**3. Biochemistry of lipid accumulation**

Lipid accumulation in oleaginous yeasts and molds has been demonstrated to occur when a nutrient in the medium (e.g. the nitrogen or the phosphorus source) becomes limited and the carbon source is present in excess. Nitrogen limitation is the most efficient condition for inducing lipogenesis. During the growth phase, nitrogen is necessary for the synthesis of proteins and nucleic acids, while the carbon flux is distributed among energetic and anabolic processes yielding carbohydrates, lipids, nucleic acids and proteins. When nitrogen gets limited, the growth rate slows down and the synthesis of proteins and nucleic acids tends to cease. In non-oleaginous species, the carbon excess remains unutilized or is converted into storage polysaccharides, while, in oleaginous species, it is preferentially channeled toward lipid synthesis, leading to the accumulation of TAG within intracellular lipid bodies (Ratledge & Wynn, 2002; Granger at al., 1993).

The biochemical pathway of lipid biosynthesis is not very different among eukaryotic organisms and does not differ in oleaginous and non-oleaginous fungi. The ability to accumulate high amounts of lipid depends mostly on the regulation the biosynthetic pathway and the supply of the precursors (i.e. acetyl-CoA, malonyl-CoA, and glycerol-3 phosphate) and the cofactor NADPH.

Most information were obtained from the model yeast *Saccharomyces cerevisiae* (Kohlwein, 2010), that does not accumulate lipids, and *Yarrowia lipolytica*, that represent a model for biooil production and is suitable for genetic manipulation (Beopoulos et al., 2009b).

#### **3.1 Fatty acids biosynthesis and modifications**

De novo synthesis of fatty acids (FA), the first step of lipid accumulation, is carried out in the cytosol by fatty acids synthetase (FAS) complex. In yeasts, FAS bears phosphopantheteine transferase activity to activate its acyl carrier protein (ACP) by loading the coenzyme pantothenate. FAS is a multimer of 6 α and 6 β subunits encoded by *fas2* and *fas1*, respectively, each subunit containing four functional domains. Therefore, FAS consists in a α6β6 molecular complex of 2.6 MDa with 48 functional centers that catalyze all reactions required for synthesis of fatty acids through cycles of multistep reactions. FAS firstly loads acetyl-CoA on its β-ketoacyl-ACP synthase (KS), then it exherts β-ketoacyl-ACP reductase (KR), β-hydroxyacyl-ACP dehydratase (DH), and enoyl-ACP reductase (EAR) activities. This set of reactions is repeated cyclically seven times to yield palmitoyl-ACP (Fig. 1) (Tehlivets et al, 2007).

commercial production of microbial lipids (Ratledge, 2004). Lipid accumulation in *M. circinelloides* has been extensively studied (Wynn et al., 2001), and its TAG have been proposed as feedstock for producing biodiesel by direct transformation of its lipids (Vicente et al., 2009). *M. circinelloides* represents an outstanding model within the *Zygomycota* phylum, based on the availability of an efficient transformation procedure (Gutierrez et al., 2011) and on the whole sequence of genome (http://genome.jgipsf.org/Mucci2/Mucci2.home.html). Also the phylogenetically related *Umbelopsis isabellina* has emerging as a promising species to convert biomass residues to biodiesel precursors (Meeuwse et al., 2011a). To the best of our knowledge, limited are the attempts to get lipids with *Aspergillus oryzae* that, conversely, is extensively studied as lipase

Lipid accumulation in oleaginous yeasts and molds has been demonstrated to occur when a nutrient in the medium (e.g. the nitrogen or the phosphorus source) becomes limited and the carbon source is present in excess. Nitrogen limitation is the most efficient condition for inducing lipogenesis. During the growth phase, nitrogen is necessary for the synthesis of proteins and nucleic acids, while the carbon flux is distributed among energetic and anabolic processes yielding carbohydrates, lipids, nucleic acids and proteins. When nitrogen gets limited, the growth rate slows down and the synthesis of proteins and nucleic acids tends to cease. In non-oleaginous species, the carbon excess remains unutilized or is converted into storage polysaccharides, while, in oleaginous species, it is preferentially channeled toward lipid synthesis, leading to the accumulation of TAG within intracellular

The biochemical pathway of lipid biosynthesis is not very different among eukaryotic organisms and does not differ in oleaginous and non-oleaginous fungi. The ability to accumulate high amounts of lipid depends mostly on the regulation the biosynthetic pathway and the supply of the precursors (i.e. acetyl-CoA, malonyl-CoA, and glycerol-3-

Most information were obtained from the model yeast *Saccharomyces cerevisiae* (Kohlwein, 2010), that does not accumulate lipids, and *Yarrowia lipolytica*, that represent a model for bio-

De novo synthesis of fatty acids (FA), the first step of lipid accumulation, is carried out in the cytosol by fatty acids synthetase (FAS) complex. In yeasts, FAS bears phosphopantheteine transferase activity to activate its acyl carrier protein (ACP) by loading the coenzyme pantothenate. FAS is a multimer of 6 α and 6 β subunits encoded by *fas2* and *fas1*, respectively, each subunit containing four functional domains. Therefore, FAS consists in a α6β6 molecular complex of 2.6 MDa with 48 functional centers that catalyze all reactions required for synthesis of fatty acids through cycles of multistep reactions. FAS firstly loads acetyl-CoA on its β-ketoacyl-ACP synthase (KS), then it exherts β-ketoacyl-ACP reductase (KR), β-hydroxyacyl-ACP dehydratase (DH), and enoyl-ACP reductase (EAR) activities. This set of reactions is repeated cyclically seven times to yield palmitoyl-ACP (Fig. 1)

oil production and is suitable for genetic manipulation (Beopoulos et al., 2009b).

producer to carry out transesterification of TAG (Adachi et al., 2011).

**3. Biochemistry of lipid accumulation** 

phosphate) and the cofactor NADPH.

(Tehlivets et al, 2007).

**3.1 Fatty acids biosynthesis and modifications** 

lipid bodies (Ratledge & Wynn, 2002; Granger at al., 1993).

Fig. 1. Reactions occurring sequentially in fatty acid synthetase: condensation of acyl-ACP and malonyl-ACP mediated by KS, NADPH-dependent reduction of the keto group to a hydroxyl group by means of KR, dehydration to create a double bond with DH and reduction of the double bond by means of EAR. R = H, CH3(CH2)2n; nmax=7.

The biosynthesis of FA requires the constant supply of acetyl-CoA as initial biosynthetic unit and of malonyl-CoA as the elongation unit, supplying two carbons at each step. Nonoleaginous yeasts receive acetyl-CoA mostly from glycolysis. In oleaginous yeasts, acetyl-CoA is mostly provided by the cleavage in the cytosol of citrate, which accumulated as a consequence of nitrogen limitation (Ratledge, 2002) (Fig. 2). In fact, lipid accumulation by oleaginous fungi does not occur under balanced nutrient conditions.

In oleaginous yeasts, nitrogen limitation activates AMP-deaminase (Ratledge & Wynn, 2002), which supply ammonium to the nitrogen-starved cell. As a consequence, mitochondrial AMP concentration decreases, causing isocitrate dehydrogenase activity to drop. The TCA cycle is then blocked at the level of isocitrate, which accumulates and equilibrates with citrate through aconitase. Excess of citrate from TCA cycle is exported out of the mitochondrion via the malate/citrate antiport. Cytosolic ATP-citrate lyase (ACL) cleaves citrate to give oxaloacetate and acetyl-CoA (Fig. 2).

ACL represents one of the key enzymes that contribute to the oleaginous trait of yeasts, whereas its activity is negligible in non-oleaginous species. ACL is composed of two subunits, encoded by *ACL1* and *ACL2* and is negatively regulated by exogenous FA.

Malonyl-CoA is produced from acetyl-CoA by acetyl-CoA carboxylase (ACC) that condensate an acetyl-CoA unit with bicarbonate:

#### Acetyl-CoA + HCO3- +ATP malonyl-CoA + ADP + Pi

ACC is also a key enzyme in *de novo* FA synthesis, since *ACC1* mutants became FA auxotrophs or maintain low levels of ACC activity (Tehlivets et al., 2007). ACC1 undergoes allosteric activation by citrate. Furthermore the transcription of *FAS1*, *FAS2*, and *ACC1* is coordinately regulated, being negatively regulated by FA.

Getting Lipids for Biodiesel Production from Oleaginous Fungi 77

Fig. 3. Biosynthesis of poly-unsaturated fatty acid. Δ9d, Δ12d and Δ15d are the most common desaturases which are present in the endoplasmic reticulum (Ratledge 2004).

oleaginous yeasts, based on production of γ and α-linolenic acids, respectively (Fig. 3).

The fatty acyl-CoA produced by *de novo* synthesis are esterified with glycerol or sterols to produce triacyl-glycerol (TAG) and steryl-esters (SE), respectively. In oleaginous fungi, the neutral lipids SE and TAG are store inside the lipid bodies (LB). TAG are mostly formed by consecutive acylation of glycerol-3-phosphate (G3P), carried out by diverse acyl transferases. G3P is formed from glycerol by glycerol kinase or can be synthesized from dihydroxyacetone phosphate (DHAP) by G3P dehydrogenase, in a reversible reaction. *S. cerevisiae* can use both G3P and DHAP as acyl-group acceptor. The addition of the first acyl group leads to 1-acyl G3P, also named lysophosphatidic acid (LPA). LPA can also be formed by reduction of acyl-DHAP, carried out by a NADPH dependent reductase. A second acyltransferase loads an other acyl group, producing 1,2-diacyl G3P (phosphatidic acid, PA). Phosphate is removed from PA by phosphatidate phosphatase isoenzymes, generating diacylglycerol (DAG). DAG can be the direct precursor of TAG, or can be channeled toward

**3.2 Biosynthesis of triacyl-glycerol** 

phospholipids biosynthesis (Fig. 4).

The final products of FAS are myristic or palmitic acids, depending on the yeast species. Reactions resulting in further elongation or desaturation occur in the endoplasmic reticulum (ER). Elongation reactions are catalyzed by elongases (such as malonyl-palmitoil transacylase, MPT) organized in a complex that requires malonyl-CoA provided by ACC. Desaturations are introduced by ER desaturases, hydrophobic membrane-bound proteins. The most common desaturases are Δ9, which inserts the first double bond onto palmitic and/or stearic acids, and Δ12, which catalyzes the insertion of the second unsaturation into oleic acid to produce linoleic acid. Δ6 and Δ15 desaturase activities have been recently described in in psychrophilic

Fig. 2. Lipid biosynthesis from excess of citrate as a consequence of nitrogen limitation. Adapted from Ratledge, 2004.

Cytosolic NADPH is required for KR and EAR functions of FAS. For each elongation step of the acyl chain, two molecules of NADPH are required. One of the major sources of cytosolic NADPH are the pentose phosphate pathway and the transhydrogenase cycle, which transforms NADH into NADPH through the activity of pyruvate carboxylase (PC), malate dehydrogenase (MDH), and malic enzyme (ME), catalyzing the following reactions:

Pyruvate + CO2 + ATP oxaloacetate + ADP + Pi (PC)

Oxaloacetate + NADH Malate + NAD+ (MDH)

Malate + NADP+ pyruvate + CO2 + NADPH (ME)

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

#### NADH + NADP+ NADPH + NAD+

ME has been found in several oleaginous fungi and it has been regarded as a key enzyme involved in lipid accumulation (Ratledge, 2002). In *Mortierella circinelloides*, overexpression of ME enhanced lipid accumulation (Zhang et al., 2011), whereas overexpression of the ME homologous in *Yarrowia lipolytica* did not result in yield improvement.

Fig. 2. Lipid biosynthesis from excess of citrate as a consequence of nitrogen limitation.

Cytosolic NADPH is required for KR and EAR functions of FAS. For each elongation step of the acyl chain, two molecules of NADPH are required. One of the major sources of cytosolic NADPH are the pentose phosphate pathway and the transhydrogenase cycle, which transforms NADH into NADPH through the activity of pyruvate carboxylase (PC), malate dehydrogenase (MDH), and malic enzyme (ME), catalyzing the following

Pyruvate + CO2 + ATP oxaloacetate + ADP + Pi (PC)

Oxaloacetate + NADH Malate + NAD+ (MDH)

Malate + NADP+ pyruvate + CO2 + NADPH (ME)

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

NADH + NADP+ NADPH + NAD+ ME has been found in several oleaginous fungi and it has been regarded as a key enzyme involved in lipid accumulation (Ratledge, 2002). In *Mortierella circinelloides*, overexpression of ME enhanced lipid accumulation (Zhang et al., 2011), whereas overexpression of the ME

homologous in *Yarrowia lipolytica* did not result in yield improvement.

Adapted from Ratledge, 2004.

reactions:

Fig. 3. Biosynthesis of poly-unsaturated fatty acid. Δ9d, Δ12d and Δ15d are the most common desaturases which are present in the endoplasmic reticulum (Ratledge 2004).

The final products of FAS are myristic or palmitic acids, depending on the yeast species. Reactions resulting in further elongation or desaturation occur in the endoplasmic reticulum (ER). Elongation reactions are catalyzed by elongases (such as malonyl-palmitoil transacylase, MPT) organized in a complex that requires malonyl-CoA provided by ACC. Desaturations are introduced by ER desaturases, hydrophobic membrane-bound proteins. The most common desaturases are Δ9, which inserts the first double bond onto palmitic and/or stearic acids, and Δ12, which catalyzes the insertion of the second unsaturation into oleic acid to produce linoleic acid. Δ6 and Δ15 desaturase activities have been recently described in in psychrophilic oleaginous yeasts, based on production of γ and α-linolenic acids, respectively (Fig. 3).

#### **3.2 Biosynthesis of triacyl-glycerol**

The fatty acyl-CoA produced by *de novo* synthesis are esterified with glycerol or sterols to produce triacyl-glycerol (TAG) and steryl-esters (SE), respectively. In oleaginous fungi, the neutral lipids SE and TAG are store inside the lipid bodies (LB). TAG are mostly formed by consecutive acylation of glycerol-3-phosphate (G3P), carried out by diverse acyl transferases. G3P is formed from glycerol by glycerol kinase or can be synthesized from dihydroxyacetone phosphate (DHAP) by G3P dehydrogenase, in a reversible reaction. *S. cerevisiae* can use both G3P and DHAP as acyl-group acceptor. The addition of the first acyl group leads to 1-acyl G3P, also named lysophosphatidic acid (LPA). LPA can also be formed by reduction of acyl-DHAP, carried out by a NADPH dependent reductase. A second acyltransferase loads an other acyl group, producing 1,2-diacyl G3P (phosphatidic acid, PA). Phosphate is removed from PA by phosphatidate phosphatase isoenzymes, generating diacylglycerol (DAG). DAG can be the direct precursor of TAG, or can be channeled toward phospholipids biosynthesis (Fig. 4).

Getting Lipids for Biodiesel Production from Oleaginous Fungi 79

Fig. 5. Model of lipid bodies biogenesis from the membrane of the ER. TAG and SE

where β-oxidation takes place, or to phospholipid biosynthesis.

**4. Metabolic engineering of oleaginous yeasts** 

of TAG and SE.

accumulate between the two leaflets of the phospholipid bilayer (i to iii). The micro-droplet generated (iii, iv) evolve to lipid bodies (v) (Figure adapted from Czabany et al., 2007).

In most oleaginous yeasts, the neutral lipids of LB consist mostly of TAG (up to 90% or more) whereas a small fraction is represented by steryl esters. The presence of significant quantity of free fatty acids (FFA) within LP has been reported only for *Y. lipolytica*. In *S. cerevisiae*, which accumulates less than 15% lipids of its biomass, LB contain similar amounts

The core of LB, consisting of neutral lipids is surrounded by a phospholipid monolayer where several proteins are embedded. These proteins exert a key role in lipid metabolism, biosynthesis, and substrate trafficking. Upon requirement, storage lipids are mobilized from this compartment by triacylglycerol lipases and steryl ester hydrolases. The respective degradation products serve as energy sources and/or building blocks for membrane formation. In fact, FA hydrolyzed from TAG or SE are either channeled to the peroxisome,

The availability of genome data and genetic tools, such as the possibility to integrate homologous or heterologous genes, opened up the possibility to use metabolic engineering to understand the molecular mechanisms involved in lipid accumulation or to increase the yield of stored lipids in *S. cerevisiae* and *Y. lipolytica*. Whereas *S. cerevisiae* 

Fig. 4. *de novo* synthesis of TAG (adapted from Czabany et al., 2007)

The last step of *de novo* synthesis of TAG can be carried either by using diverse acyl donors, such as acyl-CoA or with phospholipids. In the former case, DAG acyl transferases (DGAT), which are integral proteins of the ER, can directly load the third Acyl-CoA. A DGAT enzyme is present in *S. cerevisiae* and *Y. lipolytica* and is mostly active during the stationary phase, although it is expressed also during the exponential phase. A second DGAT, more active during the exponential growth phase, has been identified in *Y. lipolytica.* In *S. cerevisiae* the phospholipid:DAG acyltransferase (PDAT) is localized in the ER, whereas in *Y. lipolytica* it is present both in the ER and in the surface of LB (Fig. 4).

#### **3.3 Biogenesis of lipid bodies**

In eukaryotes, neutral lipids are stored in specialized compartments known as lipids bodies (LB). They are assembled at a specialized subdomain of the ER where most biosynthetic enzymes and structural proteins are located (Waltermann et al., 2005). The neutral lipids do not fit among phospholipids and are thus deposited between the two leaflets of the membrane bilayer. However, substantial amounts of neutral lipids cannot be incorporated into membrane bilayer of ER and ongoing neutral lipid synthesis leads to the formation of a bud which buds off of the ER as a mature LB after reaching the critical size (Fig. 5).

Fig. 4. *de novo* synthesis of TAG (adapted from Czabany et al., 2007)

*lipolytica* it is present both in the ER and in the surface of LB (Fig. 4).

**3.3 Biogenesis of lipid bodies** 

size (Fig. 5).

The last step of *de novo* synthesis of TAG can be carried either by using diverse acyl donors, such as acyl-CoA or with phospholipids. In the former case, DAG acyl transferases (DGAT), which are integral proteins of the ER, can directly load the third Acyl-CoA. A DGAT enzyme is present in *S. cerevisiae* and *Y. lipolytica* and is mostly active during the stationary phase, although it is expressed also during the exponential phase. A second DGAT, more active during the exponential growth phase, has been identified in *Y. lipolytica.* In *S. cerevisiae* the phospholipid:DAG acyltransferase (PDAT) is localized in the ER, whereas in *Y.* 

In eukaryotes, neutral lipids are stored in specialized compartments known as lipids bodies (LB). They are assembled at a specialized subdomain of the ER where most biosynthetic enzymes and structural proteins are located (Waltermann et al., 2005). The neutral lipids do not fit among phospholipids and are thus deposited between the two leaflets of the membrane bilayer. However, substantial amounts of neutral lipids cannot be incorporated into membrane bilayer of ER and ongoing neutral lipid synthesis leads to the formation of a bud which buds off of the ER as a mature LB after reaching the critical

Fig. 5. Model of lipid bodies biogenesis from the membrane of the ER. TAG and SE accumulate between the two leaflets of the phospholipid bilayer (i to iii). The micro-droplet generated (iii, iv) evolve to lipid bodies (v) (Figure adapted from Czabany et al., 2007).

In most oleaginous yeasts, the neutral lipids of LB consist mostly of TAG (up to 90% or more) whereas a small fraction is represented by steryl esters. The presence of significant quantity of free fatty acids (FFA) within LP has been reported only for *Y. lipolytica*. In *S. cerevisiae*, which accumulates less than 15% lipids of its biomass, LB contain similar amounts of TAG and SE.

The core of LB, consisting of neutral lipids is surrounded by a phospholipid monolayer where several proteins are embedded. These proteins exert a key role in lipid metabolism, biosynthesis, and substrate trafficking. Upon requirement, storage lipids are mobilized from this compartment by triacylglycerol lipases and steryl ester hydrolases. The respective degradation products serve as energy sources and/or building blocks for membrane formation. In fact, FA hydrolyzed from TAG or SE are either channeled to the peroxisome, where β-oxidation takes place, or to phospholipid biosynthesis.
