Improve the production of d-limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation

Author:

Hu Zhihui1,Li Hongxuan1,Weng Yanru1,Li Ping1,Zhang Cuiying1,Xiao Dongguang1

Affiliation:

1. grid.413109.e 0000 0000 9735 6249 Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process Control, Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology Tianjin University of Science and Technology 300457 Tianjin People’s Republic of China

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 (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.

Funder

National Basic Research Program of China (973 Program)

Publisher

Oxford University Press (OUP)

Subject

Applied Microbiology and Biotechnology,Biotechnology,Bioengineering

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