Engineering for the production of diterpenoid compounds.

Photosynthetic microalgae like hold enormous potential as sustainable, light-driven biofactories for the production of high-value natural products such as terpenoids. is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how can be engineered to produce the diterpene casbene-an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate-limiting enzymes in the 2-C-methyl- d-erythritol 4-phosphate pathway, 1-deoxy- d-xylulose-5-phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.