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Plant synthetic biology could drive a revolution in biofuels and medicine

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Population growth, climate change, and dwindling finite resources are amongst the major challenges which are facing the planet. Requirements for food, materials, water, and energy will soon exceed capacity. Green biotechnology, fueled by recent plant synthetic biology breakthroughs, may offer solutions. This review summarizes current progress towards robust and predictable engineering of plants. I then discuss applications from the lab and field, with a focus on bioenergy, biomaterials, and medicine. Impact statement The plant synthetic biology field has exploded in the last five years, in part driven by techniques such as CRISPR and cheap DNA synthesis. This review summarizes the current state of research in plant synthetic biology, and how it is being applied to two topics: renewable fuels and chemicals, and medicine.
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Plant synthetic biology could drive a revolution in biofuels
and medicine
Jenny C Mortimer
1,2
1
Biosciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA;
2
Joint BioEnergy Institute, Emeryville, CA
94608, USA
Corresponding author: Jenny C Mortimer. Email: jcmortimer@lbl.gov
Abstract
Population growth, climate change, and dwindling finite resources are amongst the major
challenges which are facing the planet. Requirements for food, materials, water, and energy
will soon exceed capacity. Green biotechnology, fueled by recent plant synthetic biology
breakthroughs, may offer solutions. This review summarizes current progress towards
robust and predictable engineering of plants. I then discuss applications from the lab and
field, with a focus on bioenergy, biomaterials, and medicine.
Keywords: Biofuels, gene editing, genetic engineering, plant produced vaccines, plant synthetic biology, renewable chemicals
Experimental Biology and Medicine 2018; 0: 1–9. DOI: 10.1177/1535370218793890
Introduction
The human population has grown 9-fold since the begin-
ning of the industrial revolution,
1
requiring increased
exploitation of the Earth’s natural resources. Major advan-
ces in agronomic practices and plant breeding (the Green
Revolution) resulted in vastly increased crop yields but
needed large inputs of irrigated water, synthetic nitrogen
fertilizers, and other nutrients. These practices are energy
intensive and lead to environmental problems, including
salinization of soils, erosion, and nitrification of water
bodies.
2
Further increases in crop yields are essential to
feed our future population, which is predicted to exceed
11 billion by 2100.
3
Moreover, as fossil fuel reserves are
depleted, the importance of agriculture for production of
non-food products such as fuels, fibers, and platform chem-
icals will only increase. Boosting crop yields to meet these
demands must be achieved (i) by maintaining or improving
the productivity of land currently in cultivation to avoid
further deforestation and (ii) by reducing inputs of water
and nutrients.
To achieve this, a Green Revolution version 2.0 is neces-
sary.
4
In this vision, new crops are precision engineered
for both optimal yield and environmental interactions.
Plants are not considered in isolation but, for example,
in combination with their microbiome and soil. The goals
of the synthetic biology discipline: robust, predictable and
rapid engineering of biology align well with this vision.
5
To date, much synthetic biology research has focused on
microbial engineering (with the vast bulk of that being
the model organism Escherichia coli). Plant synthetic biology
has necessarily been more limited and has faced different
challenges, such as life-cycle length.
In this review, I will briefly define synthetic biology, dis-
cuss the history of plant engineering, and summarize
the current state of the field in plant science. I will then
discuss two promising applications of plant synthetic biol-
ogy: production of renewable fuels and chemicals, and
therapeutics. While increased yields of food crops, as well
as the production of foods with increased nutritional value
is clearly important, this will not be covered here due to
space limitations, and because it has been recently
reviewed elsewhere, e.g. Tyagi et al.
6
Defining synthetic biology
What is synthetic biology? While many definitions exist,
that from the Engineering Biology Research Consortium
(EBRC; www.ebrc.org) is helpful: “Synthetic biology aims
to make biology easier to engineer.... It can be thought of
Impact statement
The plant synthetic biology field has
exploded in the last five years, in part
driven by techniques such as CRISPR and
cheap DNA synthesis. This review sum-
marizes the current state of research in
plant synthetic biology, and how it is being
applied to two topics: renewable fuels and
chemicals, and medicine.
ISSN 1535-3702 Experimental Biology and Medicine 2018; 0: 1–9
Copyright !2018 by the Society for Experimental Biology and Medicine
as a biology-based toolkit that uses abstraction, standardi-
zation, and automated construction to change how we
build biological systems and expand the range of possible
products.” The application of engineering principles to
biology requires us to be able to move from a sequenced
genome to a predictable systems output, which is very chal-
lenging.
7
The engineering concept of the design–build–
test–learn (DBTL) cycle can underpin the application of
synthetic biology to organismal engineering (Figure 1
(a)).
8,9
Synthetic biology aims to speed up the rate at
which we move round the DBTL cycle, and, combined
with systems biology, encourages us to explicitly learn
from each experiment and integrate that knowledge into
the next iteration. Engineers rely on interchangeable com-
ponents and this concept has also been adopted by synthet-
ic biologists. In microbial synthetic biology, this was
pioneered by the BioBrick standards (https://biobricks.
org/) and more recently a plant standard has been devel-
oped based on Type IIS restriction endonuclease assem-
bly
10
(Figure 1(b)).
How does synthetic biology differ from conventional
genetic engineering? It can be considered in some senses
a continuum. Whereas genetic engineering may manipu-
late the expression of a single gene, synthetic biology
aims to go much further – for example by the introduction
of a complete metabolic pathway under tight regulatory
control. While the design, synthesis and expression of the
DNA parts are important, it is also the incorporation of
systems data (such as transcriptomics, proteomics,
metabolomics) and mathematical modeling into subse-
quent experiments that really sets synthetic biology apart.
The eventual goal is to possess biological blueprints such
that we can understand and modify an organism, as an
engineer does a car. For plants, these latter goals are still
very distant. However, progress has been made, and it is
that which will be discussed in this review.
A brief history of plant engineering
Domestication of plants by artificial selection is central to
human history.
11
It began as saving seed from plants with
desirable traits, and using them for subsequent plantings.
It then developed into deliberate breeding, as evidenced by
the work of Gregor Mendel
12
and his laws of inheritance.
The discovery of heterosis in 1908 (hybrid vigor), in which
the offspring of certain crosses perform better than either
parents, and the cytoplasmic male sterility (CMS) trait in
1933 that enables the facile production of hybrids led to
great advances in yield.
13
The identification of wheat culti-
vars with traits such as short-stature, which makes plants
less prone to lodging (falling over as a result of wind-
damage), led to what is now referred to as the “Green
Revolution.” Norman Borlaug, an American agronomist,
was awarded the Nobel Peace Prize in 1970 for using
these techniques to increase wheat yields in India and
Pakistan by 60%.
Progress in the field of genetics in the first half of 20th
century suggested the promise of further yield increases.
Mutagenesis (using X-rays, radiation, and chemical muta-
gens) was routinely employed to try to produce new com-
mercial plant varieties. For example, much of the world’s
mint oil is produced from a disease-resistant cultivar
“Todd’s Mitcham” developed from a gamma irradiation
breeding program started at Brookhaven National
Laboratory in 1955.
14
In the long-term, however, these
methods have been most successful for producing new
ornamental plants, such as petunias
15
and cherry blossom
16
(Figure 2), and the number of agriculturally important
traits identified this way is relatively low, as compared to
conventional breeding.
The advent of transgenic methods in the 1980s was a
step-change. It allowed the introduction of new traits that
may not exist within the species’ gene pool, such as resis-
tance to a specific disease. Traditional breeding simply
cannot do this. Plant science led transgenic research
because of the observation that the rhizobial bacteria
Agrobacterium tumefaciens can induce crown gall tumors in
plants, and that this cellular proliferation was due to the
presence of genes transferred from the Agrobacterium Ti
(tumor inducing) plasmid and integrated into the plant
genome.
17
The DNA transferred (T-DNA) is flanked by
defined left- and right-borders. Researchers showed that
foreign genes placed between these borders could be inte-
grated into the plant genomes, and disarmed Ti plasmids,
that transferred DNA without causing tumor formation,
were developed.
18,19
T-DNA transformation rapidly
became a common tool for plant research.
20
Unfortunately, not all plants are equally susceptible to
Agrobacterium transformation (including some major crop
Figure 1. Plant synthetic biology concepts. (a) The design–build–test–learn
(DBTL) cycle underpins experimental design and analysis in synthetic biology.
Modified from Petzold et al.
9
In the Design phase, the problem is described, the
pathway selected, and the parts designed or selected. In the Build phase, the
parts are synthesized, assembled, and transformed into the host organism of
choice. During the Test phase, the modified organism is analyzed and data
collected. In the Learn phase, the data from the Test phase are analyzed, and
used to inform the next Design stage. (b) Type IIS restriction site-based DNA
parts for meeting plant synthetic biology community standards.
10
All DNA parts
should be sequenced without a BsaI restriction site (at a minimum – BpiI and
BsmBI should be avoided). Standard parts are flanked by convergent BsaI cut
sites. The plasmid backbones should be free of BsaI/BpiI/BsmBI sites, and
should not contain resistance genes for ampicillin/carbenicillin, or kanamycin,
since these are commonly used in assembly. (A color version of this figure is
available in the online journal.)
2Experimental Biology and Medicine
............................................................................................... ................................... .............................
species). For example, only a few cultivars of barley can be
transformed this way, and any modifications are then intro-
duced into elite cultivars via multiple generations of back-
crossing.
21
Biolistic transformation (the gene gun) was
developed as an alternative transformation method.
22
Heavy metal particles (usually gold or tungsten) coated
with DNA are fired at cells, and some of the payload inte-
grates into the genome. This method has been used to pro-
duce many commercialized genetically modified crops,
such as insect-resistant Bt corn and cotton, which expresses
endotoxins derived from the soil bacterium Bacillus
thuringiensis.
23
However, these transgenesis techniques have limita-
tions. For example, it is difficult to control where in the
genome the transgene integrates, or the copy number of
the introduced DNA. This can lead to variation in gene
expression, gene-silencing, or inter-generational instability,
and this unpredictably has hindered wide-spread commer-
cialization beyond a few traits. In addition, there has been
much public concern over the dangers of genetically mod-
ified organisms (GMOs), and, despite the lack of scientific
evidence to support this view,
24
it has limited applications
of these technologies.
Gene editing
While the transgenic techniques described above have been
used in early plant synthetic biology approaches (see
below), the development of precise gene editing technolo-
gies that can not only precisely alter a DNA sequence, but
also gene expression levels, has been fundamental to recent
progress.
25
Importantly, these methods do not necessarily
require the introduction of foreign DNA, which can have
regulatory advantages.
26
Here, site-specific endonucleases
are introduced into the cell. The resulting DNA double-
strand break is then repaired using methods that exist in
the cell: either non homologous end joining (NHEJ) or
homology-directed repair (HDR).
27
NHEJ is quick and
dirty: efficient but error prone. HDR is slower but more
precise. NHEJ usually results in non-functional genes,
which is important for reverse genetics experiments, as
well as some agricultural traits, e.g. powdery mildew resis-
tance in wheat.
28
HDR, by comparison, can be also be used
for the introduction of foreign DNA.
29
Three different endonuclease systems have been suc-
cessfully deployed in plant systems: zinc fingered nucle-
ases (ZFNs), transcription activator-like effector nucleases
(TALENs), and clustered regularly interspaced palindrom-
ic repeats (CRISPR) – CRIPSR-Associated (Cas) proteins
such as CRIPSR-Cas9. See Table 1 for a comparison of the
three methods. CRISPR-based systems in particular are
expanding rapidly, with new functions added via gene-
fusion with the endonuclease, such as gene repression
30
and, as has been shown in mammalian cells, epigenetic
modification.
31
Gene stacking and whole pathway engineering
Plant transformation is still a relatively slow and labori-
ous process. Recent advances in which whole metabolic
pathways can be introduced with a single transformation
event hold great promise.
32,33
COSTREL (combinatorial
supertransformation of transplastomic recipient lines)
takes advantage of both plasmid and nuclear transforma-
tion.
33
The core metabolic pathway is transformed into
the plasmid, and then these plants are then co-
transformed with a cocktail of accessory protein plas-
mids. The resulting offspring is then screened for the
abundance of the desired product. In the J-Stack
method, yeast homologous recombination, combined
with a toolbox of compatible synthetic DNA promoters
and terminators, can be used to rapidly assemble path-
ways for introduction in a single transformation step, as
was demonstrated both for in planta production of a
potential biofuel and antibiotic.
32
Both methods allow a
relatively easy and rapid way to shuffle genes in a met-
abolic pathway, and to test different combinations. This
can be essential to alleviate metabolic bottlenecks or iden-
tify potential co-factors.
Figure 2. Some examples of radiation plant breeding. (A) Nishina Otome cherry blossom tree (R) and the parent (L), created at RIKEN. In the absence of vernalization
(exposure to winter temperatures) it is still able to flower, and will produce flowers in all four seasons. If exposed to winter temperatures, it produces three-times more
flowers than the parent (B) The commercial cultivar Todd’s Mitcham mint (Mentha x piperita), which was bred at Brookhaven National Lab and has resistance to
Verticillium wilt. (A) RIKEN Nishina Center for Accelerator-Based Science (B) Colonial Creek Farm (www.colonialcreekfarm.com). (A color version of this figure is
available in the online journal.)
Mortimer Plant SynBio impacts on biofuels and medicine 3
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Applications of plant synthetic biology
Currently, most plant synthetic biology approaches have
been applied to discovery research, and this is likely to
continue in the short term. Plant research can be compara-
tively slow, and synthetic biology provides a method to
accelerate this.
34
In particular, as the cost of DNA synthesis
has plummeted, the use of synthetic genes (e.g. codon opti-
mized for expression in different species) is now within
reach of the average research laboratory. However, in the
future, it is likely that plant synthetic biology will become
increasingly important for commercial agriculture, as will
be discussed below.
Biofuels and bioproducts
Almost all transportation infrastructures in the US rely on
petroleum-derived hydrocarbons. Not only are fossil fuels
non-renewable, but their usage contributes to climate
change. Of course, petroleum is not only used for fuels. A
vast range of chemicals and materials are also produced
including plastics, paints, and fabrics. The reliance on
petroleum has resulted in an economic, environmental,
and strategic push to develop renewable alternatives.
35
This includes using plant lignocellulosic biomass (i.e. the
plant cell wall) as a carbon feedstock, and has coincided
with, and in many cases has driven forward, advances in
plant synthetic biology. This is because the economics of
producing fuels and chemicals in a sustainable manner
from plant biomass is challenging.
The cheapest and simplest method of producing biofuels
is to convert corn starch or sugar cane sucrose into ethanol
using yeast fermentation. However, this can have knock-on
consequences for food prices
36
and land use.
35
Additionally, food crops such as corn require high inputs
of water and fertilizer to produce the high yields that
modern farming expects. Many fertilizers are derived
from non-renewable resources, and their production con-
tributes to greenhouse gas emissions. They also add to pro-
duction costs. While agricultural lignocellulosic residues
such as corn stover can be (and are) used, this alone will
not be sufficient to significantly displace fossil fuels.
Therefore, dedicated bioenergy crops, such as switchgrass,
poplar, and biomass sorghum have been proposed.
35,37
There are many barriers to the widespread implementa-
tion of dedicated biomass crops. The majority of these spe-
cies have not been domesticated. Extensive improvements
are required to optimize them for deconstruction and con-
version, as well as making them sustainable to grow.
This includes tolerance to biotic and abiotic stress, and
the ability to produce high yields even in poor quality
marginal soils without inputs of fertilizer (which is often
non-renewable and requires fossil fuels to produce).
Conventional breeding methods alone simply cannot
achieve the scale of change required, at least within a rea-
sonable timeframe. Synthetic biology has accelerated both
the fundamental research required to enable predictive
engineering by describing complex metabolisms, as well
as the production of designed cultivars. A few examples
will be highlighted here.
Lignin is a complex polyphenolic network found in
some secondary plant cell walls, and it forms around 30%
dry weight of biomass. While it is important for providing
structural integrity to some cell types, such as the water-
conducting xylem vessels, it is also challenging to break
down and process as part of lignocellulosic biofuel produc-
tion. The complexity arises from its chemistry. Lignin is
made primarily from three monolignol units: p-coumaryl
alcohol, coniferyl alcohol, and sinapyl alcohol, which are
synthesized in the cytosol, and then transported to the apo-
plast. Monolignol radicals are generated, and these are then
incorporated by combinatorial radical coupling into the
lignin polymer, forming p-hydroxyphenyl (H), guaiacyl
(G), and syringyl (S) subunits.
38
The result is a highly het-
erogeneous polymer. Plant breeding programs have suc-
ceeded in developing lower-lignin plants, e.g. brown
midrib (bm or bmr), which have up to 20% reduction in
lignin compared to wild type.
39
However, if the total
lignin content is decreased much more than this, the
Table 1. Comparison of three gene editing techniques that have been successfully used in plant systems.
Mechanism Pros Cons
ZFN Fok1 endonuclease is fused to zinc finger
domain (ZFD)
ZFD interacts with DNA (each domain rec-
ognizes three DNA bases)
4–6 ZFDs required for specificity
Single strand cleavage
High specificity Difficult to use
Not all triplets have a zinc finger
Pair of ZFNs required for double
strand break
TALEN Fok1 endonuclease is fused to
TALE domain
TALE domain interacts with DNA (each
domain recognizes one DNA base)
Single strand cleavage
High specificity
Modular assembly
Very large
50of TALEN target must be a T
Off target issues
Pair of TALEN’s required for double
strand break
DNA methylation affects efficiency
CRISPR-Cas9 Cas9 endonuclease is recruited to the DNA
by single guide (sg)RNA
sgRNA (20 bp) provides specificity via
Watson-Crick pairing
Double strand cleavage
Quick to design
and assemble
Multiplexing
Off-target issues
Requirement for a PAM sequence
near target
Chromatin structure
affects efficiency
ZFN: zinc fingered nuclease; TALEN: transcription activator-like effector nuclease;
4Experimental Biology and Medicine
............................................................................................... ................................... .............................
plants will show growth defects, including lodging or
stunting. The lignin biosynthetic pathway is well character-
ized, and synthetic biology has provided the opportunity to
make “designer lignin” – lignin which is more amenable to
biotechnological applications while not affecting crop
yields. Multiple strategies have been implemented.
One approach has been to introduce more labile bonds
into the lignin. For example, the expression of a ferulolyl-
CoA:monolignol transferase (FMT) to produce ferulated
monolignol subunits, which can then be incorporated into
the lignin polymer to produce so-called Zip-lignin
TM
.
40
To do this, the authors searched for suitable FMT enzymes
in nature, and identified an FMT from Angelica sinensis,a
root used in traditional Chinese medicine which accumu-
lates coniferyl ferulate. Expression of AsFMT in poplar
indeed introduced alkali-sensitive ester bonds
40
(Figure 3
(a)). Poplar trees engineered in this manner show increased
cell-wall digestibility under mild alkaline pre-treatment
conditions.
41
An alternative approach is to specifically reduce lignin
in cell types where it is not essential, e.g. fiber cells, so it
only remains in cells where it is essential for plant function,
e.g. xylem vessels. To precisely achieve this goal is challeng-
ing using gene silencing or gene knockouts, since it usually
requires the targeting of multiple genes and results in
developmental defects.
42–44
A more effective method is
to use a gain-of-function approach. For example, expres-
sion of a plastidial-targeted, bacterially-derived enzyme,
3-dehydroshikimate dehydratase (QsuB), under the control
of the plant CINNAMATE-4-HYDROXYLASE (C4H)
promoter
45
(Figure 3(b)). The QsuB enzyme converts
3-dehydroshikimate into protocatechuate (PCA). This
reduces the availability of an intermediate in the shikimate
pathway, which in turn reduces the substrate for monoli-
gnol production, reducing total lignin synthesis. The use of
the cell-specific promoter avoids altering the lignin in the
xylem vessels, where it is essential to avoid vesicular col-
lapse. As a bonus, the plant accumulates PCA, which is a
promising bio-based platform chemical.
46
While this initial
work was performed in Arabidopsis, since the lignin and
shikimate pathways are highly conserved, it is likely that
this approach will work in biomass crop plants.
Plant biomass is composed of, depending on the species,
40% hexose (mostly derived from cellulose) and 30%
pentose (mostly derived from hemicelluloses and pectins)
sugars, with lignin as the remainder.
47
In most microbes,
hexose sugars are preferentially utilized as a carbon source,
due to carbon catabolite repression. As a result, pentoses
are only consumed once the hexose supply has been
exhausted. While much research has been directed towards
the development of microbes which can use both simulta-
neously,
48
it is not yet clear that this approach will be suc-
cessful in industrial production strains. An alternative
approach is to optimize the biomass to match the microbial
preference. In one example, the authors chose to increase
the cell wall content of the pectin galactan (which is com-
posed of the hexose galactose). Initially, they constitutively
overexpressed the Golgi-localized GALACTAN
SYNTHASE1 (GALS1), the enzyme responsible for galactan
biosynthesis,
49
achieving an increase of 50% galactan
in the leaf cell wall. They have since boosted this further
by co-expressing the cytosolic UDP-GLUCOSE/UDP-
GALACTOSE-4-EPIMERASE2 (UGE2) gene responsible
for synthesizing the GALS1 substrate, UDP-galactose.
50
Finally, a gene stacking approach combined the GALS1
and UGE2 with a UDP-galactose transporter, which
moves the substrate into the Golgi lumen for utilization
by GALS1,
51
resulting in a 50% increase in galactan in
stem cell walls.
In a powerful demonstration of plant synthetic biology,
this study went further, by combining the high-galactan
lines with cell-specific low xylan (i.e. low pentose) and
low lignin (using the QsuB strategy described above).
51
The use of cell-specific promoters avoided negative
growth effects while delivering plants which had 3 fold
increase in the hexose:pentose ration and increased release
(up to 73%) of fermentable sugars from the biomass.
51
It
remains to be seen how well this strategy will translate to
crop plants, but, at least in related species like poplar which
have conserved cell wall synthesis pathways, there is a high
chance of success.
While the previous examples demonstrate the ability of
synthetic biology to produce plant biomass which is easier
to breakdown for microbial conversion, the following
examples showcase the possibilities of producing platform
chemicals in planta. Platform chemicals are defined as the
building block molecules from which most bulk chemicals,
materials, and polymers are produced from. These
Figure 3. Examples of synthetic biology strategies to engineer lignin. (a) “Zip” lignin. Introduction of ferulated monolignols into the lignin polymer results in alkali-
sensitive ester bonds. (b) QsuB lignin. By reducing the amount of shikimate available to the lignin biosynthesis pathway, a lignin enriched in H-monomers is formed,
and the soluble produces protocatechuate accumulates. (A color version of this figure is available in the online journal.)
Mortimer Plant SynBio impacts on biofuels and medicine 5
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molecules (short-chain unsaturated hydrocarbons) are cur-
rently produced in a highly efficient manner from fossil
fuels. However, a number of alternatives have been sug-
gested as replacements which could be derived from
renewable sources.
52
One such molecule is the dicarboxylic
acid cis,cis-muconic acid. This is a precursor for bulk chem-
icals that include caprolactam, adipic acid, and terephthalic
acid, which in turn are precursors for plastics such as nylon
and polyethylene terephthalate (PET), or can be directly
incorporated into polyesters.
53
Synthetic biology has been
used successfully to produce muconic acid in a range of
microbial systems, including Escherichia coli,Pseudomonas
sp., and Sphingobium sp.
54
However, large-scale microbial
production requires access to a cheap carbon source, which
hinders the economic competitiveness of production. An
alternative is to produce muconic acid in an autotroph,
such as a biomass crop, where it can be considered an
added value product in addition to the biomass. This has
been successfully achieved in Arabidopsis using a complex
engineering strategy that required the introduction of four
bacterial enzymes.
55
In particular, by controlling the
expression of these genes using spatially- and temporally-
specific promoters, negative growth effects associated with
pathway intermediates were avoided. The muconic acid
produced could be extracted from senesced, mature bio-
mass, making it compatible with existing harvesting
strategies.
55
Polyhydroxyalkanoates (PHAs) are a class of
biodegradable, renewable plastics which include poly-3-
hydroxypropionic acid (a precursor of acrylic) and
poly-3-hydroxybutarate (PHB, a precursor of propylene).
Some microbial species produce PHAs as a stress response
in the face of nutrient limitation, where it acts as a carbon
store. Engineering of microbes to produce PHAs has been
relatively successful, and this is now entering limited com-
mercial production. However, like muconic acid, there
would be advantages to producing PHAs in an autotroph.
56
In some species, this has been highly successful. For exam-
ple, in Arabidopsis, up to 40% dry weight of PHB in leaf
tissue has been reported,
57
and 10.6% dry weight in
Phaeodactylum tricornutum (a microalgae). It has been chal-
lenging to achieve similar levels in highly productive C4
crop plants such as sugar cane
58
and switchgrass,
56
where
whole plant levels above 2% dry weight have not been
reported, despite extensive research efforts. PHB-
producing C4 plants show a strong growth phenotype,
including chlorosis and stunting, and a recent systems biol-
ogy study linked this to the physical presence of PHB in
bundle sheath cells.
59
They showed that high PHB plants
are ATP starved, and hypothesized that PHB granules may
shade the thylakoids, reducing photosynthetic rates.
59
This
suggests that future efforts to produce high-PHB C4 plants
may require a careful consideration for where in the plant
the PHB is produced, and at what point in the lifecycle. For
example, placing the enzymes under senescence associated
promoters, as has been done successfully in rice to alter
biomass composition,
60
may avoid such yield penalties.
Plant-produced therapeutics and vaccines
The concept of plant-based pharmaceutical production
(pharming) was proposed nearly 30 years ago.
61
Originally, this was conceived as the production of edible
vaccines in crop plants. These would be cheap to produce,
and avoid the expensive costs associated with manufacture,
or supply chain issues such as refrigeration.
62
However,
it rapidly became clear that this goal was unachievable.
The challenges of regulating dose are just too difficult to
overcome. As a result, the approach has been modified, and
there is now acceptance that while plant-produced vaccines
and pharmaceuticals have many benefits over other expres-
sion systems, some processing of the plant material will be
required prior to vaccination of the patient.
The major reason for choosing plants for the production
of monoclonal antibodies (mAbs), as opposed to other
industrial production systems such as bacteria, yeast,
insect or mammalian cells is a combination of cost, safety,
and the potential of plant systems to produce mAbs with
the correct post-translational modifications.
63,64
For exam-
ple, mammalian production systems, which dominate the
market, are expensive and challenging to scale up, whereas
bacteria are unable to produce most of the correct post-
translational glycosylation required for the mAb activity.
Yeast, insect cells, and plants, since they are eukaryotes,
can perform complex post-translational glycosylations.
However, in all these systems, the exact nature of the gly-
cosylation differs depending on the species, and all show
significant differences to mammalian systems (Figure 4(a)).
As a result, the concept of glycoengineering has been devel-
oped, in which the expression system is modified to
Figure 4. Glycoengineering of proteins in plants. (a) Simplified example structures of N-linked glycosylation in humans, plants, and yeast (b) N-linked glycosylation in
DXF tobacco plants, which produce humanized glycans on proteins. They lack the immunogenic b1,2-xylose and core a1,3-fucose.
6Experimental Biology and Medicine
............................................................................................... ................................... .............................
produce mammalian-like glycosylated mAbs. Plants have
proved to be particularly amenable to glycoengineering,
and this has been accelerated by synthetic biology
tools.
65–67
Perhaps the most well-known example of a plant-
produced therapeutic mAb is ZMapp
TM
, a promising
treatment for Ebola virus.
68,69
This consists of three
mouse/human chimeric mAbs which target the surface gly-
coprotein of the virions, and are produced in a strain of
Nicotiana benthamiana (tobacco) called DXF (Figure 4(b)).
DXF plants have reduced xylosyltransferase (XT) and fuco-
syltransferase (FT) activity, and produce authentic human
protein glycosylation.
66,70
Additional plant-produced thera-
peutics are currently in clinical trials (clinicaltrials.gov) or
have been approved. For example, Elelyso
TM
(taliglucer-
ase-a), a treatment for Gaucher’s disease, is produced in
carrot cell culture and was approved by the FDA in 2012.
As the global market for mAbs was reported to exceed $100
billion in 2017, the expectation is that this class of plant-
produced products will continue to grow.
Plants also hold promise for the commercial production
of pharmaceuticals or pharmaceutical precursors. One
example is the anti-malarial drug artemisinin, the main
ingredient in the ACT therapies used globally (and the
only current effective treatment). Artemesinin is found at
relatively low abundance in the Artemisia annua plant.
However, as demand for the drug increased, it became
increasingly challenging to produce it from its natural
source. The precursor, artemisinic acid, can be converted
chemically in a low-cost process to artemisinin, and there-
fore it has become an important biotechnological target.
The initial strategy used synthetic biology to engineer arte-
misinic acid production in yeast by overexpressing 14
genes, and conditionally repressing two more.
71
While
this approach was highly successful, producing yields in
excess of 25 g/L, the costs of large-scale yeast fermentation
have made this challenging to produce commercially. More
recently, a similar strategy has been used to produce arte-
mesinic acid in tobacco making use of the COSTREL gene
stacking strategy discussed above.
33
This has resulted in
yields in excess of 120 mg/kg of biomass. The authors esti-
mate that 200 km
2
of tobacco fields would be enough to
meet the current global demand of artemisinin (100 t).
Future work
As the precision with which plants can be engineered
increases, so will the range of applications. Recently, there
has been a growing interest in plants as remote sensors.
Plants can be considered sustainable and self-propagating
reporters of their environment, and can be surveyed
remotely, e.g. by drone. Suggested purposes include envi-
ronmental pollution,
72
explosives,
73
and radiation.
74
DARPA’s new Advanced Plant Technologies (APT)
research program (https://www.darpa.mil/program/
advanced-plant-technologies) which was announced in
2017, will be interesting to follow in this regard. Food
crops are also a major target, and engineering strategies
have been developed to reduce the methane emissions of
rice,
75
make low-gluten wheat,
76
and improve cold-storage
properties of potatoes.
77
This is likely to be a huge market
for growth. As humans prepare for interplanetary missions,
including to Mars in the near future, synthetic biology will
be key to developing plants which can survive the chal-
lenges of space travel, as well as providing optimized feed-
stocks for microbial manufacturing efforts.
78
The success of
all of these approaches will be reliant on increasing the
predictability of plant engineering. Some progress has
been made here, as a result of the reduced cost of DNA
synthesis. Groups such as Open Plant (https://www.open
plant.org/) and the Joint BioEnergy Institute (https://
public-registry.jbei.org/) are developing open-source regis-
tries for plant-specific DNA parts, e.g. Lao et al.
79
Next steps
include the development of better genetic regulators, such
as synthetic riboswitches, promoters, and transcription fac-
tors, as has been done for other organisms.
80,81
However,
more ambitious goals such as re-designing whole plant
genomes, as is being undertaken for yeast in the Sc2.0 proj-
ect,
82
still seem a very distant prospect. Finally, it remains to
be seen how the public will view synthetic biology and
gene-editing, in light of previous concerns about GMOs.
ACKNOWLEDGEMENTS
I would like to thank my colleagues at JBEI for fruitful dis-
cussions on this topic, especially Henrik Scheller, Aymerick
Eudes, and Yan Liang.
FUNDING
This work was part of the DOE Joint BioEnergy Institute
(http://www.jbei.org) and was supported by the U. S.
Department of Energy, Office of Science, Office of Biological
and Environmental Research, through contract DE-AC02–
05CH11231 between Lawrence Berkeley National Laboratory
and the U. S. Department of Energy.
DECLARATION OF CONFLICTING INTERESTS
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of
this article.
ORCID iD
Jenny C Mortimer http://orcid.org/0000-0001-6624-636X
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Mortimer Plant SynBio impacts on biofuels and medicine 9
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... The goals of the synthetic biology are robust, predictable, and rapid engineering of biology align well with this vision (Cameron et al. 2014a, b). According to Mortimer (2019) to date, much synthetic biology research has focused on microbial engineering. ...
... Systems and Synthetic Biology Mortimer et al. (2019) suggested that out of several definitions of synthetic biology the one emerging from the Engineering Biology Research Consortium (EBRC; www.ebrc.org) is most appropriate and is quoted here: -"Synthetic biology aims to make biology easier to engineer. . . It can be thought of as a biology-based toolkit that uses abstraction, standardization, and automated construction to change how we build biological systems and expand the range of possible products". ...
... The engineering concept of the design-build-test-learn (DBTL) cycle can underpin the application of synthetic biology to organismal engineering (Fig. 6.2) (Liu and Stewart 2015;Petzold et al. 2015;Mortimer 2019). The convergence of nextgeneration sequencing and synthetic biology, along with progress in analytic technologies such as mass spectroscopy, opens the door to the creation of large, reliable libraries of pure natural products for biofuels as well as drug discovery (Mitchell 2011). ...
... Technological advances in mass spectrometry-based platforms have facilitated the separation and identification of multiple metabolomic profiles of medicinal plants. With the implementation of the Herbal Genome Project (Chen et al., 2011;Su et al., 2022) and the development of traditional Chinese medicine synthetic biology (Mortimer, 2019), the metabolomic research of medicinal plants will accelerate the discovery of novel bioactive compounds with pharmacological potential (Bhardwaj et al., 2022;Nisar et al., 2022). ...
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Medicinal plants are natural sources to unravel novel bioactive compounds to satisfy human pharmacological potentials. The world’s demand for herbal medicines is increasing year by year; however, large-scale production of medicinal plants and their derivatives is still limited. The rapid development of modern technology has stimulated multi-omics research in medicinal plants, leading to a series of breakthroughs on key genes, metabolites, enzymes involved in biosynthesis and regulation of active compounds. Here, we summarize the latest research progress on the molecular intricacy of medicinal plants, including the comparison of genomics to demonstrate variation and evolution among species, the application of transcriptomics, proteomics and metabolomics to explore dynamic changes of molecular compounds, and the utilization of potential resources for natural drug discovery. These multi-omics research provide the theoretical basis for environmental adaptation of medicinal plants and allow us to understand the chemical diversity and composition of bioactive compounds. Many medicinal herbs’ phytochemical constituents and their potential health benefits are not fully explored. Given their large diversity and global distribution as well as the impacts of growth duration and environmental factors on bioactive phytochemicals in medicinal plants, it is crucial to emphasize the research needs of using multi-omics technologies to address basic and applied problems in medicinal plants to aid in developing new and improved medicinal plant resources and discovering novel medicinal ingredients.
... Synthetic biology uses established loop workflows [6,7] based on (i) design, (ii) build, (iii) test, and (iv) learn phases. However, before entering this loop workflow, the design must be preceded by a theoretical phase in which the engineers define biomimicry, reconstruction and/or optimization of natural mechanisms, construction of new-to-nature pathways, and a precise list of problems solvable with the downstream loop workflow (Figure 1). ...
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Although it is still in its infancy, synthetic biology has the capacity to face scientific and societal problems related to modern agriculture. Innovations in cloning toolkits and genetic parts allow increased precision over gene expression in planta. We review the vast spectrum of available technologies providing a practical list of toolkits that take advantage of combinatorial power to introduce/alter metabolic pathways. We highlight that rational design is inspired by deep knowledge of natural and biochemical mechanisms. Finally, we provide several examples in which modern technologies have been applied to address these critical topics. Future applications in plants include not only pathway modifications but also prospects of augmenting plant anatomical features and developmental processes.
... Generation of mutant with CCR1 and KCS1 knockdown may show interconnections between the different pathways that may confirm and explain these results. This analysis using high throughput omics analysis may give opportunities in synthetic biology and metabolic engineering (Mortimer, 2019). ...
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Sugarcane (Saccharum sp.) is a tropical and subtropical C4 plant with a high photosynthetic and carbon assimilation efficiency that stores sucrose. Culm biomass is also composed of bagasse fibre, a by‐product of the sugarcane industry. This high‐yielding grass, high in sucrose and lignocellulosic biomass, is considered an optimal feedstock as an alternative to fossil fuels and to produce a broad range of high‐value biomaterials. The ideal sugarcane production system would optimise the relative production of sugar and these new products. Multi‐omics correlation analysis was used to generate a global view of the essential metabolic pathways identifying critical genes involved in carbon partitioning during different stages of development. This research employed an unprecedented metabolic and transcriptomic dataset of 360 samples from a selection of 1440 culms of 24 genotypes at five different development stages. Chemical composition and metabolome analysis showed an increase through the culm development of lignin, sucrose, carbon, and amino acids such as aspartic acid, serine, alanine, methionine, threonine 3‐cyano‐L‐alanine, and citric acid. Transcriptome analysis revealed functionalities such as transcription, nucleotide transport and metabolism, and the biosynthesis of amino acids that are highly activated during the immature stage and highly down‐regulated during the most mature age. Sugarcane (Saccharum sp.) is a tropical and subtropical C4 plant with a high photosynthetic and carbon assimilation efficiency that stores sucrose. This high‐yielding grass, high in sucrose and lignocellulosic biomass, is considered an optimal feedstock as an alternative to fossil fuels and to produce a broad range of high‐value biomaterials. Multi‐omics correlation analysis was used to generate a global view of the essential metabolic pathways identifying critical genes involved in carbon partitioning during different stages of development.
... Synthetic biology proposes to go beyond genetic engineering by manipulating not only genes but whole metabolic systems and their regulatory pathways [6]. The ribosome has been a primer target of synthetic biology efforts [7], mainly due to its capability to synthesize protein at an extraordinary rate and accuracy [8]. ...
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As the centerpiece of the biomass production process, ribosome activity is highly coordinated with environmental cues. Findings revealing ribosome subgroups responsive to adverse conditions suggest this tight coordination may be grounded in the induction of variant ribosome compositions and the differential translation outcomes they might produce. In this perspective, we go through the literature linking ribosome heterogeneity to plants’ abiotic stress response. Once unraveled, this crosstalk may serve as the foundation of novel strategies to custom cultivars tolerant to challenging environments without the yield penalty.
... The synthetic biology tools applied to microorganisms offer the possibility of producing chemicals for agricultural production that can increase agricultural productivity [147], give greater nutritional value to agricultural products [148], and benefit the environment [149]. Bio-factories can also be used to make substances currently produced by plants for food [150] and medicine [151,152], among other sectors. Thus, the diversity of compounds and molecules that can be produced using synthetic living factories can revolutionize the production of biomolecules for routine and common use, entering a new era of bioeconomy, with the development of bioproducts of high economic value [153][154][155]. ...
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Despite the acknowledged relevance of renewable energy sources, biofuel production supported by food-related agriculture has faced severe criticism. One way to minimize the considered negative impacts is the use of sources of non-food biomass or wastes. Synthetic biology (SB) embraces a promising complex of technologies for biofuel production from non-edible and sustainable raw materials. Therefore, it is pertinent to identify the global evolution of investments, concepts, and techniques underlying the field in support of policy formulations for sustainable bioenergy production. We mapped the SB scientific knowledge related to biofuels using software that combines information visualization methods, bibliometrics, and data mining algorithms. The United States and China have been the leading countries in developing SB technologies. The Technical University of Denmark and Tsinghua University are institutions with higher centrality and have played prominent roles besides UC Los Angeles and Delft University Technology. We identified six knowledge clusters under the terms: versatile sugar dehydrogenase, redox balance principle, sesquiterpene production, Saccharomyces cerevisiae, recombinant xylose-fermenting strain, and Clostridium saccharoperbutylacetonicum N1-4. The emerging trends refer to specific microorganisms, processes, and products. Yarrowia lipolytica, Oleaginous yeast, E. coli, Klebsiella pneumoniae, Phaeodactylum tricornutum, and Microalgae are the most prominent microorganisms, mainly from the year 2016 onward. Anaerobic digestion, synthetic promoters, and genetic analysis appear as the most relevant platforms of new processes. Improved biofuels, bioethanol, and N-butanol are at the frontier of the development of SB-derived products. Synthetic biology is a dynamic interdisciplinary field in environmentally friendly bioenergy production pushed by growing social concerns and the emergent bioeconomy.
... reviewed application of synthetic biology for obtaining biofuels through synthetic routes. According to Mortimer (2019) to date, much synthetic biology research has focused on microbial engineering. ...
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Plant natural products have been extensively exploited in food, medicine, flavor, cosmetic, renewable fuel and other industrial sectors. Synthetic biology has recently emerged as a promising means for cost‐effective and sustainable production of natural products. Comparing to engineering microbes for the production of plant natural products, the potential of plants as chassis for producing these compounds is underestimated largely due to challenges encountered in engineering plants. Knowledge in plant engineering is instrumental for enabling the effective and efficient production of valuable phytochemicals in plants, which also paves ways for a more sustainable future agriculture. In this manuscript, we briefly recap the biosynthesis of plant natural products, focusing primarily on industrially important terpenoids, alkaloids and phenylpropanoids. We further summarize the plant hosts and strategies that have been employed to engineer the production of natural products. The challenges and opportunities of using plant synthetic biology to achieve rapid and scalable production of high‐value plant natural products are also discussed. This article is protected by copyright. All rights reserved.
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Human health faces one of the grave issues from the nutrition deficiency in crops. Therefore, it should be given prime importance to develop crops for subsiding this issue. In order to improve the nutritional quality of crops, biofortification aids in combatting nutrient deficiency by enhancing the nutrient-specific component levels. Plants have various types of complex metabolic pathways specific for the synthesis of particular nutrient compounds as well as other secondary metabolites. Plant metabolic engineering aids in the modulation of these specific biosynthetic pathways so as to introduce the new metabolic abilities by either downgrading or upgrading any particular enzyme according to the requirement for amplifying the nutritional quality. Progress and innovations made in the sequencing and bioinformatics will provide an ample opportunity about metabolic pathways and discover the best intervention point. Knowledge attained about vitamin metabolism, uptake of minerals, and relocation in plant makes it feasible today to increase micronutrient level in crop plants. While making comparison of different agronomic practices and conventional plant breeding with plant metabolic engineering and synthetic biological strategies, the latter ones have found to be more effective in synthesizing the specific nutrients, bioactive components in crops. Technological advancement and progresses made in the field of plant metabolic engineering particularly in view of research strategies of multigene stacking tool, complex metabolic engineering with prime focus on refining characters or traits associated with micronutrients, phytonutrients, and bioactive compounds will ultimately help the human race in developing the crops to meet the nutritional demand.
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Our lives are connected to climate and atmosphere. Our current circumstance turns out to be warm and polluted step by step with these coal and oil. Greenhouse gases like CO2, CFC are the fundamental explanation behind environmental change. These gases are sufficient to contribute genuine dry spells, ruinous floods, and lease less snowfalls. United States has fewer than 5% of the total populace and it demolishes 25% of oils that is equivalent to 20 million barrels for each day for just transportation. These are the reasons for diminishing the fuel sources that come from nature. One way to deal with moving back these examples is to assemble fuel sources. Numerous energy concentrated enterprises are reacting to the atmosphere evolving challenge. They consented to utilize bio powers like bioethanol rather than oil. Bioethanol is an adaptable fuel that contributes next to zero CO2 to develop greenhouse gases and further for the warm-up of the climate. Generally biofuels had been delivered from food crops. Yet, presently new innovations are empowering biofuel creation from nonedible gases, woods, and other plant squander materials. The excellence of biofuels is that they drain carbon dioxide out of the air as they developed. Simply on premise biofuels give off an impression of being zero net producers.
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The interest in human space journeys to distant planets and moons has been re-ignited in recent times and there are ongoing plans for sending the first manned missions to Mars in the near future. In addition to generating oxygen, fixing carbon, and recycling waste and water, plants could play a critical role in producing food and biomass feedstock for the microbial manufacture of materials, chemicals, and medicines in long-term interplanetary outposts. However, because life on Earth evolved under the conditions of the terrestrial biosphere, plants will not perform optimally in different planetary habitats. The construction or transportation of plant growth facilities and the availability of resources, such as sunlight and liquid water, may also be limiting factors, and would thus impose additional challenges to efficient farming in an extraterrestrial destination. Using the framework of the forthcoming human missions to Mars, here we discuss a series of bioengineering endeavors that will enable us to take full advantage of plants in the context of a Martian greenhouse. We also propose a roadmap for research on adapting life to Mars and outline our opinion that synthetic biology efforts towards this goal will contribute to solving some of the main agricultural and industrial challenges here on Earth.
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Muconic acid (MA) is a dicarboxylic acid used for the production of industrially relevant chemicals such as adipic acid, terephthalic acid, and caprolactam. Because the synthesis of these polymer precursors generates toxic intermediates by utilizing petroleum-derived chemicals and corrosive catalysts, the development of alternative strategies for the bio-based production of MA has garnered significant interest. Plants produce organic carbon skeletons by harvesting carbon dioxide and energy from the sun, and therefore represent advantageous hosts for engineered metabolic pathways towards the manufacturing of chemicals. In this work, we engineered Arabidopsis to demonstrate that plants can serve as green factories for the bio-manufacturing of MA. In particular, dual expression of plastid-targeted bacterial salicylate hydroxylase (NahG) and catechol 1,2-dioxygenase (CatA) resulted in the conversion of the endogenous salicylic acid (SA) pool into MA via catechol. Sequential increase of SA derived from the shikimate pathway was achieved by expressing plastid-targeted versions of bacterial salicylate synthase (Irp9) and feedback-resistant 3-deoxy-D-arabino-heptulosonate synthase (AroG). Introducing this SA over-producing strategy into engineered plants that co-express NahG and CatA resulted in a 50-fold increase in MA titers. Considering that MA was easily recovered from senesced plant biomass after harvest, we envision the phytoproduction of MA as a beneficial option to add value to bioenergy crops.
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Ornamental plants that have a rich variety of flower colors and shapes are highly prized in the commercial flower market, and therefore, mutant cultivars that produce different types of flowers while retaining their growth habits are in demand. Furthermore, mutation breeding is well suited for ornamental plants because many species can be easily vegetatively propagated, facilitating the production of spontaneous and induced mutants. The use of ion beams in mutation breeding has rapidly expanded since the 1990s in Japan, with the prospect that more ion beam-specific mutants will be generated. There are currently four irradiation facilities in Japan that provide ion beam irradiation for plant materials. The development of mutant cultivars using ion beams has been attempted on many ornamental plants thus far, and some species have been used to investigate the process of mutagenesis. In addition, progress is being made in clarifying the genetic mechanism for expressing important traits, which will probably result in the development of more efficient mutation breeding methods for ornamental plants. This review not only provides examples of successful mutation breeding results using ion beams, but it also describes research on mutagenesis and compares results of ion beam and gamma ray breeding using ornamental plants.
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Background Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. ResultsWe have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase and the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. Conclusion The results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.
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Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output.
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Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs) and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs) with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.
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Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.
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