## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### Lipids
#### 3.1.2 Precursor Metabolites  
Apart from G3P and FFA, acetyl-CoA is also a key lipid biosynthetic precursor with which the _de novo_ FA synthesis starts [91]. In _Y. lipolytica_, acetyl-CoA can be generated in cytosol, mitochondria and peroxisome [92]. When _Y. lipolytica_ senses nutritional limitation (e.g., nitrogen starvation with an excess of glycerol), citric acid accumulates in mitochondria and then is secreted to the cytosol. Cytosolic citric acid is then converted to acetyl-CoA and oxaloacetate by ATP-citrate lyase (ACL) (Figure 1) [91], with the former providing the first two carbons for the _de novo_ FA synthesis. The FA chain is then being elongated via the addition of malonyl-CoA (endoplasmic reticulum) or acetyl-CoA units (mitochondria). Malonyl-CoA is generated by acyl-CoA carboxylase (_ACC1_), while the mitochondrial acetyl-CoA is generated by the breakdown of accumulated lipids via the \(\beta\)-oxidation pathway [93]. Though ACL converts citrate to acetyl-CoA for lipid accumulation, _Y. lipolytica_ secretes most of the citrate into the culture medium so that the _de novo_ lipids synthesis in wild type strains is limited, and most of them accumulate less than 20% \(w/w\) lipids [60]. In most oleaginous yeasts, ACL and malic enzyme (MAE) provide for FA synthesis, acetyl-CoA and NADPH, respectively. In the study of [PERSON] et al., the corresponding genes were inactivated in _Y. lipolytica_ W29 strains to analyze the role of these key enzymes in FA synthesis. Inactivation of _ACL1_ decreased FA synthesis by 60 to 80%, corroborating its key role in FA synthesis; while, the inactivation of _MAE1_ had no effect, excluding MAE as a major key enzyme for FA synthesis [94]. Similar conclusions were reported in the study of [PERSON] et al., who expressed in _Y. lipolytica_ an _ACL_ gene from _Mus musculus_ (domestic mouse) using the mono-copy pINA1312 sp and the multi-copy pINA1292 sp integration vectors. The authors chose this heterologous gene due to its low Km value for citrate (about 0.05 mM) after their observation that the affinity of ACL for citrate significantly differs among oleaginous and non-oleaginous microorganisms. In the oleaginous _Lipomyces starkeyi_, which reaches 65% lipid content, the Km value for citrate is 0.07 mM; however, in the non-oleaginous _Aspegillus niger_, a commercial citrate producer, Km value is 2.5 mM. In their experiments with _Y. lipolytica_, a high Km value (3.6 mM) was determined, probably explaining why _Yarrowia_ can produce both fatty acid and citrate, and why homologous overexpression of _ACL_ did not improve lipid accumulation. In addition, they supported the supposition that an ACL with a low Km value may be effective for cleaving citrate, as it would then provide more acetyl-CoA for FA synthesis rather than the majority of the citrate being secreted into the culture medium. The expression of _ACL_ gene from _Mus musculus_ enhanced lipid content from 7.3% to 11-23% \(w/w\) of the CDW, while growth was slightly affected. The amount of the secreted citrate decreased by 32%, in a transformant with the highest ACL activity, suggesting that a low Km greatly enhances acetyl-CoA formation for lipid accumulation in _Y. lipolytica_[60]. Even though many engineering efforts have focused on increasing the rate of FA synthesis and the supply of pathway precursors, none of them dealt with one major bottleneck of FA flux, i.e., the allosteric inhibition of FA biosynthetic enzymes by saturated FA. The work by [PERSON] et al., identified a novel metabolic regulator through the reverse engineering of mammalian fat-storing tissue, i.e., the delta-9 stearoyl-CoA desaturase (SCD). The overexpression of this regulator enriched the intracellular monosaturated FA which in turn promoted both cell growth and FA sequestrations into neutral lipids. Simultaneous overexpression of the lipogenic _SCD_, _ACC1_, and _DGA1_ in _Y.