## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### Lipids
#### 3.1.1 TAG Biosynthetic Pathway  
[PERSON] et al., redirected carbon flux toward lipid synthesis in _Y. lipolytica_ W29 by inactivating the oxidation of G3P into DHAP (_GLT_2 deletion), thus increasing G3P availability for lipogenesis via the Kennedy pathway that synthesizes TAG by an initial acclimation of the G3P backbone with an acyl-CoA (Figure 1). The additional deletion of _POXI_-6 genes (\(\Delta\)_pox1_-6) which catalyze the limiting step of peroxisomal \(\beta\)-oxidation led to a four-fold increase in lipid content, reaching 42% of CDW. However, the \(\Delta\)_gut2_ strain did not grow on glycerol as the sole carbon source, probably because it is not likely that another enzyme catalyzes DHAP formation from G3P in glycerol medium [87]. Tofurther investigate the role of the G3P shuttle in lipid accumulation, [PERSON] et al., used different mutations in the G3P shuttle pathway (_GPD1_ overexpression or _GUT2_ deletion) to manipulate the G3P concentration. They showed that in a Po1d genetic background (Leu' Ura), _GPD1_ overexpression, _GUT2_ inactivation or both mutations together increased G3P 1.5-, 2.9-, and 5.6-fold, respectively, increasing TAG accumulation. They also showed that each strain with an increased G3P concentration also had a decreased glycerol concentration. Analysis of the genes involved in glycerol metabolism revealed that _Y. lipolytica_ does not possess a gene for glycerol-3-phosphatase (_GPP1_, _GPP2_), thus indicating that G3P synthesis competes with glycerol synthesis and that a distinctive glycerol metabolism is committed to G3P and TAG synthesis, possibly contributing to its oleaginous character. Similarly, G3P shuttle dysfunctions coupled to an inactive \(\beta\)-oxidation pathway (deletion of _POX1-6_ or _MFE1_ genes) increased TAG and FFA. Transcriptional analysis of these strains revealed that the high levels of lipids (up to 65-75% of CDW) resulted from overexpression of genes involved in TAG synthesis (_SCT1_ encoding G3P acyltransferase, _DGA1_ encoding acyl-CoA diacylglycerol acyltransferase) and the repression of genes involved in TAG degradation (_TGL3_ and _TGL4_) (Figure 1). Their findings suggest that the availability of G3P and FFA limits TAG synthesis, and that the G3P shuttle and the \(\beta\)-oxidation pathway regulate the expression of genes involved in TAG homeostasis. They conclude that the synergistic contribution of acyltransferase gene expression to G3P synthesis is required for high levels of TAGs and lipid accumulation in _Y. lipolytica_[88]. The amplification of FFA production was also achieved by engineering _Y. lipolytica_ W29 to uncouple the G3P shuttle and eliminate glycerol metabolism. Under lipogenic conditions with a limiting supply of glycerol, the triple mutant strain \(\Delta\)_gpd1\(\Delta\)gut2\(\Delta\)per10_ redirected the carbon influx towards the synthesis of unsaturated FA that accumulated in the culture medium up to 2.03 g/L [89]. For additional reading regarding TAG biosynthesis through esterification of FA to glycerol backbone in which two precursors, FA and G3P, are required, the review by [PERSON] et al., is strongly recommended. This review describes the interplay among glycerol metabolism, G3P synthesis and lipid production shedding light on how G3P affects lipid production by modulating glycerol metabolism [90]. In the study conducted by [PERSON] et al., _Y. lipolytica_ JMY3580 was constructed by over-expressing the _DGA2_ gene in the previously engineered quadruple _Adaq2 Aldga2 Alro1 Aare1_ strain to increase lipid accumulation. In _Y. lipolytica_, three diacylglycerol acyltransferases encoded by _LRO1_, _DGA1_, and _DGA2_ genes are involved in lipid formation (Figure 1). Mutant strains were successively obtained by disrupting one or more genes in the wild-type Po1d strain to construct \(\Delta\)_tro1_, \(\Delta\)_dga1_, \(\Delta\)_are1_, \(\Delta\)_dga2_, \(\Delta\)_dga2_are1_, \(\Delta\)_dga1_\(\Delta\)_tro1_are1_, and the quadruple \(\Delta\)_dga1\(\Delta\)dga2 Alro1 Aare1_.