systems are currently divided into six subtypes, I-A to I-F, each of which has its own signature gene and distinct features of operon organization [2, 3].

#### **1.2 Type II CRISPR/Cas systems**

These contain cas9 as a signature gene encoding for a multidomain protein that combines all the functions of effector complexes and the target DNA cleavage and is essential for the maturation of the crRNA. These systems use cellular (not encoded within the CRISPR/Cas loci) RNase III and tracrRNA for the processing of pre-crRNA. Type II CRISPR-Cas systems are currently classified into three subtypes such as II-A, II-B, and II-C. Type II-A encompasses an additional signature gene csn2. Protein csn2 is found to be engaged in spacer integration. Type II-B systems belong to the Cas family of proteins with 5′-single-stranded DNA exonuclease activity. The recently proposed type II-C CRISPR-Cas systems possess only three protein-coding genes (cas1, cas2, and cas9) and are common in sequenced bacterial genomes (**Figure 1**) [2, 3].

#### **1.3 Type III CRISPR/Cas systems**

All type III systems possess the signature gene cas10 which encodes a multidomain protein containing a palm domain similar to that in cyclases and polymerases of the PolB family (**Table 1**) [2, 3].

#### **2. Molecular characterization of CRISPR-Cas 12 and Cas 13**

Initially, CRISPR-Cas 9 was found to nick the DNA along with the guide RNA Cas 12a belonging to class II Type VA system, derived from *Francisella novicida* bacterium possesses enormous ability to cleave DNA at multiple targets. Cas 12 an RNA-guided DNAse, is a T-rich PAM sequence making it different from Cas 9. The positively charged central channel of a nuclease (NUC) domain determines the trans cleavage activity of the target strand after studies find that mutations in the catalytic site of

the RuvC domain of Cas12a in the bacterium *Acidaminococcus* sp. eliminate the same. CRISPR is classified into types I and II [5].

Type II is further divided into six types based on their structure and function. The Cas12a protein contains a RuvC endonuclease domain, which sequentially cleaves the non-targeting strand and the targeting strand to form DSBs (double-stranded base pairs). Compared to the CRISPR/Cas9 system, this system has several remarkable differences, including the signature protein, PAM sequence, and cleavage product [6]. CRISPR/Cas12a based sensing methods focus on fluorescence readout with reduced transduction efficiency as studies report a direct correlation between the catalysis systems with recognition elements (i.e., aptamers), thus greatly improving the working efficiency of the detection platform. Cas 13 consists of four subtypes and is involved in RNA interference activities. Off-target editing is critical to Cas 13 and requires significant attention in retrieving obstacles for protein analysis [7].

### **3. Mechanism of amplification strategy for nucleic acid and protein detection**

CRISPR/Cas systems generally play a role as RNA-guided endonucleases (crRNA). The crRNA guides cas proteins to specific DNA sequences whereby the hybridization

**Figure 2.**

*Basic components of CRISPR/Cas9, Cas12a, Cas12f, and Cas13a pink triangle indicates cis-cleavage site [9].*

#### *CRISPR Technology: Emerging Tools of Genome Editing and Protein Detection DOI: http://dx.doi.org/10.5772/intechopen.102516*

leads to cas protein activation which later results in cleavage of DNA sequence [8]. **Figure 2** shows the components of Cas9, Cas12a, Cas12f, and Cas13a.

Cas9 is an endonuclease and the single-guide RNA (sgRNA) of CRISPR-Cas9 systems contains a hairpin-rich region that binds to Cas9 and a 20-nucleotide "spacer" region that binds with the complementary "protospacer" region in the target strand of a dsDNA duplex. Binding between the sgRNA and the DNA target brings Cas9 into close proximity to the target (**Figure 2**). The His-Asn-His (HNH) domain of Cas9

#### **Figure 3.**

*Combining functional nucleic acids and molecular translators with CRISPR/Cas technology for detection of non-nucleic acids such as proteins. Adapted from Ref. [10]. Copyright 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Adapted from Ref. [11]. Copyright 2020 American Chemical Society. Adapted from Ref. [12]. Copyright 2016 Springer Nature. (A) Two copies of an aptamer lock the activator, the target ssDNA complementary to crRNA. The activator is released when the aptamer binds to a small molecule (e.g., ATP), allowing it to hybridize with crRNA and activate CRISPR-Cas12a trans-activity. (B)The activation of CRISPR-Cas12a is prevented when the target molecule binds to its aptamer. CRISPR-Cas12a is activated by an unbound aptamer. (C) Metal ions serve as co-factor(s) for an RNA-cleaving DNAzyme to generate output ssDNA for CRISPR-Cas12a activation. (D) The binding of allosteric transcription factor (aTF) to the target molecule releases output dsDNA for CRISPRCas12a activation.*

cleaves the strand that is complementary to sgRNA (target strand) and the RuvC domain of Cas9 cleaves the other strand of the dsDNA (non-target strand). Singleguide RNA (sgRNA) (**Figure 3**) [13].

Recent findings indicate that the cas12a proteins have both trans and cis cleavage activities on ssDNA regardless of the sequence. Notably, Cas12a is the first Cas protein to be identified whose ternary complex has been shown to have trans-ss DNA cleavage ability. Research shows that Cas12a may have acquired singlestranded DNA ability through evolution due to the abundance of viruses in the environment. Thus, gaining a significant role as a powerful and dominating weapon to eliminate invasion by foreign ss DNA. The well-characterized variants of cas12, cas12a, and cas12f, formerly known as cas14 lack the HNH domain but nevertheless, achieved the PAM dependant cleavage with RuvC domain alone. Recent findings have reported Cas13a also called C2Ca, an RNA-guided and RNA-targeting CRISPR effector from the class 2 type VI CRISPR system, was found to have the transcleavage activity on RNA. Additionally, the RuvC catalytic pocket of both C2c1 and Cas12a was responsible for the cleavage of both strands of targeted dsDNA [9].

#### **4. Efficient sensing mechanism of CRISPR/Cas derived biosensors**

Electrochemical biosensors register physical-chemical and biological change and possess high throughput of the biological recognition process. Depending on the type of biological recognition, sensors are classified into biocatalytic devices and affinity sensors. Biocatalytic sensors integrate enzymes and whole cells as recognition elements leading to exquisite specificity and a significant rise in the rate of reaction whereas affinity sensors make use of extreme selectivity and specificity for acquiring higher sensitivity. The electrochemical transducer responds to the binding event and converts the electrical response to an output that can be amplified, stored, and displayed [14]. Due to its signal-off architecture, these electrochemical sensors provide limited sensitivity and productivity. To overcome these limitations the CRISPR/ Cas12a based electron sensing biosensors have been developed for non-nucleic acid targets. CRISPR/Cas12a-based immobilization-free electrochemical biosensors can detect small molecules and proteins by adjusting regions for target recognition in RCA components [15]. Transcription factors (TFs) assay seems to be path-breaking as it is found to be involved directly in many diseases including cancers. CRISPR/Cas 12a based biosensors for the detection of transcription factors have been developed. The biosensing mechanism is based on the interaction of TF's with double-stranded DNA activator eliminating Cas12a/crRNA from contacting and interacting with the 14 activators, thus inhibiting Cas12a activation. As a consequence of this strategy, the DNase activity of Cas12a was controlled and several TFs with well-defined binding sites could be quantified at the picomolar level with high precision [16, 17].

#### **5. Implementation of CRISPR/Cas amplification strategy for protein detection**

Recent findings report that the implementation of various nucleic acid amplification strategies led to improvements in analytical specificity and sensitivity and the development of point of care (POC) diagnostics. For example, the best-studied reaction is the amplification employing the Cas9 nickase (Cas9nAR) which when

*CRISPR Technology: Emerging Tools of Genome Editing and Protein Detection DOI: http://dx.doi.org/10.5772/intechopen.102516*

combined with polymerase and primers may substantially duplicate double-stranded DNA (dsDNA) without requiring heat cycling as does the polymerase chain reaction (PCR) [18].

#### **5.1 iPCCA: isothermal proximity CRISPR/Cas 12a assay**

In contrast to PCR, isothermal proximity assay seems to be an effective protein quantification assay for disease biomarkers and point of care diagnostics. Recent advances in the CRISPR/Cas technique specifically combining recombinase polymerase activity (RPA) and ssDNAse activity have led to the discovery of a series of isothermal assays for protein quantification. iPCCA relies on proximity binding for target recognition due to which it holds the potential for detecting non-nucleic acid targets such as proteins. However, isothermal amplification does not necessitate the use of advanced and sophisticated thermal cyclers and hence is more commonly used in biosensing [9].

#### **5.2 Aptamer-based assay for femtomolar detection of proteins**

The most widely used bioassay, ELISA (enzyme-linked immunosorbent assay) has revolutionized the ability to detect a wide variety of antigens. Complex chemical structure and restricted catalytic efficiency of HRP has a direct correlation with poor sensitivity in picomolar and nanomolar concentration. However, conventional ELISA is still not sensitive enough to detect ultralow concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious disease. CRISPR/Cas 13a based signal amplification strategy also called CLISA has been used to develop a 10 fold high-sensitive method for detecting low abundance. Recently, CRISPR/Cas13a has been recently demonstrated to have RNA-directed RNA cleavage ability. This RNA-guided trans-endonuclease activity is highly specific, being activated only when the target RNA has the perfect complementary sequence to the crRNA and is highly efficient. This potent signal amplification ability of CRISPR/ Cas13a enables the development of direct RNA assays with a sensitivity down to the femtomolar level [19, 20].

#### **5.3 CRISPR/Cas 12a controlled aptasensor for protein detection**

Aptamer, a highly selective recognition element has been combined with various analytical techniques to increase the sensitivity of protein assay. Amongst these, an electrochemical technique using specific aptamers as recognition elements exhibits great promise in detecting protein duo to its attractive merits, such as high selectivity and sensitivity, the potential for miniaturization, and ease of integration with additional components [21]. Recent findings have demonstrated that the electrochemical aptasensor has been effectively used for the detection of thrombin in femtomolar concentration. It has been reported that once CRISPR RNA (crRNA)-directed Cas12a binds to a specific target DNA, the conserved RuvC nuclease domain in Cas12a will non-specifically cut single-stranded DNA (ssDNA). A homogeneous electrochemical aptasensor has been reported for sensitive and specific detection of thrombin by utilizing binding-induced DNA strand displacement strategy as the transduction element of thrombin and rolling circle amplification-regulated CRISPR/Cas12a for signal amplification. Importantly, this homogeneous electrochemical aptasensor can detect the femtomolar range of thrombin, and exhibited good specificity relative to

other interfering blood-relevant proteins. The BIDSD-RCA-CRISPR/Cas12a is implemented in three steps, but this electrochemical aptasensor dispenses with the need for probe surface-immobilization procedure, simplifying the preparation process, and reducing the operating cost of the analysis. The strategy further could be applied to detect another disease-related protein biomarker in early diagnosis in the future [22].

#### **6. Anti-CRISPRs: potential repressors of CRISPR/Cas**

The struggle for life between bacteria and their infecting viruses (phages) has led to the development of numerous bacterial defense mechanisms and their phageencoded opponents. Recently, anti-CRISPR proteins have been identified, which inhibits the CRISPR/Cas system. The mechanism by which anti-CRISPR proteins inhibit CRISPR/Cas provides an extensive set of valuable tools to both understand and manipulate CRISPR [21]. Several findings report that the growing number of anti-CRISPR families has a significant impact on CRISPR/Cas function and has been a driving force in the evolution of CRISPR-Cas. These Anti-CRISPR systems rely heavily on Aca proteins due to their extensive interaction with anti-CRISPRs and the presence of Aca genes has the potential to act as anti-anti CRISPR playing a vital role in CRISPR-based antibacterial technologies [22, 23]. Anti-CRISPR ranges from 50 to 150 amino acids with no sequence similarity. Recent finding demonstrated that phage carries atleast one anti-CRISPR gene to avoid elimination by competent hosts. The unique mechanism of anti-CRISPR results in sequence-specific transcriptional repression system. Type II anti-CRISPRs have more evident biotechnological uses, given the widespread usage of CRISPR-Cas9 genome editing tools. Their application could be critical for gene drive and gene therapy technologies [24].

### **7. Future perspectives of CRISPR-Cas technology**

CRISPR/Cas based technology has a lot of potential as a tool for treating a range of medical conditions that have a genetic component, including cancer, hepatitis B, or even high cholesterol [25–27]. It is likely to be many years before CRISPR/ Cas technology is used routinely in humans. CRISPR/Cas technology emerged as a versatile technology with wide application in the genome sequence editing, molecular studies of various gene functions, protein detection, gene therapy, and in the biomedical science as a diagnostic technology for detection of covid 19 like viral, bacterial, and various genetic disease [28]. Cancer is one of the fatal diseases that has severely threatened human life and caused a tremendous burden for society [29]. Early diagnosis of cancer is of great benefit to treat patients in early stages which leads to improve the survival rate of cancer patients. In body fluids detection of cancer related biomarkers is a critical kind of noninvasive technique for cancer diagnosis. Nevertheless, existing techniques of cancer biomarker detection always depends on a large-scale instruments and required sophisticated operation [30]. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR/ Cas) based in vitro diagnosis can simplify the detection procedures and improve sensitivity and specificity, with great promise as the next-generation molecular diagnosis [31]. In the future, genome-wide screening for various genetic disorders, and cancer subtypes should be conducted to identify specific genetic and epigenetic targets for CRISPR technology to be most effective. The functionality of the identified mutations *CRISPR Technology: Emerging Tools of Genome Editing and Protein Detection DOI: http://dx.doi.org/10.5772/intechopen.102516*

#### **Figure 4.**

and their related signaling pathways need to be thoroughly analyzed before they are manipulated for therapy with CRISPR technology [32]. More in vivo research on Cas9 epigenetic regulation is needed to better understand its impact on cancer epigenetics. The use of synthetic biology for Cas9 modulation can be further extended to create real-time predictive algorithms for specific metastatic pathways that update as epigenetic regulation progress and the cancer advances so that treatment can always be precisely one step ahead of cancer. Ongoing research has the potential to optimize and advance CRISPR technology, culminating in the clinical realization of its full potential for breast cancer diagnosis, modeling, and treatment [29, 33, 34]. In the future, CRISPR/Cas technology will be used as a unique promising technology to study the various genes for their function, for identification of mutations and their correction, this technology will be used in tumor angiogenesis research for cancer treatment [35], CRISPR technology also used for modification of genetic sequence to develop various organisms for the benefit of human and environmental protection. Much research is still focusing on its use in animal models or isolated human cells, with the aim to eventually use the technology to routinely treat diseases in humans (**Figure 4**).

#### **8. Conclusion**

From many years scientists have learned about genetics and gene function by studying the effects of alteration in DNA sequence. Artificially by making a change in a gene, either in a cell line or a whole organism, it is possible to study the effect of that change to understand what the function of that gene is. For a long period geneticists used chemicals or radiation to create mutations but this approach is not precise and specific and due to its randomness for several years scientists have been using 'gene targeting' to introduce changes in specific places in the genome, by deletion or insertion either whole genes or single bases. Conventional gene targeting has been very valuable for studying genes and genetics, however it takes a long time to create a mutation and is fairly expensive But the CRISPR/Cas9 system based technology currently stands out as the fastest, cheapest and most reliable system for 'editing' genes. In the last decade CRISPR/Cas is a genome editing technology that is creating a an atmosphere of excitement in the science world because of its faster, cheaper,

promising, precise, sensitive and efficient and more accurate nature than previous conventional techniques of genome engineering and it has a wide range of potential applications. CRISPR/Cas technology have made it possible to edit the genomes of most cell types precisely and efficiently hence (CRISPR)/Cas9 system is a novel, versatile and easy-to-use tool to edit genomes irrespective of their complexity, with multiple and applications in almost all branches of life science, biomedicine and facilitating the elucidation of target gene function in biology and diseases. CRISPR/ Cas technology able to detect various targets starting from nucleic acids to proteins. Incorporating CRISPR/Cas systems with numerous nucleic acid amplification strategies allows the generation of amplified detection signals, enrichment of low-abundance molecular targets, enhancements in analytical specificity and sensitivity, and development of point-of-care diagnostic techniques. It is concluded that the CRISPR/ Cas systems in association with functional nucleic acids (FNAs) and molecular translators permits the detection of non-nucleic acid targets, like proteins, metal ions, and tiny molecules. Productive integrations of CRISPR technology with nucleic acid amplification techniques lead to sensitive and fast detection of Protein.

#### **Acknowledgements**

The authors would like to thank Dr. B.A. Mehere, Principal and Head of the Department of Biochemistry and Biotechnology, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India, for providing research space and facility.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Rita Lakkakul and Pradip Hirapure\* Department of Biochemistry and Biotechnology, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India

\*Address all correspondence to: pradiphirapure@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*CRISPR Technology: Emerging Tools of Genome Editing and Protein Detection DOI: http://dx.doi.org/10.5772/intechopen.102516*

#### **References**

[1] Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, et al. Evolution and classification of the CRISPR–Cas systems. Nature Reviews Microbiology. 2011;**9**(6):467-477

[2] Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R. TALEN or Cas9–rapid, efficient and specific choices for genome modifications. Journal of Genetics and Genomics. 2013;**40**(6):281-289

[3] Liu Z, Dong H, Cui Y, Cong L, Zhang D. Application of different types of CRISPR/Cas-based systems in bacteria. Microbial Cell Factories. 2020;**19**(1):1-4

[4] Hille F, Charpentier E. CRISPR-Cas: Biology, mechanisms, and relevance. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 2016;**371**(1707):20150496. DOI: 10.1098/rstb.2015.0496

[5] Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJ, et al. Evolutionary classification of CRISPR–Cas systems: A burst of class 2 and derived variants. Nature Reviews Microbiology. 2020;**18**(2):67-83

[6] Yan F, Wang W, Zhang J. CRISPR-Cas12 and Cas13: The lesser known siblings of CRISPR-Cas9. Cell Biology and Toxicology. 2019;**35**(6):489-492

[7] O'Connell MR. Molecular mechanisms of RNA targeting by Cas13-containing type VI CRISPR–Cas systems. Journal of Molecular Biology. 2019;**431**(1):66-87

[8] Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, et al. CRISPR-Cas12a target binding unleashes indiscriminate

single-stranded DNase activity. Science. 2018;**360**(6387):436-439

[9] Feng W, Newbigging AM, Tao J, Cao Y, Peng H, Le C, et al. CRISPR technology incorporating amplification strategies: Molecular assays for nucleic acids, proteins, and small molecules. Chemical Science. 2021;**12**(13): 4683-4698

[10] Dai Y, Somoza RA, Wang L, Welter JF, Li Y, Caplan AI, et al. Exploring the trans-cleavage activity of CRISPR-Cas12a (cpf1) for the development of a universal electrochemical biosensor. Angewandte Chemie. 2019;**131**(48):17560-17566

[11] Xiong Y, Zhang J, Yang Z, Mou Q, Ma Y, Xiong Y, et al. Functional DNA regulated CRISPR-Cas12a sensors for point-of-care diagnostics of non-nucleicacid targets. Journal of the American Chemical Society. 2019;**142**(1):207-213

[12] Liang M, Li Z, Wang W, Liu J, Liu L, Zhu G, et al. A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules. Nature Communications. 2019;**10**(1):1-9

[13] Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;**156**(5):935-949

[14] Li SY, Cheng QX, Liu JK, Nie XQ, Zhao GP, Wang J. CRISPR-Cas12a has both cis-and trans-cleavage activities on single-stranded DNA. Cell Research. 2018;**28**(4):491-493

[15] Ronkainen NJ, Halsall HB, Heineman WR. Electrochemical biosensors. Chemical Society Reviews. 2010;**39**(5): 1747-1763

[16] Qing M, Chen SL, Sun Z, Fan Y, Luo HQ, Li NB. Universal and programmable rolling circle amplification-CRISPR/Cas12a-mediated immobilization-free electrochemical biosensor. Analytical Chemistry. 2021; **93**(20):7499-7507

[17] Li B, Xia A, Zhang S, Suo T, Ma Y, Huang H, et al. A CRISPR-derived biosensor for the sensitive detection of transcription factors based on the target-induced inhibition of Cas12a activation. Biosensors and Bioelectronics. 2021;**173**:112619

[18] Zhao X, Li S, Liu G, Wang Z, Yang Z, Zhang Q, et al. A versatile biosensing platform coupling CRISPR–Cas12a and aptamers for detection of diverse analytes. Science Bulletin. 2021;**66**(1):69-77

[19] Li Y, Mansour H, Watson CJ, Tang Y, MacNeil AJ, Li F. Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay. Chemical Science. 2021; **12**(6):2133-2137

[20] Chen Q, Tian T, Xiong E, Wang P, Zhou X. CRISPR/Cas13a signal amplification linked immunosorbent assay for femtomolar protein detection. Analytical Chemistry. 2019;**92**(1):573-577

[21] Bounegru AV, Apetrei C. Carbonaceous nanomaterials employed in the development of electrochemical sensors based on screen-printing technique—A review. Catalysts. 2020;**10**(6):680

[22] Marotkar S, Hirapure P, Paranjape S, Upadhye V. Crispr/Cas9 technology for crop improvement: A new weapon for Indian agricultural threats. Plant Cell Biotechnology and Molecular Biology. 2020;**21**:1-9

[23] Li H, Li M, Yang Y, Wang F, Wang F, Li C. Aptamer-linked CRISPR/ Cas12a-based immunoassay. Analytical Chemistry. 2021;**93**(6):3209-3216

[24] Pawluk A, Davidson AR, Maxwell KL. Anti-CRISPR: Discovery, mechanism and function. Nature Reviews Microbiology. 2018;**16**(1):12-17

[25] Zhang B. CRISPR/Cas gene therapy. Journal of Cellular Physiology. 2021;**236**(4):2459-2481

[26] Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: Mechanisms, advances and prospects. Signal Transduction and Targeted Therapy. 2020;**5**(1):1-23

[27] Sahel DK, Mittal A, Chitkara D. CRISPR/Cas system for genome editing: Progress and prospects as a therapeutic tool. Journal of Pharmacology and Experimental Therapeutics. 2019;**370**(3):725-735

[28] Tian X, Gu T, Patel S, Bode AM, Lee MH, Dong Z. CRISPR/Cas9—An evolving biological tool kit for cancer biology and oncology. NPJ Precision Oncology. 2019;**3**(1):1-8

[29] Alter HJ, Seeff LB. Recovery, persistence, and sequelae in hepatitis C virus infection: A perspective on long-term outcome. In: Seminars in Liver Disease. Vol. 20. New York, NY, USA: Thieme Medical Publishers, Inc.; 2000. pp. 17-36

[30] Gong S, Zhang S, Lu F, Pan W, Li N, Tang B. CRISPR/Cas-based in vitro diagnostic platforms for cancer biomarker detection. Analytical Chemistry. 2021;**93**:11899-11909

[31] Li Y, Li S, Wang J, Liu G. CRISPR/ Cas systems towards next-generation

*CRISPR Technology: Emerging Tools of Genome Editing and Protein Detection DOI: http://dx.doi.org/10.5772/intechopen.102516*

biosensing. Trends in Biotechnology. 2019;**37**(7):730-743

[32] Bondy-Denomy J, Garcia B, Strum S, Du M, Rollins MF, Hidalgo-Reyes Y, et al. Multiple mechanisms for CRISPR–Cas inhibition by anti-CRISPR proteins. Nature. 2015;**526**(7571):136-139

[33] Stanley SY, Borges AL, Chen KH, Swaney DL, Krogan NJ, Bondy-Denomy J, et al. Anti-CRISPRassociated proteins are crucial repressors of anti-CRISPR transcription. Cell. 2019;**178**(6):1452-1464

[34] Chen Y, Zhang Y. Application of the CRISPR/Cas9 system to drug resistance in breast cancer. Advanced Science. 2018;**5**(6):1700964

[35] Shah SZ, Zhao D, Hussain T, Sabir N, Yang L. Regulation of microRNAs-mediated autophagic flux: A new regulatory avenue for neurodegenerative diseases with focus on prion diseases. Frontiers in Aging Neuroscience. 2018;**10**:139

[36] Wu H, Chen X, Zhang M, Wang X, Chen Y, Qian C, et al. Versatile detection with CRISPR/Cas system from applications to challenges. TrAC Trends in Analytical Chemistry. Feb 2021;**135**:116150

Section 4

## Gene Cloning for Nonribosomal Peptide Synthesis

### **Chapter 5** Nonribosomal Peptide Synthesis

*Sadık Dincer, Hatice Aysun Mercimek Takci and Melis Sumengen Ozdenefe*

#### **Abstract**

Nonribosomal peptides (NRPs) are a type of secondary metabolite with a wide range of pharmacological and biological activities including cytostatics, immunosuppressants or anticancer agents, antibiotics, pigments, siderophores, toxins. NRPs, unlike other proteins, are synthesized on huge nonribosomal peptide synthetase (NRPS) enzyme complexes that are not dependent on ribosomal machinery. Bacteria and fungi are the most common NRPs producers. Furthermore, the presence of these peptides has been confirmed in marine microbes. Nowadays, many of these peptides are used in the treatments of inflammatory, cancer, neurodegenerative disorders, and infectious disease for the development of new therapeutic agents. The structure, function, and synthesis of NRPs, as well as producer microorganisms and their several application areas, are covered in this chapter.

**Keywords:** biological activity, nonribosomal peptide, producer microorganisms, secondary metabolite, synthesis, structure

#### **1. Introduction**

Bioprocesses, which are consisted of a series of enzymatically catalyzed biochemical reactions in all living things, are necessary for survival. They have a high potential in terms of material synthesis, which has recently been performed by chemical techniques [1]. Furthermore, the advancement of heterologous production systems and genetic engineering techniques has resulted in pioneering initiatives to manufacture usable biomaterials [2]. These advancements also enabled the successful generation of primary and secondary metabolic pathway products in physiologically and genetically well-defined hosts, such as *Escherichia coli* and *Saccharomyces cerevisiae*, by precise manipulation of the related genes. In particular, heterologous molecular hosts have been used to successfully synthesize structurally varied secondary metabolites showing unique pharmacological action [1–3]. Nonribosomal peptides (NRPs) obtained by the most extensive, appealing, and useful actively-studied bioprocesses are included among these metabolites, which are important in the discovery and development of drugs and therapeutic reagents [1, 4].

NRPs are secondary metabolites that are synthesized outside of the ribosomal machinery and have a variety of properties such as cytostatics, immunosuppressants or anticancer agents, antibiotics, pigments, siderophores, toxins [3, 5, 6]. NRPs are typically produced by marine microorganisms and invertebrates, as well as soil-inhabiting microorganisms [5, 7, 8]. The majority of natural products produced by sponges, bryozoans, mollusks, and tunicates are members of the NRP or mixed polyketide–NRP families. Several of NRPs are being used in the development of new medicines for inflammatory, cancer, neurological diseases, and infectious disease nowadays [7].

Non-ribosomal peptide synthetases (NRPSs) enzymes are poly-functional mega-synthases that biosynthesize NRPs [7, 9, 10]. NRPSs, multi-modular enzyme or enzyme complexes from common bacteria, less common eukarya, and rare archaea, are capable of producing a wide range of natural pharmaceutical products (Bacitracin, antibiotics for skin infections; Bleomycin antitumor; Cyclosporin, antifungal, and immunosuppressive drugs; Daptomycin, antibiotics) [5, 7, 11]. NRPSs use both proteinogenic and nonproteinogenic amino acids (not encoded by DNA) as building blocks for the growing peptide chain [1, 7, 11, 12]. They catalyze multiple biosynthetic processes, each of which is responsible for particular one amino acid elongation on the growing peptide chain [10]. This chapter looks at the structure, function, and synthesis of NRPs, as well as producer microorganisms and their various applications.

#### **2. Synthesis, structure, and function of nonribosomal peptide (NRP)**

NRPSs are responsible for nonribosomal peptide (NRP) synthesis. These are large multi-enzyme complexes that are modularly organized and serve as biosynthetic machinery and templates [5, 11–14]. For example, a single NRPS of 1.6 MDa synthesizes the Cyclosporine A (7). In fungal systems, such as in the case of cyclosporine A (7), a single NRPS synthesizes peptides, whereas bacteria frequently use numerous NRPSs with genes grouped in an operon. NRPSs have a modular structure [14, 15].

In a genome mining research of 2699 genomes, Wang et al. found that more than half of the NRPS enzymes were non-modular NRPS enzymes [16]. Nonmodular NRPS enzymes can be found in siderophore biosynthesis pathways, such as EntE and VibH in enterobactin and VibE in vibriobactin, or as a standalone peptidyl carrier protein, such as BlmI from the bleomycin gene cluster. NRPS enzymes are found frequently in bacteria, less frequently in eukaryotes, and infrequently in archaea. Actinobacteria, Cyanobacteria, Firmicutes, and Proteobacteria were the phyla with the greatest number of these enzymes in the bacterial domain. There was a correlation between genome size and the number of NRPS clusters [5, 17].

A module is a part of the NRPS polypeptide chain that is in charge of integrating one amino acid into the final product. Modules can further be separated into domains (**Figure 1**), which represent enzyme units that catalyze distinct steps of NRP synthesis. On the protein level, domains are defined by distinctive, greatly conserved order of patterns known as "core motifs." In certain instances, biochemical and structural data were used to confirm the involvement of greatly conserved residues in domain function (**Table 1**) [14].

There are three domains in a module. These are 1) the adenylation (A) domain, 2) the peptidyl carrier protein (PCP) or thiolation (T) domain, and 3) the condensation (C) domain, all of which are responsible for the synthesis of NRPs. A module may include additional tailoring or altering domains incorporating epimerization (E), methylation and oxidation domains or a heterocyclization (Cy) domain in place of a C-domain. Finally, most NRPS termination modules have a TE-domain, which is in charge of releasing linear, cyclic, or branching cyclic peptides [5, 9–11, 15, 18–21].

**Figure 1.** *Catalyzed reactions by various NRPS-domains [14].*

The order of the modules is frequently aligned with the sequences of the resulting peptides. NRP synthesis begins at the N-terminus and ends at the C-terminus, yielding peptides that are typically 3–15 amino acids long. The released peptides contain amino acids, that is, imino acids or ornithine and their structures are linear, cyclicmacrocyclic, branched-cyclic, branched-macrocyclic, dimers or trimers of identical structural elements [5].

The A-domain is responsible for the first step in biosynthesis, which involves recognizing and activating the amino acid substrate via adenylation with Mg-ATP, resulting in an aminoacyl adenylated intermediate. Around 550 amino acids make up domain A. It has 10 amino acid residues that serve as NRPS enzyme "codons" and are essential for substrate specificity. The D and L forms of the 20 amino acids used in ribosomal protein synthesis, as well as non-proteinogenic amino acids like imino acids, ornithine, and hydroxy acids like β-butyric acids and α-aminoadipic, are substrates recognized by the A-domain. The PCP-domain, which consists of about 80 amino acids and covalently attaches the activated amino acid to their cofactor 4′-phosphopantetheine (PP)


#### **Table 1.**

*NPRS-domains' core-motifs [14].*

arm via a thioester bond, completes the second step. And also, the active substrate and elongation intermediates are transferred to the C-domain via this domain. In the last step, C-domain, which contains approximately 450 amino acids, catalyzes the formation of peptide bonds between the carboxyl group of the incipiently synthesized peptide and the amino acid transported by the side module [5, 22]. Furthermore, this domain allows the expanding chain to be translocated to the next module. Following this step, the linear intermediate peptide is liberated in bacteria via internal cyclization or hydrolysis with the help of the Thioesterase (TE) domain. On the other hand,

#### *Nonribosomal Peptide Synthesis DOI: http://dx.doi.org/10.5772/intechopen.104722*

it appears less commonly in fungi's NRPSs. Fungi use a variety of ways to release chains. The first is a thioesterase NADP(H)-dependent reductase domain (R), which catalyzes NADPH reduction to create an aldehyde and the second is a terminal C domain, which catalyzes release by forming intermolecular or intramolecular amide bonds. By N-, C-, and O-methylation, halogenation, acylation, hydroxylation, glycosylation, or heterocyclic ring formation, the primary product of this synthesis can be changed post-synthetically to reach its mature form by additional tailoring enzymes that are not part of the NRPS. The structural diversity of NRPs is formed in part by these enzymes and their reactions [5].

Because of their extensive multidomain organization, NRPS genes are easier to identify using recent genome mining technologies, and they are also relatively easy to detect. Secondary metabolites production genes are frequently found in bacterial and fungal gene clusters. The clusters' core is thought to be NRPS genes. Nevertheless, they are linked to genes involved in building blocks synthesis, product ornamentation, self-resistance, and peptide export. For the purpose of analyzing and in silico exploration of NRPS pathways, advanced genome sequencing techniques have made genome mining methodologies available, which are assisted by a variety of bioinformatics tools, such as AntiSMASH, PRISM, and SMURF [23].

Nowadays, known NRP structures are divided into various categories, each with its own structural characteristics. Lipocyclopeptides with varied linkage patterns, such as fengycin, iturin, surfactin, and head-to-tail-cyclized peptides of varying ring sizes, such as cyclosporine, gramicidin S, maybe the largest group. There are also a lot of linear peptide configurations. They include tripeptides (such as sevadicin and bialaphos) as well as 20-mer peptides (e.g., alamethicin, peptaibols). The current highest size limit for NRPs is syringopeptin 25A, which has 25 amino acids (syringopeptin 25A). Tailoring enzymes modify the structure of some NRPs. The most structurally complicated molecules are probably bleomycins, ergopeptides, glycopeptide antibiotics, and β-lactams [23].

**Figure 2** shows some NRPs with diverse structures and a wide spectrum of activities. ACV-tripeptide (6), for example, is a precursor to antibiotics of the penicillin and cephalosporin families. Gramicidin S (4), tyrocidine A (1), and vancomycin (5) are three other antibiotic-active substances. Cyclosporin A (7), an immunosuppressive drug, is used in the post-transplantation care of patients. Cancer is treated with cytostatic agents, such as bleomycin A2 (8) and epothilone (9). Enterobactin (10), bacillibactin (11), mixochelin A (12), yersiniabactin (13), and vibriobactin (14) are examples of iron chelating agents. These compounds, known as siderophores, are created in iron-deficient environments to provide bacteria with an iron source. **Figure 2** also depicts the structures of pigments like indigodin (15), toxins like thaxtomin A (17), and peptides with uncertain functions like anabaenopeptilide 90-A (18) [14].

NRPs have a number of structural characteristics that distinguish them from ribosomal peptides. For example, non-proteinogenic amino acids, such as ornithine in 1, 2, and 4, hydroxyphenyl or dihydroxyphenyl-glycine in 5 and (4R)-4-[(E)- 2-butenyl]-4-methyl-L. -threonine (Bmt) in 7, are included. Furthermore, the structures are frequently macrocyclic (1), branched macrocyclic (2), or dimers of two (4) or trimers of three (10, 11) structural components. Smaller heterocyclic rings, such as thiazole in 9, thiazoline in 13, or oxazoline in 14, are common structural properties of nonribosomal peptides. In addition, fatty acids (3), glycosylations (5), N-methylations (7), and N-formylations (18) may also be present, as well as the addition of propionate units (8) or acetate [14].

**Figure 2.** *Some NRPs with structural diversity [14].*

#### **3. Overview of producer microorganisms for NRP**

NRPs are typically produced by marine microorganisms, soil-inhabiting microorganisms, including *Actinomycetes*, *Bacilli,* and eukaryotic filamentous fungus, and invertebrates, such as sponges, bryozoans, mollusks, and tunicates [5, 7, 11, 13, 24]. Many pharmacologically active NRPs have been effectively generated in heterologous hosts, such as *Bacillus subtilis*, *Escherichia coli*, *Saccharomyces cerevisiae,* and *Streptomyces* sp. [2]. Bacteria and fungi are the primary producers of NRPSbased metabolites. Except for bacteria and fungus, NRPS Ebony from *Drosophila melanogaster* ("fruit fly") and nemamide synthetase from the worm *Caenorhabditis elegans* have been confirmed. The distribution and occurrence of NRPS pathways and *Nonribosomal Peptide Synthesis DOI: http://dx.doi.org/10.5772/intechopen.104722*

products have been discovered, thanks to screening efforts and genome sequencing projects followed by bioinformatics research. NRPS enzymes are found frequently in bacteria, less frequently in eukaryotes, and infrequently in archaea. The phylum Actinobacteria (*Mycobacterium*, *Streptomyces*), Firmicutes (*Bacillus*, *Staphylococcus,* and *Streptococcus*), and the alpha-/beta-/gama-Proteobacteria classes (*Burkholderia*, *Escherichia*, *Erwinia, Photorhabdus, Pseudomonas*, *Salmonella*, *Serratia*, *Vibrio*, and *Yersinia*) are the most important contributors among bacteria. Nonetheless, in recent years, the phylum Cyanobacteria (*Microcystis*, *Planktothrix*, *Anabaena*, *Oscillatoria,* and *Nostoc*) and the teta-Proteobacteria (*Myxobacterium*) class have received greater attention [5, 22, 23]. NPRS genes are found predominantly in the Ascomycota (*Tolypocladium*, *Fusarium*, *Penicillium*, *Acremonium*, *Claviceps,* and *Trichoderma*) and marginally in the Basidiomycota (*Ustilago*) phylum. NRPS biosynthesis investigations in fungus are less investigated than in bacteria due to greater genome sizes, the existence of scattered introns in gene clusters, and a less established molecular biology toolbox [23].

#### **4. Application areas of NRPs**

Novel peptide products' biological functions are strictly associated with their chemical structure, which is constrained by a peptide sequence that ensures specific interaction with a specific molecular target. Chemical alterations, such as the incorporation of fatty acid chains, D-amino acids, glycosylated amino acids, and heterocyclic rings, as well as cyclization or oxidative cross-linking of side chains, add a lot to these unique interactions. Bacitracin, fengycin, pristinamycin, surfactin, tyrocidine, and vancomycin are examples of novel peptides with antibacterial and antifungal properties [25].

When the ribosomal code was deciphered in the 1960s, Tatum and coworkers discovered that ribosomes had no effect on cell-based tyrocidine production [23, 26]. The first NRPs agent is tyrocidine, a cyclic decapeptide that is biosynthesized outside of the *Bacillus brevis* ribosome. Researchers discovered that ribosome targeting antibiotics had no effect on tyrocidine production. They also discovered that *B. brevis* can synthesize gramicidin S, a cyclic decapeptide, without the use of tRNA molecules or aminoacyl-tRNA synthetases [13, 27]. Nobel Prize Laureate Fritz Lipmann and Søren Laland contributed to present essential biochemical activity insights into NRPSs, including specific ATP-dependent activation of amino acids, thioester-mediated 4′-phosphopantetheine (Ppant) binding of activated amino acids, and the directionality of the peptide synthesis and have given acceleration to the production of NRPS-based metabolites synthesized by a mechanism distinct from protein synthesis. The NRPs and NRPSs were discovered as a result of these findings associated with the synthesis of tyrocidine and gramicidin S peptides. Surprisingly, the majority of studies investigating nonribosomal NRPS-based metabolites have focused on antibacterial and antifungal action [23]. NRPS-based metabolites with antimalarial, antimicrobial, antiparasitic, antiviral, animal growth promoters, cytostatic, immunosuppressive, and natural insecticides properties are currently available on the market, and several are being studied in clinical research [28]. **Table 2** presents a summary of commercialized NRPs-based medications with antibacterial activity.

As demonstrated in **Table 2**, systemic and topical antibacterials are the most often used NRPs-based drugs, accounting for billions of dollars in the chemical and pharmaceutical industry sales. **Table 3** lists their other applications, which include



#### **Table 2.**

*Overview of NRPs-based drugs [7, 23].*


**Table 3.**

*Marketed-NRPs agents [23].*

anticancer agents, antifungals, animal feed additives, immunosuppressants (cyclosporine), obstetrics (ergometrine), and pain management (ergotamine).

In the medical field, NRP-based marketed drugs, such as Cyclosporin A and Bleomycin A2, have high selling prices. The cost of these medicines is \$107 for 25 mg of Cyclosporine A (98% purity) obtained from *T. inflatum* and \$847 for 20 mg of Bleomycin A2 (70% purity) isolated from *S. verticillus*, according to Sigma Chemical Company [5].

The 70% discovery of NRPs with antibacterial, antiviral, cytostatic, immunosuppressive, antimalarial, antiparasitic, animal growth promoters, and natural insecticides activity is mostly attributed to marine organisms [13]. NRPs obtained from marine organisms (sponges, tunicates, and their associated phyla, such as Acidobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Cyanobacteria, Nitrospira, Planctomycetes, Poribacteria, Proteobacteria, Verrucomicrobia, and Archaea) have excellent binding properties, low off-target toxicity, and high stability and these properties make them a promising molecule for the development of new therapeutics pharmacologically active in many clinical searches. **Table 4** shows the chemical structure and source of various NRPs isolated from marine sponges and tunicates.


#### *Nonribosomal Peptide Synthesis DOI: http://dx.doi.org/10.5772/intechopen.104722*



#### **Table 4.**

*Agents produced from marine sponges and tunicates which are based on NRPs [7].*

In the NCBI database, there are currently about 1.164 distinct non-ribosomal peptides that form over 500 different monomers including both proteinogenic and non-proteinogenic L- and D-amino acids, as well as amines and carboxylic acids. These complex secondary metabolites' linear, cyclic, branching, or other complicated primary structures are frequently altered to enhance clinical qualities and/or bypass resistance mechanisms. Indeed, the nucleotide sequence modification of a native NRPS gene or mixing modules from multiple NRPSs makes them more efficient with pharmacological properties. Several bioengineering and molecular techniques have been developed during the last few decades to produce modified NRPs with improved physicochemical characteristics and bioactivity [13].

#### **5. Conclusion**

In this chapter, we discussed the significance, synthesis, and application areas of NRPs-based agents, which have received a lot of interest as a new source of pharmaceutical agents. NRPs with unique chemical structures and diverse biological actions, such as antibacterials (penicillin, vancomycin), anticancer compounds (bleomycin), and immunosuppressants (cyclosporine), have been researched as novel compounds for new drug discovery and development throughout the last several decades. *In vitro* bioassays and the transfer of biosynthetic gene clusters of NRPs have been the focus of the majority of these studies. For the development of NRPs drugs with improved pharmacological properties, genetic manipulation and molecular approaches will allow the rapid construction of new NRPSs containing specific point mutations or exchanged domains.

*Nonribosomal Peptide Synthesis DOI: http://dx.doi.org/10.5772/intechopen.104722*

#### **Author details**

Sadık Dincer1 \*, Hatice Aysun Mercimek Takci<sup>2</sup> and Melis Sumengen Ozdenefe3

1 Cukurova University, Adana, Turkey

2 Kilis 7 Aralık University, Kilis, Turkey

3 Near East University, Nicosia, Northern Cyprus

\*Address all correspondence to: sdincer@cu.edu.tr

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Tsuge K, Matsui K, Itaya M. Production of the non-ribosomal peptide plipastatin in *Bacillus subtilis* regulated by three relevant gene blocks assembled in a single movable DNA segment. Journal of Biotechnology. 2007;**129**:592-603. DOI: 10.1016/j.jbiotec.2007.01.033

[2] Siewers V, San-Bento R, Nielsen J. Implementation of communicationmediating domains for non-ribosomal peptide production in *Saccharomyces cerevisiae*. Biotechnology and Bioengineering. 2010;**106**(5):841-844. DOI: 10.1002/bit.22739

[3] Zhang H, Liu Y, Wang X, Hu R, Xu G, Mao C, et al. Gene sequence diversity of the nonribosomal peptide and polyketide natural products in Changbaishan soil correlates with changes in landscape belts. Ecological Indicators. 2021;**133**:108160. DOI: 10.1016/j. ecolind.2021.108160

[4] Corpuz JC, Sanlley JO, Burkart MD. Protein-protein interface analysis of the non-ribosomal peptide synthetase peptidyl carrier protein and enzymatic domains. Synthetic and Systems Biotechnology. 2022;**7**(2):677-688. DOI: 10.1016/j.synbio.2022.02.006

[5] Martinez-Nunez MA, López y López VE. Nonribosomal peptides synthetases and their applications in industry. Sustainable Chemical Processes. 2016;**4**:13. DOI: 10.1186/ s40508-016-0057-6

[6] Duban M, Cociancich S, Leclère V. Nonribosomal peptide synthesis definitely working out of the rules. Microorganisms. 2022;**10**:577. DOI: 10.3390/ microorganisms10030577

[7] Agrawal S, Adholeya A, Deshmukh SK. The pharmacological potential of non-ribosomal peptides from marine sponge and tunicates. Frontiers in Pharmacology. 2016;**7**:333. DOI: 10.3389/fphar.2016.00333

[8] Tippelt A, Nett M. *Saccharomyces cerevisiae* as host for the recombinant production of polyketides and nonribosomal peptides. Microbial Cell Factories. 2021;**20**(1):161. DOI: 10.1186/ s12934-021-01650-y

[9] Izoré T, Candace Ho YT, Kaczmarski JA, Gavriilidou A, Chow KA, Steer DL, et al. Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity. Nature Communication. 2021;**12**(1):2511. DOI: 10.1038/s41467-021-22623-0

[10] Fortinez CM, Bloudoff K, Harrigan C, Sharon I, Strauss M, Schmeing TM. Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module. Nature Communications. 2022;**13**(1):548. DOI: 10.1038/ s41467-022-28221-y

[11] Bozhuyuk KAJ, Fleischhacker F, Linck A, Wesche F, Tietze A, Niesert CT, et al. De novo design and engineering of non-ribosomal peptide synthetases. Nature Chemistry. 2018;**10**:275-281. DOI: 10.1038/NCHEM.2890

[12] Oestreich AM, Suli MI, Gerlach D, Fan R, Czermak P. Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in *Escherichia coli*. Electronic Journal of Biotechnology. 2021;**52**:85-92. DOI: 10.1016/j.ejbt.2021.05.001

[13] Agrawal S, Acharya D, Adholeya A, Barrow CJ, Deshmukh SK. Nonribosomal *Nonribosomal Peptide Synthesis DOI: http://dx.doi.org/10.5772/intechopen.104722*

peptides from marine microbes and their antimicrobial and anticancer potential. Frontiers in Pharmacology. 2017;**8**:828. DOI: 10.3389/fphar.2017.00828

[14] Schwarzer D, Finking R, Marahiel MA. Nonribosomal peptides: From genes to products. Natural Product Reports. 2003;**20**(3):275-287. DOI: 10.1039/b111145k

[15] Kittilä T, Kittel C, Tailhades J, Butz D, Schoppet M, Büttner A, et al. Halogenation of glycopeptide antibiotics occurs at the amino acid level during non-ribosomal peptide synthesis. Chemical Science. 2017;**8**(9):5992-6004. DOI: 10.1039/c7sc00460e

[16] Wang H, Fewer DP, Holm L, Rouhiainen L, Sivonen K. Atlas of nonribosomal peptide and polyketide biosynthetic pathways reveals common occurrence of nonmodular enzymes. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**(25):9259-9264. DOI: 10.1073/ pnas.1401734111

[17] Chang Z, Flatt P, Gerwick WH, Nguyen VA, Willis CL, Sherman DH. The barbamide biosynthetic gene cluster: A novel marine cyanobacterial system of mixed polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) origin involving an unusual trichloroleucyl starter unit. Gene. 2002;**296**(1-2):235-247. DOI: 10.1016/S0378 1119(02)00860-0

[18] Bozhueyuek KAJ, Watzel J, Abbood N, Bode HB. Synthetic zippers as an enabling tool for engineering of non-ribosomal peptide synthetases. Angewandte Chemie International Edition in English. 2021;**60**(32):17531- 17538. DOI: 10.1002/anie.202102859

[19] Kries H, Niquille DL, Hilvert D. A Subdomain swap strategy for reengineering nonribosomal peptides. Chemistry & Biology. 2015;**22**:640-648. DOI: 10.1016/j.chembiol.2015.04.015

[20] Takahashi H, Kumagai T, Kitani K, Mori M, Matoba Y, Sugiyama M. Cloning and characterization of a *Streptomyces* Single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis. The Journal of Biological Chemistry. 2007;**282**(12):9073-9081. DOI: 10.1074/ jbc.M611319200

[21] Tooming-Klunderud A, Rohrlack T, Shalchian-Tabrizi K, Kristensen T, Jakobsen KS. Structural analysis of a non-ribosomal halogenated cyclic peptide and its putative operon from Microcystis: Implications for evolution of cyanopeptolins. Microbiology. 2007;**153**:1382-1393. DOI: 10.1099/mic.0.2006/001123-0

[22] Tapi A, Chollet-Imbert M, Scherens B, Jacques P. New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction. Applied Microbiology and Biotechnology. 2010;**85**(5):1521-1531. DOI: 10.1007/ s00253-009-2176-4

[23] Süssmuth RD, Mainz A. Nonribosomal Peptide Synthesis-Principles and Prospects. Angewandte Chemie International Edition in English. 2017;**56**:3770-3821. DOI: 10.1002/ anie.201609079

[24] Zhou K, Zhang X, Zhang F, Li Z. Phylogenetically diverse cultivable fungal community and polyketide synthase (pks), non-ribosomal peptide synthase (nrps) genes associated with the South China Sea sponges. Microbial Ecology. 2011;**62**:644-654. DOI: 10.1007/ s00248-011-9859-y

[25] Sieber SA, Marahiel MA. Molecular mechanisms underlying nonribosomal

peptide synthesis: Approaches to new antibiotics. Chemical Reviewes. 2005;**105**:715-738. DOI: 10.1021/ cr0301191

[26] Dell M, Dunbar KL, Hertweck C. Ribosome-independent peptide biosynthesis: The challenge of a unifying nomenclature. Natural Product Reports. 2021:1-7. DOI: 10.1039/d1np00019e

[27] Iacovelli R, Bovenberg RA, Driessen AJ. Nonribosomal peptide synthetases and their biotechnological potential in *Penicillium rubens*. Journal of Industrial Microbiology and Biotechnology. 2021;**48**(kuab045). DOI: 10.1093/jimb/kuab045

[28] Vinothkumar S, Parameswaran P. Recent advances in marine drug research. Biotechnology Advances. 2013;**31**:1826-1845. DOI: 10.1016/j. biotechadv.2013.02.006

*Edited by Sadık Dincer, Hatice Aysun Mercimek Takcı and Melis Sumengen Ozdenefe*

This book examines the fundamentals of molecular cloning and molecular cloning applications in various areas. Chapters address such topics as tools and methodologies of molecular cloning, molecular cloning for medicine, food and feed, the environment, and the future of molecular cloning.

Published in London, UK © 2022 IntechOpen © Rost-9D / iStock

Molecular Cloning

Molecular Cloning

*Edited by Sadık Dincer, Hatice Aysun Mercimek Takcı* 

*and Melis Sumengen Ozdenefe*