Journal of Experimental Botany, Vol. 70, No. 5 pp. 1425–1433, 2019
doi:10.1093/jxb/erz029  Advance Access Publication 31 January 2019
© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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EXPERT VIEW
Synthetic biology approaches for improving photosynthesis
Armin Kubis and Arren Bar-Even*,
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany
* Correspondence: bar-even@mpimp-golm.mpg.de
Received 1 November 2018; Editorial decision 8 January 2019; Accepted 8 January 2019
Editor: Christine Raines, University of Essex, UK
Abstract
The phenomenal increase in agricultural yields that we have witnessed in the last century has slowed down as we
approach the limits of selective breeding and optimization of cultivation techniques. To support the yield increase
required to feed an ever-growing population, we will have to identify new ways to boost the efficiency with which
plants convert light into biomass. This challenge could potentially be tackled using state-of-the-art synthetic biology
techniques to rewrite plant carbon fixation. In this review, we use recent studies to discuss and demonstrate different
approaches for enhancing carbon fixation, including engineering Rubisco for higher activity, specificity, and
activation; changing the expression level of enzymes within the Calvin cycle to avoid kinetic bottlenecks; introducing
carbon-concentrating mechanisms such as inorganic carbon transporters, carboxysomes, and C4 metabolism; and
rewiring photorespiration towards more energetically efficient routes or pathways that do not release CO2. We conclude
by noting the importance of prioritizing and combining different approaches towards continuous and sustainable
increase of plant productivities.
Keywords:   carbon fixation, Calvin cycle, Rubisco, C4 metabolism, carbon-concentrating mechanisms, photorespiration.
Introduction
Selective breeding and optimization of cultivation techniques
have historically driven increases in agricultural output. In the
last century,these efforts have adopted a more scientific approach
with the development of the Haber–Bosch process (Haber and
Le Rossignol,1909;Sutton et al.,2008,and,later,the‘green revolution’
(Khush, 2001). Since 1961, global rice and wheat yields
increased by 150% and 210%,respectively (FAO,2018).However,
we have recently started to witness stagnation in growth improvement
of major crops such as rice in China (Peng et al., 2009)
or wheat in the USA (Ray et al., 2012).This presents a major
problem, as further yield increases are sorely needed to feed the
human population,especially considering the global shift towards
meat-dependent diets, use of arable lands to feed bio-refineries,
deleterious effects of climate change, and continuous erosion of
agricultural land (Godfray et al.,2010;Tilman et al.,2011).
Agricultural yield can be modeled as a product of three
factors (Monteith and Moss, 1977; Fletcher et  al., 2011): (i)
efficiency of intercepting light; (ii) efficiency of converting
intercepted light into biomass; and (iii) the harvest index (i.e.
the fraction of biomass that is captured in the harvested part).In
the past,improved yields have largely been achieved by increasing
the light capture efficiency and the harvest index; however,
these two factors now appear to be approaching their practical
limits (Long et al., 2006).Therefore, the efficiency with which
plants convert light to biomass has become the prime focus
for further improvement (Long et  al., 2006). This efficiency
is determined by two main processes, the light-dependent
reactions, in which photoenergy is used for the generation of
the cellular redox and energy carriers NADPH and ATP, and
the light-independent reactions, which use these carriers to
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1426 | Kubis and Bar-Even
fix CO2 and reduce it to organic carbon. The efficiency of
both processes is unlikely to be improved by a classic selective
breeding approach—as demonstrated by a recent study exploring
80 years of soybean breeding (Koester et al., 2016)—but
could be potentially increased by dedicated engineering (Zhu
et al., 2010). The focus of this review is the use of synthetic
biology tools for boosting the efficiency and rate of carbon
fixation (Box 1). Rather than discuss the technical aspects of
synthetic biology in plants—for which we refer the readers
to other reviews (DePaoli et al., 2014; Liu and Stewart, 2015;
Boehm and Bock,2018;Piatek et al.,2018;Vazquez-Vilar et al.,
2018)—we emphasize conceptual strategies to boost carbon
Box 1. Key developments in synthetic biology approaches for improving photosynthesis
•	Assembly of Rubisco-containing carboxysomes in tobacco chloroplasts
Assembly of a simplified α-carboxysome in tobacco chloroplasts by replacing native Rubisco
with large and small subunits of Rubisco from cyanobacteria and two key structural subunits.
The introduction of carboxysomes to plant chloroplasts is a key step towards establishing a
full biophysical carbon-concentrating mechanism in higher plants (Long et al., 2018).
•	Design and in vitro realization of carbon-conserving photorespiration
A systematic search and analysis of synthetic photorespiration bypass routes that do not
release CO2 reveals that these can enhance the carbon fixation rate under all relevant
physiological conditions. Two enzymes were engineered jointly to enable the reduction
of glycolate to glycolaldehyde. The combination of these evolved enzymes with existing
enzymes supported the in vitro recycling of glycolate to RuBP without the loss of CO2,
indicating the feasibility of carbon-conserving photorespiration (Trudeau et al., 2018).
•	The synthetic malyl-CoA–glycerate pathway supports photosynthesis
An in vivo demonstration of a synthetic pathway that can support photosynthesis in two
ways. First, it can produce acetyl-CoA from C3 sugars without releasing CO2. It can also
assimilate photorespiratory glycolate without loss of carbon (Yu et al., 2018).
•	Carbon fixation via a novel pathway in vitro
An in vitro reconstruction of a synthetic carbon-fixing pathway, the CETCH cycle, based
on highly efficient reductive carboxylation. The pathway, utilizing 17 enzymes that originate
from nine organisms, was optimized by a combination of enzyme engineering and metabolic
proofreading (Schwander et al., 2016).
•	Overexpressing the H-protein of the glycine cleavage system increases biomass yield in
glasshouse- and field-grown transgenic tobacco plants
Increased biomass upon overexpression of a limiting photorespiratory protein in tobacco grown
in field conditions. This indicates that optimization of expression levels within native carbon
fixation-related pathways could be harnessed to increase productivity, and that photorespiration
could be improved even without the need for synthetic pathways (López-Calcagno et al., 2019).
•	The road to C4 photosynthesis: evolution of a complex trait via intermediary states
A case for engineering C3–C4 intermediate metabolism as a way to increase photosynthetic
efficiency and set the stage towards future realization of complete C4 metabolism. This study
suggests that a detailed and mechanistic understanding of C3–C4 intermediates could provide
valuable guidance for experimental designs aiming to boost carbon fixation (Schlüter and
Weber, 2016).
•	Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and
plant growth
A demonstration of the use of directed laboratory evolution to improve the kinetic properties
of Rubisco from an archaeal origin. The improved Rubisco variant was introduced to tobacco
chloroplasts and demonstrated to increase photosynthesis. Such protein engineering
strategies could be used to address the kinetic limitations of key enzymes, thus supporting
higher metabolic fluxes and boosting productivities (Wilson et al., 2016).
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Synthetic biology approaches for improving photosynthesis  |  1427
fixation.In particular,we discuss efforts aiming to improve carboxylation
by Rubisco, optimize expression levels of enzymes
within the Calvin cycle, introduce carbon-concentrating
mechanisms (CCMs), and rewire photorespiration. We claim
that multiple complementary strategies are paving the way
towards substantial yield increases that are not feasible using
conservative selective breeding techniques.
Engineering Rubisco
Rubisco, the key enzyme of the Calvin cycle, is probably the
most abundant protein in the biosphere (Ellis, 1979; Raven,
2013), and is responsible for assimilating the vast majority of
inorganic carbon (Raven, 2009). The enzyme catalyzes the
condensation of ribulose 1,5-bisphosphate (RuBP) with CO2
to give two molecules of glycerate 3-phosphate (G3P).Despite
its key biochemical role, Rubisco is considerably slower than
most enzymes in central metabolism (Bar-Even et al., 2011).
Moreover, Rubisco is not completely specific to CO2 and also
accepts O2, leading to the formation of 2-phosphoglycolate
(2PG) that needs to be reassimilated. In the C3 model plant
Arabidopsis thaliana, the carboxylation to oxygenation ratio was
measured to be as low as 2.3:1 under high light conditions (Ma
et al., 2014). Suppressing oxygenation reactions by cultivating
plants at elevated CO2 concentrations has repeatedly been
shown to increase productivity.For example,a meta-analysis of
70 studies showed that rice yields increased by 23% when CO2
concentrations were raised to 627  ppm (Ainsworth, 2008).
These results indicate that engineering Rubisco for higher
CO2 specificity could substantially boost yield.
Approaches to improve Rubisco catalysis by random or
site-directed mutagenesis have generally failed to yield substantial
kinetic enhancements (Somerville and Ogren, 1982;
Spreitzer et al., 2005;Whitney et al., 2011;Wilson et al., 2016).
Comparisons between Rubisco variants from a range of different
organisms have revealed a trade-off between CO2 specificity
and carboxylation velocity (Tcherkez et al., 2006; Savir
et al., 2010; Galmés et al., 2014), although several recent studies
challenge this finding (Young et al., 2016; Cummins et al.,
2018). Considering this trade-off, it actually seems that most
Rubisco variants are well adapted to their intracellular environment.
Still, as ambient CO2 concentrations are changing at
a rate faster than plants can adapt to them, it was suggested
that replacing plant Rubisco with another variant could boost
carbon fixation by up to 25% (Zhu et al.,2004;Orr et al.,2016).
Substituting one Rubisco variant with another is undoubtedly
a challenging task, but has already been demonstrated
using homodimeric Rubisco from the α-proteobacterium
Rhodospirillum rubrum (Whitney and Andrews, 2001) and,
more recently, using a fast hexadecameric Rubisco from
Synechococcus elongatus (Lin et al., 2014; Occhialini et al., 2016).
Co-expression of supporting chaperones, including the appropriate
accumulation factors, can assist in producing an active
Rubisco recombinantly,and can further facilitate efforts to enhance
the kinetics of this key enzyme via mutagenesis (Aigner
et al., 2017).
Carbon fixation via Rubisco can potentially be improved
by means other than direct engineering of its catalytic parameters.The
addition of a CO2 molecule to an active site lysine,
namely carbamylation, is a prerequisite for Rubisco activity
(Lorimer and Miziorko, 1980), but can be hindered by the
premature binding of RuBP or other sugar phosphates (Portis,
2003;Parry et al.,2008).The catalytic chaperone Rubisco activase
(Rca) removes the sugar phosphate inhibitors from an inactive
uncarbamylated enzyme or an inhibited carbamylated
Rubisco (Portis, 2003). As the thermal instability of Rca was
shown to constrain carbon fixation under moderate heat stress
(Salvucci et al., 2004), it has become an attractive target for engineering
towards enhanced photosynthesis. For example, by
increasing the thermostability of Rca in A. thaliana, improved
photosynthesis and growth rate were demonstrated under a
moderate heat stress (Kurek et al., 2007; Kumar et al., 2009).
Similarly, overexpression of maize Rca in rice led to a higher
activation state of Rubisco in low light and a faster response of
photosynthesis when light intensities increased (Yamori et al.,
2012).
Optimizing expression of Calvin cycle
enzymes
G3P produced by Rubisco needs to be metabolized by nine
enzymes of the Calvin cycle to regenerate RuBP.This regeneration
process, whose rate has to match that of Rubisco, is
known to limit the carbon fixation rate under certain conditions.
Computational models have suggested that the natural
distribution of enzymes within the Calvin cycle is not optimal
and could limit photosynthesis (Zhu et al., 2007). Specifically,
it was predicted that higher levels of sedoheptulose-1,7-­
bisphosphatase and fructose-1,6-bisphosphate aldolase, as
well as enzymes linked to sink capacity, could support higher
productivity.
Unsurprising, under elevated CO2 concentrations, the
rate of Rubisco becomes less limiting and carbon fixation
is mostly constrained by RuBP regeneration. For example,
studies of Nicotiana tabacum at 930  ppm CO2 showed that
reducing Rubisco levels by 30–50% did not inhibit growth
(Masle et al.,1993).Similar results were obtained in rice plants
in which Rubisco levels were reduced by 65% at 1000 ppm
CO2. On the other hand, overexpression of sedoheptulose-
1,7-bisphosphatase in N. tabacum at 585 ppm CO2 resulted
in a higher carbon fixation rate (Rosenthal et  al., 2011).
Similarly, at 700  ppm, increased levels of fructose-1,6-bisphosphate
aldolase in N.  tabacum led to increased biomass
(Uematsu et al., 2012).
Even at ambient CO2 concentration, overexpression of limiting
enzymes of the Calvin cycle was shown to boost carbon
fixation. In N.  tabacum, overexpression of sedoheptulose-
1,7-bisphosphatase (Lefebvre et al., 2005) and fructose-1,6-bisphosphatase
(Miyagawa et al., 2001) increased photosynthetic
rates and biomass. Similarly, the co-overexpression of sedoheptulose-1,7-bisphosphatase
and fructose-1,6-phosphate aldolase
enhanced photosynthesis and yield (Simkin et al., 2015).
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1428 | Kubis and Bar-Even
Establishing carbon-concentrating
mechanisms
To mitigate the problem of oxygenation, and further enable
the use of faster (and less specific) Rubsico, multiple organisms
have developed CCMs to concentrate CO2 at the site of
Rubisco. As C3 plants lack CCMs, it was proposed to introduce
them to increase photosynthetic efficiency.Two main approaches
are actively pursued: (i) introduction of biophysical
CCMs from cyanobacteria and green algae (Long et al., 2016;
Rae et al., 2017); and (ii) introduction of C4 anatomy and metabolism
(Hibberd et al., 2008; Schuler et al., 2016).
Biophysical CCMs are found in cyanobacteria (Kupriyanova
et al., 2013) and in green algae such as Chlamydomonas reinhardtii
(Mackinder, 2018). In such CCMs, bicarbonate is actively
transported into the cytosol in which carbonic anhydrase
is lacking. From there, bicarbonate is further transported into
specialized compartments packed with Rubisco—carboxysomes
in cyanobacteria and pyrenoids in green algae—where
it is dehydrated to CO2 by carbonic anhydrase. It is thought
that both carboxysomes and pyrenoids present a diffusion barrier
for CO2 and O2, keeping the former molecule in and the
latter molecule out,and thus enhancing carboxylation and suppressing
oxygenation (Mangan et al., 2016).
Establishing biophysical CCM in plants is a challenging task
that first requires the expression and correct localization of inorganic
carbon transporters. It was suggested that the transporters
themselves could increase the carbon fixation rate
albeit to a limited extent (McGrath and Long, 2014;Yin and
Struik, 2017). Indeed, overexpression of the putative inorganic
carbon transporter from cyanobacteria, ictB, in A. thaliana, tobacco,
rice, and soybean was reported to increase the photosynthetic
rate and biomass (Lieman-Hurwitz et al., 2003, 2005;
Yang et al., 2008; Simkin et al., 2015; Hay et al., 2017). In contrast,
expression of other transporters from cyanobacteria or
C. reinhardtii did not increase yield or improve growth, despite
correct localization within the plant cells (Atkinson et al.,2016;
Rolland et al., 2016; Uehara et al., 2016). Optimizing transporter
activity is therefore still an open challenge that needs to
be resolved before commencing with the next step: assembly
of Rubisco-containing compartments. The establishment of
these sophisticated structures would enable a further increase
in CO2 concentration at the site of Rubisco and could therefore
substantially enhance carbon fixation. Recently, simplified
carboxysome structures were introduced into the chloroplasts
of N. tabacum (Long et al., 2018).Yet, these are expected to enhance
photosynthesis only after combination with functional
inorganic carbon transporters (McGrath and Long, 2014).
Engineering C4 metabolism
As an alternative to biophysical CCMs, ongoing research
is dedicated to introducing C4 metabolism into C3 plants
(Schuler et al., 2016). C4 metabolism utilizes the most efficient
carbon fixation enzyme, phosphoenolpyruvate (PEP) carboxylase,to
capture inorganic carbon temporarily,which is then
transported to the vicinity of Rubisco (Jenkins et al., 1989).
Specifically, PEP carboxylase in the mesophyll cells ‘borrows’
PEP and converts it to oxaloacetate, which is further metabolized
to malate or aspartate.These C4 acids are transported to
the bundle sheath cells and decarboxylated to release CO2 next
to Rubsico, which is mainly localized in these cells. Pyruvate,
the product of this decarboxylation, is then transported back
to the mesophyll cells to regenerate PEP. Hence, the entire
C4 cycle, which depends on a special anatomy termed ‘Kranz
anatomy’(mesophyll cells surrounding bundle sheath cells),can
be regarded as a sophisticated CO2 pump that results in an ~10
times higher concentration of inorganic carbon in the vicinity
of Rubsico (Jenkins et al., 1989).
Engineering C4 photosynthesis in C3 plants has been outlined
as a stepwise process (Schuler et al., 2016) that includes
alteration of plant tissue anatomy, establishment of bundle
sheath morphology, as well as ensuring a cell type-specific
enzyme expression. Although challenging, engineering a C3
plant to have C4 metabolism seems to be a feasible goal as it is
known to have emerged independently at least 66 times in different
phylogentic backgrounds (Sage et al.,2012).Importantly,
C3 plants already harbor the main enzymes of C4 metabolism,
such as PEP carboxylase (Aubry et al.,2011),and are known to
shuttle carbon from the vasculature into the surrounding cells
in a way similar to C4 plants (Hibberd and Quick,2002;Brown
et al., 2010).This provides a solid basis to replicate the emergence
of C4 metabolism by direct engineering.
Nevertheless, despite international efforts, a synthetic C4
plant has yet to be reported. Following Richard Feynman’s
famous quote ‘What I cannot create, I do not understand’, it
seems that incomplete understanding of C4 metabolism hampers
its engineering. Specifically, the metabolic shuttling of
intermediates between mesophyll and bundle sheath cells and
the factors necessary to create Kranz anatomy are still not fully
clear and need to be elucidated (Schuler et al., 2016).
It might not be necessary to establish a complete C4 metabolism
in order to improve carbon fixation. It was recently
suggested that engineering a C3–C4 intermediate metabolism
could enhance productivity (Schlüter and Weber, 2016). For
example, in C3–C4 intermediate type I plants, photorespiratory
glycine is transported from the mesophyll cells to the
bundle sheath cells for decarboxylation. In the bundle sheath
cells, the mitochondria are closely associated with the chloroplast,
thereby enhancing re-assimilation of released CO2 by
nearby Rubsico (Monson and Edwards, 1984; Rawsthorne
et al., 1988). Establishing this intermediary metabolism within
C3 plants, besides being useful on its own to boost carbon fixation,
would provide a milestone towards further engineering
of complete C4 metabolism.
An interesting alternative engineering target is crassulacean
acid metabolism (CAM).While C4 metabolism increases CO2
concentration in the vicinity of Rubisco via spatial organization,
CAM accomplishes the same goal via temporal decoupling.Specifically,inorganic
carbon is temporarily fixed by the
highly efficient PEP carboxylase during the night, when the
stomata are open and CO2 can freely enter the cell. Malate,
the indirect product of the carboxylation, is stored within
the vacuole. During the day, when the stomata are closed,
malate is decarboxylated, releasing CO2 and maintaining its
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Synthetic biology approaches for improving photosynthesis  |  1429
high concentration for subsequent fixation by Rubisco and
the Calvin cycle. Besides increasing CO2 concentration in
the vicinity of Rubisco, CAM reduces water evaporation and
increase water use efficiency by 20–80% (Borland et al., 2009),
making CAM plants highly suitable for arid climates.Similarly
to C4 metabolism, CAM has arisen multiple times in a taxonomically
diverse range of plants, indicating that its necessary
components exist in C3 plants which could potentially be
engineered towards this unique carbon metabolism (DePaoli
et al., 2014). Furthermore, the existence of C3–CAM intermediate
species and plants that switch between both metabolic
modes further supports the potential of engineering C3
metabolism towards CAM (Borland et al., 2011). Such engineering
would require precise control of the activity of key
enzymes (e.g. PEP carboxylase, malic enzyme, and Rubisco),
stomatal conductance, and intracellular transport (e.g. to and
from the vacuole) (Borland et al., 2014; DePaoli et al., 2014;
Yang et al., 2015).
Rewiring photorespiration
2PG, the product of Rubisco’s oxygenation activity, is recycled
to the Calvin cycle in a process termed photorespiration.This
rather long pathway requires the shuttling of metabolites across
multiple organelles and is considered inefficient as it dissipates
energy by releasing ammonia and using oxygen as an electron
acceptor. Moreover, photorespiration releases one CO2
molecule in the recycling of two 2PG molecules and hence
directly counteracts carbon fixation by the Calvin cycle.The
inefficiencies associated with the recycling of 2PG cannot be
prevented by simply blocking photorespiration,as this pathway
plays an essential role in plant metabolism (Somerville and
Ogren, 1979) and reduction of its flux was shown to affect
photosynthesis negatively (Servaites and Ogren, 1977;Wingler
et  al., 1997; Heineke et  al., 2001). One explanation for this
lies in the inhibitory effects exerted by several photorespiratory
intermediates. For example, 2PG was shown to inhibit
triosephosphate isomerase and sedoheptulose-1,7-bisphosphate
phosphatase (Anderson, 1971; Flügel et al., 2017), glyoxylate
impairs Rubisco activation (Chastain and Ogren,1989;
Campbell and Ogren, 1990; Hausler et al., 1996; Savir et al.,
2010), and glycine interferes with Mg2+
availability (Eisenhut
et al., 2007). Based on these observations, it was suggested that
increased photorespiratory flux could prevent the accumulation
of inhibitory intermediates and enhance photosynthesis;
indeed, this was demonstrated upon overexpression of components
of the glycine cleavage system in A. thaliana (Timm et al.,
2012, 2015) and in N. tabacum (López-Calcagno et al., 2019).
While photorespiration cannot be avoided, it might be possible
to replace the natural pathway with more efficient alternatives.The
first bypass suggested in this regard was inspired by
cyanobacterial photorespiration (Eisenhut et al., 2006), where
glyoxylate is condensed and reduced to directly generate the
key photorespiratory intermediate glycerate.This pathway was
implemented in A. thaliana (Kebeish et al., 2007) and later in
Camelina sativa (Dalal et  al., 2015) using glycolate dehydrogenase,
glyoxylate carboxyligase, and tartronic semialdehyde
reductase from Escherichia coli. In both cases, this metabolic bypass,
dissipating less energy and shifting CO2 release from the
mitochondria to the chloroplast,was shown to increase photosynthesis
and biomass.
However, it was shown that expression of only the first enzyme
of the pathway, glycolate dehydrogenase, suffices to enhance
photosynthesis.Supporting this,chloroplastic expression
of glycolate dehydrogenase in Solanum tuberosum induced a
2.3-fold increase in tuber yield (Nölke et al., 2014).This suggests
that the benefits of the glycerate pathway might not stem
from more efficient recycling of 2PG but rather from oxidation
of glycolate to glyoxylate.Indeed,incubation with glyoxylate
was shown to increase carbon fixation—potentially due
to suppression of Rubisco oxygenation—in both tobacco leaf
disks (Oliver and Zelitch, 1977) and soybean mesophyll cells
(Oliver, 1980).
Another photorespiratory bypass, which was reported to increase
biomass and photosynthesis, involves the complete oxidation
of 2PG to CO2 via a catabolic pathway that consists of
glycolate dehydrogenase, malate synthase, malic enzyme, and
pyruvate dehydrogenase (Maier et  al., 2012). A  recent study
showed that a variant of this bypass can increase the productivity
of tobacco plants in the field by >40% (South et al.,2019).
However, the mechanism that underlies the beneficial effects
of the pathway remains vague as a theoretical model predicts
a negative effect when 2PG is completely oxidized (Xin et al.,
2015).
Carbon-conserving photorespiration
As the main problem associated with photorespiration is (arguably)
the release of CO2, bypasses that do not lead to the
loss of carbon could dramatically boost carbon fixation.Several
synthetic carbon-conserving bypasses have been suggested. In
the de novo 2PG salvage pathway (Ort et al., 2015), 2PG was
suggested to be reduced to 2-phosphoglycolaldehyde, which is
subsequently condensed with dihydroxyacetone phosphate to
give xylulose bisphosphate.This intermediate is then dephosphorylated
to xylulose 5-phosphate, a Calvin cycle metabolite.
The main challenges of this proposed bypass is the reversibility
of most of its reactions (resulting in a low driving force), the
low concentration of 2PG,and the inhibitory effect of xylulose
bisphosphate (Yokota, 1991; Zhu and Jensen, 1991; Parry et al.,
2008).
Recently, a systematic analysis identified multiple synthetic
routes that can bypass photorespiration without the release
of CO2. Several of these pathways involve the reduction of
glycolate (the concentration of which is considerably higher
than that of 2PG) to glycolaldehyde, which then undergoes
an aldol condensation with a phosphosugar from the Calvin
cycle to generate a longer chain phosphosugar that is reintegrated
into the Calvin cycle (Bar-Even, 2018;Trudeau et al.,
2018). A computational model indicated that these pathways
can boost photosynthesis under all physiologically relevant irradiation
and intracellular CO2 levels.
The operation of these carbon-conserving bypass routes depends
on the conversion of glycolate to glycolaldehyde, but
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1430 | Kubis and Bar-Even
this activity is not supported by any known enzyme. To establish
this activity, two enzymes were engineered (Trudeau
et al., 2018). First, acetyl-CoA synthetase from E. coli was engineered
to accept glycolate, thus generating glycolyl-CoA.
Next, propionyl-CoA reductase from Rhodopseudomonas palustris
was engineered to accept glycolyl-CoA, reducing it to
glycolaldehyde. The cofactor specificity of this latter enzyme
was switched, such that it could use NADPH—the photosynthetic
electron carrier—as an electron donor.The two engineered
enzymes were combined, in a test tube, with fructose
6-phosphate aldolase (condensing glycolaldehyde with glyceraldehyde
3-phosphate to generate arabinose 5-phosphate),
arabinose 5-phosphate isomerase, and phosphoribulokinase.
Upon addition of glycolate and glyceraldehyde 3-phosphate,
NADPH and ATP were consumed and RuBP was found to
accumulate (Trudeau et al., 2018), demonstrating the in vitro
activity of an alternative photorespiration route that does not
release CO2.
It was further proposed to go beyond carbon conservation,
and engineer a photorespiration bypass that fixes CO2 and thus
directly supports the activity of the Calvin cycle. One such
carbon-positive bypass was inspired by the 3-hydroxypropionate
bi-cycle (Shih et al., 2014). Here, glycolate is oxidized to
glyoxylate, which is then metabolized and further carboxylated
to pyruvate.Towards the implementation of this bypass,six
non-native genes from C. aurantiacus were expressed in cyanobacteria,
but no distinct growth phenotype was evident.
In another study, glycolate was not recycled to the Calvin
cycle but instead was metabolized to acetyl-CoA via the synthetic
malyl-CoA–glycerate pathway (Yu et  al., 2018). This
pathway can further be used to generate acetyl-CoA from
photosynthetic C3 sugars via an additional CO2-fixing step,
thereby bypassing CO2 release by pyruvate dehydrogenase. In
cyanobacteria, the pathway facilitated a 2-fold increase in bicarbonate
assimilation.
Conclusions
The increasing numbers of studies demonstrating improved
photosynthesis and growth by engineering different components
of the light-dependent and independent reactions indicates
that we are on the right path.Yet, many challenges are
ahead of us.Besides the technical difficulties,which we did not
discuss here and for which we refer the reader to other reviews
(DePaoli et al., 2014; Liu and Stewart, 2015; Boehm and Bock,
2018;Piatek et al.,2018;Vazquez-Vilar et al.,2018),there is one
key barrier that is worth elaborating on, which is system complexity.
Complex systems are notoriously difficult to engineer
as the effect of even small changes can have substantial effects
that cannot be easily predicted. While mathematical models
can help deal with such complexity, the lack of knowledge
regarding many of the involved components commonly hinders
accurate prediction. Plant carbon metabolism provides an
excellent example of a complex system, the response of which
to changes is hard to foretell. Previous attempts to engineer
carbon fixation demonstrate this vividly. Perhaps the best example
is the engineering of photorespiration bypass routes as
described above. While few bypasses were already shown to
enhance photosynthesis (most notably in recent field experiments;
South et al., 2019), the cause of this effect is probably
different from that originally suggested. Unraveling this mystery
would require deep understanding of the intricate interplay
between all system components, a task which we have yet
to fully achieve.
Moreover, while some engineering efforts show only minor
benefits in isolation, the key for future improvements lies in
the correct combination of multiple strategies. Indeed, first
examples of beneficial cumulative effects have been reported
(Simkin et al., 2015). It is further clear that not all strategies
can be implemented with similar ease.Overexpressing a Calvin
cycle enzyme, for example, is considerably easier than rerouting
photorespiration via a synthetic pathway that does not release
CO2. It is therefore important to choose targets carefully
for the near and medium future and progress in a way that
ensures intermediate gains. For example, establishing a C3–C4
intermediate metabolism not only provides a solid basis for
further engineering of a complete C4 metabolism, but is also
expected to boost carbon fixation by itself. Once we gain the
required proficiency in rewiring plant central metabolism, we
can aim at even bigger targets, for example replacing Rubisco
with a set of enzymes, each responsible for a different catalytic
step (Bar-Even, 2018), or replacing the Calvin cycle with a
synthetic carbon fixation pathway (Schwander et al., 2016).
Acknowledgements
The authors thank Charlie Cotton for critical reading of the manuscript.
This work was funded by the Max Planck Society and by the European
Union’s Horizon 2020 FET Programme under grant agreement no.
686330 (FutureAgriculture).
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