Efficient Biosynthesis of the Plant-Derived Diterpenoid Miltiradiene in Saccharomyces cerevisiae

Abietane-type diterpenoids, including tanshinones and triptolides, have excellent biological activities and broad clinical application values. Miltiradiene, a tricyclic diterpene, is a crucial intermediate in the biosynthesis of such diterpenes. Building an efficient platform for the synthesis of miltiradiene is crucial to provide sufficient precursors for the synthesis of downstream products. Here, we established a platform for the de novo production of miltiradiene using Saccharomyces cerevisiae BY4741 as the initial strain. First, the complete miltiradiene biosynthetic pathway was constructed by strengthening the mevalonate acetate pathway, in which the initial titer was 7.1 mg/L in shake flasks. Then, the combinations of miltiradiene synthases from different species were screened, and the site-directed mutagenesis and comprehensive enzyme engineering strategies were performed on the selected synthases to improve the titer of miltiradiene, which reached 297.8 mg/L. Furthermore, optimizing the supply of acetyl-CoA and enhancing product trafficking were applied to overcome the unbalanced metabolic networks and the pressure of product accumulation, which reached 649.3 mg/L. The engineered strain was capable of producing 6.4 g/L of miltiradiene in a 5 L bioreactor, which was the highest titer achieved to date. We expect that the engineered yeast strain constructed in this study will take the first step toward the sustainable, large-scale production of miltiradiene and its derivatives.