Metabolic engineering in Chlamydomonas reinhardtii for the production of high-value specialty sesquiterpenoids

Terpenoids are a diverse class of natural compounds with a wide range of applications in flavoring, fragrances, and medicine. However, many of these compounds are sourced from slow-growing, rare, or endangered plants. Heterologous production of terpenoids in engineered microbes offers a promising alternative which is scalable and readily accessible when production is required. Here, we investigated the production of heterologous sesquiterpenoids with applications as fragrances from Chlamydomonas reinhardtii. To channel cellular carbon towards sesquiterpenoids, we investigated the role that perturbation of the carotenoid pathway has on cytoplasmic terpenoids by expressing a β-carotene ketolase (BKT) which is known to produce ketocarotenoids. We coupled this with knock down of squalene synthase (SQS) to reduce metabolic pull on the sesquiterpene precursor farnesyl diphosphate (FDP). These modifications resulted in a synergistic effect on heterologous sesquiterpenoid production, significantly increasing patchoulol (C15-OH) production from 5.6 ± 1.4 fg/cell to 183 ± 23 fg/cell using a cytosolically localized patchoulol synthase. To further expand the range of heterologous sesquiterpenoids produced, we expressed and compared 22 sesquiterpenoid synthases from various organisms in this engineered strain of C. reinhardtii. This broad in vivo screening of enzyme variants in C. reinhardtii was facilitated by standardized in silico designs and large-scale DNA synthesis. Synthases producing aristolochene (n=4), guaiene (n=6), santalene (n=3), valencene (n=1), valerianol (n=1), zizaene (n=2), cadinol (n=1), and bisabolol (n=1) were expressed and their terpene products quantified to identify the optimal synthase for each. Our findings suggest that metabolic engineering in C. reinhardtii can be used to produce sesquiterpenoids from CO2, reducing the need to source these compounds from their natural context. This research could have implications for the fragrance, flavor, and pharmaceutical industries.

Gutiérrez, S., Overmans, S., Wellman, G., & Lauersen, K. J. (2023). Metabolic engineering in Chlamydomonas reinhardtii for the production of high-value specialty sesquiterpenoids. KAUST Research Repository.

Conference/Event Name
The 20th International Conference on the Cell and Molecular Biology of Chlamydomonas


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