CHAPTER ONE

INTRODUCTION

Synthetic biology is a scientific area that uses science and technology to build or redesign

existing biological systems or new organisms, such as enzymes, genetic circuits and cells

(Carbonell et al., 2018). It emerges from various fields of science including biology, engineering

and computer science. SB differs from conventional genetic engineering in terms of the

complexity of organisms or systems created by researchers. The aim is to design and build

biological systems at each level of organization through genetic networks and entire organism

cooperation rather than focusing on the expression of individual genes or gene components

(Carbonell et al., 2018). Modification of an organism’s genome can cause unpredictable effects

and increase the complexity of the genome. SB aims to build or reform new organisms in human

services (Han et al., 2012); produce highly sought products in medicine, energy, the environment

and agriculture. It also has a high potential for creating new jobs, boosting the global economy

and offering solutions to environmental challenges. This paper provides brief information about

the advantages, risks, applications and ethical concerns in SB.

Synthetic biologists might use synthetic genomics to partially or wholly synthesize genes of

restructured or completely novel life forms with the aims to create life forms that are

substantially different from those that already exist (Hwang et al., 2020). To achieve their goal,

synthetic biologists pursue several visible projects, like building the library for the biological

parts and devices with known functions or features. In addition, synthetic biologists have

attempted to develop microbial pathways for the production of chemical compounds. These areas

aim to produce biological systems as biochemical plants using energy, industry and medicine
(Ma et al., 2020). SB is a technology applied in all sectors, such as agriculture, food, health,

chemical and industrial production. It is used widely in nearly all science areas.

Synthetic biology techniques based on rapid design, with iterative prototyping of the gene

circuits, have enabled the creation of several innovative diagnostic approaches. Many of these

solutions are ongoing and show the growing maturation of the field for critical biomedical issues

(Schooley et al., 2017). SB offers potential benefits for immunoassay development, diagnosis,

drug screening, new antibiotics generation, drug production and sensor-effector therapeutic

development. Diagnosis of communicable and non-communicable diseases including cancer,

coronary artery disease, Ebola, Zika, tuberculosis, malaria, HIV, SARS-CoV-2, routine blood

test quantification, and water quality monitoring has been successfully performed using SB

(McCarty et al., 2019). Recent work in animal models for human diseases has shown that the use

of sensor effectors and mammalian cell reprogramming may soon pave the way for genetic and

cellular therapies (Eustáquio et al., 2004).

Microbial consortia have been used in biotechnology processes, including fermentation, waste

treatment, and agriculture, for millennia. Today, synthetic biologists are increasingly engineering

microbial consortia for diverse applications, including the bioproduction of medicines, biofuels,

and biomaterials from inexpensive carbon sources (Byrne et al., 2013). An improved

understanding of natural microbial ecosystems, and the development of new tools to construct

synthetic consortia and program their behaviors, will vastly expand the functions that can be

performed by communities of interacting microorganisms. Here, we review recent advancements

in synthetic biology tools and approaches to engineer synthetic microbial consortia, discuss

ongoing and emerging efforts to apply consortia for various biotechnological applications, and

suggest future applications.

 CHAPTER TWO

SYNTHETIC BIOLOGY

Synthetic biology is a multidisciplinary field of science that focuses on living systems and

organisms, and it applies engineering principles to develop new biological parts, devices, and

systems or to redesign existing systems found in nature (Ando et al., 2015). It is a branch of

science that encompasses a broad range of methodologies from various disciplines, such

as biotechnology, biomaterials, material science/engineering, genetic engineering, molecular

biology, molecular engineering, systems biology, membrane science, biophysics, chemical and

biological engineering, electrical and computer engineering, control

engineering and evolutionary biology.

It includes designing and constructing biological modules, biological systems, and biological

machines, or re-designing existing biological systems for useful purposes (Edgar et al., 2012).

Additionally, it is the branch of science that focuses on the new abilities of engineering into

existing organisms to redesign them for useful purposes (Hennig et al., 2015). In order to

produce predictable and robust systems with novel functionalities that do not already exist in

nature, it is also necessary to apply the engineering paradigm of systems design to biological

systems. According to the European Commission, this possibly involves a molecular assembler

based on biomolecular systems such as the ribosome.

2.1 History of Synthetic Biology

1910: First identifiable use of the term synthetic biology in Stéphane Leduc's publication Théorie

physico-chimique de la vie et générations spontanées. He also noted this term in another

publication, La Biologie Synthétique in 1912.

1944: Canadian-American scientist Oswald Avery shows that DNA is the material of

which genes and chromosomes are made. This becomes the bedrock on which all subsequent

genetic research is built.

1953: Francis Crick and James Watson publish the structure of the DNA in Nature.

1961: Jacob and Monod postulate cellular regulation by molecular networks from their study of

the lac operon in E. coli and envisioned the ability to assemble new systems from molecular

components.

1973: First molecular cloning and amplification of DNA in a plasmid is published in P.N.A.S. by

Cohen, Boyer et al. constituting the dawn of synthetic biology.

1978: Arber, Nathans and Smith win the Nobel Prize in Physiology or Medicine for the

discovery of restriction enzymes, leading Szybalski to offer an editorial comment in the

journal Gene:

The work on restriction nucleases not only permits us easily to construct recombinant DNA

molecules and to analyze individual genes, but also has led us into the new era of synthetic

biology where not only existing genes are described and analyzed but also new gene

arrangements can be constructed and evaluated.

1988: First DNA amplification by the polymerase chain reaction (PCR) using a thermostable

DNA polymerase is published in Science by Mullis et al., 2017. This obviated adding new DNA

polymerase after each PCR cycle, thus greatly simplifying DNA mutagenesis and assembly.

2000: Two papers in Nature report synthetic biological circuits, a genetic toggle switch and a

biological clock, by combining genes within E. coli cells.

2003: The most widely used standardized DNA parts, BioBrick plasmids, are invented by Tom

Knight.[14] These parts will become central to the International Genetically Engineered

Machine (iGEM) competition founded at MIT in the following year.

2003: Researchers engineer an artemisinin precursor pathway in E. coli.

2004: First international conference for synthetic biology, Synthetic Biology 1.0 (SB1.0) is held

at MIT.

2005: Researchers develop a light-sensing circuit in E. coli. Another group designs circuits

capable of multicellular pattern formation.

2006: Researchers engineer a synthetic circuit that promotes bacterial invasion of tumour cells.

2010: Researchers publish in Science the first synthetic bacterial genome, called M.

mycoides JCVI-syn1.0. The genome is made from chemically-synthesized DNA using yeast

recombination.

2011: Functional synthetic chromosome arms are engineered in yeast.

2012: Charpentier and Doudna labs publish in Science the programming of CRISPR-

Cas9 bacterial immunity for targeting DNA cleavage. This technology greatly simplified and

expanded eukaryotic gene editing.

2019: Scientists at ETH Zurich report the creation of the first bacterial genome,

named Caulobacter ethensis-2.0, made entirely by a computer, although a related viable

form of C. ethensis-2.0 does not yet exist.

2019: Researchers report the production of a new synthetic (possibly artificial) form

of viable life, a variant of the bacteria Escherichia coli, by reducing the natural number of

64 codons in the bacterial genome to 59 codons instead, in order to encode 20 amino acids.

2020: Scientists created the first xenobot, a programmable synthetic organism derived from frog

cells and designed by AI.

2021: Scientists reported that xenobots are able to self-replicate by gathering loose cells in the

environment and then forming new xenobot.

2.2 Categories of synthetic biology

Bioengineering, synthetic genomics, protocell synthetic biology, unconventional molecular

biology, and in silico techniques are the five categories of synthetic biology (Chen et al., 2009).

It is necessary to review the distinctions and analogies between the categories of synthetic

biology for its social and ethical assessment, to distinguish between issues affecting the whole

field and particular to a specific one (Ji et al., 2022).

2.2.1 Bioengineering

The subfield of bioengineering concentrates on creating novel metabolic and regulatory

pathways, and is currently the one that likely draws the attention of most researchers and

funding. It is primarily motivated by the desire to establish biotechnology as a legitimate

engineering discipline. When referring to this area of synthetic biology, the word

"bioengineering" should not be confused with "traditional genetic engineering," which involves

introducing a single transgene into the intended organism. Bioengineers adapted synthetic

biology to provide a substantially more integrated perspective on how to alter organisms or

metabolic systems (Ji et al., 2022).

A typical example of single-gene genetic engineering is the insertion of the human insulin gene

into bacteria to create transgenic proteins. The creation of whole new signalling pathways,

containing numerous genes and regulatory components (such as an oscillator circuit to initiate

the periodic production of green fluorescent protein (GFP) in mammalian cells), is known as

bioengineering as part of synthetic biology (Ji et al., 2022).

By utilising simplified and abstracted metabolic and regulatory modules as well as other

standardized parts that may be freely combined to create new pathways or creatures,

bioengineering aims to create innovative biological systems. In addition to creating infinite

opportunities for novel applications, this strategy is anticipated to make bioengineering more

predictable and controllable than traditional biotechnology (Ji et al., 2022).

2.2.2 Synthetic genomics

The formation of animals with a chemically manufactured (minimal) genome is another facet of

synthetic biology that is highlighted by synthetic genomics. This area of synthetic biology has

been made possible by ongoing advancements in DNA synthesis technology, which now makes

it feasible to produce DNA molecules with thousands of base pairs at a reasonable cost. The goal

is to combine these molecules into complete genomes and transplant them into living cells,

replacing the host cell's genome and reprogramming its metabolism to perform different

functions (Ji et al., 2022).

Scientists have previously demonstrated the potential of this approach by creating infectious

viruses by synthesising the genomes of multiple viruses. These significant advances in science

and technology triggered the initial public concerns concerning the risks associated with this

technology (Ji et al., 2022).

A simple genome might also work as a "chassis genome" that could be enlarged quickly by gene

inclusion created for particular tasks. Such "chassis creatures" would be more suited for the

insertion of new functions than wild organisms since they would have fewer biological pathways

that could potentially conflict with the new functionalities in addition to having specific insertion

sites. Synthetic genomics strives to create creatures with novel "architectures," much like the

bioengineering method. It adopts an integrative or holistic perspective of the organism. In this

case, the objective is the creation of chassis genomes based on necessary genes and other

required DNA sequences rather than the design of metabolic or regulatory pathways based on

abstract criteria (Ji et al., 2022).

2.2.3 Protocell synthetic biology

The in vitro generation of synthetic cells is the protocell branch of synthetic biology. Lipid

vesicles, which have all the necessary components to function as a complete system, can be used

to create these artificial cells. In the end, these synthetic cells should meet the requirements for

being deemed alive, namely the capacity for self-replication, self-maintenance, and evolution.

The protocell technique has this as its end aim, however there are other intermediary steps that

fall short of meeting all the criteria for a living cell. In order to carry out a specific function,

these lipid vesicles contain cell extracts or more specific sets of biological macromolecules and

complex structures, such as enzymes, nucleic acids, or ribosomes. For instance, liposomes may

carry out particular polymerase chain reactions or synthesise a particular protein (Ji et al., 2022).

Protocell synthetic biology takes artificial life one step closer to reality by eventually

synthesizing not only the genome but also every component of the cell in vitro, as opposed to the

synthetic genomics approach, which relies on coercing a natural cell to carry out the instructions
encoded by the introduced synthetic genome. Synthetic biologists in this field view their work as

basic study into the conditions necessary for life to exist and its origin more than in any of the

other techniques. The protocell technique, however, also lends itself well to applications; similar

to other synthetic biology byproducts, protocells could be employed for the manufacture of

biopolymers and medicines (Ji et al., 2022).

2.2.4 Unconventional molecular biology

The objective of the "unnatural molecular biology" strategy is to create new varieties of life that

are based on a different kind of molecular biology, such as new types of nucleic acids or a new

genetic code. The creation of new types of nucleotides that can be built into unique nucleic acids

could be accomplished by changing certain DNA or RNA constituents, such as the bases or the

backbone sugars (Ji et al., 2022). The normal genetic code is being altered by inserting

quadruplet codons or changing some codons to encode new amino acids, which would

subsequently permit the use of non-natural amino acids with unique features in protein

production. It is a scientific and technological problem to adjust the enzymatic machinery of the

cell for both approaches (Ji et al., 2022).

A new sort of life would be formed by organisms with a genome built on synthetic nucleic acids

or on a totally new coding system for synthetic amino acids. This new style of life would have

some benefits but also some new dangers. On release into the environment, there would be no

horizontal gene transfer or outcrossing of genes with natural species. Furthermore, these kinds of

synthetic organisms might be created to require non-natural materials for protein or nucleic acid

synthesis, rendering them unable to thrive in the wild if they accidentally escaped.[38]
On the other hand, if these organisms ultimately were able to survive outside of controlled space,

they might have a particular benefit over natural organisms because they would be resistant to

predatory living organisms or natural viruses, that could lead to an unmanaged spread of the

synthetic organisms (Ji et al., 202).

2.2.5 In silico technique

Synthetic biology in silico and the various strategies are interconnected. The development of

complex designs, whether they are metabolic pathways, fundamental cellular processes, or

chassis genomes, is one of the major difficulties faced by the four synthetic-biology methods

outlined above. Because of this, synthetic biology has a robust in silico branch, similar to

systems biology, that aims to create computational models for the design of common biological

components or synthetic circuits, which are essentially simulations of synthetic organisms

(Petzold et al., 2015).

The practical application of simulations and models through bioengineering or other fields of

synthetic biology is the long-term goal of in silico synthetic biology. Many of the computational

simulations of synthetic organisms up to this point possess little to no direct analogy to living

things. It is sensible to integrate the five areas under the umbrella of synthetic biology as an

unified area of study. Even though they focus on various facets of life, such as metabolic

regulation, essential elements, or biochemical makeup, these five strategies all work toward the

same end: creating new types of living organisms. Additionally, the varied methodologies begin

with numerous methodological approaches, which leads to the diversity of synthetic biology

approaches (Petzold et al., 2015).

Synthetic biology is an interdisciplinary field that draws from and is inspired by many different

scientific disciplines, not one single field or technique. Synthetic biologists all have the same

underlying objective of designing and producing new forms of life, despite the fact that they may

employ various methodologies, techniques, and research instruments. Any evaluation of

synthetic biology, whether it examines ethical, legal, or safety considerations, must take into

account the fact that while some questions, risks, and issues are unique to each technique, in

other circumstances, synthetic biology as a whole must be taken into consideration (Petzold et

al., 2015).

2.3 Applications of Synthetic Biology

Synthetic biology initiatives frequently aim to redesign organisms so that they can create a

material, such as a drug or fuel, or acquire a new function, such as the ability to sense something

in the environment. Examples of what researchers are creating using synthetic biology include:

 Utilizing microorganisms for bioremediation to remove contaminants from our water, soil,

and air.

 Production of complex natural products that are usually extracted from plants but cannot be

obtained in sufficient amounts, e.g. drugs of natural origin, such

as artemisinin and paclitaxel.

 Beta-carotene, a substance typically associated with carrots that prevents vitamin A

deficiency, is produced by rice that has been modified. Every year, between 250,000 and

500,000 children lose their vision due to vitamin A deficiency, which also significantly

raises their chance of dying from infectious infections.

 As a sustainable and environmentally benign alternative to the fresh roses that perfumers use

to create expensive smells, yeast has been created to produce rose oil.
2.3.1 Biosensors

A biosensor refers to an engineered organism, usually a bacterium, that is capable of reporting

some ambient phenomenon such as the presence of heavy metals or toxins. One such system is

the Lux operon of Aliivibrio fischeri, (Yehl et al., 2019) which codes for the enzyme that is the

source of bacterial bioluminescence, and can be placed after a respondent promoter to express

the luminescence genes in response to a specific environmental stimulus (Yehl et al., 2019). One

such sensor created, consisted of a bioluminescent bacterial coating on a

photosensitive computer chip to detect certain petroleum pollutants. When the bacteria sense the

pollutant, they luminesce (Zhang et al., 2020). Another example of a similar mechanism is the

detection of landmines by an engineered E.coli reporter strain capable of detecting TNT and its

main degradation product DNT, and consequently producing a green fluorescent protein (GFP).

Modified organisms can sense environmental signals and send output signals that can be detected

and serve diagnostic purposes. Microbe cohorts have been used (Ye et al., 2014). Biosensors

could also be used to detect pathogenic signatures—such as of SARS-CoV-2—and can

be wearable (Perrino et al., 2021). For the purpose of detecting and reacting to various and

temporary environmental factors, cells have developed a wide range of regulatory circuits,

ranging from transcriptional to post-translational. These circuits are made up of transducer

modules that filter the signals and activate a biological response, as well as carefully designed

sensitive sections that attach analytes and regulate signal-detection thresholds. Modularity and

selectivity are programmed to biosensor circuits at the transcriptional, translational, and post-

translational levels, to achieve the delicate balancing of the two basic sensing modules (Perrino

et al., 2021).
2.3.2 Food and drink

This section is an excerpt from Cellular agriculture. Cellular agriculture focuses on the

production of agricultural products from cell cultures using a combination

of biotechnology, tissue engineering, molecular biology, and synthetic biology to create and

design new methods of producing proteins, fats, and tissues that would otherwise come from

traditional agriculture (Wu et al., 2015). Most of the industry is focused on animal products such

as meat, milk, and eggs, produced in cell culture rather than raising and slaughtering farmed

livestock which is associated with substantial global problems of detrimental environmental

impacts (e.g. of meat production), animal welfare, food security and human health (Wu et al.,

2015). Cellular agriculture is a field of the biobased economy. The most well known cellular

agriculture concept is cultured meat.

2.3.3 Materials

Photosynthetic microbial cells have been used as a step to synthetic production of spider silk (Ye

et al., 2014).

2.3.4 Biological computers

A biological computer refers to an engineered biological system that can perform computer-like

operations, which is a dominant paradigm in synthetic biology. Researchers built and

characterized a variety of logic gates in a number of organisms and demonstrated both analog

and digital computation in living cells. They demonstrated that bacteria can be engineered to

perform both analog and/or digital computation (Zhu et al., 2012). In 2017, in human cells,

research demonstrated a universal logic evaluator that operates in mammalian

cells. Subsequently, researchers utilized this paradigm to demonstrate a proof-of-concept therapy

that uses biological digital computation to detect and kill human cancer cells in 2011 (Zhu et al.,

2012). In 2016, another group of researchers demonstrated that principles of computer

engineering can be used to automate digital circuit design in bacterial cells (Benítez‐Chao et al.,

2020).

2.3.5 Cell transformation

Cells use interacting genes and proteins, which are called gene circuits, to implement diverse

function, such as responding to environmental signals, decision making and communication.

Three key components are involved: DNA, RNA and Synthetic biologist designed gene circuits

that can control gene expression from several levels including transcriptional, post-

transcriptional and translational levels.

Traditional metabolic engineering has been bolstered by the introduction of combinations of

foreign genes and optimization by directed evolution. This includes engineering E.

coli and yeast for commercial production of a precursor of the antimalarial drug, Artemisinin

(Breitling and Takano, 2015).

Entire organisms have yet to be created from scratch, although living cells can

be transformed with new DNA. Several ways allow constructing synthetic DNA components and

even entire synthetic genomes, but once the desired genetic code is obtained, it is integrated into

a living cell that is expected to manifest the desired new capabilities or phenotypes while

growing and thriving (Chen et al., 2014). Cell transformation is used to create biological circuits,

which can be manipulated to yield desired outputs (Dedrick et al., 2019).

By integrating synthetic biology with materials science, it would be possible to use cells as

microscopic molecular foundries to produce materials whose properties were genetically

encoded. Re-engineering has produced Curli fibers, the amyloid component of extracellular

material of biofilms, as a platform for programmable nanomaterial. These nanofibers were

genetically constructed for specific functions, including adhesion to substrates, nanoparticle

templating and protein immobilization (El Karoui et al., 2019).

Natural proteins can be engineered, for example, by directed evolution, novel protein structures

that match or improve on the functionality of existing proteins can be produced. One group

generated a helix bundle that was capable of binding oxygen with similar properties

as hemoglobin, yet did not bind carbon monoxide (El Karoui et al., 2019). A similar protein

structure was generated to support a variety of oxidoreductase activities while another formed a

structurally and sequentially novel ATPase. Another group generated a family of G-protein

coupled receptors that could be activated by the inert small molecule clozapine N-oxide but

insensitive to the native ligand, acetylcholine; these receptors are known as DREADDs (Hennig

et al., 2015). Novel functionalities or protein specificity can also be engineered using

computational approaches. One study was able to use two different computational methods: a

bioinformatics and molecular modeling method to mine sequence databases, and a computational

enzyme design method to reprogram enzyme specificity. Both methods resulted in designed

enzymes with greater than 100 fold specificity for production of longer chain alcohols from

sugar (Hwang et al., 2020).

Another common investigation is expansion of the natural set of 20 amino acids. Excluding stop

codons, 61 codons have been identified, but only 20 amino acids are coded generally in all

organisms. Certain codons are engineered to code for alternative amino acids including:
nonstandard amino acids such as O-methyl tyrosine; or exogenous amino acids such as 4-

fluorophenylalanine. Typically, these projects make use of re-coded nonsense suppressor tRNA-

Aminoacyl tRNA synthetase pairs from other organisms, though in most cases substantial

engineering is required (Kim et al., 2016).

2.3.6 Designed nucleic acid systems

Scientists can encode digital information onto a single strand of synthetic DNA. In 2012, George

M. Church encoded one of his books about synthetic biology in DNA. The 5.3 Mb of data was

more than 1000 times greater than the previous largest amount of information to be stored in

synthesized DNA (Kim et al., 2016). A similar project encoded the complete sonnets of William

Shakespeare in DNA (Lin et al., 2012). More generally, algorithms such as NUPACK,

ViennaRNA, Ribosome Binding Site Calculator, Cello, and Non-Repetitive Parts

Calculatorenables the design of new genetic systems.

2.3.7 Space exploration

Synthetic biology raised NASA's interest as it could help to produce resources for astronauts

from a restricted portfolio of compounds sent from Earth (Moosmann et al., 2020). On Mars, in

particular, synthetic biology could lead to production processes based on local resources, making

it a powerful tool in the development of occupied outposts with less dependence on Earth

(McCarty et al., 2019). Work has gone into developing plant strains that are able to cope with the

harsh Martian environment, using similar techniques to those employed to increase resilience to

certain environmental factors in agricultural crops.

2.3.7 Drug delivery platforms

In therapeutics, synthetic biology has achieved significant advancements in altering and

simplifying the therapeutics scope in a relatively short period of time. In fact, new therapeutic

platforms, from the discovery of disease mechanisms and drug targets to the manufacture and

transport of small molecules, are made possible by the logical and model-guided design

construction of biological components (Opgenorth et al., 2019).

Synthetic biology devices have been designed to act as therapies in therapeutic treatment. It is

possible to control complete created viruses and organisms to target particular pathogens and

diseased pathways. Thus, in two independent studies 91,92, researchers utilised genetically

modified bacteriophages to fight antibiotic-resistant bacteria by giving them genetic features that

specifically target and hinder bacterial defences against antibiotic activity (Wu et al., 2015). In

the therapy of cancer, since conventional medicines frequently indiscriminately target tumours

and normal tissues, artificially created viruses and organisms that can identify and connect their

therapeutic action to pathological signals may be helpful. For example, p53 pathway activity in

human cells was put into adenoviruses to control how they replicated (Yehl et al., 2019).
 CHAPTER THREE

SYNTHETIC BIOLOGY TOOLS IN MICROBIAL COMMUNITIES

Microbial consortia or communities are ubiquitous in nature and useful in many areas of the

bioeconomy (Yim et al., 2016). Natural consortia are important in the production of foods, the

recycling of micronutrients, and in maintaining the health of humans, animals, and plants. Such

microbial communities consist of member organisms that, together, are more robust to

environmental challenges, exhibit reduced metabolic burden due to a division of labor (DOL)

and exchange of resources, possess expanded metabolic capabilities relative to monocultures,

and can communicate (chemically or physically) between species 3.

These unique features of microbial consortia make them an attractive platform for synthetic

biologists that aim to modify microorganisms for biotechnological applications. Therefore, the

field of synthetic biology is increasingly expanding from a focus on the engineering of single

organisms to a discipline that, in addition, aims to engineer multiple species within dynamic

communities. This comes, in part, due to the limitations inherent on engineering a single

cellular chassis. For example, the incorporation of large or complex heterologous pathways in a

single strain is limited by the ability to transfer long amounts of DNA efficiently into the selected

microorganism, the requirement of a high level of pathway characterization, the presence of

precursors and cofactors in the host cell, and the metabolic burden caused by the expression of
the heterologous enzymatic machinery (Meng et al., 2017). Moreover, monocultures are often

more sensitive to system perturbations, such as environmental changes or contaminations, which

necessitates highly controlled culture conditions and sterilization protocols (Palazzotto et al.,

2019).

Such limitations can be surmounted by the construction of synthetic microbial communities, here

defined as those composed of two or more genetically engineered cell populations (Song et al.,

2017). This transition from engineering static monocultures to dynamic consortia presents unique

challenges, but ultimately relies upon existing synthetic biology tools and approaches.

Synthetic Biology Tools to Engineer and Control Microbial Communities

Synthetic biologists use engineering approaches, including computational models

and modular DNA ‘parts’, to rationally engineer living organisms (Shomar et al., 2018).

Advancements in genetic engineering (including CRISPR/Cas systems for efficient gene

deletions, insertions, and transcriptional control (Sakai et al., 2012) and rapid methods to

assemble DNA fragments enables modular components to be interconnected to build metabolic

pathways and construct biological circuits to control cellular behavior (Lubkowicz et al., 2018).

Some synthetic biology tools, enabled by genetic engineering and DNA assembly methods, are

specifically useful for controlling organisms within microbial consortia. These tools include

intercellular signaling to construct communication systems between organisms, exogenous

molecules to control specific population behaviors, and syntrophic interactions to build

codependent networks of microorganisms.

3.1 Intercellular Signaling to Coordinate Communication between Organisms

Synthetic microbial consortia can be constructed by adapting existing biological communication

systems, such as chemical or physical communication networks, adhesion molecules, ion

channels, and electricity (Kim et al., 2016;Lubkowicz et al., 2017). Quorum sensing (QS) is one

such biological communication system, in which cells produce autoinducer molecules that act as

a proxy for cell density in prokaryotic species 20, 21. As a cell population grows, cells sense the

increased autoinducer concentration and regulate gene expression to control population-level

behaviors. Despite being the most common method to engineer synthetic consortia, QS systems

are limited in their use as communication networks because of crosstalk at the promoter and

signal levels (Ma et al., 2017). Gram-negative organisms use N-acyl-L-homoserine lactones

(HSL) as communication chemicals, while gram-positive organisms use autoinducing peptides.

The lux and las systems, derived from Vibrio fischeri and Pseudomonas aeruginosa,

respectively, are both HSL-based and are commonly used in synthetic biology due to their

compatibility with E. coli, a model gram-negative organism. One organism (termed the sender

strain) is engineered to synthesize a HSL molecule, which diffuses across cell membranes and

activates gene expression in an organism (called the receiver strain) that has been engineered to

express the corresponding receptor/responsive promoter pair. QS-based systems can also be

linked to the expression of antibiotic resistance genes or toxins, either of which can be used to

modify communication between organisms in a consortium.

3.2 Exogenous Inputs to Control Cellular Behaviors

Organisms not only communicate with one another, but also sense their environment to

coordinate behavior. Consequently, tools to engineer living organisms need not confine

themselves to genomic modifications; synthetic biologists can also tune environmental

conditions or add exogenous molecules to control gene expression and cell populations. Inducer
molecules, such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) or anhydrotetracycline (aTc),

can be used to exogenously regulate transgene expression in organisms that express a

corresponding, responsive promoter. Since genetic circuits and pathways distributed between

members of a consortium are difficult to independently control due to a lack of gene regulatory

systems without interference, orthogonal gene regulatory systems with minimal crosstalk have

been developed (Makitrynskyy et al., 2021). To minimize crosstalk between an inducer and its

regulated promoter, a transcriptional regulator can be engineered, or the IPTG-inducible

promoter sequence to which it binds mutated.

Microorganisms can also be controlled exogenously with antibiotics and bacteriocins, which are

peptides produced by some bacteria that inhibit the growth of closely related bacterial

strains (Bikard et al., 2014). Each of these molecules can be genetically encoded or exogenously

added to a growth medium, meaning they can be used to regulate cell populations within a

microbial consortium in a cell–cell or environment–cell manner. At the same time, the required

machinery to resist these molecules can be genetically encoded and its expression controlled by

inducers or signaling molecules, which can be used to generate positive or negative interactions

and produce desired social interactions between consortium members (Arafat et al., 2021). In

addition to antibiotics, toxins, and inducer molecules, population-specific gene expression can be

altered by tuning nutrient concentrations and culture conditions (Carbonell et al., 2018).

3.3 Engineered Syntrophies to Build Codependent Strains

Metabolic interdependencies and cross-feeding is ubiquitous in natural microbial communities.

Similarly, codependent synthetic consortia can be engineered via syntrophic interactions, in

which organisms feed off of metabolites produced by other consortium members (Carbonell et
al., 2018). Typically, these mutually dependent consortia consist of co-auxotrophic strains whose

survival is each dependent upon supplementation of the missing metabolite by another

consortium member.

While co-auxotrophies are a viable approach for generating large communities of unique strains

in a consortium, not all auxotrophs exhibit comparable growth patterns (Edgar et al., 2012).

Specifically, a 14-member E. coli polyculture containing strains auxotrophic for different amino

acids suggests that arginine, lysine, methionine, and threonine auxotrophs dominate a consortium

after only a few days. Organisms auxotrophic for nucleotide biosynthesis genes can also be used

to construct codependent communities of E. coli.

Engineering eukaryotic synthetic consortia with co-auxotrophies is more challenging. Two

nonmating strains of S. cerevisiae with lysine and adenine auxotrophies grow when cocultured,

but demand that each strain is engineered to overproduce the metabolite required by the

other (Edgar et al., 2017). To overcome the challenges associated with co-auxotrophic

interactions between yeast, a self-established, metabolically cooperating yeast community

(SeMeCo) was developed, in which metabolic auxotrophies were randomly introduced into a

yeast population by loss of plasmids expressing genes involved in amino acid biosynthesis.

These randomly introduced auxotrophies can be used to develop yeast communities that enter a

state of syntrophic metabolic cooperation. Co-auxotrophic interactions have also been

engineered between a respiration-deficient yeast (via deletion of cox2, a mitochondrial gene

encoding a subunit of cytochrome c oxidase) and endosymbiotic E. coli. Here, the yeast provides

thiamin to an E. coli auxotrophic for this vitamin and the E. coli shares ATP with the yeast host.
3.4 Synthetic Biology Tools Enable the Construction of Microbial Consortia with Defined
Behaviors

An understanding of the synthetic biology tools used to modify living organisms can also be

applied to the engineering of yet more complex functions and behaviors in microbial consortia.

Intercellular signaling, exogenous inputs, and syntrophic interactions are all tools that, together,

can be applied to tune population levels, distribute or compartmentalize tasks, and define spatial

morphologies in synthetic consortia.

Figure 1.

Engineering Behaviors of Synthetic Microbial Consortia. (A) The population of consortium

members can be regulated by genetic circuits and feedback control. By linking quorum sensing
(QS) systems (rhl and lux) to the repression of a toxin (ccdB) by an antitoxin (ccdA), strains in a
consortium can be made to maintain a stable population ratio. In this example, addition of
Isopropyl β-D-1-thiogalactopyranoside (IPTG) activates transcription of the Plac promoter in
strain 1, which increases the expression of ccdA and decreases the expression of ccdB in strain 2.
This enables IPTG-inducible tuning of population ratios between the strains. Adapted from
McCardell and colleagues. Strain 2 similarly regulates the growth of strain 1. Unannotated
promoters are constitutively expressed. (B) Heterologous pathways can be divided between
consortium members. A five-gene heterologous pathway can be divided between two strains, the
first of which expresses two of the heterologous genes to produce an intermediate metabolite.
This intermediate may either diffuse or be transported to the other strain, which converts the
intermediate to a desired end-product. (C) Spatial programming of at least two E. coli strains in a
synthetic consortium can be achieved by engineering each to express either a nanobody (Nb) or
corresponding antigen (Ag). An N-terminus (N-term) fusion mediates expression of the
nanobody or antigen outside of the cell for cell–cell adhesion. TetR is constitutively expressed
by both strains, which inhibits expression of the adhesion constructs until repressed by
anhydrotetracycline (aTc). Expression of different Nb or Ag proteins can be used to form desired
patterns between consortium members, such as layered or spheroid shapes (bottom). Adapted
from Glass and Riedel-Kruse .
 CHAPTER FOUR

APPLICATION OF SYNTHETIC BIOLOGY IN MICROBIAL ENGINEERING FOR

INDUSTRIAL PROCESSES

With the increase in clinical cases of bacterial infections with multiple antibiotic resistance, the

world has entered a health crisis. Overuse, inappropriate prescribing, and lack of innovation of

antibiotics have contributed to the surge of microorganisms that can overcome traditional

antimicrobial treatments (Ventola, 2015). In 2017, the World Health Organization published a

list of pathogenic bacteria, including Enterococcus faecium (E. faecium), Staphylococcus

aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A.

baumannii), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli) (ESKAPE)

(World Health Organization, 2017). These bacteria have the ability to adapt to multiple

antibiotics and transfer their resistance to other organisms; therefore, studies to find new

therapeutic strategies are needed. One of these strategies is synthetic biology geared toward

developing new antimicrobial therapies.

Synthetic biology is founded on a solid and well-established theoretical framework that provides

tools for conceptualizing, designing, and constructing synthetic biological systems (Perrino et

al., 2021). Recent developments in synthetic biology provide tools for engineering synthetic

control systems in microbial cells. Applying protein engineering, DNA synthesis, and in
silico design allows building metabolic pathways and biological circuits to control cellular

behavior (McCarty and Ledesma-Amaro, 2019). Thus, synthetic biology advances have

permitted the construction of communication systems between organisms where exogenous

molecules can control specific population behaviors, induce intracellular signaling, and establish

co-dependent networks of microorganisms (Hennig et al., 2015; McCarty and Ledesma-Amaro,

2019).

The applications of synthetic biology systems (artificial circuits and functions within biological

systems) include producing biologically based products in agriculture, industry, environmental,

and healthcare studies. In this review, we highlight recent works of the impact of synthetic

biology on genetic engineering to modify antibiotics and enhance antibiotic production,

engineered phages, microbial control systems as an alternative to fight antibiotic resistance, and

some other synthetic biology tools to engineer microbial communities (Figure 1).

4.1 Genetic Engineering to Modify Antibiotics

Organisms are naturally capable of producing metabolites with antimicrobial characteristics.

Unfortunately, “wild-type” antibiotic producers have poor production and low titers; however,

with the unravel of novel biosynthesis mechanisms of that produce antibiotics and the tools

provided by synthetic biology, it is possible to engineer the capacities of organisms to over-

produce and diversify these metabolites (Breitling and Takano, 2015; El Karoui et al.,

2019; Zhang et al., 2020).

One of the most interesting antibiotic synthesis mechanism is the multimodal enzymatic complex

presented in biosynthetic cluster genes (BCG). It is composed of non-ribosomal peptide

synthetases (NRPS), polyketide synthases (PKS), and combinations. NRPS and PKS are

multimodal enzymatic complexes that are responsible for assembling non-ribosomal peptides

(NRP) and polyketides, respectively, and giving them an active chemical structure (Martínez-

Núñez and López, 2016; Nivina et al., 2019; Hwang et al., 2020). With the tools provided by

synthetic biology and applying some engineering to these enzyme complexes, it is possible to

attack the problem of low production and limited diversity of antibiotics.

As a first approach to tackle low production and diversity, antimicrobial compounds members of

the NRP group have been addressed especially the family of antibiotics known as glycopeptides

(Yim et al., 2014). Some authors had focused their efforts on diversifying these antibiotics, such

as Yim et al., who used combinations of 13 scaffold-modifying enzymes from 7 GPA BCGs to

be introduced into Streptomyces coelicolor (S. coelicolor). Among these combinations, nine new

compounds were reported. Interestingly, eight of those compounds had antimicrobial activity

against vancomycin-resistant Enterococcus faecalis (E. faecalis), exhibiting a MIC between 0.5–

4 μg/ml (Yim et al., 2016). One of the promising newcomers in the glycopeptide family is

corbomycin. Despite its outstanding clinical performance, natural production with Streptomyces

sp. has poor performance (Culp et al., 2020). Xu et al. used a glycopeptide antibiotic

heterologous expression system (GPAHex) to enhance the expression of genes for corbomicyn

synthesis in S. coelicolor, obtaining a 19-fold increase in titers using this platform (Xu et al.,

2020).

It is also possible to find that combinations between NRPS and PKS modules can give rise to

more classes of antibiotics, such as lipopeptides. Daptomycin is a clinically significant

lipopeptide antibiotic used primarily against methicillin-resistant Staphylococcus

aureus (MRSA), naturally produced in Streptomyces roseosporus (S. roseosporus).

Unfortunately, similar to other antibiotic natural producers, “wild-type” S. roseosporus has low

daptomycin titers (Ye et al., 2014). Recently, Ji et al., using a “top-down” synthetic biology

approach, achieved an increase in total lipopeptide production up to ∼2,300%, where up to 40%

was daptomycin (Ji et al., 2022).

Not only do combinations between NRPS and PKS give rise to the production of antibiotics, in

some microorganisms, these enzyme complexes are joined by other families of enzymes such as

fatty acid synthases (FAS). Initially discovered in Dickeya zeae, the new family of zeamines is

one example of naturally occurring antibiotics resulting from the interaction between NRPS,

PKS, and FAS (Wu et al., 2010). Zeamines drew the scientific community’s attention for their

potent microbicidal activity against Gram-positive and Gram-negative bacteria.

Unfortunately, Dickeya zeae has poor production of these antibiotics (Liao et al., 2014).

Therefore, different authors, such as Masschelein et al., have studied other natural producers of

zeamines, such as Serratia plymuthica (S. plymuthica) RVH1. Using in-frame deletion of

biosynthetic genes, some of the mechanisms involved in synthesizing these antibiotics have been

uncovered. This has been an outstanding contribution to synthetic biology for future

combinatorial biosynthesis and bioengineering to produce new antibacterial compounds

(Masschelein et al., 2013; Masschelein et al., 2015).

In addition to the approaches made by the combination of enzyme complexes, metabolic

engineering offers another perspective to improve and diversify the production of antibiotics.

Metabolic engineering aims to understand the networks of cellular metabolism and redesign

them to improve productive capacities (Olano et al., 2008; Kim et al., 2016; Palazzotto et al.,
2019). The efforts provided by metabolic engineering usually focus on redirecting metabolic

fluxes, regulating BCGs and enzymes causing bottlenecks.

Discovering the relationships between intermediate metabolites and the biosynthesis of

antibiotics have allowed the scientific community to improve yields in antibiotic production. An

example of this is the regulation of the metabolite S-Adenosylmethionine (SAM). Although

SAM is an essential precursor in methylation processes, this compound is involved in antibiotic

biosynthesis in different species (Huh et al., 2004; Wang et al., 2007). Authors, such as Cai et

al., manage to increase the yields in bacitracin production in Bacillus licheniformis (B.

licheniformis), analyzing the SAM synthetic pathway. Authors, such as Cai et al., through

analyzing the SAM synthetic pathway, have managed to increase the yields in bacitracin

production up to 28.97% in B. licheniformis with a combination of different synthetic biology

techniques such as heterologous expression, deletion, and overexpression of different genes (Cai

et al., 2019; Cai et al., 2020).

Up to this point, the described studies have shown that the elucidation of the different

mechanisms involved in the biosynthesis of antibiotics and the tools provided by synthetic

biology have allowed the redesign of organisms by engineering them to improve their productive

capacities and thus contributing to the fight against antimicrobial resistance. Although there are

still some critical challenges for the continuous application of synthetic biology strategies to

diversify antibiotics, the next few years promise to be rewarding for discovering new antibiotic

compounds.

4.2 Genetic Engineering to Enhance Antibiotic Production

Different microorganisms are sources of other compounds used as antimicrobial agents. A

variety of genetic material codes those metabolites and accessing this genetic diversity could

increase the possibility of finding new or better ways to fight resistant microorganisms. Genetic

techniques like gene mutation and the ability to control cellular functions at the genetic level

could improve antimicrobial biosynthesis (Miao et al., 2006; Ma et al., 2017). Arafat et al.

(2021) observed changes in antibiotic production, exposing Streptomyces graminofaciens (S.

graminofaciens) to UV light. Studying the cellular function of various genes have allowed

researchers to produce higher amounts and even better antibiotics. Studies of the gene cluster of

the nucleoside antibiotic A201A in Marinactinospora thermotolerans (M. thermotolerans)

SCSIO 00652 (Zhu et al., 2012) have led to improving its production; this has also been

accomplished through genetic modification for the biosynthesis of amphotericin analogs

in Streptomyces nodosus (S. nodosus) by disruption of amphDIII and amphL (Byrne et al.,

2003). Makitrynskyy developed a manipulation of AdpA regions of Streptomyces ghangensis (S.

ghangensis) (Makitrynskyy et al., 2021), giving researchers the understanding of a metabolic

pathway to improve a specific antimicrobial biosynthesis. By understanding metabolic pathways

and applying different gene engineering techniques, research can improve antimicrobial

production through punctual modifications to regulate specific genes, demonstrating that genetic

modifications to control biosynthesis pathways could improve antimicrobial production and its

activities (Song et al., 2017; Li D. et al., 2021; Li Y.-P. et al., 2021).

Similarly, intended gene modifications can improve antimicrobial production, improve the

metabolic flux to precursor availability, and enhance biosynthesis (Meng et al., 2017; Moosmann

et al., 2020). These modifications could increase the concentration of antimicrobial precursors,

such as (Shomar et al., 2018) expressing the gene cluster for carbapenem in E. coli producing
antibiotics with a 60-fold increase. Also, modifying metabolic pathways, like carbon flux from

the pentose phosphate pathway (PPP) to glycolysis by modifying zwf1 and zwf2 on Streptomyces

lividans (S. lividans) (Butler et al., 2002) have led to increased glycolysis intermediates needed

for antibiotic production.

A better bio-factory could be achieved by introducing genes in or from a different host taking

advantage of its synthesis route (Han et al., 2012; Sakai et al., 2012; Makitrynskyy et al., 2021).

Chen et al. (Chen et al., 2009) studied the expression of a gene cluster of Streptomyces

cacaoi (S. cacaoi) in S. lividans TK24. However, polyoxin production by S. lividans TK24 was

entirely the polyoxin H derivate due to the lack of genes for hydroxylation/carboxylation, leading

to the formation of polyoxin A/F derivates. Eustáquio et al. (Eustáquio et al., 2004) expressed

the gene cluster to synthesize novobiocin in S. coelicolor. Two S. coelicolor mutants were

obtained by modifying the novO gene, an 8′-unsubstituted novobiocin by inactivation of novO,

and a chlorine atom at C-8′ by expressing the clo-hal gene from the clorobiocin gene cluster.

Wang et al. (Wang et al., 2021) expressed CYP genes of the mushroom Ganoderma

lucidum in Saccharomyces cerevisiae (S. cerevisiae) to produce a derivate of 3,28-dihydroxy-

lanosta-8,24-dien-26-oic acid. By improving the copy number of two resistance plasmids, the

production of this new antibiotic was increased 8.2 folds compared to the control strain. The

expression of non-ribosomal peptide synthetases in Bacillus subtilis (B. subtilis) was studied by

Eppelmann et al. (Eppelmann et al., 2001) by introducing the bacitracin biosynthesis gene

cluster of B. licheniformis. B. subtilis showed comparable self-resistance to bacitracin due to the

gene replacement and increased bacitracin A production due to the high-level expression of the

bacitracin synthetase and the higher growth rate compared to B. licheniformis. Similarly, Wu et
al. (Wu et al., 2015) expressed the gene ram29 of the ramoplanin producer Actinoplanes sp.

into Streptomyces fungicidius (S. fungicidius)to produce monomannosylated enduracin derivates.

The main goal of creating an engineered microorganism is the increased production of a specific

antimicrobial. Nevertheless, when genetic engineering is focused on manipulating foreign genes

in an organism, various reasons could affect its biosynthesis. Modifying genes of a particular

biosynthesis or metabolic pathway may be enhanced in production but could decrease its

antimicrobial activity. Even in the same genre, the differences between strains give the

possibility of obtaining derivates of the intended antimicrobial, which could also modify its

action. Thus, knowing how a microorganism’s genetic material helps it survive in the

microbiome can help us acquire the tools to manipulate it and use it to our advantage, increasing

the possibility of obtaining new and better antibiotics.

4.3 Engineered Phages to Fight Superbugs

Phages are viruses that infect specific bacteria and are the most abundant organisms on Earth (up

to 2.5 × 108 phages per mL in water) (Bergh et al., 1989). Phages need bacteria to replicate and

survive; thus, naturally, they work as bacteria controllers. Phages were discovered at the

beginning of the XX century by Frederick Tort and Felix d’Herelle (Sharma et al., 2017), but

antibiotics rapidly replaced phages in treating microorganism infections. However, due to the

emergence of multi-antibiotic resistant bacterial pathogens, phages are considered an alternative

way to treat multi-resistant bacteria.

Nowadays, researchers are studying single phages, discovering new phages, and designing phage

cocktails as therapies to treat multi-resistant bacterial infections. Natural phages could be enough
to fight superbugs, just like the works reporting on the administration of phage cocktails to

patients infected by a multi-drug resistant A. baumannii strain, with results showing complete

recovery after phage therapy (Schooley et al., 2017). Nevertheless, synthetic biology can

improve or extend phage’s abilities to generate variants with unique properties (Yacoby et al.,

2007; Edgar et al., 2012). One of the strategies is to design and build phages with a broad host

range (Lin et al., 2012). Phages provide a highly specific target of bacterial strains; thus,

cocktails are required to fight infections and reduce the possibility that bacterium acquires

resistance to any phage. Yehl et al. (2019) have developed a high-throughput strategy to engineer

host-range-determining regions (HRDRs) in T3 phage by site-directed mutagenesis. Inspired by

antibody specify engineering, this approach reduces disruptions in tail structure, and they call it

“phage bodies”.

Following this strategy could reduce the number of phages in cocktails. Using the monophages

approach reduces the preparation and purification efforts and there is less potential for

complications derived from using phages cocktails, such as phage purification, phage

compatibility, making it easier to use as treatment. In addition, cocktails could make bacteria

develop “broad-spectrum” mechanisms of phage resistance, such as capsules that avoid phage

binding (Schooley et al., 2017).

Furthermore, Ando et al. (2015) swapped tail fiber genes to allow a genetically E. coli to

target Klebsiella and vice versa. The authors also demonstrated that synthetic phage cocktails

with the same scaffold, but different tails selectively remove bacteria from multi-specie

communities. Bacterial biofilms are difficult to eradicate since the physical properties of matrix,

the physicochemical properties of the exopolysaccharides, and the heterogeneity of the bacterial
cells within the biofilm confers them a strong resistance against chemicals (antibiotics,

immunological approaches, and phages). Nevertheless, phages genetically engineered could be

used to overcome this problem. Chen et al. (2021) constructed an engineered T4 called T4 Rn11

that exhibits antibiofilm activity against Streptococcus mutants (S. mutants). A reduction in

biofilm biomass and formation of microcolonies was achieved using this phage. In a similar

work, Pei and Lamas-Samanamud (2014) constructed a T7 phage that produces a lactonase

enzyme with broad-range activity for quenching quorum sensing. The modified phage effectively

degrades acyl-homoserine lactones (AHLs) from many bacteria. In addition, it inhibited P.

aeruginosa and E. coli biofilm formation.

Reducing biofilm is crucial in the treatment of infection caused by microorganisms in cystic

fibrosis disease (Dedrick et al., 2019) or in wound infections, but also it is useful in reducing

biofilm in medical devices such as catheters (Fu et al., 2010). These devices are responsible for

substantial morbidity and mortality among patients.

As we described above, combining phage’s natural abilities with synthetic biology tools can

improve the potential for phages by expanding their range of infection and mediating bacterial

signaling or expressing enzymes (Bikard et al., 2014) that help eliminate multi-resistant bacteria.

4.4 Microbial Control Systems

In the last decade, synthetic biology tools have allowed the construction of microbial control

systems by engineering whole living cells to act as biosensors and detect and respond to internal

and external signals [quorum sensing (QS)] secreted by pathogens (Benítez-Chao et al.,

2021; Perrino et al., 2021). Some QS-based phenotypical behaviors in bacteria are sporulation
formation, virulence factors related to invasion, bioluminescence, and population control

(Benítez-Chao et al., 2021).

In numerous studies, synthetic genetic circuits have been developed to analyze biological

systems and guide their design based on QS. A group of researchers genetically modified E.

coli to detect wild-type P. aeruginosa (PAO1), specifically via its QS molecule. Their results

demonstrated that engineered E. coli sentinels successfully inhibit PAO1 growth by secreting a

novel pathogen-specific engineered chimeric bacteriocin (Gupta et al., 2013). Later,

a Lactococcus lactis (L. lactis) to detect E. faecalis was designed in another study. L. lactis was

able to produce and secrete peptides that inhibit enterococcal growth and reduce the viability of

enterococci in the surrounding area of L. lactis. This engineered system was demonstrated to be

effective against multidrug-resistant E. faecium strains (Borrero et al., 2015). Holowko et al.

(2016) designed and created a synthetic genetic sensing system using nonpathogenic E. coli as

the host based on CRISPRi technology. They moved proteins used by Vibrio cholerae (V.

cholerae) for QS into E. coli and showed high sensitivity to the presence of V.

cholerae supernatant with tight control of expression of output GFP protein (Holowko et al.,

2016). Lubkowicz et al. (2018) developed a probiotic lactic acid bacteria (Lactobacillus reuteri)

engineered to detect autoinducer peptide-I (AIP-I), a quorum-sensing molecule produced

by Staphylococcus sp. during pathogenesis. Their results showed that the engineered biosensor

could detect AIP-I levels under various strenuous conditions in the S. aureus (Lubkowicz et al.,

2018). Recently, Wu et al. created a novel whole-cell biosensor to detect bacterial pathogens (P.

aeruginosa and Burkholderia pseudomallei) by responding to the relevant QS signal molecules.

Results showed that engineered whole-cell biosensors provide rapid and cost-effective detection

of waterborne pathogens (Wu et al., 2021).

As described above, quorum sensing is a process in which bacteria communicate with their own

species or across species to coordinate cellular behavior. In the last decade, synthetic biology has

used the properties of quorum sensing as a tool to build and develop genetic circuits for

population control.

Synthetic biology is a recently emerging discipline used to design or redesign biological systems

and give them improved or new qualities. One of the SynBio engineering principles used to

create new biological systems is the Design-Build-Test-Learn (DBTL) cycle (Whitford et al.,

2021). DBTL cycle is an increasingly adopted metabolic engineering framework that helps

systematize cellular activities and increase their efficacy and generalizability (Opgenorth et al.,

2019). Thus, the design of biological circuits and high-throughput screening (HTS) technologies

have begun to speed up modern drug discovery cycles and produce new medicines. Each phase

of the DBTL cycle is a fundamental component of the cycle. The Design component identifies

the problem and selects the desired pathway and host. The Build component selects, synthesizes,

and assembles parts for the incorporation into the host. The Test component validates the

engineered constructs and strains for target molecule production, transcripts, proteins, and

metabolites. This phase generates a significant amount of data. The Learn component analyzes

the Test data, and the learnings are used to create a novel testable hypothesis and incorporate

them into the next cycle (Petzold et al., 2015; Carbonell et al., 2018). With the DBTL cycle,

researchers can rapidly construct new biological systems. Coupling the DBTL cycle with

engineering technologies can deliver solutions in drug discovery and solve global problems such

as antimicrobial resistance.

4.5 Protein Engineering Against Multidrug-Resistant Bacteria

Like endolysins (encoded by bacterial viruses), proteins act by disrupting the bacterial cell wall

or other proteins that inhibit the expression of genes related to antibiotic resistance (gene

regulators) and leading to cell death. Some proteins alter microbial behavior, the bacterial cell

wall and interrupt signals and genes confering multi-drug resistance. Based on these kinds of

proteins, synthetic biology techniques are helpful for engineering proteins into making them

more stable or efficient. Moreover, synthetic proteins can be created and manufactured such is

the case of the Briers et al. (2014) group that engineered endolysins to act as artilysins (outer

membrane-penetrating endolysins Briers et al., 2014). One drawback is that endolysins do not

have activity against Gram-negative bacteria because of the impermeable lipopolysaccharide

layer surrounding their cell wall. Still, artilysins are highly bactericidal against Gram-negative

pathogens, including P. aeruginosa and A. baumannii.

Another example is dCas9 which is a “dead” catalytical protein. The system is the same used in

CRISPR-Cas9 technology, with the difference that dCas9 can bind to the DNA but not cut the

strands. In this way, dCas9 is targeted to suppress gene transcription. Wang and Nicholaou

(2017) designed two CRISPR-dCas9 systems to target the mecA promoter in MRSA to repress

the gene’s transcription (Wang and Nicholaou, 2017). Although these alternatives are not applied

to clinical, they are promising approaches to use alone or combined with antibiotics or another

technology.

4.6 Logic Gates

An interesting line of research in synthetic biology is that of making microorganisms work as

control elements, sending and receiving signals through logic gates. Logic gates are widely used

in electronic devices. These devices have electronic circuits that transform an input signal into
another output signal or convert two input signals into one output signal (Wang et al., 2011). In

synthetic biology, living cells act as control elements; therefore, living cells can be programmed

to produce the precisely desired behavior in response to intra or extracellular signals (Morris et

al., 2010). Gene regulatory network that cells use to interact and respond to the environmental

signals, can be used to program logic circuits to link cellular sensors and actuators. Genetic logic

gates rely on direct control of the transcription, activating a promoter. One example is using

these genetic elements to program a cell to recognize quorum-sensing signals. Benítez-Chao et

al. (2021) designed a whole-cell biosensor to sense and kill MRSA. Using genetic logic gates,

they design a genetic circuit where E. coli can recognize quorum-sensing molecules from MRSA

and triggers the expression of a bacteriocin that kills MRSA. Genetic circuits can also be

designed to disrupt a metabolic pathway in multi-drug resistant bacteria or to create whole-cell

biosensors, responding to a molecule produced by pathogen organisms and producing an

antimicrobial compound. Whole-cell biosensors are still in research but have a promising future

in controlling pathogen bacteria.

CHAPTER FIVE
CONCLUSION
Synthetic biology is a technological science discipline used to create and reshape biological

molecules, such as enzymes, genetic circuits and cells without the use of living cells. Synthetic

biologists may employ synthetic genomics to partially or wholly synthesize genes of restructured

or completely novel life forms. Genome minimization is one of the applications of this

technology performed either via the top-down approach, which seeks to shorten the genome by

identifying the lowest number of genes necessary to survive in laboratory conditions, or the
bottom-up method to create new types of self-reproducing minimal cellular life. Some research

institutions and companies work on microbes to optimize their synthetic metabolic pathways,

biofuel production, enzyme production and the development of engineered microbes. Progress in

synthetic genomics has led to a debate on ethics for the synthesis of natural pathogens, which

improves their virulent properties. One starting point may be the development of strategies for

the identification and assessment of the different values at stake. Microbial consortia have been

used in biotechnology processes, including fermentation, waste treatment, and agriculture, for

millennia. Today, synthetic biologists are increasingly engineering microbial consortia for

diverse applications, including the bioproduction of medicines, biofuels, and biomaterials from

inexpensive carbon sources. An improved understanding of natural microbial ecosystems, and

the development of new tools to construct synthetic consortia and program their behaviors, will

vastly expand the functions that can be performed by communities of interacting

microorganisms.

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