## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### Terpenoids  
_Y. lipolytica_ possesses a native cytosolic mexulonate pathway (MVA) which supplies the C5 isopenoid precursors for terpenoid biosynthesis, i.e., isopentenyl diphosphate (IPP) and dimethyllyl diphosphate (DMAPP). The synthesis of farnesyl diphosphate (FPP), the C15 direct precursor of sesquiterpenes, follows; thus, only one heterologous gene is required for sesquiterpene biosynthesis. Apart from this, and due to its oleaginous character, high culture density, and large intracellular acetyl-CoA pool, _Y. lipolytica_ is a promising sesquiterpene chassis producer. Farnesene is a sesquiterpene typically used as fragrance, flavor and vitamin E/K1 synthetic precursor. Farnesene biosynthesis has been widely studied in _E. coli_ and _S. cerevisiae_. Using engineered _S. cerevisiae_, Amyris produced 130 g L\({}^{-1}\)\(\beta\)-farnesene in industrial bioreactors [110]. Meanwhile, in _E. coli_, [PERSON] et al., reported _a_-farnesene production up to 1.1 g L\({}^{-1}\) in fed-batch fermentation, which is the highest production in _E. coli_ cells to date [111]. Using the engineered _Y. lipolytica_ strain P7, [PERSON] et al., produced approximately 55 mg L\({}^{-1}\)\(a\)-farnesene in shake flasks and 260 mg L\({}^{-1}\) in a bioreactor. Different overexpression vectors harboring combinations of truncated HMG-CoA reductase (_tHMG1_), TPP isomerase (_IDI_), FPP synthase (_ERG20_) and codon-optimized _a_-farnesene synthase (_OptFS_) genes were constructed and integrated into the genome of _Y. lipolytica_ Po1h (Figure 3). The engineered strain produced 57.08 mg L\({}^{-1}\)\(\alpha\)-farnesene, which is a 20.8-fold increase over the initial production of 2.75 mg L\({}^{-1}\) in YPD medium in shake flasks. Bioneactor scale-up in PM medium led to _a_-farnesene concentration of 260 mg L\({}^{-1}\) with _a_-farnesene to biomass ratio of 34 mg g\({}^{-1}\), which was a 94.5-fold increase over the initial production. This was the first report on _a_-farnesene synthesis in _Y. lipolytica_, albeit using glucose [112]. In their later work, a series of metabolic engineering strategies were employed to facilitate _a_-farnesene accumulation including optimization of the MVA pathway, engineering intracellular acetyl-CoA availability and promoter replacement, leading to engineered _Y. lipolytica_ strains producing up to 90 mg L\({}^{-1}\)_a_-farmesene in shake flask culture. The best metabolic engineered strain was used in shake flasks to determine the optimal carbon source, with glycerol also included (mannitol, glycerol, citric acid, maltose, sucrose, fructose, glucose). The results showed that _a_-farmesene production from glycerol was higher than that from any other carbon source reaching up to 90 mg L\({}^{-1}\). Therefore, glycerol was used for feeding after 48 h batch fermentation with glucose in 5 L bioreactors. _A_-farmesene production was significantly enhanced, reaching 2.57 g L\({}^{-1}\) final concentration and 34 mg g\({}^{-1}\) yield on biomass, more than 60% higher than those obtained when glucose was used for feeding [79]. It should be noted that the highest terpenoid titer reported in _Y. lipolytica_ is 25.55 g L\({}^{-1}\) after 288 h of fed-batch fermentation with glucose as initial and feeding carbon source. The engineering strategy applied in this case was based on the design of a highly efficient non homologous end-joining (NHEJ)-mediated genome integration, and the enhancement of the intracellular mevalonate synthesis [113]. _Y. lipolytica_ has also been used for limonene production, a valuable monocyclic monoterpene. Limonene biosynthesis has been widely studied in _E. coli_[114; 115] and _S. cerevisiae_[116; 117]. _Y. lipolytica_ possesses a native MVA pathway; however, it cannot produce limonene due to the lack of limonene synthase (LS). [PERSON] et al., constructed a limonene biosynthesis pathway in _Y. lipolytica_ and then further engineered this strain to achieve efficient production.