To resolve the mechanistic research gaps of biflorane formation, we sought to perform extensive labeling studies. We first proposed two possible cyclization cascades of PcTS1 (Supplementary Figure ). Initially, GGPP is isomerized to geranyllinalyl diphosphate (GLPP) or 2Z-GGPP within the enzyme's pocket, resulting in the formation of a 2Z double bond, which is crucial for the subsequent cyclization steps. This isomerization step is consistent with mechanisms observed in other TSs, such as CpPS and EcTPS1 . In the first pathway (pathway I), a 1,10-cyclization is followed by either a 1,3-hydride shift or two 1,2-hydride shifts, leading to a 1,6-ring closure. This is then followed by another set of hydride shifts (either a 1,3-hydride shift or two 1,2-hydride shifts) to form intermediate carbocation, culminating in deprotonation. Conversely, in the second pathway (pathway II), the process begins with a 1,6-cyclization, followed by a 1,10 cyclization (Supplementary Figure ). We first investigated the isomerization hypothesis pathway for the formation of the 2Z double bond. GGPP, GLPP, and 2Z-GGPP were chemically synthesized (Supplementary Figure ) and incubated with purified PcTS1 (Supplementary Figure ) in vitro. The results revealed that both GGPP and GLPP yielded the same compound 3 (Fig. ). However, PcTS1 failed to cyclize 2Z-GGPP into compound 3, in contrast to MicA, which can catalyze the cyclization of both GLPP and 2Z-GGPP to form the eunicellane compound microeunicellene . The incapacity of PcTS1 to catalyze the cyclization of 2Z-GGPP may be attributed to its inability to recognize 2Z-GGPP as a substrate or to initiate carbocation formation by removing the pyrophosphate group.