Improving Marine Algae for Bioenergy 281
Accepting that the Rubisco oxygenase activity is inescapable; the 2-phosphoglycolate metabolism
may be redesigned to diminish its cost in terms of nutrients (since the conventional photorespiratory
pathway releases nitrogen in the form of ammonia) and energy (Peterhansel and Maurino 2011). Toward
that end, the bacterial glycolate pathway—which converts glycolate to glycerate in three steps, was
engineered into the chloroplast of a higher plant (Arabidopsis thaliana) (Kebeish et al. 2007). The final
product of this pathway (glycerate) can enter the Calvin cycle after phosphorylation. With a similar
aim but following a different strategy, a new pathway was created also in Arabidopsis to achieve full
oxidation of glycolate to CO 2 . This approach was based on the conversion of glycolate to glyoxylate,
followed by two sequential decarboxylations carried out by malate synthase and the NADP malic enzyme
to yield pyruvate (Peterhansel and Maurino 2011). In both cases, the new routes skipped ammonia loss
(and subsequent expense of re-fixation energy), released CO 2 in the chloroplast (raising the local CO 2 /O 2
ratio that governs Rubisco partitioning), and produced additional NADH and/or NADPH. Consequently,
the engineered plants showed improved growth and produced higher leaf biomass (up to 30% more in
case of overexpression of the glycolate pathway) (Kebeish et al. 2007). In principle, this strategy could
work equally well in the case of algae, although possession of a strong CCM may leave less room for
metabolic improvement.
Under intense light, radiant energy to biomass conversion can be limited by the speed of RuBP
regeneration through the Calvin cycle. Theoretical analyses have shown that this pathway still allows
for a potential increase of photosynthetic rate, highlighting sedoheptulose 1,7-bisphosphatase (SBPase)
as the key enzyme controlling RuBP regeneration and main target for biotechnological manipulation
of the metabolic flux through the cycle (Raines 2003; Zhu et al. 2007). In fact, overexpression of a
bifunctional fructose 1,6-bisphosphatase/SBPase from Synechococcus in tobacco chloroplasts resulted
an increase in photosynthesis and growth (Miyagawa et al. 2001). A further comparison of the effect of
expressing either fructose 1,6-bisphosphatase or SBPase alone showed that both of them could separately
contribute to increase photosynthetic rate, with SBPase being the most important for RuBP regeneration
(Tamoi et al. 2006). Similarly, overexpression of an Arabidopsis cDNA coding SBPase in transgenic
tobacco promoted sucrose and starch accumulation and increased biomass up to 30% (Lefebvre et al.
2005). These results demonstrate that further improvements in photosynthetic efficiency for selected
algal species could result from metabolic control analysis and engineering of Calvin cycle enzymatic
activities. Indeed, significant enhancements of photosynthetic activity and biomass production have been
reported through transgenic expression of a fructose 1,6-bisphosphate aldolase in C. reinhardtii (Yang
et al. 2017) and a bifunctional fructose 1,6-bisphosphatase/SBPase in Euglena gracilis (Ogawa et al.
2015). In both cases, the cyanobacterial enzymes were fused to plastid transit peptides in order to ensure
expression inside the chloroplast.
Finally, a radical solution for eliminating photorespiratory losses has been proposed (but still not
successfully achieved) by replacing the Calvin cycle altogether by another different carbon fixation
pathway, as found in bacteria and archaea (Blankenship et al. 2011). Most of these routes cannot be easily
transferred to photosynthetic organisms performing oxygenic photosynthesis because the relevant enzymes
(which are to be expressed in the host) are strongly oxygen-sensitive. However, the 3-hydroxypropionate/
malyl-CoA cycle, present in the green nonsulfur bacterium Chlorofexus, has been found not to be inhibited
by oxygen (Thauer 2007) and could, in principle, be used (Blankenship et al. 2011). Another possibility
would be to design a new (“synthetic”) fixation pathway from existing enzymatic activities collected
from different organisms. In this regard, a new pathway converting 2-phosphoglycolate into pyruvate
(bypassing photorespiration) has been implemented in the cyanobacterium Synechococcus elongatus
by expressing enzymes from Accumulibacter phosphatis and from the 3-hydroxypropionate/malyl-CoA
cycle from Chlorofexus. This new route has been demonstrated to work although it did not result in faster
growth, probably because S. elongatus possesses already a highly active CCM that curtails the metabolic
benefit (Shih et al. 2014). Alternative synthetic routes for carbon fixation, employing the superior
phosphoenolpyruvate carboxylase instead of Rubisco, have also been proposed (Bar-Even et al. 2010).
Nevertheless, it should be kept in mind that all carbon fixation pathways face similar thermodynamic
constraints, which determine the free energy and cellular resources that should be invested (Bar-Even
et al. 2012). Therefore, a detailed analysis of the expected costs and gains should be carried out before
Accepting that the Rubisco oxygenase activity is inescapable; the 2-phosphoglycolate metabolism
may be redesigned to diminish its cost in terms of nutrients (since the conventional photorespiratory
pathway releases nitrogen in the form of ammonia) and energy (Peterhansel and Maurino 2011). Toward
that end, the bacterial glycolate pathway—which converts glycolate to glycerate in three steps, was
engineered into the chloroplast of a higher plant (Arabidopsis thaliana) (Kebeish et al. 2007). The final
product of this pathway (glycerate) can enter the Calvin cycle after phosphorylation. With a similar
aim but following a different strategy, a new pathway was created also in Arabidopsis to achieve full
oxidation of glycolate to CO 2 . This approach was based on the conversion of glycolate to glyoxylate,
followed by two sequential decarboxylations carried out by malate synthase and the NADP malic enzyme
to yield pyruvate (Peterhansel and Maurino 2011). In both cases, the new routes skipped ammonia loss
(and subsequent expense of re-fixation energy), released CO 2 in the chloroplast (raising the local CO 2 /O 2
ratio that governs Rubisco partitioning), and produced additional NADH and/or NADPH. Consequently,
the engineered plants showed improved growth and produced higher leaf biomass (up to 30% more in
case of overexpression of the glycolate pathway) (Kebeish et al. 2007). In principle, this strategy could
work equally well in the case of algae, although possession of a strong CCM may leave less room for
metabolic improvement.
Under intense light, radiant energy to biomass conversion can be limited by the speed of RuBP
regeneration through the Calvin cycle. Theoretical analyses have shown that this pathway still allows
for a potential increase of photosynthetic rate, highlighting sedoheptulose 1,7-bisphosphatase (SBPase)
as the key enzyme controlling RuBP regeneration and main target for biotechnological manipulation
of the metabolic flux through the cycle (Raines 2003; Zhu et al. 2007). In fact, overexpression of a
bifunctional fructose 1,6-bisphosphatase/SBPase from Synechococcus in tobacco chloroplasts resulted
an increase in photosynthesis and growth (Miyagawa et al. 2001). A further comparison of the effect of
expressing either fructose 1,6-bisphosphatase or SBPase alone showed that both of them could separately
contribute to increase photosynthetic rate, with SBPase being the most important for RuBP regeneration
(Tamoi et al. 2006). Similarly, overexpression of an Arabidopsis cDNA coding SBPase in transgenic
tobacco promoted sucrose and starch accumulation and increased biomass up to 30% (Lefebvre et al.
2005). These results demonstrate that further improvements in photosynthetic efficiency for selected
algal species could result from metabolic control analysis and engineering of Calvin cycle enzymatic
activities. Indeed, significant enhancements of photosynthetic activity and biomass production have been
reported through transgenic expression of a fructose 1,6-bisphosphate aldolase in C. reinhardtii (Yang
et al. 2017) and a bifunctional fructose 1,6-bisphosphatase/SBPase in Euglena gracilis (Ogawa et al.
2015). In both cases, the cyanobacterial enzymes were fused to plastid transit peptides in order to ensure
expression inside the chloroplast.
Finally, a radical solution for eliminating photorespiratory losses has been proposed (but still not
successfully achieved) by replacing the Calvin cycle altogether by another different carbon fixation
pathway, as found in bacteria and archaea (Blankenship et al. 2011). Most of these routes cannot be easily
transferred to photosynthetic organisms performing oxygenic photosynthesis because the relevant enzymes
(which are to be expressed in the host) are strongly oxygen-sensitive. However, the 3-hydroxypropionate/
malyl-CoA cycle, present in the green nonsulfur bacterium Chlorofexus, has been found not to be inhibited
by oxygen (Thauer 2007) and could, in principle, be used (Blankenship et al. 2011). Another possibility
would be to design a new (“synthetic”) fixation pathway from existing enzymatic activities collected
from different organisms. In this regard, a new pathway converting 2-phosphoglycolate into pyruvate
(bypassing photorespiration) has been implemented in the cyanobacterium Synechococcus elongatus
by expressing enzymes from Accumulibacter phosphatis and from the 3-hydroxypropionate/malyl-CoA
cycle from Chlorofexus. This new route has been demonstrated to work although it did not result in faster
growth, probably because S. elongatus possesses already a highly active CCM that curtails the metabolic
benefit (Shih et al. 2014). Alternative synthetic routes for carbon fixation, employing the superior
phosphoenolpyruvate carboxylase instead of Rubisco, have also been proposed (Bar-Even et al. 2010).
Nevertheless, it should be kept in mind that all carbon fixation pathways face similar thermodynamic
constraints, which determine the free energy and cellular resources that should be invested (Bar-Even
et al. 2012). Therefore, a detailed analysis of the expected costs and gains should be carried out before