Outdoor cultivation and metabolomics exploration of Chlamydomonas engineered for bisabolene production

[Display omitted] •Chlamydomonas as a one-cell two-wells (engineered and natural products) biorefinery.•Autotrophic cultivation with added bicarbonate improved outdoor strain performance.•Successful outdoor cultivation of engineered Chlamydomonas at incremental scales.•Engineered strain produced 906 mg/L bisabolene and 54 mg/L carotenoids.•Metabolomics and PAM fluorometry confirmed improved cellular metabolism. Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineeredC. reinhardtii,establishing it as a one-cell two-wells biorefinery.