Construction of isopentenol utilization pathway and artificial multifunctional enzyme for miltiradiene synthesis in Saccharomyces cerevisiae.

Miltiradiene serves as a pivotal precursor for the synthesis of numerous abietane-type diterpenes with important pharmacological activities. The endogenous mevalonate (MVA) pathway is tightly regulated in Saccharomyces cerevisiae, which limits the availability of precursors for the heterologous production of miltiradiene. In this study, the orthogonal isopentenol utilization pathway (IUP) was constructed and investigated for its adaptability with mitochondria and peroxisomes in S. cerevisiae for the synthesis of miltiradiene. Compartments combinatorial engineering was used to enhance precursor supply and miltiradiene synthesis, thereby elevating the production of miltiradiene to 146.1 mg/L in S. cerevisiae. Furthermore, an artificial multifunctional enzyme, tSmCPS-tSmKSL-PvPT, was constructed by mimicking the natural multifunctional enzyme to enhance the biosynthesis of miltiradiene in S. cerevisiae strain PCM-MT1, which is capable of producing 414.4 mg/L miltiradiene. Finally, the titer of miltiradiene was increased to 1.02 g/L by fed-batch fermentation in a 5 L bioreactor. This study broadens the application of the IUP in S. cerevisiae by integrating compartmentalization and artificial multifunctional enzymes for the synthesis of diterpenes.