Synthetic biology, a relatively new scientific discipline has been developed in the last two decades. However, a very pertinent question arises: Why is synthetic biology essential for our future? Currently, humans are facing many serious challenges on earth that can be overcome by biological technologies. From the very inception of life, the biological systems are associated and pervade in every aspect of human life. Biological systems have enormously helped in the development of complex biological machines by a marriage between eukaryotic cells and prokaryotic (cyanobacteria) systems during evolution, for instance, the development of plant cells having chloroplast and mitochondria both. Therefore, plant cells become great biological factories and provide enormous amounts of oxygen, food, medicine, and raw materials essential to support life on earth. At the beginning of human civilization followed by the initiation of agriculture, humans domesticated bacteria, plants, and animals, as a result humans were immensely benefited in the various aspects of industries and life. For example, many microorganisms (bacteria & yeast), plants, and animals are used in the development of beer, wine, food, medicines, pharmaceuticals, and costly value-added products.
Another classic example is exploitation of store forms of carbon i.e., petroleum and coal products that helped in the expansion of the human population and economies on earth. These are a few instances of how biological systems are exploited to sustain life on earth.
Currently, a huge amount of data has been generated in the field of biology and technologies. It has realized that biological systems are very complex but comparatively in-efficient in output, and their industrial exploitation for human needs is relatively restricted. Therefore, synthetic biology/biological engineering approaches are used to engineer living systems (cells, animals, and cell-free systems) to achieve great functional efficiency, robustness, predictability, and scalability, therefore now, “Biology turned into a true technology”. New biological technologies can be applied to diverse areas of industry, agriculture, medicines, pharmaceuticals, clinics, academics, and other areas. Hence, synthetic biology helps to enhance the optimization of industrial biotechnologies that will further promote a bio-based economy.
Before the inception of synthetic biology, recombinant DNA technology (rDNA) played a crucial role in the improvements of crops, pharmaceuticals, to cure diseases and the production of plant-based foods. Being a reductionist approach, genetic engineering is a “skilled based” or “artisan technology” not sufficient to address the issues of cost-effective production of bio-based products at the industrial level. Moreover, it is unable to unravel the complexity and predictability of genetically manipulated organisms in the view of genetic factors on biological systems such as pleiotropism. Therefore, understanding of biological complexity is another aim of SynBio. Currently, increased computational power has tremendously enhanced quantitative and deep molecular understanding of biological processes, that will be helpful to established new biological design principles for biological parts (biomolecules) and modules (switches, cascades, pulse generators, time delay circuits, oscillators), and devices (engineered gene circuits) by using the DBTL cycle approach.
The three most important inter-linked problems of the world i.e. food, energy, and environment revolve around living organisms, and their rational designing by using synthetic biology approaches can address these emerging problems. In the last two decades, synthetic biology has made tremendous progress in the most promising areas such as crops, foods, clinics, biofuels, bioremediations, and environmental issues. In the future, SynBio will help to achieve United Nations Sustainable Development Goals 2030, which are mainly focused to preserved and maintaining the earth’s natural resources and ecosystem by using new sustainable technologies.Therefore, a hydrocarbon-based economy can replaced with alternative bio-based and reduced the problem of environmental, starvation, use of hydrocarbon-based fuels.
Recently many biological parts, genetic regulatory elements, modules, and devices are synthesized e.g., promoters, activators, repressors, ribosome binding sites, and transcriptional repressors, which are further assembled in the form of small sized modules used to regulate the expression of genes/genome. Moreover, advanced types of regulatory modules are also used to introduce novel functionalities in biological systems via controlling gene expression, protein function, metabolism, and cell-cell interactions. So far, many genetic circuits are characterized and standardized in the cellular environment by using engineering biological processes, including transcription (transcriptional repressor, Lacl), translation (engineered RNA devices), and post-translational (scaffold protein phosphorylation system). Alternatively, combinations of biological parts and modules are used to construct unique genetic circuits to ensure optimal performance of microorganisms, plants, and animals using directed evolution approaches to circumvent complex biological problems.
Application of Synthetic biology
❖ Synthetic Genomics
Synthetic genomics can be defined as “Synthetic genomics is a nascent field of synthetic biology that uses aspects of genetic modification on pre-existing life forms, or artificial gene synthesis to create new DNA or entire life form”. The genome (blueprint of life) of any organism is just like software of a cell, analogically, a computer contains a software (program) that instructs the operating system to execute the specific function by using computer hardware. Similarly, the genome also executes a wide variety of biological functions by using cellular hardware (cellular components). Therefore, synthetic biology aims to rationally design the genome of an organism to introduce novel functions in the biological system. Recently simple organisms like coli and yeast are biologically engineered through synthetic genomics principles such as restructuring, recoding, minimization, chimerism, organelle re-engineering, and genome resurrection. It is now possible that megabase-sized (MB) chromosomes can easily be synthesized from nucleotides. Alternatively, with the creation of a hybrid genome containing significant parts from natural genomes and few synthetic parts, therefore synthetic genomics can produce a synthetic genetic code. Initially, a few small-sized viral genomes were synthesized like, hepatitis C13, polio14, ΦX17415, a SARS-like coronavirus16, herpes type 117, and coronavirus that causes COVID-19.
In the last decade, nucleotide synthesis facilities have significantly improved leading to chromosomal level synthesis being possible in the cell. Firstly, a human pathogenic bacterium Mycoplasma genitalium genome was synthesized, how many bacterial chromosomes are synthesized, for example, Mycoplasma mycoides, Caulobacter crescentus, and Escherichia coli including Saccharomyces cerevisiae. Genome minimization is a major aim of synthetic genomics, thus, chromosomes must be re-engineered in such a manner so that non-essential genes removed from a genome e.g., minimal bacterial genome.Therefore, organism can be trained or domesticated for the execution of specific tasks.
Globally, agriculture is facing many complex challenges including decreasing crop production, rapid urbanization, salinization, contamination of soil, and decreasing arable land. Today’s rapid population growth and climate changes lead to the major challenges of food, fuel, and chemical production that are closely linked with the agriculture sector. Synthetic biology, short of disruptive technology, has a great potential to transform agriculture in a highly sustainable manner that can overcome drawbacks of the Green Revolution 1.0 in the 1960s. Currently, cell-free agriculture has opened new avenues to produce high value-added products exclusively coming from plants.
SynBio will support agriculture, firstly in a re-design of genetic circuits, metabolic pathways, anatomy and physiology, and plant architects to design future crops. Secondly, to ensure sustainable agriculture to support a circular economy, Thirdly, to exploit the direct evolution to mold plant cells and ultimately fulfill the increasing demand for food, biofuels, feedstocks, and specialty chemical and pharmaceuticals.
There are certain thrust areas where plant synthetic biology can greatly contribute in the future. Such as photosynthesis, a life support system on earth, is less explored, and so far, restricted to plants and microorganisms, but SynBio design cycles offer various 28 theoretical possibilities in plant cells to improve the efficiency of photosynthesis and CO2 fixation. This can be achieved by designing new proteins, enzymes, enzymatic reactions, and metabolic pathways by using the potential of directed evolution. Sustainable agriculture is the “need of today” to feed a huge population and preserve the ecosystem of the earth, especially from climatic changes. Synthetic biology can support the improvement of various feedstocks, harvested yields, and agro-ecosphere in overexploited arable land, for example, rational design of carbon partitioning pathways in leaves and roots of plants offer a better yield and sucking of environmental CO2. The proof of concept experiment has been shown in the case of tobacco plants. Another synthetic biological approach is CO2 concentrate by reducing carbon loss in the photorespiration pathway i.e., engineering of carboxylase. SynBio can offer help in the production of a “Smart Plant” soon which takes climatic cues ranging from nutrients to pollutants levels from its environment and conveys it to the genetic circuitry of plant cells so that plants would be able to save water and protect during abiotic stresses.
Nitrogen fixation is another most important aspect of crop plants. Generally, a huge amount of nitrogen is provided to plants via inorganic nitrogen fertilizers, which causes a lot of nitrogen pollution. Therefore, synthetic biology offers an appropriate solution to the nitrogen fixation process by targeting three points, (1) to improve overall nitrogen fixation in root noodles by engineering plant-root microbiome, (2) induce symbiotic nitrogen fixation in non-legumes, for example, wheat and rice, and (3) introduced nitrogen fixation pathway as de novo in plants. Plant synthetic biology can be used to reduce the applications of pesticides and chemicals in crop production by inducing the endogenous production of secondary metabolites, which are mainly responsible for plant defense under biotic stress.
Currently the world’s transportation system relies on hydrocarbon-based fossil Moreover, many industries such as chemicals, materials plastics, paints, and fabrics are also produced from petroleum products. The excessive usage of fossil fuels has adversely affected our environment and led to serious climate changes. Additionally, the economies of non-oil producing countries are also badly suffering due to the fluctuation of oil price. Therefore, synthetic biology can be used to produce renewable green biofuel for a sustainable green and circular economy in the world, so adverse impacts of fossil fuels can be overcome shortly. Synthetic biology is used to engineer recombinant microorganisms, which are capable of efficiently converting renewable plant biomass to biofuels such as long-chain alcohols. However, the first-generation biofuel was produced by converting starch-based feedstocks into free glucose then changed into alcohols but this process is highly water and energy intensive so need to develop advanced green biofuel from the conversion of renewable lignocellulosic material (plant cell wall) into biofuel and chemicals and medicines through synthetic biology in biorefinery.
But there are many hurdles faced during the deconstruction of cellulosic material into free glucose residues. Hence, many SynBio tools, for example, cell-specific novel promoters, metabolic pathways by using the DBTL cycle, are used to engineer the lignin and cellulosic material in plants e.g., Zip-lignin. Manipulated lignocellulosic material containing a large amount of fermentable sugar got high yield of alcohol. However, this process still costly, but the introduction of biorefinery concept, cost of second-generation biofuel can be reduced substantially due to side by production of other value-added products, for example, muconic acid, Polyhydroxyalkanoates (PHAs), bioplastic. Shortly, more efficient C4 plants such as sugar cane are engineered so more biofuel and products are produced. The second most important source of next-generation biofuels is obtained from microbial sources owing to the availability of deep knowledge about their genetic makeup.
SynBio technologies are available to efficiently genetically programmed or rationally designed microorganisms to produce alcohols, isoprenoids, and fatty acid derivative alkanes and alkenes, these are good alternatives of petrol, gasoline, and diesel. Currently, traditional genetic manipulation allows us to redesign metabolic pathways (Alcohols, coenzyme A (CoA) dependent pathway, keto-acid pathway, Isoprenoids, fatty acids, butanol pathway and Ehrlich pathway in lignocellulolytic and autotrophic bacteria and native biofuel producing microorganisms. Many model’ organisms such as E. coli, B. subtilis, Clostridium acetobutylicum, Clostridium beijerinckii, C. acetobutylicum, Zymomonas mobilis, Corynebacterium glutamicum, C. glutamicum, Thermoanaerobacter, and Thermoanaerobacterium sp. Ralstonia eutropha and yeast. The third major source of biofuel production is algae, an emerging source that does not compete with arable land and water and nutrients rather is based on the exploitation of highly renewable photosynthesis and photobioreactors strategies. Algae synthesize oils (TAGs) by using sunlight, carbon dioxide, and water. However, algal photosynthetic transduced many polar lipids such as glycolipids, phospholipids, phospholipids, glycolipids and sterols, stored in noticeable amount in cellular membrane.These lipids are converted into biodiesel and gasoline (petrol) or jet fuel with efficiently. But there are several challenges, for example, agricultural and industrial production, screening of good strains, strain optimization, to improve photosynthetic efficiency, increased lipid and fuel quality. Moreover, Synthetic biology must applied to nutrient use, protection algal strains from pathogens and improved harvesting of important species such as Chlamydomonas, Nannochloris, Nannochloropsis, Chlorella Vulgaris, Porphyridium cruentum (a red alga), Chaetoceros calcitrans, C. Vulgaris, C. Vulgaris, and C. reinhardti,i green alga, Ostreococcus Dunaliella and Nannochloropsis.
A new branch of synthetic biology, which deals with material production is Material Synthetic Biology. “In materials synthetic biology, living systems are used to produce dynamic and responsive materials for user-defined applications. These materials can be endowed with new functions using programmable features, such as self-regeneration, remodeling in response to environmental cues, and evolution” (Tang et al., 2021).
Recently, manufacturing machines and robotics are computationally programmed to manufacture a specific type of material. This type of analogy can compare with car manufacturer plants or toy molding machines. Therefore, biological machines are also engineered by using computational tools in sync with genome sequencing, gene synthesis, and genome editing (CRISPR–Cas system) techniques to produce specific biological material. Scientists are embarking on the idea of application of mimicking and synthetic biology in the production of smart material, environment responsive stuff, self-organizing and multifunctional materials, engineered biofilms, hybrid materials, sensors, smart building material, therapeutics, electronics, and energy-conversion materials.
In the recent past, engineering principles have been applied in the development of novel genetic circuits with predictable behaviors by using the genetic toggle switch, repressilator, logic gates, modular parts to tune gene expression and biomolecular interactions of the biological system. Synthetic biology is now able to allow organisms to perform sophisticated complex synthetic processes just like industrial processes. Biologically engineered material has far-reaching implications in the near future because of climate changes and environmental upheavals. Furthermore prospective applications are numerous with main applications, in the area of medicine, civil, environmental engineering, architecture, and product design.
In material science a pertinent question arises that how an atomic pattern (geometrical patterns) and functionalities in a specific material can be introduced by synthetic biology? The development of biofilm in the E coli poses a suitable example, where this could achieved by deep studies of cell-to-cell interactions in the mammalian cells.Therefore, scientists are very hopeful that they will be succeed to produce biomaterials with characteristics like self-regeneration, reorganized in response of extraneous perturbation, and evolution. Currently many bioengineered synthetic or hybrid materials such as protein-based adhesives, bioplastics, natural marine glues, biofilms, and bacterial cellulose, are synthesized in organisms. But there is a great challenge that synthetic materials must show a great level of self-assembly capability and a wide range of diverse biological engineered materials by using synthetic biology technologies.
Biological systems including bacteria, fungi, algae, plants, and animal cells are best prefabricated biological factories that can be used for the synthesis of pharmaceuticals or biopharmaceuticals, for example, proteins or bio-peptides. Synthetic biology protocols are available to engineered biological systems that produce huge amounts of bio-peptides. Recently, many proof-of-concept experiments have shown the reprogramming of genetic circuits in microorganisms to produce pharmaceuticals, high-value small molecules, small-molecule drugs, flavors and fragrances, and other high-value chemicals. Two great stories of biopharmaceuticals production by using bio-engineering methods are aspirin to artemisinin which has been promoted to industrial biotechnology. Currently synthetic biology is targeting or engineering metabolic pathways which are responsible for the synthesis of various metabolites used as valuable bioactive compounds. In the future, rapid genome and metagenome sequencing and genome editing will provide great opportunities to explore biosynthetic capabilities of microorganisms, fungi, and plants.
❖ Environmental issues
The intense anthropological activities have caused a substantial level of environmental pollution that is a major threat to human existence on earth. The increasing pollution is not only affecting the human’s health (carcinogenic) but also adversely affects the ecosystem and brings climatic changes. The World Health Organization (WHO) reported that “an estimated 6 million people died as a result of living or working in an unhealthy environment in 2012, nearly 1 in 4 of total global deaths”. There are numerous sites of environmental degradation including the fresher water bodies, soil, and oceans that are contaminated with extremely harmful non-degradable synthetic organic compounds, Currently, hydrocarbons from soils and spilling sites, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), heterocyclic compounds (such as pyridine or quinoline), pharmaceutical substances, radionuclides and heavy metals ( Pb, Cd, Cu, Hg, Sn, and Zn) have been remediating by using different advanced strains of extremophilic microorganisms which produced from different industrial activities. To circumvent this problem various chemical, physical and biological strategies were adopted in past, among them bioremediation has proved the most potential and cost-effective and sustainable approach.
“Bioremediations are the applications of living organisms including microorganisms, plants, fungi, and algae to remove or degrade the contaminants from the pollutant sites” However, bioremediation is not new science to monitoring and cleaning of hazardous chemicals and pollutants from the environment. But, after the emergence of systems, biological methodologies have created a wealth of knowledge in terms of genomics, proteomics, and metabolic data related to natural scavenging bacteria, plants, algae, and fungi. This allows us to rewrite or redesign the genetic programming of subject organisms by using synthetic biology tools and techniques. Therefore, these organisms can convert harmful organic compounds into less harmful or valuable products. The revolution in the area of synthetic genetic circuit construction can help us detect pollutants but also in their cleaning from the ecosystems. A few of them are Pseudomonas, Aeromonas, Moraxella, Beijerinckia, Flavobacteria, chlorobacteria, Nocardia, Corynebacteria, Atinetobacter, Mycobactena, Modococc, Streptomyces, Bacilli, Arthrobacter, Aeromonas, Cyanobacteria Dehalococcoides, Dehalobacter, and Desulfitobacterium, etc. which can detoxify petroleum products. Recently Uranium degrading bacteria Pseudomonas sp., Pantoea sp. and Enterobacter sp., Geobacter, and Thermus scotoductus have been identified from the sites of uranium mining sites.
SynBio can contribute to the efficient detection process by using several biosensors-based methods that rely on the enzyme, nucleic acid, immunogens, quorum sensing-based biosensors, RNA aptamer-based, Peptide-based biosensors, Whole-cell biosensors. Under the iGEM project effort arsenic biosensor has been developed. The future development of future generation biosensors will be based on multitasking and multi-detection systems in single biosensors to design biosensors, enzymes with unique activities towards persistent organic xenobiotics, organisms that are resistant to challenging environmental conditions, robust biopolymers, artificial storage organelles for toxic metals, and much more. The use of synthetic biology technologies for bioremediation is still in its infancy but already offers exciting possibilities towards the use of engineered organisms to provide a cleaner, safer environment.
❖ Clinical Application
Synthetic biology has been now entered into clinics. It is mainly aimed to understand the mechanisms of disease that affects humans and animals. Consequently, SynBio offers novel diagnostic tools and techniques that will provide novel, effective, and economic therapeutic agents for several fatal diseases e.g., cancer, heart disease, diabetes, gout, immunological diseases, many metabolic disorders, and infectious diseases. Today, SynBio efforts aim to develop effective diagnostic methods as well as therapeutic agents simultaneously to treat the above-said ailments by using engineered biomolecules, synthetic gene networks, and programmable organisms. Additionally, efforts are also made to improve the development of many vaccines, microbiome engineering, cell therapy, and regenerative medicine. But the prerequisite to cure diseases is an appropriate method of diagnosis in real-time.
Recently, bioengineers have developed many Theragnostic cells which are capable of detecting disease in biological samples or within the human body or by non-invasive methods. Because bacteria can be used as sensors, actuators, and chemical factories. Synthetic biology has enabled the engineering of increasingly complex and accurate control genetic devices that perform sensing and delivery functions. New synthetic genetic circuits provide means which are suitable for biomedical challenges. Therefore, synthetic biology can improve overall human health, diagnostic, disease preventive strategies, drug discovery, and their design and delivery to the human body. Currently, clinical synthetic biology helps in the understanding of various diseases, for example, host-pathogen interactions. The current strategies have been applied to understand the virus-based pandemic such as synthesis and analysis of H1N1 viruses and severe acute respiratory syndrome (SARS), coronavirus, post-Lyme-disease syndrome hepatitis C, and immunodeficiency virus (HIV).
Synthetic Biology has also proved helpful to understand the functioning of Immune systems as well as its turn berserk that causes many autoimmune diseases, so it is important for etiology, diagnostics, and designing therapeutic agents that counter autoreactive immune cells.
A more aggressive strategy against any disease is vaccine development, and vaccinology is another area where SB is performing very well, for example, many attenuated pathogens are used as vaccines. Recently, poliovirus attenuated by using the iterative DBTL cycle for genome-scale manipulations to execute specific antigens. Moreover, vaccine delivery also improved by using the immunostimulatory liposomes, therefore, genetically programmable synthetic vaccines or next-generation vaccine development is more rapid and accurate in their applications.
Many vector- borne diseases are spread by insects as a vector population, and their reproduction must be checked. Hence many transgenic viral strains have been developed and used its conditional dominant-lethal synthetic circuitry against malaria and dengue causing mosquitoes.
Cancer is a fatal disease that causes millions of deaths every year, and millions of dollars are pumped every year in return for meager success in the prevention of cancer-related mortality rates. So synthetic circuitry can be engineered to arrest the cell division at the G stage by synthetic circuitry that acts on the expression of the cyclin-dependent kinase inhibitor p27. SB can help in the development of anticancer agents may be chemical agents, programmed bacteria, and viruses. Engineered bacteria specifically target growing tumor tissues by releasing toxic agents therefore, tumor growth is checked. Simultaneously, many reporter proteins are developed for the real-time monitoring of tumor regression.
Recently synthetic pathways have been produced by combining many enzymatic reactions which can be used for the production of large-scale novel metabolites or value-added products in bacteria, yeast, and plants that can be used as a high-value drug, precursor molecules, secondary metabolites, for example, complex polyketides, halogenated alkaloids, and antimalarial drug artemisinin. SB-based therapies are smart drugs, specific, dynamic, efficacious, and safe to treat many diseases.
However, synthetic Biology is still in its infancy stage, and there is huge potential to improve tools and techniques and protocols for circuit transformation and chassis development. Because, SynBio community is working on the suitable chassis for material, pharma and utility chemical production, SB will need to be given priority to make the world more sustainable in the future. But, Synthetic biology also raises some serious ethical and social issues which must be addressed in a very balanced manner.