Algae are diverse group of organisms that inhabit a vast range of ecosystems, from the ex- tremely cold (Antarctic) to extremely hot (desert) regions of the Earth (Guschina and Harwood, 2006; Round, 1984). Algae account for more than half the primary productivity at the base of the food chain (Hoek et al., 1995). Lipid metabolism (the biosynthetic pathways of fatty acids and triacylglycerol, or TAG synthesis), particularly in algae, has been less stud- ied than in higher plants (Fan et al., 2011). Based on the sequence homology and some shared biochemical characteristics of a number of genes and/or enzymes isolated from algae and higher plants that are involved in lipid metabolism, it is generally believed that the basic path- ways of fatty acid and TAG biosynthesis in algae are directly analogous to higher plants (Fan et al., 2011). Thede novosynthesis of fatty acids in algae occurs primarily in the thylakoid and stromal region of the chloroplast (Liu and Benning, 2012). Algae fix CO2during the day via photophosphorylation (thylakoid) and produce carbohydrate during the Calvin cycle (stroma), which converts into various products, including TAGs, depending on the species of algae or specific conditions pertaining to cytoplasm and plastid (Liu and Benning, 2012). Microalgae are proficient at surviving and functioning under phototrophic or hetero- trophic conditions or both. A schematic illustration of algal-based lipid biosynthesis by a pho- toautotrophic mechanism is given inFigure 8.1. The biosynthetic pathway of lipid in algae occurs through four steps: carbohydrates accumulating inside the cell, formation of acetyl- CoA followed by malony-CoA, synthesis of palmitic acid, and finally, synthesis of higher fatty acid by chain elongation.
Acetyl CoA Pyruvate Glucose, 3PGS
Cytosol
Photo- synthesis CO2
TAG
Lipid biosynthesis
Plastid Chloroplast
Lipid Droplet
Glucose
FIGURE 8.1 Localization of various components of the lipid biosynthetic pathway in an algal cell
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8.2 CELLULAR BIOCHEMISTRY TOWARD LIPID SYNTHESIS
8.2.1 Glucose Accumulation Inside the Cell
Accumulation of energy-rich compounds is the primary step for microalgal lipid biosynthesis. However, this carbon accumulation varies with both autotrophic and hetero- trophic organisms. Autotrophs synthesize their own carbon (photosynthates) through photosynthesis, whereas heterotrophic organisms assimilate it from outside the cell.
In photoautotrophs, the chloroplast is the site of photosynthesis where, light reaction takes place at the thylakoid followed by CO2fixation to carbohydrates in the stroma of the chloroplast. These photosynthates provide an endogenous source of acetyl-CoA for further lipid biosynthetic pathways. Heterotrophic nutrition is again light-dependent and light-independent, where the carbon uptake will be through an inducible active hexose symport system from outside the cell (Perez-Garcia et al., 2011; Tanner, 1969;
Komor, 1973; Komor and Tanner, 1974), and in this process the cell invests energy in the form of ATP (Tanner, 2000). However, carbon assimilation is more favorable in the case of light-independent processes (dark heterotrophic) over light-dependent ones (photoheterotroph). In dark heterotrophic algae, light inhibits the expression of the hexose/Hþsymport system (Perez-Garcia et al., 2011; Kamiya and Kowallik, 1987), which decreases glucose transport inside the cell. Algae can also accumulate carbon in the presence of light through photoheterotrophic nutrition. Once carbon enters the cytosol, it follows cytosolic conversion of glucose to pyruvate through glycolysis and leads to the generation of acetyl-CoA, similar to photoautotrophs, followed by the pathway of lipid biosynthesis. In mixotrophic nutrition, both the biochemical process of autotrophs and het- erotrophs occur simultaneously, and the preference of substrate uptake depends on the substrate availability in addition to other environmental conditions.
8.2.2 Formation of Acetyl-CoA/Malonyl-CoA
Photosynthates provide an endogenous source of acetyl-CoA by activated acetyl-CoA synthetase in the stroma, from free acetate, or from the cytosolic conversion of glucose to pyruvate during glycolysis (Somerville et al., 2000; Schwender and Ohlrogge, 2002). This acetyl-CoA is preferentially transported from the cytosol to the plastid, where it is converted to the fatty acid and subsequently to TAG, which again is transported to the cy- tosol and forms the lipid bodies (Figure 8.1). The acetyl-CoA pool will be maintained through the Calvin cycle, glycolysis and pyruvate kinase (PK) mediated synthesis of py- ruvate from PEP, which occur in the chloroplast in addition to the cytosol. The first reaction of the fatty acid biosynthetic pathway towards the formation of malonyl-CoA from acetyl- CoA and CO2is catalyzed by the enzyme Acetyl-CoA carboxylase (ACCase). (Ohlrogge and Browse, 1995). Figure 8.2 illustrates the conversion of acetyl-CoA to malonyl-CoA by utilizing ATP. During this process, seven molecules of acetyl-CoA and seven molecules of CO2form seven molecules of malonyl-CoA. This malonyl Co-A undergoes synthesis of long carbon-chain fatty acids through repeating multistep sequences, as represented in Figures 8.2 and 8.3. A saturated acyl group produced by this set of reactions becomes the substrate for subsequent condensation with an activated malonyl group (Ohlrogge and Browse, 1995).
158 8. ALGAE OILS AS FUELS
8.2.3 Synthesis of Palmitic Acid
After the formation of seven malonyl-CoA molecules, a four-step repeating cycle (exten- sion by two carbons/cycle), i.e., condensation, reduction, dehydration, and reduction, takes place for seven cycles and forms the principal product of the fatty acid synthase systems, i.e., palmitic acid, which is the precursor of other long-chain fatty acids (Fan et al., 2011;
8 Acetyl-CoA + 7ATP + 14NADPH + 14H+ Acetyl-CoA + 7 malonyl-CoA + 14NADPH +14H+ 7 Acetyl-CoA + 7CO2+ 7ATP®7 malonyl-CoA + 7ADP+ 7Pi
Seven cycles of condensation and reduction
Overall reaction
Formation of seven malonyl-CoA molecules
® palmitate + 8 CoA + 7ADP + 7Pi +14NADP
® Palmitate + 7CO2+ 8 CoA + 14NADP +6H2O
FIGURE 8.2 Cascade of reac- tions involved in microalgae lipid biosynthesis
CH2
CH3
CH2 CH2 CH2
CH2
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2
CH2 CH2 CH2 CH2 CH2 C=O CH2
CH2 CH2 CH2 CH2 C=O CH2
CH2 CH2 C=O
C=O
CO2
CO2
CO2 C=O
C=O
C=O
C=O
COO- COO-
COO-
COO- S
S
FAS FAS
FAS
FAS S
S
S
C
O O
Palmitate
HS HS
FAS Inactive
FAS Enzyme 4 more additions S S
+ S 4e- +
4e- 4H+
4H+
+ 4e- 4H+
FIGURE 8.3 Sequential chain elongation steps and formation of precursor molecules (palmitic acid) from CO2
159
8.2 CELLULAR BIOCHEMISTRY TOWARD LIPID SYNTHESIS
Alban et al., 1994). With each course of the cycle, the fatty acyl chain is extended by two carbons.Figures 8.2and8.3illustrate the palmitic acid formation and chain elongation. When the chain length reaches 16 carbons, the product (palmitate) leaves the cycle (Liu and Benning, 2012). All the reactions in the synthetic process are catalyzed by a multienzyme com- plex, i.e., fatty acid synthase (FAS).
8.2.4 Synthesis of Higher Fatty Acids
Palmitate is the precursor of stearate and longer-chain saturated fatty acids as well as palmitoleate and oleate (Pollard and Stumpf, 1980). The palmitic acid gets modified further and lengthened to form stearate (18:0) or even to longer saturated fatty acids (oleiceate, linealate, etc.) by further additions of acetyl groups through the action of fatty acid elongation systems present in the smooth endoplasmic reticulum (ER) and in mitochondria (Thelen and Ohlrogge, 2002). The mechanism of elongation in the ER is identical to palmitate synthesis, which involves donation of two carbons by malonyl-CoA, followed by reduction, dehydra- tion, and reduction to the saturated 18-carbon product, stearoyl-CoA.Figure 8.4shows the formation of higher fatty acids from the palmitic acid through different steps of chain elonga- tion. In algae, oleate (from stearoyl-CoA) gets converted to theaandglinolenates (Thelen and Ohlrogge, 2002). a-linolenate further getsconverted to other polyunsaturated fatty acids, whileg-linolenate converts to the eicosatrienoate and further arachidonate. Mammals cannot
Palmitate
Desaturation
Desaturation Desaturation
Desaturation
Elongation
Elongation Elongation
Repetitive Desaturation
Repetitive Desaturation
Palmitoleate
Longer saturated fatty acid Stearate
Oleate
Linoleate
Eicosatrienoate
Arachidonate Other polyunsaturated fatty acid
g-Linolenate a-Linolenate
FIGURE 8.4 Schematic represen- tation of long-chain fatty acid forma- tion from palmitic acid
160 8. ALGAE OILS AS FUELS
convert oleate to linoleate or linolenate because of the lack of enzymes to introduce double bonds at carbon atoms beyond C9 (Nelson and Cox, 2009). All fatty acids containing a double bond at positions beyond C9 have to be supplied in the diet and are called essential fatty acids.