322 Marine Macro- and Microalgae: An Overview
chloroplast-localized proteins requires targeting peptide sequences across membranes. In certain classes
of algae, that is, the Eustigmatophytes, this can be across four membranes (i.e., two ER membranes and
two chloroplast membranes, Ott and Oldham-Ott 2003). Therefore, metabolic engineering of proteins to
complement or supplement chloroplastic functions requires identification of not only nuclear promoters
and terminators, but appropriate targeting sequences.
In addition to enhancing the biochemical pathway to lipids, partitioning in that direction can be
increased by eliminating alternate routes of carbon flux. In algae that accumulate storage carbohydrates
(such as starch) in place of, or in addition to lipids, the synthesis of the carbohydrates can be attenuated
or blocked by targeted inactivation or down-regulation of appropriate biosynthetic enzymes. Likewise,
carbon flux to metabolic energy generation, other biosynthetic pathways and waste carbon products can
be interrupted, with the carbon channeled to additional lipid. The limitation on these manipulations is the
impact they can have on strain robustness. There must be sufficient energy reserves available for dark
metabolism to make it through the night.
The artificial chromosome
The two previous sections describe some of the attempts that different groups have carried out to either
increase the PE of microalgae or the proportion of fixed carbon that is destined to, for example, lipid
metabolism. A common outcome of bioengineering molecular pathways one enzyme at a time is that as
one bottleneck is resolved, another one downstream is revealed. A possible solution to this problem is the
introduction of multiple changes in a biochemical pathway at once.
It is anticipated that multiple metabolic modifications will be required to generate algae capable of
cost effective production of biofuels. Genes for improved photosynthetic efficiency, fatty acid synthesis,
triacylglycerol synthesis, attenuation of alternative carbon sinks, and to improve agronomic properties
suggest that we may be targeting as many as fifty modifications. This is not far from the exemplary twenty
six genetic modifications utilized by the Genencor/DuPont team to engineer production of 1,3-propanediol
in Escherichia coli (Nakamura and Whited 2003).
Current strategies for introduction of genetic modifications into eukaryotes algae rely on random
integration of the exogenous DNA into the chromosome (Coll 2006; Hallmann 2007) and the number of
genetic changes attempted is typically one gene at a time. Random integration results in an unpredictable
number of gene copies due to the tendency of the cellular machinery to concatenate exogenous DNA.
Also, genetic marker availability limits the number of genes that can be introduced. This results in the need
to screen many colonies to identify the few with stable and desired levels of gene expression. Synthetic
chromosomes are preferred when multiple genetic elements are required for metabolic modification. The
synthetic chromosome provides a single copy number and stable, autonomous inheritance with the ability
to accommodate a large number of genes (Zieler et al. 2009).
Early eukaryotic synthetic chromosome constructs in yeast suggested that there are three required
elements for maintenance and stability during replication; a centromere, replication origins, and telomeres
(Murray and Szostak 1983). Significant work has since been directed at the construction of synthetic
chromosomes in mammals, plants, and in some algae strains (Carlson et al. 2007; Houben et al. 2008;
Zieler et al. 2009; O’Neill et al. 2011). Strategies for establishing synthetic chromosomes are described as
Top-down or Bottom-up. Top-down strategies involve reducing functional chromosomes to the minimal
number of elements that are required for synthetic genomic applications. Although it has been reported
that irradiation of Chlorella with electron beams results in minichromosomes (Yamada et al. 2003),
a more typical approach for chromosome reduction is based on the observation that introduction of a
telomere sequence in an ectopic location results in truncation of the distal portion of the chromosome
(Farr et al. 1991). Bottom-up strategies require the isolation of a centromere, origins of replication, and
for linear constructs, telomeres which when combined function as a synthetic chromosome scaffold.
Based on the diversity of reported performance, it appears that the nature of the sequences required to
establish centromere function and the ability of circular constructs to function as synthetic chromosomes
are species-specific.
chloroplast-localized proteins requires targeting peptide sequences across membranes. In certain classes
of algae, that is, the Eustigmatophytes, this can be across four membranes (i.e., two ER membranes and
two chloroplast membranes, Ott and Oldham-Ott 2003). Therefore, metabolic engineering of proteins to
complement or supplement chloroplastic functions requires identification of not only nuclear promoters
and terminators, but appropriate targeting sequences.
In addition to enhancing the biochemical pathway to lipids, partitioning in that direction can be
increased by eliminating alternate routes of carbon flux. In algae that accumulate storage carbohydrates
(such as starch) in place of, or in addition to lipids, the synthesis of the carbohydrates can be attenuated
or blocked by targeted inactivation or down-regulation of appropriate biosynthetic enzymes. Likewise,
carbon flux to metabolic energy generation, other biosynthetic pathways and waste carbon products can
be interrupted, with the carbon channeled to additional lipid. The limitation on these manipulations is the
impact they can have on strain robustness. There must be sufficient energy reserves available for dark
metabolism to make it through the night.
The artificial chromosome
The two previous sections describe some of the attempts that different groups have carried out to either
increase the PE of microalgae or the proportion of fixed carbon that is destined to, for example, lipid
metabolism. A common outcome of bioengineering molecular pathways one enzyme at a time is that as
one bottleneck is resolved, another one downstream is revealed. A possible solution to this problem is the
introduction of multiple changes in a biochemical pathway at once.
It is anticipated that multiple metabolic modifications will be required to generate algae capable of
cost effective production of biofuels. Genes for improved photosynthetic efficiency, fatty acid synthesis,
triacylglycerol synthesis, attenuation of alternative carbon sinks, and to improve agronomic properties
suggest that we may be targeting as many as fifty modifications. This is not far from the exemplary twenty
six genetic modifications utilized by the Genencor/DuPont team to engineer production of 1,3-propanediol
in Escherichia coli (Nakamura and Whited 2003).
Current strategies for introduction of genetic modifications into eukaryotes algae rely on random
integration of the exogenous DNA into the chromosome (Coll 2006; Hallmann 2007) and the number of
genetic changes attempted is typically one gene at a time. Random integration results in an unpredictable
number of gene copies due to the tendency of the cellular machinery to concatenate exogenous DNA.
Also, genetic marker availability limits the number of genes that can be introduced. This results in the need
to screen many colonies to identify the few with stable and desired levels of gene expression. Synthetic
chromosomes are preferred when multiple genetic elements are required for metabolic modification. The
synthetic chromosome provides a single copy number and stable, autonomous inheritance with the ability
to accommodate a large number of genes (Zieler et al. 2009).
Early eukaryotic synthetic chromosome constructs in yeast suggested that there are three required
elements for maintenance and stability during replication; a centromere, replication origins, and telomeres
(Murray and Szostak 1983). Significant work has since been directed at the construction of synthetic
chromosomes in mammals, plants, and in some algae strains (Carlson et al. 2007; Houben et al. 2008;
Zieler et al. 2009; O’Neill et al. 2011). Strategies for establishing synthetic chromosomes are described as
Top-down or Bottom-up. Top-down strategies involve reducing functional chromosomes to the minimal
number of elements that are required for synthetic genomic applications. Although it has been reported
that irradiation of Chlorella with electron beams results in minichromosomes (Yamada et al. 2003),
a more typical approach for chromosome reduction is based on the observation that introduction of a
telomere sequence in an ectopic location results in truncation of the distal portion of the chromosome
(Farr et al. 1991). Bottom-up strategies require the isolation of a centromere, origins of replication, and
for linear constructs, telomeres which when combined function as a synthetic chromosome scaffold.
Based on the diversity of reported performance, it appears that the nature of the sequences required to
establish centromere function and the ability of circular constructs to function as synthetic chromosomes
are species-specific.