Part A | 5.4
56 Part A Marine Flora and Fauna
transform mammalian cells with plasmids. The transformation efficiency was significantly decreased for
plant cells due to their thick cell walls. Electroporationbased gene transformation has only been achieved with
the Chlamydomonas reinhardtii cell wall-deficient mutant and Dunaliella salina cells, which have no cell
wall [5.46, 57, 82]. However, in the studies mentioned
above, the transformation efficiency was tenfold higher
than the gene gun method applied to the corresponding
strains [5.83].
The glass beads method is a relatively simple transformation procedure that has a higher transformation
efficiency than biolistics but it is only capable of transforming cells without cell walls. Both the cell-wall
deficient C. reinhardtii mutant and D. salina have been
reported to have been successfully transformed by the
glass bead method with a higher efficiency than with
the gene gun method [5.56].
Agrobacterium-mediated transformation is based
on the characteristic of the soil bacterium Agrobacterium tumefaciens that it naturally transfers and inserts
its genes into plant chromosomes. Exogenous genes can
be transferred into plant cells through Agrobacterium
transformation using target gene inserted agrobacterium transfer DNA (T-DNA). Although reports of microalgae transferred by agrobacterium transformation
are few, Kathiresan et al. achieved a twofold transformation efficiency with Haematococcus pluvialis over
the gene gun method [5.60].
In the transformants generated by the methods mentioned above, it is not rare to find the continuous
expression of the target genes in the chloroplast and
(or) mitochondria due to the insertion of the target
genes into their organelle genome. By using a specific vector containing a homologous sequence in the
organelle genome, stable chloroplast and (or) mitochondria transformation can be expected. On the other
hand, the target genes are usually found to be randomly
inserted into the nucleic genome and even homologous recombination occurs. Thus, it is hardly possible
to control the insertion site and the number of the target genes inserted into the nucleic genome, which has
made gene functional analysis via gene knock-out difficult. With further consideration of the dual nature of
the microalgal life cycle as either haploid or diploid,
the possibility of complete knock-out dwindled significantly in diploid cells. The homologous recombination
has been applied to the transformation of C. reinhardtii and Volvox carteri, which maintain an asexual
haploid zoospore during the life cycle; their recombination efficiency, however, was inferior [5.84, 85].
Recently, highly efficient homologous recombination
was reported in Nannochloropsis sp., which suggested
the possible use in microalgal gene functional analysis [5.68]. For those diploid microalgae, the knockdown of the target gene via RNAi has been reported and
considered as the substitute for knock-out [5.40, 44, 55,
64].
So far, six microalgae including Phaeodactylum tricornutum, Thalassiosira pseudonana, Chlamydomonas
reinhardtii, Chlorella vulgaris, Volvox carteri, and
Cyanidioschyzon merolae have not only obtained stable transformants but also the whole genome sequence.
P. tricornutum has been widely used for the studies of
metabolic engineering towards enhanced lipid production. Yet, most research in this field has focused on the
established stable transformant rather than on high oilproducing strains whose transformation method have
not been determined.
5.4.2 Metabolic Engineering
Enhanced production of valuable primary or secondary
metabolites in microalgae can be rendered possible by
high density cultivation and/or application of genetic
manipulation. Recent pharmaceutical interest in unsaturated fatty acids has triggered the search for sources
of these valuable compounds. Several eukaryotic microalgae are known to produce highly unsaturated fatty
acids such as EPA and DHA, which are valuable dietary components [5.16, 19]. Genetic engineering has
been applied to produce EPA in the marine cyanobacterium Synechococcus sp. [5.71]. Cyanobacteria do not
have the biosynthetic pathway to produce them. The
EPA synthesis gene cluster (ca. 38 kbp) isolated from
a marine bacterium Shewanella putrefaciens SCRC2738 was cloned to the marine cyanobacterium using
a broad-host cosmid vector. The content of EPA grown
at 2
ı C increased to 0:64 mg g
1 dry cells after 24 h incubation at 17
ı C. Furthermore, EPA production was
improved by partial deletion of the EPA gene cluster to stabilize its expression and maintenance in host
cyanobacterial cells [5.86].
Genetic engineering of microalgae for industrial
purposes has also been performed in freshwater
cyanobacteria where the ketocarotenoid astaxanthin, an
extremely efficient antioxidant, was synthesized by the
introduction of the ˇ-c-4-oxygenase gene (crtO) from
the green alga Haematococcus [5.87]. Ethylene production was also demonstrated in the cyanobacterium
Synechococcus elongates PCC7942 by chromosomal
insertion of an ethylene forming enzyme [5.88]. How-
56 Part A Marine Flora and Fauna
transform mammalian cells with plasmids. The transformation efficiency was significantly decreased for
plant cells due to their thick cell walls. Electroporationbased gene transformation has only been achieved with
the Chlamydomonas reinhardtii cell wall-deficient mutant and Dunaliella salina cells, which have no cell
wall [5.46, 57, 82]. However, in the studies mentioned
above, the transformation efficiency was tenfold higher
than the gene gun method applied to the corresponding
strains [5.83].
The glass beads method is a relatively simple transformation procedure that has a higher transformation
efficiency than biolistics but it is only capable of transforming cells without cell walls. Both the cell-wall
deficient C. reinhardtii mutant and D. salina have been
reported to have been successfully transformed by the
glass bead method with a higher efficiency than with
the gene gun method [5.56].
Agrobacterium-mediated transformation is based
on the characteristic of the soil bacterium Agrobacterium tumefaciens that it naturally transfers and inserts
its genes into plant chromosomes. Exogenous genes can
be transferred into plant cells through Agrobacterium
transformation using target gene inserted agrobacterium transfer DNA (T-DNA). Although reports of microalgae transferred by agrobacterium transformation
are few, Kathiresan et al. achieved a twofold transformation efficiency with Haematococcus pluvialis over
the gene gun method [5.60].
In the transformants generated by the methods mentioned above, it is not rare to find the continuous
expression of the target genes in the chloroplast and
(or) mitochondria due to the insertion of the target
genes into their organelle genome. By using a specific vector containing a homologous sequence in the
organelle genome, stable chloroplast and (or) mitochondria transformation can be expected. On the other
hand, the target genes are usually found to be randomly
inserted into the nucleic genome and even homologous recombination occurs. Thus, it is hardly possible
to control the insertion site and the number of the target genes inserted into the nucleic genome, which has
made gene functional analysis via gene knock-out difficult. With further consideration of the dual nature of
the microalgal life cycle as either haploid or diploid,
the possibility of complete knock-out dwindled significantly in diploid cells. The homologous recombination
has been applied to the transformation of C. reinhardtii and Volvox carteri, which maintain an asexual
haploid zoospore during the life cycle; their recombination efficiency, however, was inferior [5.84, 85].
Recently, highly efficient homologous recombination
was reported in Nannochloropsis sp., which suggested
the possible use in microalgal gene functional analysis [5.68]. For those diploid microalgae, the knockdown of the target gene via RNAi has been reported and
considered as the substitute for knock-out [5.40, 44, 55,
64].
So far, six microalgae including Phaeodactylum tricornutum, Thalassiosira pseudonana, Chlamydomonas
reinhardtii, Chlorella vulgaris, Volvox carteri, and
Cyanidioschyzon merolae have not only obtained stable transformants but also the whole genome sequence.
P. tricornutum has been widely used for the studies of
metabolic engineering towards enhanced lipid production. Yet, most research in this field has focused on the
established stable transformant rather than on high oilproducing strains whose transformation method have
not been determined.
5.4.2 Metabolic Engineering
Enhanced production of valuable primary or secondary
metabolites in microalgae can be rendered possible by
high density cultivation and/or application of genetic
manipulation. Recent pharmaceutical interest in unsaturated fatty acids has triggered the search for sources
of these valuable compounds. Several eukaryotic microalgae are known to produce highly unsaturated fatty
acids such as EPA and DHA, which are valuable dietary components [5.16, 19]. Genetic engineering has
been applied to produce EPA in the marine cyanobacterium Synechococcus sp. [5.71]. Cyanobacteria do not
have the biosynthetic pathway to produce them. The
EPA synthesis gene cluster (ca. 38 kbp) isolated from
a marine bacterium Shewanella putrefaciens SCRC2738 was cloned to the marine cyanobacterium using
a broad-host cosmid vector. The content of EPA grown
at 2
ı C increased to 0:64 mg g
1 dry cells after 24 h incubation at 17
ı C. Furthermore, EPA production was
improved by partial deletion of the EPA gene cluster to stabilize its expression and maintenance in host
cyanobacterial cells [5.86].
Genetic engineering of microalgae for industrial
purposes has also been performed in freshwater
cyanobacteria where the ketocarotenoid astaxanthin, an
extremely efficient antioxidant, was synthesized by the
introduction of the ˇ-c-4-oxygenase gene (crtO) from
the green alga Haematococcus [5.87]. Ethylene production was also demonstrated in the cyanobacterium
Synechococcus elongates PCC7942 by chromosomal
insertion of an ethylene forming enzyme [5.88]. How-