Effects of The Constructed (+)-Limonene Synthesis Pathway in Sake Yeast on The Brewing Flavor of Sake

Author(s): Hao Xu, Nuo Xu, Yanhui Li, Jie Sun, Tingheng Zhu, Shijin Wu

Sake is a traditional alcoholic beverage in Japan, and the improvement of sake flavour and quality can be achieved by metabolic engineering modification of strains. Limonene, a terpenoid compound with an aromatic odour, has been detected in plant-based alcoholic products such as wine and brandy, and has flavour-modifying and bioactive effects. As limonene has not been investigated in sake, we used CRISPR/Cas9 technology to construct a limonene synthesis pathway in an industrial sake yeast strain and analysed the modification of sake flavour and composition. Eight strains with limonene yields ranging from 2 μg/L to 2.6 mg/L were obtained using the strategies of increasing the copy of the limonene expression cassette, weakening the competitive pathway, and enhancing the mevalonate (MVA) pathway. Comparative analyses of the sake brewing performance of the starting strain SY00 and the engineered bacteria with different limonene yields, as well as the composition of the sake samples, were carried out using gas chromatography-mass spectrometry (GC-MS) and other analytical methods. The result showed that the total amount of higher alcohols decreased, the phenylethanol content increased, and the flavour was significantly improved in the samples brewed with the limonene synthesising strains. Limonene was detected in the production of sake by two-phase fermentation of the engineered limonene producing strain SY08 (2.6 mg/L), but it was not detected in the final sake samples after fermentation, probably due to volatilisation. In conclusion, this study provides a new strategy for the construction of limonene producing strains by metabolic modification and its application to sake brewing.