Synthetic Biology: Extraordinarily Advanced Systems

Zehra Nur Koyuncu^T., Nida Dereli Çalışkan^E.

With the advancement of technology, there has been a tremendous expansion of scientific knowledge. This flow of information has led to the emergence of interdisciplinary fields such as “Synthetic Biology (SB)”. This integrates concepts from biological sciences, engineering, and computational sciences. SB is an approach that aims to integrate the principles of biological systems with concepts from other disciplines. In SB, the goal is to redesign, reprogram, and create new biological systems by manipulating the natural biological systems found within genetic circuits. Genetic circuits are functional combinations of basic biological entities (DNA, RNA, proteins) created using typical design, build, test, and learn (DBTL) cycles^1-4. SB is a multidisciplinary approach that utilizes various disciplines to construct new organisms within the human body and produce useful products in energy, the environment, agriculture, and medicine⁵ (Figure 1)⁶. This review will delve into the fundamental components of SB and discuss its potential applications, highlighting recent advancements in the field.

Figure 1. Overview of synthetic biology⁶.

1. Synthetic Biology Approaches

SB is based on two different approaches. The first one is the “top-down” approach, where cells are modified using molecular biology and metabolic/genetic engineering techniques. An alternative approach involves the construction of cell-like structures called artificial cells (proto-cells or synthetic cells) from non-living building blocks, which is referred to as the “bottom-up” approach⁷. The top-down approach aims to derive synthetic cells from biologically modified cells, for example, by manipulating genes and protein content. These synthetic cells are typically alive but still closely related to their biological ancestors. A well-known example of SB through the top-down approach is the minimal genome projects, which aim to identify the core genes of a specific species. Such genomes have been successfully initiated within living cells^{8, 9}. On the other hand, the bottom-up approach in cell design starts with inanimate matter. A synthetic cell is a compartment of cellular size. Cell-like functionality is achieved by reorganizing modules made from natural or artificial molecular building blocks. The complexity of a synthetic cell is gradually increased by incorporating more and more components. This strategy has been successfully used to uncover the biological role of individual proteins^{10, 11}.

2. Applications of Synthetic Biology

2.1. Genetic Manipulation

Genetic manipulation is the most well-known application of SB. In this approach, the two main targets are the genome and extrachromosomal plasmids in cells. Plasmids are preferred as transferable elements for basic applications. As genomic manipulation tools have grown and become more user-friendly (e.g., CRISPR), genetic manipulations in SB are increasingly performed at the genome level¹².

2.2. Synthetic Microbial Consortia

The scientific community has become interested in microbial consortia (or communities) of microbes because of their diverse natural distribution and crucial roles in biochemical cycles. The competitive interactions between species and strains within microbial consortia and their cooperative approaches to survival enable these communities to form more complex phenotypes¹³. Synthetic microbial consortia refer to simple microbial communities designed with a defined composition of two or more (typically 2-3) species/strains and are tailored for biotechnological applications. Synthetic biologists apply design principles such as top-down and bottom-up approaches to create synthetic microbial consortia with numerous real-life applications in healthcare, disease prevention, and environmental remediation^14-16. Creating synthetic microbial consortia relies on fundamental cell-cell communication mechanisms, one of which is quorum sensing (QS). QS, which offers dynamic solutions for metabolic engineering, involves signal molecules known as autoinducers that are released from the intracellular to the extracellular environment. When a specific threshold (often at high cell density) is reached, these molecules trigger or coordinate the expression of target genes¹⁷. In addition, in the context of consortium studies, the CRISPR/Cas9 gene editing system, single-cell technologies, and tools from systems biology are also used for DNA and genetic circuit assembly^{13, 18, 19}.

2.3. Sustainable Production and Climate Crisis

The production of thousands of chemicals derived from fossil sources, such as fuels, plastics, and industrial chemicals like petroleum and natural gas, has become a significant target for reducing global greenhouse gas emissions in the face of the current climate crisis. Sustainable production of gases with industrial value, such as acetone and isopropanol (IPA), through fermentation, is crucial for material engineering. The current sugar fermentation method presents sustainability challenges. Modified bacteria through SB offer an environmentally friendly model where waste gases, including carbon dioxide, can be converted into valuable chemicals, such as acetone and IPA (isopropyl alcohol), on a large scale, contributing to the energy cycle²⁰.

2.4. Agriculture and Nutrition

Agriculture, another industry sector negatively impacted by population growth and the climate crisis, requires alternative solutions due to inadequate technologies to meet increasing demands and the declining productivity of agricultural products due to the use of harmful chemicals like pesticides and herbicides. Therefore, the Green Revolution, aiming to mass apply and develop synthetic and natural fertilizers (phosphorus, nitrogen, and potassium), as well as cultivation strategies to maximize plant architecture and harvest, has been an approach resulting in higher yields²¹. Additionally, producing food-based cell factories or biofortification of foods through SB, enabling bio-synthesis, has emerged as an essential area of study for sustainable food supply worldwide^{22, 23}.

2.5. Industrial Biotechnology

Approximately 20% of SB start-ups focus on industrial biotechnology objectives. Industrial biotechnology (IB), defined as the use of enzymes and microorganisms for the production of bio-based products in sectors such as food, chemicals, feed, paper, detergents, bioenergy (biofuels), and textiles, primarily involves maximizing and optimizing the biochemical pathways used in production through working with natural systems²⁴. A Biological Foundry (BioFAB) refers to an integrated molecular biology facility with high-throughput processing and analytical equipment, the software, personnel, and data management systems required to operate this equipment, and broader biological foundry capabilities. It combines automation engineering with SB to create new high-efficiency biological solutions that assist in developing and empowering a DBTL approach to biological engineering while offering a suitable bioeconomy model for industrial advancement (Figure 2)²⁵.

Figure 2. DBTL diagram²⁵. The cycle depicted in the figure is iterated through the stages of design, build, test, and learning, which involve the computational design of genetic regions, circuits, and regulatory and metabolic processes.

2.6. Metabolic Engineering

Metabolic engineering is an approach that allows for developing microbial strains that efficiently produce chemicals and materials. Still, it requires considerable time, effort, and cost to make the strains industrially competitive²⁶. The integration of SB into this approach has facilitated the design of biosynthetic microorganisms and the production of valuable products from them²⁷. SB has enabled the development of artificial regulatory circuits for applications in metabolic engineering²⁸.

• Tissue Engineering and Living Biomaterial Production

The principles of tissue engineering are based on repairing and replacing damaged or lost tissues. While advances in biomaterial synthesis and stem cell technologies contribute to in vitro applications in this field, the manipulation of tissue-specific cells faces challenges that can be addressed with the principles of SB²⁹. Genetic circuits created with SB technologies, along with the control of intracellular signaling, offer an alternative potential for designing next-generation biomaterials by enabling the manageability of the extracellular environment³⁰. The collaboration between SB and materials science in the biologicalization of materials involves the reprogramming of living systems such as microorganisms to function as dynamic and responsive materials, using sensor-regulated genetic circuits, thus defining the field of Materials Synthetic Biology^31-33.

• Disease Diagnosis and Drug Discovery

SB, combined with simultaneous advancements in protein engineering, allows for the design of cells that can activate signalling pathways in living systems using synthetic receptors. These systems enable the construction of theragnostic cells that can serve as both diagnostic tools and therapeutic delivery systems³⁴. SB technologies have facilitated the development of several innovative approaches, primarily in the preclinical stage, for the diagnosis of non-infectious diseases such as enzymatic functions, infectious diseases, and coronary artery disease (e.g., paper-based RNA switch sensors, CRISPR-based diagnostics, etc.)³⁵. The integration of sensitive nanoparticles or molecular probes with SB principles is expected to open up new diagnostic pathways specific to many diseases, including cancer. Studies are being conducted to detect the altered transcriptional state found in cancer cells by designing synthetic circuits based on transcriptional control, incorporating molecular components such as synthetic promoters with enhanced cell-state specificity (SPECS)³⁶.

Advancements in SB enable the reprogramming of synthetic cells as intelligent agents to target tumors and achieve controlled release of anti-cancer drugs³⁷. Research on designing SB-based living biotherapeutics is increasing, particularly in various human pathologies such as inflammatory and metabolic diseases³⁸. SB has already made a significant impact on the development and therapeutic production of pharmaceuticals throughout the drug discovery process. Thanks to, The organization of synthetic DNA, along with the increased understanding of gene regulation and genome details, is expected to form the basis for current successes and potential future applications, accompanied by reduced costs. Rapid advancements are being made in fields such as immunotherapy, cell therapy, and biotherapeutics, including vaccines, thanks to SB^{35, 39}.

• Biosensors

Cell-based biosensors utilize the natural ability of a cell to perceive and respond to its environment by repositioning its detection mechanisms in new genetic contexts. They aim to produce cells capable of detecting specific target molecules and generating a response to them. Cell-based biosensors have gained significant interest as an alternative detection method due to various advantages such as detection sensitivity, cost-effectiveness, portability, and the absence of specialized equipment and trained personnel compared to traditional methods. The use of directed evolution is highly preferred for constructing or improving SB approaches in biosensor structures⁴⁰. Recombinases provide the opening/closing function in logic gates in the field of SB, enabling the design of genetic circuits that can compute and remember. Recombinase-based whole-cell biosensors are developed for the purpose of detecting environmental signals^41,42.

• Protein Engineering

Protein-protein interactions (PPIs) govern many cellular functions in living systems. Designing new PPIs to elucidate molecular interactions poses a fundamental challenge in protein engineering⁴³. By leveraging SB technologies, reversible PPI-based protein circuits or synthetic biological toggle switches that respond within seconds can be designed, providing insights into new biological interactions and enabling the construction of cellular functional networks^{44, 45}.

• Beyond the Laboratory: Applications of Synthetic Biology

Most scientific advancements are conducted in well-controlled laboratory settings and cannot be immediately translated into “off-laboratory” applications. The use of SB tools in such applications is considered a potential problem solver for producing various compounds, therapeutics, and materials in unconventional remote environments, as well as meeting the needs of the drug, fuel, or general resource storage in military and space missions. It is believed that SB applications capable of going beyond the confines of a laboratory can provide lasting improvements for human health and environmental applications⁴⁶.

• Synthetic Cells and Biomedical Applications

Cells have the ability to come together as the building blocks of everything around us, forming tissues and other complex structures. This has led to the development of synthetic cells (SCs) that can mimic live cells, particularly for clinical applications. SCs can mimic live cells while providing advantages for cellular stability studies under conditions incompatible with life. Building functional synthetic cells from the bottom up remains an ongoing effort for scientists worldwide, although there are various limitations⁴⁷. Two recently published studies have shed light on the path to life-like human-made systems in SCs. In the first study that explains the integration of cell division mechanisms into SC, a successful reconstruction of active bacterial division machinery mimicking the mechanism in Escherichia coli was achieved by integrating the relevant proteins onto a synthetic membrane called Janus dendrimers. This study represents a step towards designing synthetic entities that can leverage mechanisms provided by nature⁴⁸. In the second study, functional DNA-based cell skeletons were incorporated into cell-sized compartments, allowing them to perform functions such as molecular transport or assembly and disassembly on specific triggers⁴⁹. SCs are considered potential structures for various biomedical applications, including therapeutic discovery, nanomaterial synthesis, photochemical control of intracellular functions, and artificial enzyme production^{47, 50}.

Consequently, SB, an approach to integrating biology with more engineering applications, aims to design existing or novel systems by creating various biological components within a specific standardization framework. While the development of application-oriented tools and interdisciplinary approaches has led to advancements in the field of SB, the lifelong developmental capacity of living systems poses a major challenge in the synthetic design of biological systems. However, it has contributed to enhancing our understanding of fundamental cellular processes through the DBTL method, leading to a wide range of diverse applications in academia, industry, and the clinic. The rapid advancements in SB hold the potential to improve the quality of human life, but they also come with certain risks. While synthetic biology plays a key role in realizing futuristic ideas such as designing functional cells, genetic manipulation, and more, addressing ethical concerns is important to prevent the misuse of such technologies and similar ones.

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