Investigating factors that influence microalgae production platforms for diterpenoids in laboratory and outdoor conditions
Photosynthetic microalgae offer a biotechnological solution to current climate concerns and the growing demand for food, bioproducts and bulk chemicals. As naturally efficient producers of carbohydrates, lipids, proteins and pigments, microalgae are a sustainable platform in which to realise the demand for high-value compounds. Moreover, they are amenable to metabolic engineering to produce other such molecules, including those with medicinal or therapeutic potential. The challenge however is to find solutions to the current bottlenecks, a major one of which is the challenge of microalgal scale-up. This project uses strains of the green microalga *Chlamydomonas reinhardtii*, UVM4 which is cell-wall deficient and RSW2 that has an intact cell wall. Both strains had previously been engineered to produce the diterpenoid, casbene, by introduction of the *Jatropha curcas* gene encoding casbene synthase. Two constructs were used, PM50, where the casbene synthase gene is under the control of the HSP70/RBCS2 promoter and includes a chloroplast transit peptide, and PM51, which in addition includes a thiamine dependent riboswitch (CrTHI4_4N). Casbene-producing *C. reinhardtii* strains were characterised under standard laboratory conditions of 25ºC and 90 μmol s-1 m-2 in Tris-Acetate Phosphate media (TAP) and found to reliably produce casbene when measured by gas-chromatography mass-spectrometry. Casbene production in strains ranged from 0.064 ± 0.074 mg l-1 to 16.7 ± 0.66 mg l-1 with RSW2::PM50#B08 being the best. The results of this work provided a reference against which to compare diterpenoid production in outdoor conditions. Casbene-producing strains RSW2::PM50#B08, UVM4::PM50#B12 and UVM4::PM51#E09 were taken forward for experiments in the outdoor facility located in the Botanic Garden at the University of Cambridge. Strains were inoculated in 10 l hanging bags, and the impact of environmental conditions during Summer and Autumn, the walled versus cell wall deficient phenotype, and the ability of a repressible riboswitch system to regulate diterpenoid production were investigated by measuring a range of abiotic and biotic factors alongside casbene production. During the Summer, casbene titer reached a maximum of 0.17 ± 0.05 mg l-1 compared to 0.061 ± 0.008 mg l-1 in Autumn. Cell walled RSW2::PM50#B08 produced 2.3-fold more casbene than cell wall deficient UVM4::PM50#B12. To prevent any impact on biomass by engineered pathways, a construct was made where casbene synthase was regulated by a thiamine repressible riboswitch. Preliminary experiments showed that the riboswitch worked to turn casbene synthase off, however it was not possible to show casbene production. Observations of microalgal growth and casbene production in natural settings informed further investigations. Temperature, light intensity, media type and algae-bacteria co-cultures were further investigated under controlled laboratory conditions. Increasing temperature improved growth and casbene production, although this decreased when the upper limit of temperature and light thresholds were tested, 38ºC and 200 μmol s-1 m-2, respectively. Casbene titer increased when grown at lower light intensity (45 μmol s-1 m-2) and when grown heterotrophically in TAP in the dark, strain UVM4::PM50#B12 produced 2.3 ± 0.4 mg l-1 of casbene. When grown in High Salt Medium (HSM) under normal laboratory conditions, growth was much slower and casbene production was an order of magnitude lower than in TAP medium. Preliminary investigations of mutualistic microalgae-bacteria co-cultures suggested that bacteria can improve casbene production two-fold, compared to axenic *C. reinhardtii* cultures. The experience of moving through scales of cultivation revealed challenges and solutions for translation and commercialization of algae biotechnology beyond the lab. This work highlighted the success of engineered strains at scale and identified that many abiotic and biotic factors can strongly influence the success or failure of cultures in outdoor settings. This work has provided new insights into microalgae cultivation practices, which are key to developing microalgae as next-generation, low-cost, sustainable, scalable, and high-productivity systems.
biotechnology, diterpenoid, microalgae, scale-up, synthetic biology
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