Enhancing Lipase Production from Potato Peel Fermentation Through CRISPR-

Cas9 Genome Editing of Non-Pathogenic E. coli

 CHAPTER TWO

2.0 LITERATURE REVIEW

Genome editing technology has transformed genetic and biological research

by providing a unique capability to precisely manipulate and modify the genomes of

living organisms, thereby accelerating the study of functional genomics. Over recent

years, various genome editing tools have been employed to investigate both simple

and complex genomes (Mangwar et al., 2019).

The inception of genome editing technology dates back to the 1990s, and since

then, several methods have been devised for targeted gene editing. In general, three

main systems, each with its distinct advantages, have gained widespread use in cells

and animals. These systems include transcription activator-like effector nucleases

(TALEN), zinc finger nucleases (ZFN), and clustered regularly interspaced short

palindromic repeats (CRISPR) (Tavakoli et al., 2021).

TALENs induce double-stranded breaks (DSBs) in target sequences,

triggering DNA damage response pathways that ultimately lead to repair (Tavakoli et

al., 2021). However, it's worth noting that despite their extensive application from

2002 to 2012, TALENs encounter certain limitations that hinder their effective use

(Tavakoli et al., 2021). These limitations include challenges associated with cloning

large modules in series, reduced efficiency in screening positive targeted cells, and

efficient ordered affiliation by ligase (Tavakoli et al., 2021).

On the other hand, ZFNs are confronted with issues related to complexity and

lack of specificity (Tavakoli et al., 2021).

 In 1987, a revolutionary genome editing system known as CRISPR-Cas9

emerged, earning recognition as one of the most remarkable genetic tools of the

century, as highlighted by Martinez-Lage et al. in 2018. CRISPR-Cas9 technologies

quickly surpassed earlier methods like TALENs and ZFNs due to a multitude of

advantages, including cost-effectiveness, simplicity, high efficiency, and rapidity. A

comparison of these differences is presented in Table 1.

 Table 1: Main differences between genome editing techniques (Tavakoli et al.,

2021)

Feature CRISPR-Cas TALEN ZFN

Cost Low High Low
Ease of design Simple A little complex Moderate
Specificity High Intermediaate Low
Pros Modifies multiple Highly effective Highly effective
sites in tandem and specific and specific
Cons PAM motif Time consuming Time consuming
required next to
target sequence
Multiplex genome High-yield Few models Few models
editing multiplexing
2.1 Genesis, evolution, and molecular mechanism of CRISPR-Cas9

The concept of CRISPR was initially introduced by Yushizumi Ishino, as

discussed by Tavakoli et al. in 2021. However, its biological applications were not

understood at the time. This system has been classified into two main classes with six

subtypes based on effector proteins, as elucidated by Magwhar et al. in 2019.

Among these, the Type 2 CRISPR-Cas9 system stands out as the most

extensively utilized tool in genome editing. It comprises three primary components: a

CRISPR RNA (cRNA), an endonuclease known as Cas9, and a transactivating crRNA

(tracrRNA). This system operates through two crucial elements: firstly, the Cas9

protein, responsible for DNA cleavage, and secondly, the guide RNA, which

identifies the specific DNA sequence for correction.(Tavakoli et al., 2021)

To employ CRISPR-Cas9, researchers begin by identifying the target genome

sequences they wish to modify. Subsequently, they customize the guide RNA to

recognize a specific sequence of nucleotides (A, T, G, or C) within the DNA. The

guide RNA binds to the DNA-cutting enzyme, Cas9, forming a complex that is then

introduced into the target cells. Cas9 precisely locates the target nucleotide and

initiates a cut in the DNA at that specific point. This process enables the alteration of

the existing genome by either making modifications or adding new sequences (refer to

Figure 1). (Tavakoli et al., 2021)

In essence, CRISPR-Cas9 functions as a molecular cut-and-paste tool for

DNA editing, allowing for precise modifications of any genomic sequence specified

by a short guide RNA. Notably, this system was first applied to the human genome in

2013. Since then, CRISPR-Cas9 has found widespread use in creating gene edits in
plants, animals, and human samples. Its versatile applications extend across various

scientific disciplines, including medical science, therapeutics, as well as plant and

animal sciences, making it a ubiquitous and transformative technology (Tavakoli et

al., 2021).
Figure 1: Schematic of CRISPR-Cas9 genome editing system
2.2 Applications of CRISPR-Cas9 technology

2.2.1 Human science

CRISPR-Cas9 is currently employed in the investigation of various genetic

disorders, including but not limited to hemophilia, Duchenne muscular dystrophy, α1-

antitrypsin deficiency, hearing impairment, and hematologic diseases. Among these,

hematologic conditions have historically presented challenges for treatment through

genome manipulation. However, recent studies have demonstrated the capability of

CRISPR-Cas9 to rectify genetic anomalies in hematopoietic stem cells, which are

responsible for the development of hematologic diseases. This breakthrough has

paved the way for the application of CRISPR-Cas9-based therapies involving

hematopoietic stem and progenitor cells (HSPCs). Notably, one of the most promising

strategies for treating hematologic diseases involves the precise editing of the HBB

mutation using CRISPR-Cas9 technology. Remarkably, this approach has already

yielded successful results in induced pluripotent stem cells (iPSCs) derived from

patients. In a survey conducted by Park et al., CRISPR-Cas9 technology was

employed to correct the HBB gene mutation in HSPCs. The final outcomes of this

study revealed a significant reduction in both hemoglobin levels and sickle cells,

highlighting the potential efficacy of this approach in treating hematologic diseases

(Tavakoli et al., 2021)

2.2.2 Plant science

Historically, biotic stresses in agriculture have been managed through the

dissemination of pathogen-resistant plant varieties and the application of

agrochemicals. However, the utilization of agrochemicals carries the potential for

environmental contamination and adverse effects on human health. Moreover, plant

pathogens exhibit an ongoing capacity to evolve and develop resistance to these

control measures.

To combat this, efforts were made to introduce resistance genes from wild

plant species into cultivated varieties through breeding and genetic transformation

technologies that involved the integration of large genomic regions. Nevertheless, this

approach had its drawbacks, as the lack of specificity in the introduced genomic

regions sometimes resulted in the unintentional incorporation of undesirable traits

alongside the desired resistance traits in elite cultivars.

In contrast, the CRISPR-Cas9 system has emerged as a more precise method

for genetic modification in plants. Since its introduction to the field, CRISPR-Cas9

has demonstrated its effectiveness in addressing various agricultural challenges,

including enhancing resistance to biotic stresses such as fungal and bacterial diseases

and viral infections. For instance, researchers have successfully employed CRISPR-

Cas9 to knock out the mitogen-activated protein kinase-5 (OsMPK5) gene in rice,

thereby bolstering disease resistance.

Notably, plant viruses can infect a wide range of plant species, posing a threat

to crop fertility and yield (Tavakoli et al., 2021).

2.2.3 Animal breeding

CRISPR-Cas9 stands out as the preeminent gene-editing technology of our

time, playing a pivotal role in the advancement of livestock improvement. This

technique is instrumental in effecting desired alterations by either augmenting the

frequency of advantageous alleles or eliminating detrimental ones. The impact of

CRISPR-Cas9 extends beyond the realm of livestock production, as it has also made

significant contributions to the field of biomedicine through the creation of transgenic

and cloned animals.

Furthermore, genome editing tools, including CRISPR-Cas9 and PiggyBac

transposon, hold considerable promise in immunology and vaccine development due

to their inherent safety and minimal risk to human health. These tools have the

potential to play a crucial role in preventing the transmission of viruses and enhancing

our understanding of immunological processes (Tavakoli et al., 2021).

2.3 Genetic engineering in micro organism

Microbial genetic engineering involves the utilization of genetic tools to

manipulate target genes through cutting, splicing, and integration processes,

subsequently introducing them into chassis cells. This facilitates the transfer of

recombinant genes into desired products or the conferment of new phenotypic traits to

bacteria (Liu et al., 2022). Recent advancements in sequencing technologies and

bioinformatics have led to the discovery of a growing repertoire of functional genes

and gene clusters from previously nonculturable microorganisms. Consequently, the

focus of research in the field of Genetically Engineered Bacteria (GEBs) has shifted

toward the challenge of expressing these functional genes or gene clusters within

chassis cells (Liu et al., 2022).

 The construction of GEBs involves two key stages: upstream, which

encompasses the acquisition of functional genes, and downstream, involving the

heterogeneous expression of these genes (Liu et al., 2022). The availability of

potential genes has been greatly expanded through national microbial genome projects

initiated in various countries. The choice of Molecular Genetic Engineering (MGE)

methods for GEB construction is influenced by the size of the target genes. Smaller

gene fragments (<10 kb) can be acquired and modified using general or long PCR

methods, direct DNA synthesis, and restriction enzyme digestion (Fahnøe and Bukh,

2019).

However, when dealing with genes exceeding 50 kb in size, recombination

techniques such as CRISPR–Cas9 and the Red/ET recombination system become the

preferred methods for altering, replacing, deleting, or adding bases or gene fragments

within plasmids or genomes (Strain-Damerell et al., 2021). Specifically, CRISPR–

Cas9 technology has the capability to edit bacterial DNA fragments up to 100 kb in a

single step. This process involves the RNA-guided Cas9 nuclease targeting and

cleaving DNA fragments, with the final assembly of large gene fragments achieved

through Gibson assembly (Liu et al., 2022). The precision and efficiency brought

about by CRISPR–Cas9 technology have significantly improved DNA manipulation,

greatly benefiting the extraction and heterologous expression of gene clusters within

chassis cells, particularly when dealing with genes exceeding 50 kb in size (Liu et al.,

2022).

2.4 CRISPR/Cas Technologies and Their Applications in Escherichia coli

 Escherichia coli (E. coli) stands as one of the most extensively employed

cellular platforms for the production of various biofuels and bulk chemicals,

encompassing ethanol, higher alcohols, fatty acids, amino acids, shikimate-

derivatives, terpenoids, polyketides, and essential polymer precursors like 1,4-

butanediol (Yang et al., 2021). Achieving the metabolic engineering required for the

synthesis of these biochemicals involves substantial modifications to the cellular

metabolism to enhance productivity. Efficient tools for genome editing are essential to

facilitate time-saving sequential or multiplex manipulations.

Numerous gene-editing techniques are available for E. coli, each possessing

distinct advantages and limitations. For instance, recombineering with double-

stranded DNA (dsDNA) often necessitates the use of selectable markers, which

subsequently need to be removed in the subsequent steps for further modifications

(Datsenko and Wanner, 2000; Sharan et al., 2009). In contrast, single-stranded DNA

(ssDNA)-mediated recombineering offers significantly improved efficiency and has

been developed into gene-editing tools for multiplex modifications, such as Multiplex

Automated Genome Engineering (MAGE) and trackable multiplex recombineering.

However, these methods may not be suitable for inserting multiple targeted genes

over 20 bp without the use of selectable markers and often require robust high-

throughput screening techniques.

Recently, the Clustered Regularly Interspaced Short Palindromic Repeats

(CRISPR)/CRISPR-associated protein (Cas) system has gained widespread use in

genetic engineering of E. coli, greatly facilitating its application. In this system, a

mature CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) duplex (or a

single synthetic guide RNA, sgRNA) guide the Cas nuclease(s) to cleave a target
DNA sequence at a specified protospacer adjacent motif (PAM). Notably, the

CRISPR/Cas system continues to cleave the target site until it is either successfully

edited or the unedited cell undergoes cell death, eliminating the need for selectable

markers.

Based on the structure and function of the Cas protein, CRISPR/Cas systems

are classified into two classes (class I and class II) and six types (types I ∼VI). Class I

comprises types I, III, and IV, whereas class II encompasses types II, V, and VI.

Types I, II, and V are recognized for their DNA cleavage capabilities, type VI can edit

RNA, and type III can edit both DNA and RNA (Wang et al., 2019). Due to their

relatively simple structure, type II and type V systems are widely employed in E. coli.

Additionally, endogenous type I and type III systems have been adapted into efficient

genome engineering tools for E. coli (Figure 2) (Dong et al., 2021).

Figure 2: Schematic overview of CRISPR/Cas systems as genome engineering

tools. (A) Type II (Cas9), (B) Type Ⅴ (Cas12a), (C) Type I (Cas3), (D) Type III

(Cas10).
2.5 Lipase

Lipases are highly versatile enzymes that have garnered significant attention in

various industrial processes. They can be sourced from a range of origins, including

animals, plants, and microorganisms. Lipases, classified as triacylglycerol

acylhydrolases, act on carboxylic ester bonds and belong to the hydrolase enzyme

family (Chandra et al., 2020). Notably, they do not necessitate cofactors and fall

within the category of serine hydrolases (Chandra et al., 2020). These enzymes play a

pivotal role in hydrolyzing triglycerides into diglycerides, monoglycerides, fatty

acids, and glycerol naturally (Fig. 3). It's worth mentioning that esterases can also

hydrolyze carboxylic ester bonds alongside lipases (Pascoal et al., 2018).

Lipases are widespread in nature, with animals, plants, and microorganisms

serving as sources for their production. Among these sources, microorganisms hold

particular significance in commercial lipase production. Various microorganisms

produce diverse lipase variants, each possessing unique activity, stability, and

substrate preferences tailored to their specific biological functions. The challenge lies

in identifying the most suitable lipase variants for specific catalytic reactions from this

wide array of natural lipases (Pascoal et al., 2018).

Continuous efforts are underway to discover new lipases through extensive

screening programs involving microorganisms that can be cultivated in laboratory

settings or by initiating searches within metagenomics libraries. At times, promising

lipase candidates may be found in microorganisms that may not perform optimally in

large-scale production. In such cases, heterologous production becomes imperative to

harness the diversity of lipases (Pascoal et al., 2018).

 Escherichia coli and Komagataella phaffii (formerly known as Pichia pastoris)

are the most commonly utilized hosts for recombinant lipase production. Nonetheless,

there is a growing interest in other species with robust secretory capabilities.

Filamentous fungi, such as Aspergillus spp. and Trichoderma reesei, possess highly

efficient secretory pathways, enabling them to yield more than 30 grams of protein

per liter of culture medium. Additionally, various Bacillus species serve as viable

alternatives for the recombinant production of prokaryotic enzymes, achieving

production levels of up to 20 grams of protein per liter of medium. Although some

successes have been reported in overproducing lipases using these organisms, there

remains substantial potential for exploration in the realm of recombinant lipase

production using these hosts (Contesini et al., 2020).

Lipases typically exhibit molecular weights ranging from 19 to 60 kDa and are

generally reported as monomeric proteins. The physical properties of lipases depend

on factors such as the position of the fatty acid in the glycerol backbone, the chain

length of the fatty acid, and its degree of unsaturation (Carpen et al., 2019). Many

lipases are capable of catalyzing a variety of useful reactions, including esterification,

due to their activity in organic solvents. Their enzymatic activities are often pH-

dependent, with many lipases exhibiting optimal activity at a neutral pH of 7.0 or

within the range of pH 4.0 to 8.0. Some lipases produced by microorganisms like

Chromobacterium viscosum, A. niger, and Rhizophus sp. remain active at acidic pH

levels, while P. nitroaeducens produces an alkaline lipase active at pH 11.0. Under

specific experimental conditions, lipases can even catalyze reversible reactions,

leading to esterification and interesterification in the absence of water. Importantly,

lipase activities do not typically require cofactors, although calcium is a divalent

cation that can stimulate their activity. Conversely, other ions such as Co, Ni2+,
Hg2+, and Sn2+ can substantially inhibit lipase activities, while Zn2+, Mg2+, EDTA,

and SDS may inhibit them to a lesser extent (Carpen et al., 2019)

Figure 3: (A) Hydrolysis of triglyceride converts into glycerol and fatty acid, (B)

Representation of a molecule of lipase with its features(Chandra et al., 2020)

2.6 Structural characteristic of lipase

Understanding the structure of an enzyme holds paramount importance, not only for

enhancing enzyme production but also for facilitating protein engineering through

site-directed mutagenesis. Regarding the structural characteristics of lipases, they

possess a catalytically active core domain known as the α/β-hydrolase fold, which

shares similarities with other hydrolases (Contesini et al., 2020). This core domain

houses the catalytic triad and the oxyanion hole. Some lipases may also feature

additional structural elements, such as a lid or flap-like structure. The core domain of

most lipases predominantly comprises eight parallel β strands, forming a centrally

twisted β sheet, which is encircled by a varying number of α helices. It's important to

note that the number and arrangement of β strands can differ among lipases. For

instance, the canonical fold of Bacillus subtilis lipase lacks the β1 and β2 strands

(Contesini et al., 2020). Furthermore, the first X-ray structure of a triglyceride lipase,

Mucor miehei, was reported by Brady et al. (Contesini et al., 2020). Figure 4 provides

a visual representation of the three-dimensional structure of two microbial lipases,

illustrating the lid in both open and closed

conformations.

Figure 4: An overview of the three-dimensional structure of two microbial lipases is

presented. (A) The crystallographic depiction of Candida rugosa lipase reveals its α/β-

hydrolase fold shown in gray, illustrating the open configuration of the lid (PDB

access number 1CRL). The closed position of the lid is depicted with a wheat tint

(PDB access number 1TRH). (B) The crystallographic portrayal of Rhizomucor

miehei lipase shows its α/β-hydrolase fold in gray, representing the lid in its open

state (PDB access number 4TGL). The closed configuration of the lid is depicted in

cyan (PDB access number 3TGL). In both structures, the lids are highlighted in red.

These illustrations were generated using molecular visualization software, PYMOL.

(Contesini et al., 2020)

2.7 Industrial application of lipase

2.7.1 fat and oleo-chemical industry

The current trend in the oleo chemical industry involves the use of immobilized lipase

to initiate various reactions such as hydrolysis, alcoholic’s, and glycolysis using

mixed substrates. Thus, the use immobilized enzyme ensures high productivity as

well as continuous running of the processes. This offers a greatest hope for successful

fat splitting/modification without substantial investment in expensive equipment as

well as in expenditure of large amounts of thermal energy. (Choudhury et al., 2015)

2.7.2 Detergent industry

Lipases in the detergent industry have gained momentum following the

success of proteases as detergent additives. These enzymes are evaluated for

compatibility with various commercial detergents and bleaching agents to eliminate

fats. Enzyme use is prevalent in detergent formulations in developing countries,

making laundry detergents increasingly popular due to their fabric softening,

antistatic, and water-dispersible properties. Notably, Thermomyces sp., particularly

Lipolase by Novozymes, and Pseudomonas spp. formulations like Lumafast and

Lipomax are widely used detergent lipases. Their optimal performance at alkaline pH

and elevated temperatures makes them attractive choices for detergent applications.

Numerous Pseudomonas species are explored for effective lipase applications in

various fields. Additionally, crude lipolytic extracts from P. aeruginosa exhibit

substantial stability in the presence of certain nonionic surfactants and local

commercial detergents, further enhancing their utility. (Choudhury et al., 2015)

2.7.3 Production of biodegradable polymer

Lipases are pivotal in organic syntheses due to their versatile applications,

particularly in the production of biodegradable compounds. Recent studies have

highlighted their role in esterifying butanol to produce 1-Butyl lactate and in reducing

the viscosity of biodiesel, especially during winter conditions. Additionally, lipases

facilitate the synthesis of trimethylol-propane esters as lubricants through direct

esterification. These enzymes also serve as catalysts for ester syntheses and

transesterification reactions in organic solvent systems, offering the potential for bio-

catalyzed biodegradable polyester production. Notably, lipase biocatalysts can be

employed in the synthesis of aromatic polyesters. (Choudhury et al., 2015)

2.7.4 Food processing, flavor development and improving quality

Lipases play a pivotal role in the food processing industry, primarily in biomaterial

modification and breakdown. They are extensively employed in various food

applications, including dairy products, meat, vegetables, fruits, baked goods, milk

products, and beer (Raveendran et al., 2018). In the dairy sector, lipases hydrolyze

milk fat, altering the fatty acid chain lengths to enhance cheese flavors. They

accelerate cheese ripening and facilitate the lipolysis of fatty products like cheese and

cream, contributing to flavor development. Various microbial lipases, such as those

from M. miehei, A. niger, and A. oryzae, are utilized in cheese manufacturing.

Additionally, lipases are employed to modify flavor in food by synthesizing esters of

short-chain fatty acids and alcohols, known as flavor and fragrance compounds.

(Choudhury et al., 2015).

2.7.5 Pulp and paper industry

Wood serves as the primary raw material for the paper and pulp industry, yet its

content of hydrophobic components, chiefly triglycerides and waxes (known as pitch),

poses significant challenges during production. To address this issue, lipases are

employed in the papermaking process to effectively eliminate pitch from the pulp. For

example, in Japan, Nippon Paper Industries has successfully implemented a pitch

control system utilizing a lipase derived from C. rugosa to remove the majority of

wood triglycerides. (Choudhury et al., 2015)

2.7.6 Lipase in bio diesel

Lipases, recognized for their exceptional biochemical and physiological attributes, are

gaining prominence as biocatalysts in various biotechnological applications. These

hydrolytic enzymes find utility in a range of industrial processes, including

alcoholysis, acidolysis, aminolysis, and hydrolysis reactions. A noteworthy

application is their role as biocatalysts in biodiesel production, a practice initiated

around 2012. Lipase-catalyzed biodiesel production involves a two-step process

encompassing ester bond hydrolysis and esterification with a secondary substrate (Ali

et al., 2023). While lipases from diverse sources can catalyze the same reaction,

bacterial and fungal lipases are predominantly favored in biodiesel production.

Notable examples include A. niger, C. antarctica, C. rugosa, C. viscosum, M. miehei,

P. cepacia, P. fluorescens, P. lipolyticum, Rhizopus oryzae, Streptomyces sp., and T.

lanuginose. Among these, C. rugosa, sourced from yeast, stands out as the most

frequently employed microorganism for lipase production. More recently,

Streptomyces sp. has emerged as a promising lipase-producing microbe for biodiesel

production, marking an innovative step in the field of sustainable biodiesel.

(Choudhury et al., 2015)

2.8 Production of lipase

2.8.1 Microbial sources of lipase

Microbial lipases are enzymes sourced from microorganisms, including

bacteria, yeasts, and filamentous fungi. In contrast to lipases derived from animals,

such as pancreatic lipases, and lipases found in plants like Carica papaya and various

seeds, microbial lipases have gained significant prominence in industrial applications.

This heightened recognition is primarily attributed to their suitability for cultivation in

straightforward and cost-effective growth media, ease of handling, scalability in

production, and the availability of diverse tools for both genetic and protein

engineering. Additionally, the substantial knowledge base pertaining to the genetics

and physiology of these microorganisms, particularly well-studied model organisms

such as E. coli, K. phaffii, and Saccharomyces cerevisiae, makes them highly

desirable candidates for lipase production (Contesini et al., 2020).

Microorganisms thriving in extreme environments characterized by conditions

such as extreme pH levels, salinity, and temperature variations serve as valuable

sources of lipases with significant industrial applications. However, it can be

challenging to culture these microorganisms under laboratory conditions,

complicating the harnessing of their lipases for practical use. Extremophile bacteria,

which include thermophiles residing in hot springs, psychrophiles inhabiting deep-sea

sediments, and those found in exceptionally cold regions like Antarctica, have

evolved to survive in such harsh environments. These extremophiles produce a

diverse array of lipases known for their distinctive tolerance properties (Contesini et

al., 2020)

2.8.2 Production of recombinant lipases

 In situations where the native microorganism producing lipases can be

efficiently cultured in a bioreactor, homologous expression emerges as a viable

option. This approach often simplifies the production process since lipase production

is naturally optimized within the host organism. There is often a desire to boost lipase

yields, which can be accomplished by employing stronger promoters to drive the

expression of the lipase gene. Additionally, hosts can be equipped with extra copies of

the lipase gene through the use of plasmids containing the lipase gene or by

integrating lipase genes into the host's genome. Some studies have investigated the

production of recombinant lipases through homologous expression in bacteria like

Serratia marcescens and Burkholderia cepacia, as well as in the filamentous fungus A.

niger. However, native lipases may not always meet the technical requirements for

industrial applications, such as rapid growth, high protein production levels, or

optimal physiological properties needed in bioreactors. Therefore, heterologous

expression serves as an alternative approach, making use of well-established and

efficient host organisms.

In technical terms, heterologous expression systems typically involve three

primary steps: (i) cloning the gene of interest into a vector that contains a selection

marker, (ii) introducing the constructed plasmid into the host strain, and (iii)

expressing the gene of interest under the control of either an inducible or constitutive

promoter, along with a known terminator. Heterologous expression systems

encompass both prokaryotic and eukaryotic hosts, with examples being E. coli and K.

phaffii, respectively. This approach has also been employed in numerous studies

involving recombinant lipases produced by microorganisms from extreme

environmental conditions that cannot be readily cultured. In such cases, the genes

responsible for encoding their lipases can be isolated and expressed in heterologous
expression systems by constructing functional metagenomics libraries. Functional

metagenomics offers the advantage of not requiring individual genome sequencing or

the cultivation of unknown producer microorganisms. For a more comprehensive

exploration of metagenomics studies related to lipases with biotechnological potential,

Almeida and collaborators provide an informative review (Contesini et al., 2020)

2.9 Potato peel waste

The potato crop holds great significance globally, ranking as the third-largest

crop and serving as a crucial source of nutritious food, especially in developing

nations. The substantial growth in potato production worldwide has played a pivotal

role in ensuring global food security. However, this increase in potato production has

also led to the generation of substantial quantities of potato peel waste, accounting for

approximately 8% of the potato's weight (Almuhayawi et al., 2023). It is estimated

that the annual worldwide production of potato peel waste amounts to around 370

million tons. This escalating volume of waste generated by the potato industry

presents notable environmental and disposal challenges (Almuhayawi et al., 2023).

A significant portion of these solid organic wastes, commonly found in urban

areas, is disposed of in landfills, resulting in the loss of valuable land resources and

the release of organic chemicals such as methane (CH4) during anaerobic

fermentation processes. This release of methane is a concern because methane is a

potent greenhouse gas with significant energy value, possessing a global warming

potential over 300 times greater than that of CO2 (Almuhayawi et al., 2023)
2.9 E coli as model organism

Escherichia coli (E. coli) is a Gram-negative bacterium characterized by its

rod-shaped morphology and is commonly found in the lower intestine (colon) of

warm-blooded organisms (Idalia and Bernado, 2017). Model organisms are non-

human species strategically chosen for laboratory research due to their ease of

maintenance, breeding, and experimental utility. E. coli stands out as an exceptionally

well-suited model organism, having been employed for over six decades in scientific

investigations. Its cost-effective and straightforward cultivation methods have made it

a subject of extensive study, and it occupies a central position in both biotechnology

and microbiology research (Idalia and Bernado, 2017).

E. coli possesses the remarkable ability to acquire nutrients from its

surroundings and synthesize essential nutrients when they are unavailable. This

adaptability allows it to thrive in a minimal medium containing only essential

nutrients. Consequently, researchers can identify mutants that are unable to grow in

such minimal conditions but can grow when specific nutrients are supplemented

(Idalia and Bernado, 2017).

Several key attributes contribute to E. coli's suitability as a model organism, including

its single-celled nature, rapid reproduction rate, ease of cultivation, adaptability to

varying growth conditions, the presence of naturally occurring harmless strains, and

its amenability to genetic manipulation (Idalia and Bernado, 2017).

2.9.1 Genetic Manipulation of E. coli

Escherichia coli has been particularly valuable to molecular biologists due to

its relatively short doubling time, simplicity, and ease of genetic modification (Tuttle
et al., 2017). E. coli can be easily propagated and studied in laboratory settings. Its

genome, comprising approximately 4.6 million base pairs and encoding about 4,000

different proteins, contrasts sharply with the human genome, which is nearly a

thousand times more complex, with approximately 3 billion base pairs and encoding

around 100,000 different proteins. The compact size of the E. coli genome offers clear

advantages for genetic analysis, and the complete sequencing of the E. coli genome

has been accomplished (Tuttle et al., 2017). Notably, researchers have endeavored to

develop an E. coli K-12 derivative with the ability to metabolize sucrose by

introducing the SacC enzyme from Mannheimia succiniciproducens (M.

succiniciproducens), thus conferring sucrose-utilizing capabilities to other organisms

lacking this ability. This approach holds significant promise for various applications.

(Tuttle et al., 2017)

2.9.2 Applications of Escherichia coli in Biotechnology

Escherichia coli, commonly referred to as E. coli, plays a crucial role in the

field of industrial microbiology and modern biological engineering due to its well-

established history of laboratory cultures and ease of genetic manipulation

(Sadominoco et al., 2020). It has been extensively utilized for the production of

heterologous proteins, and various protein expression systems have been developed to

facilitate recombinant protein production in E. coli. One notable milestone in the

history of recombinant DNA technology involved modifying E. coli to produce

human insulin (Sadominoco et al., 2020). However, certain limitations are associated

with using E. coli for recombinant biopharmaceutical production, primarily linked to

the absence of critical post-translational modifications (PTMs) such as glycosylation,

phosphorylation, proteolytic processing, and disulfide bond formation, which are

essential for the biological activity of many proteins (Sadominoco et al., 2020).

Recent advancements have addressed some of these limitations, enabling the

successful expression of proteins in folded forms that were previously considered

challenging or unattainable. In cases where proteins depend on glycosylation for

function or stability, researchers have engineered the N-linked glycosylation system

from Campylobacter jejuni into E. coli for expression purposes (Sadominoco et al.,

2020). This innovative approach allows for the production of glycosylated

heterologous proteins in E. coli. Modified E. coli cells also find applications in

diverse areas, including vaccine development and biofuel production (Sadominoco et

al., 2020).
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