280 Marine Macro- and Microalgae: An Overview
appears to require very specific chaperones) in the hosting chloroplast (Whitney et al. 2001). Nevertheless,
full expression in algae which are phylogenetically nearer the donor species might be plausible, and
will surely be attempted as new transformation procedures become suitable for an extended number of
microalgae.
Artificial directed evolution of Rubisco is also possible, as demonstrated by a clever scheme
designed to improve Rubisco features (Parikh et al. 2006). The Calvin cycle was partially reconstructed
in an engineered strain of E. coli, which still required Rubisco and phosphoribulokinase for growth in a
medium with a pentose as the sole source of carbon supplemented with a pentose. After random mutation
of the gene encoding the large subunit of the Synechococcus Rubisco, the resulting library was coexpressed together with the small subunit of Rubisco and the phosphoribulokinase from Synechococcus
in the engineered E. coli, and screened for hypermorphic variants. After three rounds of random
mutagenesis the selected strains exhibited 5-fold improvement in Rubisco specific activity relative to
the wild type enzyme. The selected Rubisco displayed higher catalytic efficiency and folding capacity
(i.e., better interaction with the chaperones from E. coli), but showed no improvement in the specificity
factor (Greene et al. 2007). Further attempts of evolving a better Rubisco have been carried out in E.
coli starting from enzymes of cyanobacteria (Synechococcus) (Mueller-Cajar and Whitney 2008a) or
archea (Methanococcoides burtoni) (Wilson et al. 2016). These approaches yielded enzyme variants with
higher turnover number (Durao et al. 2015), but no significant increase of substrate specificity. While
directed evolution of Rubisco appears as highly promising strategy, it is possible that an increase of the
natural specificity factor (without compromising the catalytic activity itself during the change) requires
an array of simultaneous mutations that cannot be explored, even using high-throughput techniques,
without a better understanding of the complex structure-function relationships and the catalytic chemistry
of the enzyme (Mueller-Cajar and Whitney 2008b). Regardless the moderate specificity factor may be
tolerated in organisms that possess mechanisms to concentrate CO 2 at the Rubisco location, thereby
achieving a high carboxylation to oxygenation rate. C4 metabolism is a well-known strategy used by
land plants. Evidence suggesting a C4-like metabolism has been also reported in diatoms and other algae
(Reinfelder 2011), although its possible physiological significance is still under discussion (Raven 2010;
Haimovich-Dayan et al. 2013; Raven and Giordano 2017). However, the most common device found in
aquatic organism to favour carboxylation is the active pumping of bicarbonate into cellular compartments
containing Rubisco, followed by the local release of CO 2 from bicarbonate by carbonic anhydrase. Many
marine microalgae employ this procedure or variants thereof (Spalding 2008; Reinfelder 2011). All these
strategies are collectively known as carbon concentrating mechanisms (CCM). The existence of a CCM
seems to be more the rule than the exception, since the alternative (i.e., diffusive entry of CO 2 ) appears
to be present only in a small minority of eukaryotic algae (Raven 2010). As CCMs from different species
display variable efficiency, it has been suggested that the identification of genes coding for high-activity
versions of carbonic anhydrases and bicarbonate transporters would be of interest in order to introduce
them into suitable hosts as a mean to improve Rubisco carboxylation to oxygenation ratio (Work et al.
2012).
Besides, cyanobacteria possess their own CCM in the form of carboxysomes, which are selfassembling particles containing mostly Rubisco and carbonic anhydrase inside a proteinaceous shell (Rae
et al. 2013). A hypothetical assembly of carboxysomes inside the chloroplast could potentially raise the
carboxylation/oxygenation ratio of Rubisco (McGrath and Long 2014; Hanson et al. 2016). The correct
assembly of functional carboxysomes in E. coli (Bonacci et al. 2012) and the expression of a functional
cyanobacterial Rubisco in the chloroplast of higher plants (Lin et al. 2014) have been hailed as first steps
towards the introduction of carboxysomes in photosynthetic eukaryotes.
Redesigning the carbon assimilation metabolism
A different approach to enhance biomass production is to modify the natural metabolic routes of algae to
permit a more efficient processing of fixed carbon to fuels or fuel precursors. Several attempts have been
reported along these lines.
appears to require very specific chaperones) in the hosting chloroplast (Whitney et al. 2001). Nevertheless,
full expression in algae which are phylogenetically nearer the donor species might be plausible, and
will surely be attempted as new transformation procedures become suitable for an extended number of
microalgae.
Artificial directed evolution of Rubisco is also possible, as demonstrated by a clever scheme
designed to improve Rubisco features (Parikh et al. 2006). The Calvin cycle was partially reconstructed
in an engineered strain of E. coli, which still required Rubisco and phosphoribulokinase for growth in a
medium with a pentose as the sole source of carbon supplemented with a pentose. After random mutation
of the gene encoding the large subunit of the Synechococcus Rubisco, the resulting library was coexpressed together with the small subunit of Rubisco and the phosphoribulokinase from Synechococcus
in the engineered E. coli, and screened for hypermorphic variants. After three rounds of random
mutagenesis the selected strains exhibited 5-fold improvement in Rubisco specific activity relative to
the wild type enzyme. The selected Rubisco displayed higher catalytic efficiency and folding capacity
(i.e., better interaction with the chaperones from E. coli), but showed no improvement in the specificity
factor (Greene et al. 2007). Further attempts of evolving a better Rubisco have been carried out in E.
coli starting from enzymes of cyanobacteria (Synechococcus) (Mueller-Cajar and Whitney 2008a) or
archea (Methanococcoides burtoni) (Wilson et al. 2016). These approaches yielded enzyme variants with
higher turnover number (Durao et al. 2015), but no significant increase of substrate specificity. While
directed evolution of Rubisco appears as highly promising strategy, it is possible that an increase of the
natural specificity factor (without compromising the catalytic activity itself during the change) requires
an array of simultaneous mutations that cannot be explored, even using high-throughput techniques,
without a better understanding of the complex structure-function relationships and the catalytic chemistry
of the enzyme (Mueller-Cajar and Whitney 2008b). Regardless the moderate specificity factor may be
tolerated in organisms that possess mechanisms to concentrate CO 2 at the Rubisco location, thereby
achieving a high carboxylation to oxygenation rate. C4 metabolism is a well-known strategy used by
land plants. Evidence suggesting a C4-like metabolism has been also reported in diatoms and other algae
(Reinfelder 2011), although its possible physiological significance is still under discussion (Raven 2010;
Haimovich-Dayan et al. 2013; Raven and Giordano 2017). However, the most common device found in
aquatic organism to favour carboxylation is the active pumping of bicarbonate into cellular compartments
containing Rubisco, followed by the local release of CO 2 from bicarbonate by carbonic anhydrase. Many
marine microalgae employ this procedure or variants thereof (Spalding 2008; Reinfelder 2011). All these
strategies are collectively known as carbon concentrating mechanisms (CCM). The existence of a CCM
seems to be more the rule than the exception, since the alternative (i.e., diffusive entry of CO 2 ) appears
to be present only in a small minority of eukaryotic algae (Raven 2010). As CCMs from different species
display variable efficiency, it has been suggested that the identification of genes coding for high-activity
versions of carbonic anhydrases and bicarbonate transporters would be of interest in order to introduce
them into suitable hosts as a mean to improve Rubisco carboxylation to oxygenation ratio (Work et al.
2012).
Besides, cyanobacteria possess their own CCM in the form of carboxysomes, which are selfassembling particles containing mostly Rubisco and carbonic anhydrase inside a proteinaceous shell (Rae
et al. 2013). A hypothetical assembly of carboxysomes inside the chloroplast could potentially raise the
carboxylation/oxygenation ratio of Rubisco (McGrath and Long 2014; Hanson et al. 2016). The correct
assembly of functional carboxysomes in E. coli (Bonacci et al. 2012) and the expression of a functional
cyanobacterial Rubisco in the chloroplast of higher plants (Lin et al. 2014) have been hailed as first steps
towards the introduction of carboxysomes in photosynthetic eukaryotes.
Redesigning the carbon assimilation metabolism
A different approach to enhance biomass production is to modify the natural metabolic routes of algae to
permit a more efficient processing of fixed carbon to fuels or fuel precursors. Several attempts have been
reported along these lines.