## 2 Glycerol-Based Biomanufacturing Using _Yarrowia_  
In nature, glycerol is a constituent of phospholipids in cell membranes and of TAG found in storage lipids. Many microorganisms can naturally utilize glycerol as the sole carbon and energy source through enzymatic degradation catalyzed by native lipases. In particular, yeasts catabolize glycerol using two different pathways. The first is the phosphorylative glycerol catabolic pathway. In this, glycerol is phosphorylated to glycerol-3-phosphate (G3P) by a glycerol kinase (GK; _GUT1_ gene) and then converted to dihydroxy-acetone phosphate (DHAP) by a mitochondrial bound G3P dehydrogenase (mGPDH; _GUT2_ gene) which acts in conjunction with a freely dispersed cytosolic one (cGPDH; _GPD1,2_ gene) using FAD\({}^{+}\)/FADH\({}_{2}\) and NAD\({}^{+}\)/NADH as cofactors, respectively. The second pathway includes oxidation of glycerol to dihydroxyacetone (DHA) by an NAD\({}^{+}\)-dependent glycerol dehydrogenase (GDH; _GCY1_ gene) and, after this, phosphorylation to DHAPby a dihydroxyacetone kinase (DHAK; _DAK1_ gene) [26] (Figure 1). It was reported that overexpression of the _GUT1_ gene in _Y. lipolytica_ results in rapid glycerol assimilation, which is beneficial for downstream processing [27]. The higher degree of chemical reduction compared to e.g., glucose, sucrose, and starch, makes glycerol a suitable substitute for traditional carbon sources used in various biotechnological processes. [PERSON] et al., quantified the cellular performance during growth on glycerol, glucose or a mixture of both in 1 L batch cultivations. The growth rate was 0.24 h\({}^{-1}\) and 0.30 h\({}^{-1}\) when glucose or glycerol was the sole carbon, respectively, with the utilization of the latter causing higher oxygen uptake rates, indicating that the import mainly occurred via phosphorylation into G3P [14].  
Glycerol of abiotic origin is primarily generated from industrial soap and biodiesel manufacturing, where saponification and transesterification of animal and vegetable fats and oils takes place, respectively [26]. Biodiesel-derived glycerol is produced at a ratio of 1:10 crude glycerol to biodiesel, with a varying concentration of pure glycerol from 70 to 98%, and the remaining containing impurities such as fatty acid methyl esters (FAME), fatty acids (FA), methanol, water, soap and ash. Due to the increasingly frequent exploitation of inexpensive substrates for biodiesel production (e.g., waste frying oils), the surplus of glycerol further hampers the biodiesel market [28; 29; 30]. Since pure glycerol is laboriously  
Figure 1: Metabolic pathways and cellular compartments involved in glycerol and glucose metabolism, triacylglycerlecrol (TAG) biosynthesis and degradation. Yeasts catabolize glycerol using two pathways. In the phosphorylative glycerol catabolic pathway, glycerol is phosphorylated to glycerol-3-phosphate (G3P) by a glycerol kinase (GK; _GUT1_ gene) and then converted to dihydroxyacetone phosphate (DHAP) by a FAD\({}^{+}\)/FADH\({}_{2}\)-dependent mitochondrial bound G3P dehydrogenase (mGDPH; _GUT2_ gene). The second pathway includes oxidation of glycerol to dihydroxyacetone (DHA) by an NAD\({}^{+}\)-dependent glycerol dehydrogenase (GDH; _GCY1_ gene) and, after this, phosphorylation to DHAP by a dihydroxyacetone kinase (DHAK; _DAK1_ gene). The red arrows indicate deleted reactions while the green arrows indicate gene overexpression as discussed in this study. Abbreviations of metabolites are 6-PGL, 6-phosphogluconolactone; 6-TGA, 6-phosphogluconate; Ribu-5-P, ribulose-5-phosphate; Cit, citrate; Isocit, isocitrate; \(a\)-KG, \(a\)-ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; OAA, oxaloacetate; MAG, monoacylglycerolglycerol. Abbreviations of metabolic pathways are TCA, tricarboxylic acid cycle; MVA, mexolonate.  
obtained via an uneconomic purification process, many biodiesel companies are trying to establish new strategies to valorize this valuable feedstock [13; 31; 32; 33].