Background: Limonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. The objective of this work was to engineer Rhodosporidium toruloides as a cell factory for the production of limonene. Results: By overexpressing the limonene synthase (LS), neryl pyrophosphate synthase (NPPS)/geranyl pyrophosphate synthase and the native hydroxy-methyl-glutaryl-CoA reductase (HMGR), we established a baseline for limonene production based on the mevalonate route in Rhodosporidium toruloides. To further enhance the limonene titer, the acetoacetyl-CoA thiolase/HMGR (EfMvaE) and mevalonate synthase (EfMvaS) from Enterococcus faecalis, the mevalonate kinase from Methanosarcina mazei (MmMK) and the chimeric enzyme NPPS-LS were introduced in the carotenogenesis-deficient strain. The resulting strains produced a maximum limonene titer of 393.5 mg/L. Conclusion: In this study, we successfully engineered the carotenogenesis yeast R. toruloides to produce limonene. This is the first report on engineering R. toruloides toward limonene production based on NPP and the fusion protein SltNPPS-CltLS. The results demonstrated that R. toruloides is viable for limonene production, which would provide insights into microbial production of valuable monoterpenes.

Background: Resveratrol is a plant-derived phenylpropanoid with diverse biological activities and pharmacological applications. Plant-based extraction could not satisfy ever-increasing market demand, while chemical synthesis is impeded by the existence of toxic impurities. Microbial production of resveratrol offers a promising alternative to plant- and chemical-based processes. The non-conventional oleaginous yeast Rhodotorula toruloides is a potential workhorse for the production of resveratrol that endowed with an efficient and intrinsic bifunctional phenylalanine/tyrosine ammonia-lyase (RtPAL) and malonyl-CoA pool, which may facilitate the resveratrol synthesis when properly rewired. Results: Resveratrol showed substantial stability and would not affect the R. toruloides growth during the yeast cultivation in flasks. The heterologus resveratrol biosynthesis pathway was established by introducing the 4-coumaroyl-CoA ligase (At4CL), and the stilbene synthase (VlSTS) from Arabidopsis thaliana and Vitis labrusca, respectively. Next, The resveratrol production was increased by 634% through employing the cinnamate-4-hydroxylase from A. thaliana (AtC4H), the fused protein At4CL::VlSTS, the cytochrome P450 reductase 2 from A. thaliana (AtATR2) and the endogenous cytochrome B5 of R. toruloides (RtCYB5). Then, the related endogenous pathways were optimized to affect a further 60% increase. Finally, the engineered strain produced a maximum titer of 125.2 mg/L resveratrol in YPD medium. Conclusion: The non-conventional oleaginous yeast R. toruloides was engineered for the first time to produce resveratrol. Protein fusion, co-factor channeling, and ARO4 and ARO7 overexpression were efficient for improving resveratrol production. The results demonstrated the potential of R. toruloides for resveratrol and other phenylpropanoids production.

Microbial lipids (MLs) are potential alternatives to vegetable oils and animal fats for production of biofuels and oleochemicals. It remains critical to improve ML production efficiency and costs for further commercial development. In the present study, we overexpressed a gene encoding phosphotransacetylase (Pta) in the oleaginous yeast Rhodosporidium toruloides for enhanced cell growth and lipid production. Compared with the parental strain R. toruloides NP11, the engineered strain NP-Pta-15 showed significant improvement in glucose consumption, cell growth and lipid accumulation when cultivated under nitrogen limited conditions in an Erlenmeyer flask as well as a stirred tank bioreactor. The introduction of Pta may establish an additional acetyl-CoA formation route by utilization of acetylphosphate. Our results should inspire more engineering efforts to facilitate the economic viability of ML technology.

Persimmon wine has various nutritional elements and high commercial potential. However, the high content of methanol, which is derived from the fruit’s pectin, always hinders persimmon wine production. To reduce the methanol level in the wine, the effects of persimmon cultivar, starter, pectinase, and pretreatment methods were investigated via single-factor and orthogonal experiments. The persimmon cultivar ‘MaoKui’ was finally used throughout the study owing to its lowest pectin concentration (24.5 g/kg). The best treatment conditions against the persimmon pulp were pectinase (0.04 g/kg) at 30 °C for 4 h, then boiled at 115 °C for 15 min before fermentation started. The optimized fermentation conditions for wine production were pectinase (0.03 g/kg), 250 mg/kg starter (BO213 and SPARK with equal amounts), at 28 °C for 6 d. The obtained wine had 77.7 mg/L methanol and a 68.4% raw juice yield. The fruit wine had 111.4 mg/L methanol and a 90.6 sensory evaluation score. Forty-nine volatile aromas were identified. Ethyl acetate content was the highest, followed by 3-methyl-1-butanol, 2,3-butanediol, and lactate ethyl ester. The persimmon wine had a unique style with transparent color, elegant aroma, and pure taste.