Abstract:[Background] Isoamyl alcohol is the main higher alcohol synthesized by yeast via the catabolic Ehrlich pathway or by anabolic amino acid metabolism during Chinese spirit fermentation, and the content of isoamyl alcohol influences the drinkability of Chinese spirit. [Objective] To analyze and compare the ability of yeasts isolated from fermented grains to synthesize isoamyl alcohol and reveal the synthetic pathways of isoamyl alcohol in the yeasts.[Methods] Isoamyl alcohol-synthesizing yeast strains were isolated from fermented grains of strong-aroma Chinese spirit, and their capability of synthesizing isoamyl alcohol at different growth phases was compared. Moreover, the synthetic pathways of isoamyl alcohol in these strains were detected based on the metabolism of its precursors. [Results] The capability of synthesizing isoamyl alcohol by the five yeast strains isolated from the fermented grains was in the order of Naumovozyma castellii JP3-1>Saccharomyces cerevisiae JP3>Pichia fermentans JP22>P. kudriavzevii JP1>Naumovozyma dairenensis CBS421. Overall, isoamyl alcohol was synthesized mainly at the logarithmic phase, and it was also synthesized at the stationary phase of N. castellii JP3-1, P. fermentans JP22, and N. dairenensis CBS421. In addition, it was synthesized via the Harris pathway in the whole growth periods of S. cerevisiae JP3, N. castellii JP3-1, and N. dairenensis CBS421 and via the Ehrlich pathway in the whole growth period of P. kudriavzevii JP1. At the logarithmic phase, the isoamyl alcohol-synthesizing ability of P. fermentans JP22 through Harris pathway was identical to that through the Ehrlich pathway. However, P. fermentans JP22 at the stationary phase mainly synthesized isoamyl alcohol via the Harris pathway. [Conclusion] This study revealed the relationship of the synthesis ability and pathways of isoamyl alcohol in five yeast strains isolated from fermented grains with their growth phase. The result lays a theoretical basis for analyzing the mechanism of isoamyl alcohol synthesis and accumulation in the fermentation process of strong-aroma Chinese spirit and the precisely regulating isoamyl alcohol synthesis during the fermentation of strong-aroma Chinese spirit.