Biotransformation of natural products to the natural flavoring, gamma-decalactone (GDL), has attracted con-siderable attention. However, improving its yield is challenging due to its high feedback inhibition of yeast cells, which lowers the productivity of the biotransformation process. In this study, we compared two in situ separation processes established by adding either resin (HZ-816) or cyclopentasiloxane (DC345) to a biotransformation medium and investigated their efficiency and effect on yeast metabolism. Compared with a control, yields from the medium with HZ-816 and DC345 increased by 140% and 175%, respectively. However, after 84 h of biotransformation, the protein leakage in the medium with HZ-816 and DC345 was respectively 2.04 times and 1.43 times that of the control. Meanwhile, the mortality of yeast cells was 32.8% and 24.0% in the medium with HZ-816 and DC345, respectively, whereas that in the control was 20.1%. Our findings indicate that a cyclase is involved in the final step of the biotransformation. The activity of the yeast cyclase in the DC345 system was 3.33 times greater than that in the HZ-816 system. The DC345 system was superior to the HZ-816 resin system in this separation process because its yield was 30.8% greater and it had less cellular damage. Thus, we showed that the DC345 system has potential as a new separation system for the production of GDL by biotransformation.