d-Limonene, a cyclized monoterpene, possesses citrus-like olfactory property and multi-physiological functions, which can be used as a bioactive compound and flavor to improve the overall quality of alcoholic beverages. In our previous study, we established an orthogonal pathway of d-limonene synthesis by introducing neryl diphosphate synthase 1 (tNDPS1) and d-limonene synthase (tLS) in Saccharomyces cerevisiae. To further increase d-limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes tHMGR1, ERG12, IDI1, and IDI1^WWW, respectively, or co-overexpressing. The results showed that strengthening the MVA pathway significantly improved d-limonene production, while the best strain yielded 62.31 mg/L d-limonene by co-expressing tHMGR1, ERG12, and IDI1^WWW genes in alcoholic beverages. Furthermore, we also studied the effect of enhancing the MVA pathway on the growth and fermentation of engineered yeasts during alcoholic beverage fermentation. Besides, to further resolve the problem of yeast growth inhibition, we separately investigated transporter proteins of the high-yielding d-limonene yeasts and the parental strain under the stress of different d-limonene concentration, suggesting that the transporters of Aus1p, Pdr18p, Pdr5p, Pdr3p, Pdr11p, Pdr15p, Tpo1p, and Ste6p might play a more critical role in alleviating cytotoxicity and improving the tolerance to d-limonene. Finally, we verified the functions of three transporter proteins, finding that the transporter of Aus1p failed to transport d-limonene, and the others (Pdr5p and Pdr15p) could improve the tolerance of yeast to d-limonene. This study provided a valuable platform for other monoterpenes’ biosynthesis in yeast during alcoholic beverage fermentation.

d-柠檬烯是一种环化的单萜，具有类似柑橘的嗅觉特性和多种生理功能，可以用作生物活性化合物和调味剂，以改善酒精饮料的整体质量。在我们以前的研究中，我们建立的正交途径D-通过引入橙花基二磷酸合成酶1苎烯合成（吨NDPS1）和D-柠檬烯合酶（吨LS）在酿酒酵母。为了进一步增加丁香烯的形成，甲羟戊酸（MVA）途径的代谢通量通过过表达关键基因t HMGR1，ERG12，IDI1和IDI1来增强^WWW或共同过表达。结果表明，加强MVA途径可显着提高d-柠檬烯的产量，而最佳菌株可通过在酒精饮料中共表达t HMGR1，ERG12和IDI1 ^WWW基因产生62.31 mg / L d-柠檬烯。此外，我们还研究了在酒精饮料发酵过程中增强MVA途径对工程酵母生长和发酵的影响。此外，为进一步解决酵母生长抑制问题，我们分别研究了高产d-柠檬烯酵母和亲本菌株在不同d-胁迫下的转运蛋白。柠檬烯浓度，表明Aus1p，Pdr18p，Pdr5p，Pdr3p，Pdr11p，Pdr15p，Tpo1p和Ste6p的转运蛋白在减轻细胞毒性和提高对d-柠檬烯的耐受性方面可能起更关键的作用。最后，我们验证了三种转运蛋白的功能，发现Aus1p转运蛋白未能转运d-柠檬烯，而其他转运蛋白（Pdr5p和Pdr15p）可以提高酵母对d-柠檬烯的耐受性。该研究为酒精饮料发酵过程中酵母中其他单萜的生物合成提供了有价值的平台。