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.

Limonene is a valuable monoterpenoid with diverse applications in food, medicine, agriculture, etc. Currently, the commercial production of limonene from citrus pomace cannot meet the evergrowing market demand. With the development of synthetic biology, microbial biosynthesis is offering a sustainable alternative to plant-based extraction. However, the performance of engineered strains can be affected by many factors including the microbial host, the cultivation conditions, etc. Therefore, in the present study, potential factors that influence the limonene production of the engineered strains were investigated through single-factor and orthogonal experiments. The optimized medium was MM, with a working volume of 50 mL, inoculum OD₆₀₀ = 0.6, 22 °C, pH 6, and a dodecane coverage (v/v) of 20%. The final limonene titer was improved from 52.5 to 358.1 mg/L, a 586% net increase in titer and 34.8-fold improvement in production efficiency (mg/OD₆₀₀) on 250-mL flasks. The results here demonstrated that R. toruloides could be explored as an efficient limonene producer and could facilitate the study of the biosynthesis of other terpenes using R. toruloides.

De novo biosynthesis of (−)-limonene, (−)-carveol and (−)-carvone was achieved via rewiring the mevalonate pathway in the oleaginous yeast Rhodotorula toruloides. Strategies including enzyme selection, protein fusion, pathway optimization (MVA upstream enhancement, cofactor supply), and fermentation condition optimization were applied to improve the monoterpenoids production. Finally, the (−)-limonene titer of 1.6 g/L and (−)-carveol of 668.0 mg/L were achieved through fed-batch fermentation in a 3-L bioreactor. Furthermore, the feasibility of biosynthesis of (−)-carvone was also demonstrated. This study represents an example for de novo microbial production of (−)-limonene, (−)-carveol and (−)-carvone with the oleaginous red yeast R. toruloides, demonstrating its potential as a versatile chassis for monoterpenoid biosynthesis.

Limonene is a valuable monoterpenoid that with diverse applications. Microbial biosynthesis offers a sustainable alternative to plant-based extraction, which is prone to hinderance by climate change and acreage. In the present study, the effects on limonene production of carbon source, inoculum, working volume, temperature, pH, dodecane coverage, and media were investigated with engineered strains of Rhodosporidium toruloides. Through single-factor and orthogonal experiments, the limonene titer was improved from 52.2 to 358.1 mg/L on 250-mL flask, a 586% increase. The optimized medium was MM, with a working volume of 50 mL, inoculum OD600=0.6, 22 °C, pH 6, and dodecane coverage (v/v) of 20%. The results here demonstrated that the R. toruloides could be explored as an efficient limonene producer and facilitate other monoterpenoids biosynthesis with R. toruloides.