Modular Pathway Engineering (MOPE) in Yeast for Diterpenoids

Nature has developed two chemical strategies for the cyclization step in isoprenoid production. In one strategy, the cyclization reaction is catalyzed by a bifunctional enzyme; in the other strategy, it is catalyzed by two consecutive enzymes involving a stable intermediate. To facilitate a heterogeneous production of phytoterpenoids in microorganisms, strategies remain to be developed to reduce diffusion, degradation and deviation of intermediates along their biosynthetic pathway. In the cases of miltiradiene, a key precursor to the diterpenoid Tashionones in the Chinese medicinal herb Salvia miltiorrhiza, two enzymes, copalyl diphosphate synthase (SmCPS) and kaurene synthase-like (SmKSL), have been identified. In the present study, we realized an effective production of miltiradiene in Saccharomyces cerevisiae at a concentration of 178 mg/L upon rapid construction of rationally designed pathways by the modular pathway engineering (MOPE) strategy. In particular, we found that the pathway with independently expressed SmCPS and SmKSL was less efficient than those with a fusion protein, and that the pathway with the SmKSL-SmCPS fusion brought two active sites closer than the SmCPS-SmKSL fusion led to substantially higher miltiradiene production. Moreover, the fusion of two key endogenous protein (E,E,E)-geranylgeranyl diphosphate synthase (BTS1) and farnesyl diphosphate (FPP) synthase (ERG20) led to reduced cellular by-product farnesol level and improved the efficiency of miltiradiene production. Lastly, we constructed a super producer for another diterpenoid, sclareol, and confirmed the generality of our strategy. Our results suggested that the diterpene synthase engineering was key to efficient heterologous production, and that the MOPE strategy was a powerful tool for constructing pathways involving multiple genes.