Improve the production of d -limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic
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METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY - ORIGINAL PAPER
Improve the production of d‑limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation Zhihui Hu1 · Hongxuan Li1 · Yanru Weng1 · Ping Li1 · Cuiying Zhang1 · Dongguang Xiao1 Received: 24 July 2020 / Accepted: 2 November 2020 © Society for Industrial Microbiology and Biotechnology 2020
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 IDI1WWW, 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 IDI1WWW 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. Keywords d-Limonene · Transporter proteins · CRISPR/Cas9 system · Saccharomyces cerevisiae · Metabolic engineering
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10295-020-02329-w) contains supplementary material, which is available to authorized users. * Cuiying Zhang [email protected] * Dongguang Xiao [email protected] 1
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, Tianjin 300457, People’s Republic of China
China is a big country in the production and consumption of alcoholic beverages. With the development of the economy and t
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