Improving Marine Algae for Bioenergy 283
of nascent acyl chains requires the sequential entrance of malonyl CoA until reaching a 16-carbon
acyl-CoA. The first desaturation step for fatty acids is catalyzed by a plastidial stearoyl-acyl carrier
protein (ACP) desaturase. Termination of fatty acid chain elongation in the plastids is catalyzed by (acylACP) thioesterases (TE), which hydrolyze acyl chains from ACP. After termination, free fatty acids are
activated to CoA esters, exported from the plastid, and assembled into glycerolipids at the endoplasmic
reticulum (ER) (Thelen and Ohlrogge 2002). Genetic modification of genes involved in TAG assembly
in the ER produced better results toward the increase of the amount of lipids than genetic manipulation
of ACCase in the chloroplasts. For example, the overexpression of genes involved in TAG assembly,
such as glycerol-3-phosphate dehydrogenase (G3PDH) and diacylglycerol acyltransferase (DGT),
results in significant increases in plant lipid production (Lardizabal et al. 2008). An increase of TAGs
accumulation was also accomplished in C. reinhardtii, using the type-2 diacylglycerol acyltransferase
(DGTT2) with a phosphorus starvation–inducible promoter, which was up-regulated during P starvation
(Iwai et al. 2014). Further lipid modifications (desaturation, hydroxylation, and elongation) occur in the
ER (Thelen and Ohlrogge 2002). The low polarity of triacylglycerols (TAG), which are neutral lipids,
causes accumulation of these lipids between bilayer leaflets (termed oil bodies or oleosomes) in the
cytosol that is the general lipid storage form.
It has been known for a long time that nitrogen starvation, which stops protein (and starch)
synthesis, can induce lipid synthesis. Based on this, a possible approach to improve cell lipid contents
is via blocking metabolic pathways that lead to the accumulation of storage compounds such as starch
(Radakovits et al. 2010a). Chlamydomonas reinhardtii has a mutant collection defective for starch
biosynthesis—sta1-1, sta6, and sta7 (see Ball and Deschamps 2009), which can be good candidates to
improve lipid production. When the sta6 strain is nitrogen starved in acetate and then was “boosted” with
additional acetate, the cells become “obese”. The genes of G3PDH, DGTT2, and lipases were selectively
upregulated in this condition (Goodenough et al. 2014). Within the genus Chlorella, C. pyrenoidosa
has fewer lipid contents than the other species (Mata et al. 2010)—which may be due to the presence
of pyrenoid, a structure rich in starch and Rubisco. It should be noted that a starch-less mutant of
C. pyrenoidosa accumulated much higher polyunsaturated fatty acids per dry weight than the wild type
(Ramazanov and Ramazanov 2006). Rubisco could be a potential target because modifications in the
enzyme cause changes in carbon allocation in terms of membrane fatty acid composition and storage lipid
accumulation (Esquivel et al. 2017). On the other hand, the identification of genes that are up-regulated
upon nitrogen depletion resulted in the elucidation of a putative transcription factor that triggers lipid
accumulation in C. reinhardtii, as well as the identification of three acyltransferases that are implicated
in nitrogen starvation-induced TAG accumulation (Boyle et al. 2012). Those genes are potential targets
for manipulating TAG hyperaccumulation in marine microalgae. Another strategy was patented to
increase lipid production using homologous recombination in the alga Nannochloropsis to knock out the
alternative oxidase (AOX). With mitochondrial respiration inhibited, a high concentration of lipids inside
the cells was achieved (Bailey et al. 2011).
Engineering microalgae is also desirable to improve the quality of algal lipids to be used as biodiesel.
The carbon chain length of fatty acids affects the cold flow properties of biodiesel, with shorter chain
lengths being preferred. By resorting to genetic engineering approaches, a shorter fatty acid chain was
attained with the diatom Phaeodactylum tricornutum. Two medium-chain acyl-ACP thioesterases from
Umbellularia californica and Cinnamomum camphora were overexpressed in this diatom, resulting in
increased production of lauric acid (12:0) and myristic acid (14:0) (Radakovits et al. 2010b). Interestingly,
the overexpression of these thioesterases also resulted in an increased production of total fatty acids on a
per cell basis. Another approach to improve the production of shorter fatty acid chain was achieved with a
transgenic strain of Chlamydomonas overexpressing thioesterase (TE) in the algal chloroplast. This effect
is thought to be due to protein-protein interactions between the fatty acid acyl carrier protein (ACP) and
the TE, resulting in short-circuiting of fatty acid chain elongation—which leads to increasing myristic
acid content by 2.5fold, compared with the wild-type (Blatti et al. 2012). These results unfold a new tool
to manipulate chain lengths in fatty acid biosynthesis through protein-protein interactions.
of nascent acyl chains requires the sequential entrance of malonyl CoA until reaching a 16-carbon
acyl-CoA. The first desaturation step for fatty acids is catalyzed by a plastidial stearoyl-acyl carrier
protein (ACP) desaturase. Termination of fatty acid chain elongation in the plastids is catalyzed by (acylACP) thioesterases (TE), which hydrolyze acyl chains from ACP. After termination, free fatty acids are
activated to CoA esters, exported from the plastid, and assembled into glycerolipids at the endoplasmic
reticulum (ER) (Thelen and Ohlrogge 2002). Genetic modification of genes involved in TAG assembly
in the ER produced better results toward the increase of the amount of lipids than genetic manipulation
of ACCase in the chloroplasts. For example, the overexpression of genes involved in TAG assembly,
such as glycerol-3-phosphate dehydrogenase (G3PDH) and diacylglycerol acyltransferase (DGT),
results in significant increases in plant lipid production (Lardizabal et al. 2008). An increase of TAGs
accumulation was also accomplished in C. reinhardtii, using the type-2 diacylglycerol acyltransferase
(DGTT2) with a phosphorus starvation–inducible promoter, which was up-regulated during P starvation
(Iwai et al. 2014). Further lipid modifications (desaturation, hydroxylation, and elongation) occur in the
ER (Thelen and Ohlrogge 2002). The low polarity of triacylglycerols (TAG), which are neutral lipids,
causes accumulation of these lipids between bilayer leaflets (termed oil bodies or oleosomes) in the
cytosol that is the general lipid storage form.
It has been known for a long time that nitrogen starvation, which stops protein (and starch)
synthesis, can induce lipid synthesis. Based on this, a possible approach to improve cell lipid contents
is via blocking metabolic pathways that lead to the accumulation of storage compounds such as starch
(Radakovits et al. 2010a). Chlamydomonas reinhardtii has a mutant collection defective for starch
biosynthesis—sta1-1, sta6, and sta7 (see Ball and Deschamps 2009), which can be good candidates to
improve lipid production. When the sta6 strain is nitrogen starved in acetate and then was “boosted” with
additional acetate, the cells become “obese”. The genes of G3PDH, DGTT2, and lipases were selectively
upregulated in this condition (Goodenough et al. 2014). Within the genus Chlorella, C. pyrenoidosa
has fewer lipid contents than the other species (Mata et al. 2010)—which may be due to the presence
of pyrenoid, a structure rich in starch and Rubisco. It should be noted that a starch-less mutant of
C. pyrenoidosa accumulated much higher polyunsaturated fatty acids per dry weight than the wild type
(Ramazanov and Ramazanov 2006). Rubisco could be a potential target because modifications in the
enzyme cause changes in carbon allocation in terms of membrane fatty acid composition and storage lipid
accumulation (Esquivel et al. 2017). On the other hand, the identification of genes that are up-regulated
upon nitrogen depletion resulted in the elucidation of a putative transcription factor that triggers lipid
accumulation in C. reinhardtii, as well as the identification of three acyltransferases that are implicated
in nitrogen starvation-induced TAG accumulation (Boyle et al. 2012). Those genes are potential targets
for manipulating TAG hyperaccumulation in marine microalgae. Another strategy was patented to
increase lipid production using homologous recombination in the alga Nannochloropsis to knock out the
alternative oxidase (AOX). With mitochondrial respiration inhibited, a high concentration of lipids inside
the cells was achieved (Bailey et al. 2011).
Engineering microalgae is also desirable to improve the quality of algal lipids to be used as biodiesel.
The carbon chain length of fatty acids affects the cold flow properties of biodiesel, with shorter chain
lengths being preferred. By resorting to genetic engineering approaches, a shorter fatty acid chain was
attained with the diatom Phaeodactylum tricornutum. Two medium-chain acyl-ACP thioesterases from
Umbellularia californica and Cinnamomum camphora were overexpressed in this diatom, resulting in
increased production of lauric acid (12:0) and myristic acid (14:0) (Radakovits et al. 2010b). Interestingly,
the overexpression of these thioesterases also resulted in an increased production of total fatty acids on a
per cell basis. Another approach to improve the production of shorter fatty acid chain was achieved with a
transgenic strain of Chlamydomonas overexpressing thioesterase (TE) in the algal chloroplast. This effect
is thought to be due to protein-protein interactions between the fatty acid acyl carrier protein (ACP) and
the TE, resulting in short-circuiting of fatty acid chain elongation—which leads to increasing myristic
acid content by 2.5fold, compared with the wild-type (Blatti et al. 2012). These results unfold a new tool
to manipulate chain lengths in fatty acid biosynthesis through protein-protein interactions.