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.
J Ind Microbiol Biotechnol. 2020 Jul;47(6-7):511-523. doi: 10.1007/s10295-020-02284-6. Epub 2020 Jun 3.
d-Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d-limonene synthase (tLS) 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 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 (tNDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20 could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a tLS-tNDPS1 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.
柠檬烯是一种环状单萜，具有柑橘般的气味特性和多种生理功能。本研究对来源于柠檬的柠檬烯合酶（tLS）进行密码子优化，并在酿酒酵母中异源表达。通过过表达内源性牻牛儿基二磷酸合酶基因（ERG20）及其变体 ERG20，强化了基于经典途径的代谢通量，以提高中国白酒中柠檬烯的产量。为了进一步提高产量，我们通过引入番茄中的香叶基二磷酸合酶 1（tNDPS1）建立了正交途径。结果表明，表达 ERG20 和 ERG20 可以增强柠檬烯的合成，而表达异源 NPP 合酶基因则显著增加了柠檬烯的形成。此外，我们构建了 tLS-tNDPS1 融合蛋白，通过优化氨基酸接头和融合顺序，最佳菌株在白酒发酵过程中产生了 9.8mg/L 的柠檬烯，比游离酶提高了 40%。最后，在优化的发酵条件下，菌株 AY12α-L9 中柠檬烯的最大含量达到 23.7mg/L，这是中国白酒中报道的最高产量。此外，我们还研究了柠檬烯浓度对酵母生长和发酵的影响。本研究为中国白酒发酵中其他有价值的单萜类生物合成提供了有意义的见解。
