120
Rhodosporidium toruloides are also emerging as promising candidates for PUFA
production mainly owing to their ability to utilize a range of agro-industrial wastes
as substrates and their high level of lipid accumulation, thus enabling a more economic and eco-friendly production of PUFA. However, genetic engineering in these
species is still a challenging endeavour as not much is known about their cellular
regulatory mechanisms. However, many emerging technologies such as CRISPR/
Cas9 can be exploited in future for potential manipulation of these species (McNeil
and Stuart 2018; Park et al. 2018).
7.2.1 Induction of Lipogenesis in Oleaginous Fungi
Lipogenesis in oleaginous fungi is generally achieved by cultivating these organisms in a medium containing excess carbon with limiting amounts of any essential nutrient such as vitamin or minerals. However, the most commonly employed
strategy is nitrogen limitation. Besides, culture conditions such as temperature,
pH and moisture content also directly account for the amount of lipid that can be
accumulated (Bellou et al. 2016). In oleaginous yeast, the activity of NAD
+
isocitrate dehydrogenase is absolutely dependent on AMP. However, this dependency is not general in all oleaginous fungi as NAD
+
isocitrate dehydrogenases
of Mortierella alpina and Mucor circinelloides are not completely ATP dependent (Laoteng et al. 2011). Under nitrogen stress conditions, AMP is converted
to ammonium ions in the cell, and as a result NAD
+
isocitrate dehydrogenase
activity is reduced. NAD
+
isocitrate dehydrogenase catalyzes the conversion of
isocitrate to α-ketoglutaric acid, and as a result of its reduced activity, an
increased amount of isocitrate accumulates in the mitochondria. Isocitrate exists
in equilibrium with citrate in mitochondria mediated via aconitase enzyme. The
accumulated citrate is then transported out of mitochondrial membrane via a
citrate-malate shunt and converted to acetyl-CoA. The malate inside the mitochondria is subsequently converted to pyruvate via malic enzyme generating
NADPH which can later be utilized for fatty acid synthesis. The ability to convert citrate in cytoplasm to acetyl-CoA by ATP-citrate lyase (ACL) is often
regarded as a marker of oleaginous fungi. The acetyl-CoA is then converted to
malonyl- CoA by the action acetyl-CoA carboxylase (ACC) enzyme. ACC activity is the first committed step towards fatty acid synthesis in yeast and fungi
(Akpinar- Bayizit 2014; Laoteng et al. 2011). A diagrammatic representation of
the above process is given in Fig. 7.2. Lipid biosynthesis is an energy-extensive
process and requires huge amount of NADPH. Important sources of NADPH in
oleaginous fungi include malic enzyme, pentose phosphate pathway, folate
metabolism and isocitrate dehydrogenase in tricarboxylic acid (TCA) cycle
(Wang et al. 2020).
R. Gupta and S. Gaur
Rhodosporidium toruloides are also emerging as promising candidates for PUFA
production mainly owing to their ability to utilize a range of agro-industrial wastes
as substrates and their high level of lipid accumulation, thus enabling a more economic and eco-friendly production of PUFA. However, genetic engineering in these
species is still a challenging endeavour as not much is known about their cellular
regulatory mechanisms. However, many emerging technologies such as CRISPR/
Cas9 can be exploited in future for potential manipulation of these species (McNeil
and Stuart 2018; Park et al. 2018).
7.2.1 Induction of Lipogenesis in Oleaginous Fungi
Lipogenesis in oleaginous fungi is generally achieved by cultivating these organisms in a medium containing excess carbon with limiting amounts of any essential nutrient such as vitamin or minerals. However, the most commonly employed
strategy is nitrogen limitation. Besides, culture conditions such as temperature,
pH and moisture content also directly account for the amount of lipid that can be
accumulated (Bellou et al. 2016). In oleaginous yeast, the activity of NAD
+
isocitrate dehydrogenase is absolutely dependent on AMP. However, this dependency is not general in all oleaginous fungi as NAD
+
isocitrate dehydrogenases
of Mortierella alpina and Mucor circinelloides are not completely ATP dependent (Laoteng et al. 2011). Under nitrogen stress conditions, AMP is converted
to ammonium ions in the cell, and as a result NAD
+
isocitrate dehydrogenase
activity is reduced. NAD
+
isocitrate dehydrogenase catalyzes the conversion of
isocitrate to α-ketoglutaric acid, and as a result of its reduced activity, an
increased amount of isocitrate accumulates in the mitochondria. Isocitrate exists
in equilibrium with citrate in mitochondria mediated via aconitase enzyme. The
accumulated citrate is then transported out of mitochondrial membrane via a
citrate-malate shunt and converted to acetyl-CoA. The malate inside the mitochondria is subsequently converted to pyruvate via malic enzyme generating
NADPH which can later be utilized for fatty acid synthesis. The ability to convert citrate in cytoplasm to acetyl-CoA by ATP-citrate lyase (ACL) is often
regarded as a marker of oleaginous fungi. The acetyl-CoA is then converted to
malonyl- CoA by the action acetyl-CoA carboxylase (ACC) enzyme. ACC activity is the first committed step towards fatty acid synthesis in yeast and fungi
(Akpinar- Bayizit 2014; Laoteng et al. 2011). A diagrammatic representation of
the above process is given in Fig. 7.2. Lipid biosynthesis is an energy-extensive
process and requires huge amount of NADPH. Important sources of NADPH in
oleaginous fungi include malic enzyme, pentose phosphate pathway, folate
metabolism and isocitrate dehydrogenase in tricarboxylic acid (TCA) cycle
(Wang et al. 2020).
R. Gupta and S. Gaur