Abstract：Cembratriene-ol is a biodegradable green pesticide component that has significant application value in sustainable agriculture. However, the chemical synthesis of this substance is inefficient, and the extraction of the substance from plants is challenging. The precise mechanisms of biosynthetic processes and the identification of key intermediate metabolites remain to be elucidated. In this study, a multi-gene integration expression strategy was employed to heterologously express the geranylgeranyl diphosphate synthase (crtE) from Pantoea ananatis and cembratriene-ol synthase (cbts) from Nicotiana tabacum in Escherichia coli, while integrating the isopentenol utilization pathway (IUP) by introducing the choline kinase gene (chk) from Saccharomyces cerevisiae, the isopentenyl phosphate kinase gene (ipk), and E. coli's isopentenyl diphosphate isomerase gene (idi) to enhance the supply of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). SDS-PAGE analysis confirmed that the successful expression of all 5 target proteins in E. coli, with observed molecular weights aligning precisely with theoretical predictions. GC-MS analysis demonstrated the successful establishment of a heterologous E. coli synthesis pathway from isopentenol to cembratriene-ol, yielding a target product of 19.7 mg/L. Furthermore, the potential intermediate for cembratriene-ol synthesis from geranylgeranyl pyrophosphate (GGPP) was identified as cembrene, marking a significant discovery as it was the first time this intermediate was identified. This study provides fundamental evidence for elucidating the biosynthetic mechanism of cembratriene-ol, optimising its microbial synthesis pathway to promote the industrialisation of green pesticides, and also offers basic data for heterologous biosynthesis research on agricultural specialty secondary metabolites.
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