Part A | 5.4
54 Part A Marine Flora and Fauna
fied eukaryotic microalgal genome. Up until November
2012, the whole genome sequence of 11 strains of
microalgae had been sequenced, including 2 diatoms
(Thalassiosira pseudonana [5.24] and Phaeodactylum
tricornutum [5.25]), a red alga (Cyanidioschyzon merolae [5.26]), and 8 green algae (Chlamydomonas reinhardtii [5.27], Ostreococcus lucimarinus [5.31], Ostreococcus tauri [5.32], Chlorella variabilis [5.28], Volvox
carteri [5.30], Coccomyxa subellipsoidea [5.33], Micromonas pusilla [5.29], and Micromonas sp. [5.29]),
see Table 5.1. In addition, the draft genome sequences
of 17 strains of microalgae can been found in the NCBI
GenBank databases [5.34]. With next generation technology, the draft genome sequence of the biodiesel
producing microalga Nannochloropsis gaditana strain
CCMP526 were also identified recently [5.35]. The
identified microalgal whole genome sequences provide a powerful tool for the discovery of genes and
metabolic pathways. Even though most of the predicted
microalgal pathways have been proved to be similar to
corresponding pathways in higher plants, the urea cycle
identified from genomes of diatoms is absent in higher
plants but present in animals [5.24]. The existence of
an animal metabolic pathway in microalgal cells further highlights the importance of genome analysis for
microalgae.
5.4 Genetic Engineering of Microalgae
5.4.1 Genetic Transformation Methods
Genetic studies on microalgae have been redirected
mainly toward analysis of photosynthesis and metabolic
pathways. A limited number of microalgae such as
cyanobacteria have been used in biotechnological applications. The development of molecular techniques for
physiological analysis and enhancement of biotechnological applications is necessary. Many attempts at gene
transfer have been made in eukaryotic and prokaryotic microalgae. Genetic manipulation in prokaryotic
microalgae cyanobacteria was studied extensively after several transformable unicellular strains were discovered. First, the freshwater cyanobacterium Synechococcus PCC7942 was reported to have the ability
to take up DNA. Subsequently, several other naturally transformable freshwater strains were found. Gene
transfer has been developed mainly in the freshwater
strains Synechococcus, Synechocystis, Anabaeba, and
Nostoc [5.69]. Several marine cyanobacterial strains
of the genus Synechococcus have been also used for
heterogeneous gene expression and other genetic applications [5.70, 71]. There are two commonly used gene
transfer procedures: transformation using naturally occurring or artificially competent cells, e.g., conjugation
with Escherichia coli, or physical methods for gene
introduction, e.g., electroporation and particle bombardment. Natural transformation has been reported
for Synechococcus sp. PCC7002 [5.72]. Other strains
have been transformed successfully by electroporation
or conjugation. Further, plasmids harvested from several marine microalgal species have been used as vector
DNA for gene transfer. Marine plasmids have been
found in Synechococcus sp. NKBG042902, which has
a high phycocyanin content and a rapid growth rate.
This strain contains more than three cryptic endogenous plasmids, and one of these, the plasmid pSY10
has the unique replication characteristic that its copy
number increases under high salinity conditions [5.73].
Plasmids are maintained at a high copy number in
cyanobacteria, which suggests the possibility that they
act as a shuttle vector between cyanobacteria and E.
coli. In fact, a shuttle vector with E. coli has been
constructed using pSY10. Conjugative gene transfer
using a broad-host range vector pKT230 was successful for the marine cyanobacterium Synechococcus
sp. NKBG 15041C [5.74]. It has been demonstrated
that this plasmid is stably maintained in cyanobacterial cells [5.75]. In marine cyanobacteria, in addition to
the plasmid vector system, the construction of a phage
vector system is also required to enable the cloning of
large DNA fragments in specific cyanobacterial hosts.
Since cyanophages were first reported by Safferman
and Morris [5.76], various types of cyanophages have
been found in seawater [5.77, 78] and characterized
according to their genetic diversity and phylogenetic affiliations [5.79].
Due to the advance of genome, proteome, and
metabolome analyses of microalgae, many attempts at
gene transfer to eukaryotic microalgae have been made
to enhance the production of useful compounds and
biomass. However, because of the stiff cell wall of
microalgae, the introduction of exogenous genes into
microalgal cells could be challenging. The additional
frustules and coccoliths surrounding some species of
microalgae cells further increases the difficulty. Thus,
54 Part A Marine Flora and Fauna
fied eukaryotic microalgal genome. Up until November
2012, the whole genome sequence of 11 strains of
microalgae had been sequenced, including 2 diatoms
(Thalassiosira pseudonana [5.24] and Phaeodactylum
tricornutum [5.25]), a red alga (Cyanidioschyzon merolae [5.26]), and 8 green algae (Chlamydomonas reinhardtii [5.27], Ostreococcus lucimarinus [5.31], Ostreococcus tauri [5.32], Chlorella variabilis [5.28], Volvox
carteri [5.30], Coccomyxa subellipsoidea [5.33], Micromonas pusilla [5.29], and Micromonas sp. [5.29]),
see Table 5.1. In addition, the draft genome sequences
of 17 strains of microalgae can been found in the NCBI
GenBank databases [5.34]. With next generation technology, the draft genome sequence of the biodiesel
producing microalga Nannochloropsis gaditana strain
CCMP526 were also identified recently [5.35]. The
identified microalgal whole genome sequences provide a powerful tool for the discovery of genes and
metabolic pathways. Even though most of the predicted
microalgal pathways have been proved to be similar to
corresponding pathways in higher plants, the urea cycle
identified from genomes of diatoms is absent in higher
plants but present in animals [5.24]. The existence of
an animal metabolic pathway in microalgal cells further highlights the importance of genome analysis for
microalgae.
5.4 Genetic Engineering of Microalgae
5.4.1 Genetic Transformation Methods
Genetic studies on microalgae have been redirected
mainly toward analysis of photosynthesis and metabolic
pathways. A limited number of microalgae such as
cyanobacteria have been used in biotechnological applications. The development of molecular techniques for
physiological analysis and enhancement of biotechnological applications is necessary. Many attempts at gene
transfer have been made in eukaryotic and prokaryotic microalgae. Genetic manipulation in prokaryotic
microalgae cyanobacteria was studied extensively after several transformable unicellular strains were discovered. First, the freshwater cyanobacterium Synechococcus PCC7942 was reported to have the ability
to take up DNA. Subsequently, several other naturally transformable freshwater strains were found. Gene
transfer has been developed mainly in the freshwater
strains Synechococcus, Synechocystis, Anabaeba, and
Nostoc [5.69]. Several marine cyanobacterial strains
of the genus Synechococcus have been also used for
heterogeneous gene expression and other genetic applications [5.70, 71]. There are two commonly used gene
transfer procedures: transformation using naturally occurring or artificially competent cells, e.g., conjugation
with Escherichia coli, or physical methods for gene
introduction, e.g., electroporation and particle bombardment. Natural transformation has been reported
for Synechococcus sp. PCC7002 [5.72]. Other strains
have been transformed successfully by electroporation
or conjugation. Further, plasmids harvested from several marine microalgal species have been used as vector
DNA for gene transfer. Marine plasmids have been
found in Synechococcus sp. NKBG042902, which has
a high phycocyanin content and a rapid growth rate.
This strain contains more than three cryptic endogenous plasmids, and one of these, the plasmid pSY10
has the unique replication characteristic that its copy
number increases under high salinity conditions [5.73].
Plasmids are maintained at a high copy number in
cyanobacteria, which suggests the possibility that they
act as a shuttle vector between cyanobacteria and E.
coli. In fact, a shuttle vector with E. coli has been
constructed using pSY10. Conjugative gene transfer
using a broad-host range vector pKT230 was successful for the marine cyanobacterium Synechococcus
sp. NKBG 15041C [5.74]. It has been demonstrated
that this plasmid is stably maintained in cyanobacterial cells [5.75]. In marine cyanobacteria, in addition to
the plasmid vector system, the construction of a phage
vector system is also required to enable the cloning of
large DNA fragments in specific cyanobacterial hosts.
Since cyanophages were first reported by Safferman
and Morris [5.76], various types of cyanophages have
been found in seawater [5.77, 78] and characterized
according to their genetic diversity and phylogenetic affiliations [5.79].
Due to the advance of genome, proteome, and
metabolome analyses of microalgae, many attempts at
gene transfer to eukaryotic microalgae have been made
to enhance the production of useful compounds and
biomass. However, because of the stiff cell wall of
microalgae, the introduction of exogenous genes into
microalgal cells could be challenging. The additional
frustules and coccoliths surrounding some species of
microalgae cells further increases the difficulty. Thus,