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immunosuppressors), lipids (dietary supplements, pharmaceuticals and biodiesels) and isoprenoids (perfumes,
biodiesels, antimalarial drugs,
antibiotics, rubber, dietary supplements, food ingredients and vitamins).
3.1.4
Cell-free synthetic biology
An alternative and emerging field is cell-free biology, which is defined as the “activation of complex biological
processes without using intact living cells” (Harris and Jewett 2012). It features the ability to focus on
production of a single compound without physical barriers, facilitates substrate addition, product removal and
rapid sampling, provides direct access to reaction conditions, and utilises the entire reactor volume. Another
advantage is that there is no conflict between microbial growth and engineering design objectives. The most
prominent example is cell-free protein synthesis (CFPS), which allows for the synthesis of proteins containing
non-natural amino acids (NNAA, see also 3.2.3.). Major application-oriented advantages of cell-free systems
are that gene expression of multiple products may be tuned to facilitate proper product interactions, reaction
and product stability may be optimised by adjusting DNA template concentrations and controlling the redox
potential for optimal disulphide bond formation, the possibility for recombinant expression of toxic proteins,
and the production of complex biocatalysts (Smith et al. 2014). They overcome inherent limitations of living
cells, enable control of gene expression, chassis optimisation,
in situ monitoring, and automation. Cell-free
systems are used to develop new reaction pathways that significantly increase productivity, e.g. to microbe-
based systems. It also allows for the faster and more predictable development of modular gene circuits, as
control of reaction environment and components, and access to the reaction is greatly facilitated –
applications are the engineering of minimalistic artificial cells and cell-like microdevices.
3.2
Construction of a novel cell
Designed and synthesised DNA segments that encode novel functions need to be implemented into a suitable
organism by one of the many available genome engineering techniques or by novel mega-size cloning
strategies (Heinemann and Panke 2006). Complexity, the major problem, is desirable to be reduced. One
option is to reduce the genome of the host into which the new sequence is implemented, which would
eliminate many possibilities for interference. Since numerous genes are involved in cell-cell communication
while others have been shown to be non-essential to cell function it was early suggested that it would be
possible to reduce genome complexity to a minimal set of genes able to sustain cell life and reproduction (Sole
et al. 2007). No matter how small, cell genomes must contain all the information necessary for the cells to
perform essential functions allowing them to maintain metabolic homeostasis (self-maintenance), reproduce
(self-reproduction) and evolve (evolvability) – the three main properties of living cells.
It is important that the synthetic device or system is either decoupled from the metabolic processes inherent
to the viability of the cell or does not adversely affect these processes.
“Microbial synthesis of any plant natural product can be achieved by „precursor-mediated product synthesis“,
in which an existing host pathway is altered to incorporate a heterologous pathway, or by „de novo synthesis“,
in which new-to-host biosynthetic routes are imported. Global approaches have been applied to improve for
example the terpenoid pathway flux in microbial host” (Moses et al. 2013).
3.2.1
Minimal genomes – top-down approach
A minimal genome is the minimum set of genes that is necessary for a cell to propagate under specific
environmental conditions (Heinemann and Panke 2006).
According to theoretical considerations, growth in the presence of a rich but synthetic and defined medium
requires as few as 206 genes, basically comprising the DNA replication, transcriptional and translational
machinery, rudimentary DNA repair functions, protein processing and degradation, cell division and
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rudimentary metabolic and energy functions (Heinemann and Panke 2006). Relatively large genomes of
established models can, on the one hand, be substantially reduced to reach this goal or, on the other hand,
one can work on the already very small genome of other organisms in exchange for the requirement to
develop novel molecular biology tools (Heinemann and Panke 2006). The smallest genome sizes have been
detected in prokaryotic cells living in symbiosis with other organisms. Notable examples are the human
parasite
Mycoplasma genitalium, the archaeal exosymbiont
Nanoarchaeum equitans, the insect
endosymbionts
Buchnera aphidicola BCc,
Candidatus Carsonella ruddii and
Candidatus Sulia muelleri. All of
these microorganisms are heterothroph host-dependent bacteria. They are dependent on the chemically
complex environment represented by their respective host cells. As an adaptation to the symbiotic lifestyle,
their genomes underwent a reductive process in which genes that were unnecessary in the new protected
environment or redundant because their functions were provided by the host tended to be lost (Moya et al.
2009).
Which are the essential genes?
The first step of making a minimal cell is to answer the question about how many genes are necessary to
support cellular life. Whether a gene is essential depends on the environmental conditions. Esvelt and Wang
(2013) define a set of useful traits for a biological chassis as:
-
Fast growing in minimal media
with glucose
-
Capable of fermentation
-
Amenable to genetic manipulation
-
Minimally sufficient such that removal of any additional gene negatively affects the
other three stated
considerations
One way to approach the gene composition of a minimal genome is by
comparative genomics. The underlying
hypothesis is that genes shared between distantly related species are likely to be essential. It has to be
mentioned that there is an intrinsic limitation of this method: Many essential cellular functions can be
performed by several alternative and unrelated proteins, and will be misled by comparative approaches. The
comparative minimal core (common set of genes) only retrieves those genes involved in functions for which
there is no alternative in nature. The task of determining if there is a minimal deletion core is difficult because
of three critical issues:
1.
Redundancy might only be apparent: Inactivation of single genes does not detect essential functions
encoded by redundant genes, and some individually dispensable genes may not be simultaneously
dispensable, a phenomenon called synthetic lethality (Moya et al. 2009).
2.
The mechanism of cell division is not yet completely understood: Theoretical considerations of self-
reproduction (transcription/translation machinery, replication machinery and regulation) apply well to
automatic schemes, but it is not guaranteed that this also works in an artificial cell (Noireaux et al. 2011).
Life
is not only a program; it relies also on other fundamental
nongenetic properties,
for example molecular
self-organisation and molecular crowding.
3.
Each part of the genome is loosely connected with the rest: when designing novel constructs or cells one
should also bear in mind that there are also fundamental questions on nongenetic processes which are not
defined in a DNA program. A living organism is an open system made of hundreds of chemical reactions
whose properties go beyond the DNA program. The assembly of a synthetic cell unfolds the importance of
physical aspects that are,
in vivo, regulated by already evolved gene networks (Noireaux et al. 2011).
Another way is performing
large-scale inactivation studies in order to define which genes are essential for cell
survival in several well-characterised bacterial models (
Escherichia coli,
Bacillus subtilis,…) (Moya et al. 2009),