De novo accumulation of cellular lipids is an anabolic biochemical process in which, by virtue of quasi-inverted ^-oxidation reaction series, acetyl-CoA issued by the intermediate cellular metabolism, generates the synthesis of intra-cellular fatty acids. Fatty acids will be then esterified in order to synthesize structural (phospholipids, sphingolipids, etc.) and reserve lipids (TAGs and SEs) (Moreton, 1988; Ratledge, 1988, 1994; Davies and Holdsworth, 1992; Ratledge and Wynn, 2002; Papanikolaou and Aggelis, 2009). In oleaginous microorganisms in which de novo lipid accumulation is conducted, acetyl-CoA that constitutes the precursor of intra-cellular fatty acids, derives from breakdown of citric acid that under some circumstances cannot be catabolized through the reactions performed in the Krebs cycle, but it is accumulated inside the mitochondria. This occurs when its concentration becomes higher than a critical value resulting in citric acid transportation into the cytosol (Ratledge, 1988, 1994; Ratledge and Wynn, 2002; Wynn and Ratledge, 2006; Fakas et al., 2009b). The key-step for citric acid accumulation inside the mitochondrion matrix is the change of intra-cellular concentration of various metabolites, conducted after exhaustion of some nutrients (mainly nitrogen) in the culture medium (Ratledge, 1988, 1994; Ratledge and Wynn, 2002; Wynn and Ratledge, 2006). This exhaustion provokes a rapid decrease of the concentration of intra-cellular AMP, since, by virtue of AMP — desaminase, the microorganism cleaves AMP into IMP and NH4+ ions in order to utilize nitrogen, in the form of NH4+ ions, as a complementary nitrogen source, necessary for synthesis of cell material (Evans and Ratledge, 1985).

The excessive decrease of intra-cellular AMP concentration alters the Krebs cycle function; the activity of both NAD+ and NADP+-isocitrate dehydrogenases, enzymes responsible for the transformation of iso-citric to a-ketoglutaric acid, lose their activity, since they are allosterically activated by intra-cellular AMP, and this event results in the accumulation of citric acid inside the mitochondrion (studies performed in the oleaginous microorganisms Candida sp. 107, Rhodosporidium toruloides, Y. lipolytica, Mortierella isabellina, Mortierella alpina, Mucor circinelloides and Cunningamella echinulata) (Botham and Ratledge, 1979; Evans and Ratledge, 1985; Wynn et al, 2001; Finogenova et al, 2002; Papanikolaou et al., 2004b). When the concentration of citric acid becomes higher than a critical value, it is secreted into the cytosol. Finally, in the case of lipogenous (lipid — accumulating) microorganisms, cytosolic citric acid is cleaved by ATP-citrate lyase (ACL), the key-enzyme of lipid accumulation process in the oil-bearing microorganisms, in acetyl-CoA and oxaloacetate, with acetyl-CoA being converted, by an inversion of b-oxydation process, to cellular fatty acids. In contrast, non­oleaginous microorganisms (e. g. various Y. lipolytica and Aspergillus niger strains) secrete the accumulated citric acid into the culture medium (Ratledge, 1994; Anastassiadis et al., 2002; Papanikolaou et al., 2002b) instead of accumulating significant quantities of reserve lipid. In general, production of citric acid by citrate-producing strains is a process carried out when extra — and hence intra­cellular nitrogen is depleted [overflow metabolism phenomenon (see Anastassiadis et al, 2002)], while studies of the intra-cellular enzyme activities and co-enzyme concentrations have somehow identified and clarified the biochemical events leading to citric acid biosynthesis (Finogenova et al., 2002; Morgunov et al., 2004; Makri et al, 2010) and indeed it has been demonstrated that citric acid secretion and SCO accumulation are processes indeed identical into their first steps.

In a third category of microorganisms, the accumulated (inside the cytosol) citric acid provokes inhibition of the enzyme 6-phospho-fructokinase, and the above fact results in the intra-cellular accumulation of polysaccharides based on the 6-phospho-glucose (Evans and Ratledge, 1985). Schematically, the intermediate cellular metabolism resulting in the synthesis of either citric acid or storage lipid is presented in Figure 8.3 (Ratledge, 1994; Ratledge and Wynn, 2002; Papanikolaou and Aggelis, 2009).

After the biosynthesis of intra-cellular fatty-CoA esters, an esterification with glycerol takes place in order for the reserve lipids to be stocked in the form of TAGs (Ratledge, 1988, 1994). This synthesis in the oleaginous microorganisms is conducted by virtue of the so-called pathway of a-glycerol phosphate acylation (Ratledge, 1988; Davies and Holdsworth, 1992; Athenstaedt and Daum, 1999; Mullner and Daum, 2004; Fakas et al., 2009b). In this metabolic pathway, free fatty acids are activated by coenzyme A and are subsequently used for the acylation of the glycerol backbone to synthesize TAGs. In the first step of TAGs assembly, glycerol-3-phosphate (G-3-P) is acylated by G-3-P acyltranferase (GAT) at the sn-1 position to yield 1-acyl-G-3-P (lysophospatidic acid-LPA), which is then

8.3 Pathways involved in the breakdown of glucose by microbial strains capable of producing SCO and/or citric acid in nitrogen-limited conditions. FFA: free-fatty acids; TRSP: citric acid transporting system; a, b, c: systems transporting pyruvic acid from cytosol to mitochondrion and inversely; d: system transporting citric and malic acid from cytosol to mitochondrion and inversely; ACL: ATP-citrate lyase; FAS: fatty acid synthetase; ICDH: iso-citrate dehydrogenase; MDc: malate dehydrogenase (cytoplasmic); MDm: malate dehydrogenase (mitochondrial); ME: NADPH+-malic enzyme; PD: pyruvate dehydrogenase; CS: citrate synthase; ICL: iso-citrate lyase; EMP: Embden-Mayerhoff-Parnas pathway. Pathways described by Ratledge (1994), Ratledge and Wynn (2002), Papanikolaou and Aggelis (2009).

further acylated by lysophosphatidic acid acyltransferase (also named 1-acyl-G — 3-P acyltransferase-AGAT) in the sn-2 position to yield phosphatidic acid (PA). This is followed by dephosphorylation of PA by phosphatidic acid phosphohydrolase (PAP) to release diacylglycerol (DAG). In the final step DAG is acylated either by diacylglycerol acyltransferase or phospholipid diacylglycerol acyltransferase to produce TAGs (Ratledge, 1988; Davies and Holdsworth, 1992; Athenstaedt and Daum, 1999; Mullner and Daum, 2004; Fakas et al, 2009b).

As far as the structure of the microbial TAGs produced is concerned, although their final composition could theoretically be a random substitution of acyl-CoA

groups into glycerol, in the case of the oleaginous microorganisms that have been examined, the glycerol sn-2 position is almost always occupied by unsaturated fatty acids [production of vegetable-type TAGs (see Ratledge, 1988; 1994; Guo and Ota, 2000)]. Therefore, various oleaginous microorganisms (principally yeasts belonging to the species Rhodosporidium toruloides, Apiotrichum curvatum and Y. lipolytica) have long been considered as promising candidates for the production of equivalents of exotic fats (fats that are principally saturated but containing unsaturated fatty acids esterified in the sn-2 glycerol position) (Moreton 1985, 1988; Moreton and Clode 1985; Ykema et al, 1989, 1990; Davies et al., 1990; Lipp and Anklam, 1998; Papanikolaou et al., 2001, 2003; Papanikolaou and Aggelis 2003b; Papanikolaou and Aggelis, 2010).