# Biosynthesis of Edible Terpenoids: Hosts and Applications
## 2 Microbial Chassis as Cell Factories for Edible Terpenoids
### Eukaryotic microorganisms
#### 2.2.2 _Yarrowia lipolytica_  
In order to avoid the overflow of carbon metabolism and generation of by-product ethanol, scientists have focused their attention on Crabtree-negative species. _Y. lipolytica_, as a model strain of oleaginous yeasts, is Crabtree-negative and GRAS [78]. It possesses high levels of the tricarboxylic acid (TCA) cycle and the pentose phosphate (PP) pathway. Through these two pathways, a large amount of acetyl-CoA, ATP, and NADPH can be accumulated, making it a promising and superior chassis for terpenoid compounds [79].  
The level of precursors IPP and DMAPP determines the yield of subsequent metabolites. To increase the precursor level, key enzymes CHK and IPK in the IUP pathway were introduced into _Y. lipolytica_, resulting in a 15.7-fold increase in IPP and DMAPP. By overexpressing ACC1 and DGA1 to promote lipid accumulation, the intracellular hydrophobicity was modulated, thereby increasing the storage capacity for lycopene. With exogenous palmitic acid supplementation, the degradation blocks acetyl-CoA from \(\beta\)-oxidation further enhanced the natural MVA pathway. The final lycopene titer was 4.2 g/L in 3 L batch bioreactors [55]. This indicates that the hydrophobic environment of oleaginous yeast is beneficial for the accumulation of terpenoids. In geraniol production, [PERSON] et al. expressed the truncated plant-derived geraniol synthase (tCrGES) in _Y. lipolytica_ and then integrated ERG10, HMGS, tHMG1, and IDI into the chromosome to enhance precursor GPP supply. The resulting strain S5 produced geraniol with a titer of 1 g/L shake-flask fermentation [56]. [PERSON] et al. co-expressed the key enzymes tSsLPs and _Ss_SCS of sclareol synthesis pathway in _Y. lipolytica_ with tHMG1 and ERG20 overexpression, enabling the host to synthesize sclareol. They further designed a two-layer GGPP accumulation pathway, introducing _Pa_GGGPPS and _Ss_GGPPS to simultaneously utilize IPP, DMAPP, and FPP to increase GGPP supply. Then, by using protein scaffold peptide tags RIAD and RIDD, they assembled tSsLPS and _Ss_SCS into multi-enzyme complexes to facilitate substrate transport and reduce the production of by-products geranylgeraniol (GGOH) and labdendiol (LOH). The engineered strain produced sclareol as much as 12.9 g/L [57]. To facilitate genetic modification in _Y. lipolytica_, [PERSON] and co-workers knocked out the non-homologous end joining protein Ku70 and co-expressed the key homologous recombination proteins RAD52 and RAD59, achieving a homologous recombination efficiency of 68% without affecting the growth. They also systematically reshaped metabolism by enhancing MVA pathway flux, increasing the supply of acetyl-CoA and NADPH, and weakening the pathway for squalene degradation to ergosterol. In a 5 L fed-batch fermentation, the engineered strain achieved a squalene titer of 35 g/L [58]. In another case, [PERSON] et al. transformed a carotenoid cleavage dioxygenase (_Of_CCD1) with a strong multi-round promoter iteratively into _Y. lipolytica_ to construct a \(\beta\)-ionone-producing strain. Then, they further broaden the substrate range by overexpressing the key enzymes for xylose metabolism, xylose reductase (_Ss_XR), xylitol dehydrogenase (_Ss_XDH), and endogenous xylulose kinase (XK). The final engineered strain could synthesize \(\beta\)-ionone using lignocellulosic hydrolysate as the carbon source [59]. Itindicates that _Y. lipolytica_ has the potential to utilize low-cost raw materials for the green production of high-value terpenoids.