Improving Marine Algae for Bioenergy 275
metabolism to promote synthesis of the target compound, and this approach appears the most promising
to support further developments in this field.
Transformation in marine algae
Eukaryotic microalgae include cells resulting from a primary endosymbiotic event (as land plants) and
cells derived from subsequent secondary and/or tertiary endosymbiotic events. Consequences of multiple
endosymbiotic events include (1) the endosymbiotic gene transfer (EGT) which enriched the nuclear
genome. Most EGT-derived gene products have complex targeting mechanisms to be imported back
into the plastid and (2) coordination of enzymes, from “symbiogenetic” genes, derived from different
originator cells (host or various prokaryotes), to form complex metabolic pathways that include responses
to stress, phototropism, and adaptation to nutrients limitations (Parker et al. 2008; Brodie et al. 2017). Due
to the complex and unique gene organization of algae, genetic engineering approaches must frequently
consider the methodologies already in use for both prokaryotic and green lineage organisms.
The number of marine algae successful transformed has been increasing in recent times. These
include members of the chlorophytes, such as Chlorella vulgaris (Dawson et al. 1997; Hawkins and
Nakamura 1999; Cha et al. 2012), Chlorella ellipsoidea (Chen et al. 2001; Liu et al. 2012), Ulva lactuca
(Huang 1996), Dunaliella salina (Tan et al. 2005; Feng et al. 2009), and Ostreococcus tauri (van Ooijen
et al. 2012); from the phaeophytes, such as Laminaria japonica (Qin et al. 1999) and Undaria pinnatifada
(Qin et al. 2003); from the rhodophytes, such as Porphyra yezoensis (Cheney et al. 2001; Hirata et al.
2011; Uji et al. 2013), Porphyratenera (Son et al. 2012), Gracilaria changii (Gan et al. 2003), and
Porphyridium sp. (Lapidot et al. 2002); and from diatoms, such as Thalassiosira pseudonana (Poulsen
et al. 2006), Cylindrotheca fusiformis (Poulsen and Krӧger 2005), Fistulifera sp. (Muto et al. 2013),
and Phaeodactylum tricornutum (Zaslavskaia et al. 2001; Niu et al. 2012). Special attention must be
paid to the industrially relevant oleaginous marine green alga Nannochloropsis sp. Different methods of
transformation are available for Nannochloropsis, a species which is easily transformed (Cha et al. 2011;
Kilian et al. 2011; Radakovits et al. 2012). In the future, the marine algae Nannochloropsis may emerge as
significant new model algal system due to their naturally high production of biomass and lipids that could
be used as feedstock for biofuel. However, the green alga from soil and fresh water Chlamydomonas
reinhardtii is still the most significant experimental model, and the most comprehensively studied (Harris
et al. 2009)—thus remaining as a reference point in all works of genetic transformation in algae.
In general, the transformation efficiencies of the marine algae are low, which may be due to the
composition and thickness of the cell wall of these species. For example, the efficiencies for marine
Synechococcus strains were ten times lower than those for freshwater Synechococcus strains. Unique
polysaccharides, which surround the cell wall, may prevent DNA uptake (Qin et al. 2012).
Currently, the main model species of algae – the green alga C. reinhardtii, the diatom Phaeodactylum
tricornutum and the oleaginous algae Nannochloropsis sp., had their genome sequenced (Merchant et al.
2007; Bowler et al. 2008; Radakovits et al. 2012), each one has well established transformation methods
(Zaslavskaia et al. 2001; Harris et al. 2009; Radakovits et al. 2012) and the CRISPR/Cas9 gene editing is
available for the three organisms (Nymark et al. 2016; Shin et al. 2016; Wang et al. 2016).
The biolistic method (micro-particle bombardment) proved to be an efficient and highly reproducible
method for delivering exogenous DNA into algal chloroplasts. C. reinhardtii chloroplasts were the
first to be transformed by high velocity microprojectiles, 30 years ago (Boynton et al. 1988). Biolistic
procedures were used to transform other chlorophytes, such as the genus Chlorella (Dawson et al. 1997)
and Dunaliella salina (Tan et al. 2005); rhodophytes such as Gracilaria changii (Gan et al. 2003) and
Porphyra tenera (Son et al. 2012); phaeophytes, such as Laminaria japonica (Qin et al. 1999); and
diatoms, such as Phaeodactylum tricornutum (Zaslavskaia et al. 2001), Cylindrotheca fusiformis (Poulsen
and Krӧger 2005), and Fistulifera sp. (Muto et al. 2013).
Glass beads continue to be the simplest and most convenient method for algal nuclear transformation
(Harris et al. 2009). This method was first used in Chlamydomonas reinhardtii, and achievied a highfrequency of nuclear transformation (Kindle 1990). Dunaliella salina was also successfully transformed
with glass beads (Feng et al. 2009).
metabolism to promote synthesis of the target compound, and this approach appears the most promising
to support further developments in this field.
Transformation in marine algae
Eukaryotic microalgae include cells resulting from a primary endosymbiotic event (as land plants) and
cells derived from subsequent secondary and/or tertiary endosymbiotic events. Consequences of multiple
endosymbiotic events include (1) the endosymbiotic gene transfer (EGT) which enriched the nuclear
genome. Most EGT-derived gene products have complex targeting mechanisms to be imported back
into the plastid and (2) coordination of enzymes, from “symbiogenetic” genes, derived from different
originator cells (host or various prokaryotes), to form complex metabolic pathways that include responses
to stress, phototropism, and adaptation to nutrients limitations (Parker et al. 2008; Brodie et al. 2017). Due
to the complex and unique gene organization of algae, genetic engineering approaches must frequently
consider the methodologies already in use for both prokaryotic and green lineage organisms.
The number of marine algae successful transformed has been increasing in recent times. These
include members of the chlorophytes, such as Chlorella vulgaris (Dawson et al. 1997; Hawkins and
Nakamura 1999; Cha et al. 2012), Chlorella ellipsoidea (Chen et al. 2001; Liu et al. 2012), Ulva lactuca
(Huang 1996), Dunaliella salina (Tan et al. 2005; Feng et al. 2009), and Ostreococcus tauri (van Ooijen
et al. 2012); from the phaeophytes, such as Laminaria japonica (Qin et al. 1999) and Undaria pinnatifada
(Qin et al. 2003); from the rhodophytes, such as Porphyra yezoensis (Cheney et al. 2001; Hirata et al.
2011; Uji et al. 2013), Porphyratenera (Son et al. 2012), Gracilaria changii (Gan et al. 2003), and
Porphyridium sp. (Lapidot et al. 2002); and from diatoms, such as Thalassiosira pseudonana (Poulsen
et al. 2006), Cylindrotheca fusiformis (Poulsen and Krӧger 2005), Fistulifera sp. (Muto et al. 2013),
and Phaeodactylum tricornutum (Zaslavskaia et al. 2001; Niu et al. 2012). Special attention must be
paid to the industrially relevant oleaginous marine green alga Nannochloropsis sp. Different methods of
transformation are available for Nannochloropsis, a species which is easily transformed (Cha et al. 2011;
Kilian et al. 2011; Radakovits et al. 2012). In the future, the marine algae Nannochloropsis may emerge as
significant new model algal system due to their naturally high production of biomass and lipids that could
be used as feedstock for biofuel. However, the green alga from soil and fresh water Chlamydomonas
reinhardtii is still the most significant experimental model, and the most comprehensively studied (Harris
et al. 2009)—thus remaining as a reference point in all works of genetic transformation in algae.
In general, the transformation efficiencies of the marine algae are low, which may be due to the
composition and thickness of the cell wall of these species. For example, the efficiencies for marine
Synechococcus strains were ten times lower than those for freshwater Synechococcus strains. Unique
polysaccharides, which surround the cell wall, may prevent DNA uptake (Qin et al. 2012).
Currently, the main model species of algae – the green alga C. reinhardtii, the diatom Phaeodactylum
tricornutum and the oleaginous algae Nannochloropsis sp., had their genome sequenced (Merchant et al.
2007; Bowler et al. 2008; Radakovits et al. 2012), each one has well established transformation methods
(Zaslavskaia et al. 2001; Harris et al. 2009; Radakovits et al. 2012) and the CRISPR/Cas9 gene editing is
available for the three organisms (Nymark et al. 2016; Shin et al. 2016; Wang et al. 2016).
The biolistic method (micro-particle bombardment) proved to be an efficient and highly reproducible
method for delivering exogenous DNA into algal chloroplasts. C. reinhardtii chloroplasts were the
first to be transformed by high velocity microprojectiles, 30 years ago (Boynton et al. 1988). Biolistic
procedures were used to transform other chlorophytes, such as the genus Chlorella (Dawson et al. 1997)
and Dunaliella salina (Tan et al. 2005); rhodophytes such as Gracilaria changii (Gan et al. 2003) and
Porphyra tenera (Son et al. 2012); phaeophytes, such as Laminaria japonica (Qin et al. 1999); and
diatoms, such as Phaeodactylum tricornutum (Zaslavskaia et al. 2001), Cylindrotheca fusiformis (Poulsen
and Krӧger 2005), and Fistulifera sp. (Muto et al. 2013).
Glass beads continue to be the simplest and most convenient method for algal nuclear transformation
(Harris et al. 2009). This method was first used in Chlamydomonas reinhardtii, and achievied a highfrequency of nuclear transformation (Kindle 1990). Dunaliella salina was also successfully transformed
with glass beads (Feng et al. 2009).