Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways enables high-level production of miltiradiene in yeast

Miltiradiene is a key intermediate in the biosynthesis of many important natural diterpene compounds with significant pharmacological activity, including triptolide, tanshinones, carnosic acid and carnosol. Sufficient accumulation of miltiradiene is vital for the production of these medicinal compounds. In this study, comprehensive engineering strategies were applied to construct a high-yielding miltiradiene producing yeast strain. First, a chassis strain that can accumulate 2.1 g L-1 geranylgeraniol was constructed. Then, diterpene synthases from various species were evaluated for their ability to produce miltiradiene, and a chimeric miltiradiene synthase, consisting of class II diterpene synthase (di-TPS) CfTPS1 from Coleus forskohlii (Plectranthus barbatus) and class I di-TPS SmKSL1 from Salvia miltiorrhiza showed the highest efficiency in the conversion of GGPP to miltiradiene in yeast. Moreover, the miltiradiene yield was further improved by protein modification, which resulted in a final yield of 550.7 mg L-1 in shake flasks and 3.5 g L-1 in a 5-L bioreactor. This work offers an efficient and green process for the production of the important intermediate miltiradiene, and lays a foundation for further pathway reconstruction and the biotechnological production of valuable natural diterpenes.