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European Biopharmaceutical Review

Synthetic Biology

Synthetic biology is the engineering of biology which enables a rational, bottom-up approach to design and construction of artificial biological systems, as well as the redesign of natural biological systems. It merges advanced techniques in computer-aided design with biotechnology, microbiology, evolutionary biology, molecular biology, systems biology and biophysics, and has been hailed as a game changer for the fields of health, energy, medicine and industrial biotechnology.

Developments driven by synthetic biology are described with their own dedicated nomenclature, and typically involve iterative cycles of design, production and testing. These innovation cycles create biobricks – for example, synthetic promoters, regulatory elements, coding sequences or terminators – which are assembled into circuits, such as metabolic pathways, that are integrated into engineered cellular production systems called chassis.

Synthetic Gene Regulation

Biopharmaceutical manufacturing poses specific technical challenges which are critical for its success. Firstly, high production levels are needed to both simplify downstream processing and minimise overall production costs. Secondly, robust and reproducible production systems are required that are amenable to scale-up. Finally, the biopharmaceutical products themselves should display maximal biochemical and biophysical uniformity in protein folding, monodispersity and post-translational modification. When it comes to meeting these specific hurdles, the range of functionalities across available biobricks, synthetic circuits and associated molecular biology, as well as computational tools for designing and assembling these DNA elements, have exponentially increased in recent years (1,2).

Efficient and controllable transcription is a critical element contributing to production yield, reproducibility and saleability. Consequently, synthetic promoters – in addition to a range of complementary transcriptional control elements for both prokaryotes and eukaryotes – have been developed and implemented in a wide range of biopharmaceutical manufacturing applications (3,4). Similarly, RNA control elements are available that modulate splicing, and a collection of RNA interference-based parts and devices have been developed, providing an additional level of control over production (5).

Protein turnover control elements provide yet another channel for manipulating cellular metabolism and production characteristics – for example, by modulating protein degradation (6). Additionally, a broad variety of methods are being developed to sense the biopharmaceutical production cell’s natural flux in its cycle, in energy precursors or oxygenation state, to maximally channel the cell's resources toward product formation.

Taken together, a wide array of synthetic biology tools are being rolled out that are optimised to positively impact biopharmaceutical manufacturing, and it is inevitable that we will increasingly witness these technologies in upcoming generations of production platforms.

Modular Assembly

The entire revolution of synthetic biology has, to a large extent, been made possible by exponential increases in the efficiency of DNA sequencing and gene synthesis technologies. However, in spite of these advances, available gene synthesis techniques cannot produce DNA fragments of the same length as the synthetic circuits required for synthetic biology applications, which are in the region of thousands, or tens of thousands, of bases. Therefore, technologies are required that allow for the modular assembly of multiple individual DNA fragments produced by gene synthesis techniques. This has posed a considerable technical challenge that has only recently seen significant developments.

Traditional cloning methodologies have been replaced by more advanced methods, including isothermal assembly of linear DNA parts (7,8) or DNA recombination-based assembly of multiple expression cassettes (9,10). One of the central tenets of electronic circuit design, which is also applied to synthetic biology circuits, is the need to get a design from the drawing board into the laboratory with maximum speed, ease and flexibility. This is essential in enabling the user to rapidly assemble and test a synthetic circuit with minimum turnaround time, allowing for optimisation of the pathway with a new and improved combination of parts.

Exchanging one part for another, or designing new parts for shuttling into a cascade, can then be implemented quickly to improve performance of the cascade as a whole. Common goals include modulating concentrations of individual components of the cascade, and creating synthetic links between components. On a DNA level, these processes should ideally be automatable and high-throughput compatible. These requirements are finally being addressed by emerging technologies.

Circuit Delivery

The use of modern gene synthesis techniques – combined with DNA assembly technologies – can produce complex synthetic DNA circuits, but this still leaves the considerable task of getting these circuits into the cellular production system, and a further and separate complication of integrating the synthetic DNA circuits at site-specific locations in the host genome. To this end, truly revolutionary advances have been made in the last few years in available techniques, making small, site-specific sequence edits in the host genome of bacteria, yeast and even mammalian cells.

However, one of the major unsolved hurdles for application of synthetic biology to biopharmaceutical manufacturing is that no robust methods have been developed – to date – allowing for targeted delivery of large DNA circuits into bacteria, yeast or mammalian cells, combined with site-specific integration of the circuit into the genome at a locus of choice. This unmet challenge remains one of the major areas of investigation for the synthetic biology community.

Genome Engineering

The major cellular workhorses for biopharmaceutical manufacturing are Escherichia coli, yeast and mammalian cells, with the latter forming the most critical production system for market-dominating products such as monoclonal antibodies. Synthetic biology methods had their beginnings with the simpler life forms such as viruses and prokaryotic cells, in large part due to the relatively low complexity of their genomes. Viruses range in size from thousands to a few 100,000 base pairs (bps), with the E. coli genome at 5 x 106 bps, Saccharomyces cerevisiae measuring at 1.2 x 107 bps, and the human genome at 3.3 x 109 bps. Because of this genome size factor, prokaryotes and lower eukaryotes have been traditionally more accessible to rational and precise genetic engineering, and pioneering advances in synthetic biology techniques were typically first made in viruses and bacteria, followed later by yeast and then mammalian cells.

A remarkable series of advances in genome editing technologies began with zinc-finger nucleases, followed by transcription activator-like nucleases, which finally allowed site-specific editing in mammalian cells (11,12). However, their broad application in biotechnology has thus far been hampered by low efficiency. A more recently developed and greatly promising approach, the CRISPR/Cas9 technology, displays much higher efficiency than earlier methods, but still suffers from a significant drawback as it allows only small sequence edits (13).

None of these methods have been adapted for efficient chromosomal delivery of large and complex DNA cargos, such as synthetic circuits. Given the limitations of available large DNA cargo delivery technologies, the major advances in cell-line engineering programmes at the moment are carried out by iterative small, local edits in genomes which are utilised to stepwise improve production characteristics.

Success Stories

The industrial biotech community presents the most numerous and notable examples for effective application of synthetic biology to cell-line development. Arguably, the most impressive, currently marketed synthetic biology success story comes from American biotech company, Amyris. Its scientists have developed highly engineered Saccharomyces cerevisiae strains which process sugar into high titres of building block chemicals (farnesene). These building blocks are then converted, via specially engineered downstream metabolic pathways integrated into the same yeast genome, into a broad range of fine chemicals, flavours, fragrances and biofuels.

A number of genome engineering success stories are also emerging. For example, post-translational modifications (PTM) present a critical challenge for biopharmaceutical production applied to human health. The majority of therapeutic proteins contain post-translational modifications, with glycosylation being the most common and, at the same time, the most complex PTM (14). Yeasts can perform typical eukaryotic PTMs, but final glycosylation patterns of yeasts and humans differ considerably. As differences in glycosylation patterns can trigger significant adverse immune reactions, yeast genome engineering to alter glycosylation patterns of yeast-produced biopharmaceuticals has been a highly active area of investigation – and one that is seeing heavy implementation of synthetic biology methods (3).

Furthermore, similar genome engineering efforts have been carried out in insect cell lines used for biopharmaceutical production (15). Even mammalian cell lines – such as the monoclonal antibody production workhorse, Chinese hamster ovary (CHO) cells – do not create fully uniform and complete humanised glycosylation patterns, and glycoengineering CHO cells for improved biopharmaceutical production is also seeing a plethora of activity (16,17).

New Era

Scientists today still often resort to time-consuming and unpredictable screening of randomly generated cell libraries to select biopharmaceutical production strains, rather than custom designing and engineering the desired cell factories from the bottom up. However, given the rapid advance of tools and technologies being provided by the synthetic biology community, it is clear that fundamental change in the way we produce biopharmaceuticals will soon lead to a new era of artificial biology.

References


1. Ausländer S and Fussenegger M, Dynamic genome engineering in living cells, Science 346: pp813-814, 2014
2. Weber W and Fussenegger M, Engineering of synthetic mammalian gene networks, Chemistry & Biology 16: pp287-297, 2009
3. Vogl T, Hartner F and Glieder A, New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris, Curr Opin Biotechnol 24: pp1,094-1,101, 2013
4. Nishikata K et al, Database construction for PromoterCAD: Synthetic promoter design for mammals and plants, ACS Synth Biol 3: pp192-196, 2014
5. Leinert F et al, Synthetic biology in mammalian cells: Next generation research tools and therapeutics, Nature Reviews Molecular Cell Biology 15: pp95-107, 2014
6. Nishimura K et al, An auxin-based degron system for the rapid depletion of proteins in nonplant cells, Nature Methods 6: pp917-922, 2009
7. Torella J et al, Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications, Nature Protocols 9: pp2,075-2,089, 2014
8. Leinert F et al, Two- and three-input TALE-based and logic computation in embryonic stem cells, Nucleic Acids Res 41: pp9,967-9,975, 2013
9. Vijayachandran L et al, Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering, Bioengineered 4: pp279-287, 2013
10. Trowitzsch S et al, MultiBac complexomics, Expert Rev Proteomics 9: pp363-373, 2012
11. Urnov F et al, Genome editing with engineered zinc finger nucleases, Nature Reviews Genetics 11: pp636-646, 2010
12. Joung J and Sander J, TALENs: A widely applicable technology for targeted genome editing, Nature Reviews Molecular Cell Biology 14: pp49-55, 2013
13. Cong L et al, Multiplex genome engineering using CRISPR/Cas Systems, Science 339: pp819-823, 2013
14. Walsh G, Post-translational modifications of protein biopharmaceuticals, Drug Discov Today 15: pp773-780, 2010
15. Aumiller J et al, A new glycoengineered insect cell line with an inducibly mammalianized protein N-glycosylation pathway, Glycobiology 22: pp417-428, 2012 16. Steentoft et al, Precision genome editing – a small revolution for glycobiology, Glycobiology 24: pp663-680, 2014
17. Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool, Curr Opin Biotechnol 30: pp80-86, 2014


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Dominik Schelshorn received his PhD in Neurophysiology from the University of Heidelberg, Germany, and has a background in cellular assays for drug discovery and development, with a focus on G protein-coupled receptor signalling. 

Sanda Ljubicic
was a Swiss National Foundation fellow at Harvard Medical School in Boston, US. She has more than 10 years of experience in the fields of metabolic diseases and diabetes.

Imre Berger
received his PhD from the Massachusetts Institute of Technology, US, and carried out his postgraduate studies at ETH Zürich, Switzerland. He is presently group leader at EMBL Grenoble, France, specialising in the development of cutting-edge protein production technologies.

Daniel Fitzgerald received his PhD at Purdue University, US, and carried out his postgraduate studies at ETH Zürich, Switzerland. He has a 15-year track record in publishing, patenting and marketing novel protein production technologies.
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Dominik Schelshorn
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Sanda Ljubicic
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Imre Berger
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Daniel Fitzgerald
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