# Biosynthesis of Edible Terpenoids: Hosts and Applications
## 2 Microbial Chassis as Cell Factories for Edible Terpenoids
### Eukaryotic microorganisms
#### 2.2.3 Methylotrophic Yeasts  
Methylotrophic microorganisms can utilize one-carbon compounds as a carbon source for growth. _Ogataea polymorpha_ and _Pichia pastoris_, which are Crabtree-negative, have been receiving wide attention as hosts due to their ability to use methanol as a carbon and energy source. They possess high protein expression and secretion capabilities and allow for high-density fermentation [80]. The properties make them beneficial for biomanufacturing processes and widely used for efficient protein production. Since they are GRAS species, enzymes produced by these two yeasts, such as lipases, have been used as enzyme preparations in the food industry [81] already. Their robust five-carbon assimilation capacity makes them potential hosts to use lignocellulosic hydrolyses as raw materials. In addition, their endogenous MVA pathway can provide abundant precursors IPP and DMAPP for terpenoids, indicating that _O. polymorpha_ and _P. pastoris_ hold significant potential as cell factories for terpenoids [80].  
[PERSON] et al. screened a farnesene synthase (_Aa_FS) with high activity and introduced it into the fatty acid-producing _O. polymorpha_ strain M16 in their previous research. By further increasing the flux of the MVA pathway and the supply of precursor acetyl-CoA, the optimized engineering strain produced \(\beta\)-farnesene with a yield of 46 mg/g methanol, which is the highest reported production from one carbon [69]. [PERSON] and co-authors identified an efficient germacrene A synthase, _Ls_LTC2, in _O. polymorpha_. Furthermore, they enhance the acetyl-CoA, MVA, and NADPH flux and downregulate the sterol competition flux. Through spontaneous Cope rearrangement from germacrene A. They obtained \(\beta\)-elemene at a titer of 4.7 g/L in the fed-batch fermentation [61].  
In another methylotrophic yeast, _P. pastoris_, researchers have successfully constructed cell factories capable of producing terpenoid compounds such as germacrene A, santalene, and lycopene by introducing corresponding terpene synthases and performing conventional metabolic engineering [62; 63; 64]. In the case of producing \(\alpha\)-farnesene, [PERSON] et al. adopted the heterologous IUP pathway and a peroxisome-cytoplasm dual regulation strategy to enhance the \(\alpha\)-farnesene synthetic flux. Firstly, they screened and utilized a high-activity farnesene synthase (_MdA_FS) and strengthened the MVA and acetyl-CoA pathway. Secondly, they targeted the more efficient IUP pathway to peroxisomes to promote the accumulation of IPP and DMAPP. Finally, by simultaneously regulating the IPP and \(\alpha\)-farnesene synthetic pathway in the cytoplasm and peroxisomes, they obtained 1.4 g/L of \(\alpha\)-farnesene in the medium co-feeding with oleic acid and sorbitol as carbon sources [65]. Peroxisome compartmentalization not only alleviates the burden on growth caused by the accumulation of cytotoxic intermediates such as IPP but also separates IPP from competitive pathways in the cytoplasm and has now been used for the construction of various cell factories. In another case, _P. pastoris_ has been used to synthesize the precursor of a more complex ginseng precursor, dammarenediol-II. To facilitate the transfer of substrates and alleviate the interference from competing ergosterol pathway, researchers expressed squalene epoxidase (ERG1) and dammarenediol synthase (_Pg_PDD) separately using PDZ domain and its corresponding ligand (PDZlig), respectively. Ultimately, the yield of dammarenediol-II in the self-assembling multi-enzyme complex strain was 2.1-fold higher than that of the free expression strain [66].