## 2 Glycerol-Based Biomanufacturing Using _Yarrowia_
### Bioconversion of Glycerol to Lipids  
2.1.1. Lipogenesis in Oleaginous Microorganisms  
Oleaginous microorganisms utilize both hydrophilic and hydrophobic substrates and accumulate lipids via two different pathways i.e., _de novo_ by metabolizing hydrophilic substrates and _ex novo_ by fermenting hydrophobic substrates. The _de novo_ FA biosynthesis involves the synthesis of FA precursors, such as acetyl-CoA and malonyl-CoA, their elongation, and finally their integration into the Kennedy biosynthetic pathway to form TAG and SE (Figure 1). In yeast, the _de novo_ FA synthesis is catalyzed by the cytosolic multidomain FA synthase and the products consist mainly of C16 and C18 FAs that supply the physiological requirements for membrane phospholipid formation [34]. The Kennedy pathway converts diacylglycerols (DAG) into TAG either with phospholipids by the phospholipid:diacylglycerolglycerol acyltransferase (PDAT) or with acyl-CoA by diacylglycerol acyltransferase type 1/2 (DGAT1/2), whereas SEs are formed from acyl-CoA by acyl-CoA:sterol acyltransferases (ASAT) [35]. The Kennedy pathway starts with G3P, which is mainly formed by DHAP reduction. G3P is acylated by G3P acyltransferase (GPAT) to lysophosphatidic acid (LPA), after which a second acyl-CoA provides a FA chain to form phosphatidic acid (PA). PA is then dephosphorylated by a phosphatidic acid phosphatase (PAP) to release DAG. Finally, DAG is acylated either by DAG acyltransferase (DGAT1 with acyl-CoA as an acyl donor) or by phospholipid:DAG acyltransferase (LRO1 with glycerophospholipids as an acyl donor) to produce TAG (Figure 1) [36]. On the other hand, the _ex novo_ pathway takes up and transports FA, oils, and TAG from the culture medium, and stores them intracellularly either intact or modified. Due to lipotoxicity which can be caused by the accumulation of FFA and sterols, cells have developed mechanisms to store these lipids in their inert forms as TAG and SE, respectively, in the specialized LB [8; 37; 38; 39]. More than 90% of _Y. lipolytica_ lipids accumulate in TAG synthesized via the _de novo_ biosynthetic pathway. In oleaginous microorganisms, _de novo_ lipogenesis and accumulation is generally induced when an element in the culture medium becomes limiting and the present carbon source is in excess. The most efficient way to control and induce lipid accumulation is that of nitrogen limitation. When _Y. lipolytica_ cells sense nitrogen limitation in their environment, the growth rate rapidly decelerates, while the rate of carbon assimilation gradually decreases, redirecting the carbon flux into lipid synthesis and TAG accumulation within LB [40]. Briefly, under nitrogen limitation, a rapid decrease of intracellular adenosine monophosphate (AMP) takes place due to the activity of the AMP deaminase (AMPD), which temporarily provides NH\({}_{4}\)+. Due to the removal of the AMP allosteric activation, the NAD\({}^{+}\) dependent isocitrate dehydrogenase, which converts isocitric acid to _a_-ketoglutate in the TCA cycle, loses its activity, thereby leading to intra-mitochondrial citric acid accumulation. When the concentration of this intra-mitochondrial citric acid exceeds a critical value, it is secreted into the cytosol. In oleaginous yeasts, cytosolic citric acid is cleaved by ATP-citrate lyase (ACL, encoded by _ACL1_ and _ACL2_ genes), a key enzyme for FA biosynthesis, into acetyl-CoA and oxaloacetate, with acetyl-CoA being converted by quasi-inverted \(\beta\)-oxidation to FA [41; 42]. The FA biosynthesis continues by acetyl-CoA carboxylase (ACC1), which carboxylates acetyl-CoA to malonyl-CoA, and by malic enzyme (MAE) or the pentose phosphate pathway (PPP), both of which generate the required NADPH (Figure 1) [43; 44]. Acetyl-CoA and malonyl-CoA are condensed into FA (Claisen condensation), which are then activated to fatty-acyl-CoA, which is responsible for the acylation of the glycerol backbone to produce TAG [45]. _Y.