# Biosynthesis of Edible Terpenoids: Hosts and Applications
## 2 Microbial Chassis as Cell Factories for Edible Terpenoids
### Eukaryotic microorganisms
#### 2.2.4 Rhodosporidium toruloides  
_R. toruloides_ is a famous lipid yeast that can accumulate a large amount of lipids under nutrient-limited conditions. Its lipid level exceeds 70% of the dry cell weight [82], and its red phenotype is related to the natural ability to synthesize carotenoids. The high levels of lipids and carotenoids indicate that _R. toruloides_ has sufficient acetyl-CoA precursors, making it an ideal candidate for terpenoid cell factories. In addition, it also has advantages such as high-density growth, excellent stress resistance, and a wide substrate spectrum, allowing for large-scale fermentation using inexpensive raw materials [83]. Although it does not possess an efficient endogenous homologous recombination system like model microorganisms such as _S. cerevisiae_, lots of gene manipulation tools have been developed recently [84], enhancing the convenience and efficiency of homologous recombination significantly. Currently, different terpenoid compounds have been synthesized by _R. toruloides_. Apart from _R. toruloides_, other oleaginous red yeasts have also been modified to synthesize terpenoids successfully [85; 86].  
In order to directly utilize the excellent performance of _R. toruloides_ in accumulating carotenoids, [PERSON] et al. obtained a mutant strain, G17, with increased astaxanthin content by using gamma irradiation mutagenesis. They used response surface methodology to optimize the large-scale culture conditions, enabling _R. toruloides_ to produce astaxanthin with a content reaching 1.26 mg/L using molasses as a carbon source [67]. In the case of limonene, [PERSON] and co-workers introduced an orthogonal limonene synthesis pathway based on nervl pyrophosphate (NPP) into _R. toruloides_. They used carotenoid-deficient _R. toruloides_ as the host to block competing pathways, enhanced the MVA pathway flux, and simultaneously expressed the key enzyme nervl pyrophosphate synthase and limonene synthase for the downstream limonene synthesis pathway. The engineered strain achieved a final limonene yield of 393.5 mg/L [68]. Furthermore, they identified several peroxisomal targeting signals (PTSs) and used them to target MVA and limonene biosynthetic pathways to peroxisomes. Then, they carried out dual-metabolic regulation in the cytoplasm and peroxisome to enhance the supply of acetyl-CoA. A higher yield of limonene was finally reached at 1.05 g/L [69]. Similarly, [PERSON] et al. utilized metabolic engineering and Design-Build-Test-Learn (DBTL) strategies to introduce the ent-kaurene biosynthesis pathway and precursor GGPP supplying pathway into _R. toruloides_. The resultant strain obtained ent-kaurene at a titer of 1.4 g/L in a 2 L bioreactor feeding corn stover hydrolysates [70]. In order to further expand the application of _R. toruloides_ in the production of terpenoid compounds using corn stover hydrolysate, [PERSON] and colleagues introduced bisabolene synthase (BIS) and amorphadiene synthase (ADS) into _R. toruloides_ to produce bisabolol and amorphadiene, and also introduced GPPS and 1,8-cineole synthase to produce 1,8-cineole. Through metabolomic and proteomic analysis, they identified the rate-limiting steps and balanced the MVA pathway and downstream pathway. Finally, they obtained strains for bisabolene, amorphadiene, and 1,8-cineole with titers of 36 mg/L, 2.6 g/L, and 1.4 g/L, respectively [71; 72].