Engineering Saccharomyces cerevisiae for production of the valuable monoterpene d-limonene during Chinese Baijiu fermentation. (1st July 2020)

• Record Type: Journal Article
• Authors: Hu, Zhihui; Lin, Liangcai; Li, Hongxuan; Li, Ping; Weng, Yanru; Zhang, Cuiying; Yu, Aiqun; Xiao, Dongguang
• Is Part Of: Journal of industrial microbiology & biotechnology. Volume 47:Number 6/7(2020)
• Page Start: 511
• Page End: 523
• Publication Date: 2020-07-01
• Subjects: d-Limonene -- d-Limonene synthase -- Neryl diphosphate synthase 1 -- CRISPR/Cas9 system -- Chinese baijiu -- Saccharomyces cerevisiae; Industrial microbiology -- Periodicals; 660.62
• DOI: 10.1007/s10295-020-02284-6
• Languages: English
• ISSNs: 1367-5435

Abstract

Abstract: d- Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d- limonene synthase (t LS ) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae . The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene ( ERG20 ) and its variant ERG20 F96W−N127W was strengthened for improvement d- limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (t NDPS1 ) from Solanum lycopersicum . The results showed that expressing ERG20 and ERG20 F96W−N127W could enhance d- limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d- limonene formation. Furthermore, we constructed a t LS –t NDPS1 fusion protein, and the best strain yielded 9.8 mg/L d- limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d- limonene content of 23.7 mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d- limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.