282 Marine Macro- and Microalgae: An Overview
embarking in such an ambitious project, which would probably need an extensive re-design of the whole
metabolism.
Besides all such possibilities to increase the efficiency of carbon fixation, the metabolism of
photosynthates can also be steered to boost the synthesis of end products that can be used as fuel.
Accordingly, the production of alcohols, sugars starch, fatty acids, and other metabolites has been
enhanced in different photosynthetic organisms (especially cyanobacteria) either by overexpression of
key enzymes in their corresponding biosynthetic pathways, or by avoiding them (bypassing unnecessary
intermediaries and flux branching points), or by engineering suitable secretion routes (reviewed by
Rosgaard et al. 2012). Advances in the specific production of oils and ethanol will be considered in a
separate section below. Besides improving the existing metabolic pathways, another strategy consists of
creating new ones that lead to the desired final product. In a pioneer work back in 1999, genes coding
for pyruvate decarboxylase and alcohol dehydrogenase from the bacterium Zymomonas mobilis were
introduced in Synechococcus in order to deviate pyruvate to the synthesis of ethanol (Deng and Coleman
1999). An extension of this idea, which has been termed the “photanol” approach (Hellingwerf and
Teixeira de Mattos 2009), may be used to produce a variety of fuels by linking metabolites generated by
the carbon fixation pathway (particularly glyceraldehyde 3-phosphate and pyruvate) to the fermentative
pathways of chemoautotrophic bacteria (Anemaet et al. 2010). The authors propose cyanobacteria (in
particular, Synechococcus) to host the bacterial genes coding the enzymes required to implement the
fermentative route. However, this approach may in principle be also extended to those algae that can
be transformed. In a different strategy carried out in E. coli, 2-ketoacid decarboxylase and alcohol
dehydrogenase were introduced in the bacterium in order to synthesize several branched-chain higher
alcohols (which are superior to ethanol as biofuel) from 2-ketoacids that are naturally produced from
glucose through the amino acid biosynthetic pathways (Atsumi et al. 2008). A similar approach may
again be attempted in photosynthetic organisms like cyanobacteria, and perhaps eukaryotic algae; these
are, most likely, just the first examples of promising developments derived from the synthetic biology
approach to biofuel production.
Production of specific organic biofuels
Generation of oils, storage lipids, and triacylglycerides for biodiesel production
One of the most abundant forms of reduced carbon chains on Earth is fatty acids—plant seeds and algae
are the largest sources of these compounds.
Biodiesel is made by a mixture of fatty acid alkyl esters obtained by transesterification (ester exchange
reaction) of triacylglycerides (TAGs) from oils or fats. Transesterification comprises a few reactions
where triacylglycerides and a short chain alcohol react in the presence of a strong base catalyst (usually
NaOH) to form a mixture of fatty acid alkyl esters (Mata et al. 2010). This conversion of fatty acids to
biodiesel is a rapid and technically straightforward process that renders triacylglycerides attractive for
bioenergy generation.
Lipid biosynthesis and catabolism was first studied in detail in oil seed plants. Because homologues
can be found in algal genomes for the majority of genes related to lipid metabolism in land plants, the
strategies used to improve lipid production in higher plants are expected to also be effective with microalgae.
In higher plants and eukaryotic algae, the reactions for de novo fatty acid synthesis are located in plastids
and, consequently, the synthase machinery is similar to prokaryotes in that the enzymatic components
are separable polypeptides rather than large multifunctional proteins as found in animals and fungi. This
sequence of reactions is called the “prokaryotic pathway”. The priming reaction, the conversion of acetylcoenzyme A (CoA) to malonyl-CoA, is catalyzed by the plastidic acetyl-CoA carboxylase (ACCase),
which is considered the first control step in fatty acid biosynthesis in many organisms. However, several
attempts to utilize ACCase overexpression to increase lipid content in various systems have failed. The
overexpression of ACCase in two diatom species increased the activity of ACCase, but enhancement
of lipid production was not observed (Dunahay et al. 1995). After the priming reaction, the elongation
embarking in such an ambitious project, which would probably need an extensive re-design of the whole
metabolism.
Besides all such possibilities to increase the efficiency of carbon fixation, the metabolism of
photosynthates can also be steered to boost the synthesis of end products that can be used as fuel.
Accordingly, the production of alcohols, sugars starch, fatty acids, and other metabolites has been
enhanced in different photosynthetic organisms (especially cyanobacteria) either by overexpression of
key enzymes in their corresponding biosynthetic pathways, or by avoiding them (bypassing unnecessary
intermediaries and flux branching points), or by engineering suitable secretion routes (reviewed by
Rosgaard et al. 2012). Advances in the specific production of oils and ethanol will be considered in a
separate section below. Besides improving the existing metabolic pathways, another strategy consists of
creating new ones that lead to the desired final product. In a pioneer work back in 1999, genes coding
for pyruvate decarboxylase and alcohol dehydrogenase from the bacterium Zymomonas mobilis were
introduced in Synechococcus in order to deviate pyruvate to the synthesis of ethanol (Deng and Coleman
1999). An extension of this idea, which has been termed the “photanol” approach (Hellingwerf and
Teixeira de Mattos 2009), may be used to produce a variety of fuels by linking metabolites generated by
the carbon fixation pathway (particularly glyceraldehyde 3-phosphate and pyruvate) to the fermentative
pathways of chemoautotrophic bacteria (Anemaet et al. 2010). The authors propose cyanobacteria (in
particular, Synechococcus) to host the bacterial genes coding the enzymes required to implement the
fermentative route. However, this approach may in principle be also extended to those algae that can
be transformed. In a different strategy carried out in E. coli, 2-ketoacid decarboxylase and alcohol
dehydrogenase were introduced in the bacterium in order to synthesize several branched-chain higher
alcohols (which are superior to ethanol as biofuel) from 2-ketoacids that are naturally produced from
glucose through the amino acid biosynthetic pathways (Atsumi et al. 2008). A similar approach may
again be attempted in photosynthetic organisms like cyanobacteria, and perhaps eukaryotic algae; these
are, most likely, just the first examples of promising developments derived from the synthetic biology
approach to biofuel production.
Production of specific organic biofuels
Generation of oils, storage lipids, and triacylglycerides for biodiesel production
One of the most abundant forms of reduced carbon chains on Earth is fatty acids—plant seeds and algae
are the largest sources of these compounds.
Biodiesel is made by a mixture of fatty acid alkyl esters obtained by transesterification (ester exchange
reaction) of triacylglycerides (TAGs) from oils or fats. Transesterification comprises a few reactions
where triacylglycerides and a short chain alcohol react in the presence of a strong base catalyst (usually
NaOH) to form a mixture of fatty acid alkyl esters (Mata et al. 2010). This conversion of fatty acids to
biodiesel is a rapid and technically straightforward process that renders triacylglycerides attractive for
bioenergy generation.
Lipid biosynthesis and catabolism was first studied in detail in oil seed plants. Because homologues
can be found in algal genomes for the majority of genes related to lipid metabolism in land plants, the
strategies used to improve lipid production in higher plants are expected to also be effective with microalgae.
In higher plants and eukaryotic algae, the reactions for de novo fatty acid synthesis are located in plastids
and, consequently, the synthase machinery is similar to prokaryotes in that the enzymatic components
are separable polypeptides rather than large multifunctional proteins as found in animals and fungi. This
sequence of reactions is called the “prokaryotic pathway”. The priming reaction, the conversion of acetylcoenzyme A (CoA) to malonyl-CoA, is catalyzed by the plastidic acetyl-CoA carboxylase (ACCase),
which is considered the first control step in fatty acid biosynthesis in many organisms. However, several
attempts to utilize ACCase overexpression to increase lipid content in various systems have failed. The
overexpression of ACCase in two diatom species increased the activity of ACCase, but enhancement
of lipid production was not observed (Dunahay et al. 1995). After the priming reaction, the elongation