Improve the production of d-limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation. (1st December 2020)

• Record Type: Journal Article
• Authors: Hu, Zhihui; Li, Hongxuan; Weng, Yanru; Li, Ping; Zhang, Cuiying; Xiao, Dongguang
• Is Part Of: Journal of industrial microbiology & biotechnology. Volume 47:Number 12(2020)
• Page Start: 1083
• Page End: 1097
• Publication Date: 2020-12-01
• Subjects: d-Limonene -- Transporter proteins -- CRISPR/Cas9 system -- Saccharomyces cerevisiae -- Metabolic engineering; Industrial microbiology -- Periodicals; 660.62
• DOI: 10.1007/s10295-020-02329-w
• Languages: English
• ISSNs: 1367-5435

Abstract

Abstract: 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 (t NDPS1 ) and d- limonene synthase (t LS ) in Saccharomyces cerevisiae . To further increase d- limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes t HMGR1, 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 t HMGR1, 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.