Glaucocalyxin A (GLA), a well-known ent-kaurene diterpenoid, has emerged as a high-value bioactive natural product in pharmaceutical research owing to its potent and broad-spectrum activities. To date, its total synthesis has proven extremely challenging due to poor yield and intricate routes, and the biosynthetic pathway of GLA remains poorly understood. Herein, we identified two enzymes, IamCYP71D761 and IamCYP706V18, in Isodon amethystoides, which specifically mediate the C3α- and C15β-hydroxylation of ent-kaurene with high stereoselectivity and regioselectivity, respectively, generating 3α,15β-dihydroxy-ent-kaurene (3), a key precursor in the biosynthesis of GLA. In addition, we established an efficient de novo biosynthesis platform for 3 in Saccharomyces cerevisiae. Briefly, high-efficiency isozyme screening, protein engineering, increased acetyl-CoA synthesis, and copy number enhancement were applied to the ent-kaurene biosynthesis module. Subsequently, promoter optimization, competitive pathway knockout, and electron transfer optimization were introduced, resulting in production of 3 to 24.0 ± 0.2 mg/L in shake flask. In summary, this work highlights the pivotal roles of IamCYP71D761 and IamCYP706V18 in the heterologous biosynthesis of 3, offering valuable insights for the further pathway reconstruction and de novo production of GLA, GLB, and other ent-kaurene diterpenoids.
Yanying Chen, Xinqi Song, Ya-Nan Wang et al.· Synthetic and Systems Biotec...· 0 citations
Forskolin, a labdane-type diterpenoid isolated from Coleus forskohlii, exhibits therapeutic potential for osteoporosis, cardiovascular diseases, and metabolic syndrome. Its rising nutraceutical demand and limited natural availability have driven synthetic biology approaches for sustainable production. Although significant efforts have been devoted to upstream pathway optimization, the improvement of the downstream pathway still faces challenges due to the complex metabolic network and the low catalytic activity of cytochrome P450s (P450s). In this study, we elucidated the biosynthetic network involved in forskolin production, in which three P450s mediate multi-site oxidation, providing critical pathway insights for forskolin biosynthesis. Based on this, we reconstructed an efficient biosynthetic pathway of forskolin in yeast and subsequently optimized its production efficiency through multidimensional engineering strategies including central carbon flux optimization, rate-limiting enzyme engineering, P450 electron transfer chain reinforcement, and fermentation optimization. The final strain achieved the production of 2.7 g/L forskolin in a 5-L bioreactor, which represents the highest titer reported to date. This study establishes a microbial platform for forskolin production and provides advancements in the complex network of plant natural product biosynthesis.
Meiling Jiang, Hao Tang, Ying Ma et al.· Bioresource Technology· 0 citations
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