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European Biopharmaceutical Review
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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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