Genetic engineering of the fast-growing cyanobacterium Synechococcus sp. PCC 11901: introduction of astaxanthin biosynthesis, generation of homoplasmic lines and optimization of extraction methods

Astaxanthin is a high-value ketocarotenoid with red pigmentation known for its powerful antioxidant properties. This pigment is known to have wide applications in the nutraceutical, food, feed and cosmetic industries, pushing many companies to move into this market. The main producer of natural astaxanthin is the microalga Haematococcus lacustris, but, due to several complications in the production process, alternative solutions are sought. In this context, a fast-growing cyanobacterium, Synechococcus sp. PCC 11901 represents a promising system to be exploited as a platform for astaxanthin production, specifically because of the short doubling time, the ease to grow under high salinity and light intensity, the facilitated extraction of pigments compared to Haematococcus lacustris and the possibility to be genetically engineered. In this study, we engineered Synechococcus sp. PCC 11901 to produce non-native astaxanthin by constitutively heterologous expression of β-carotene 4-ketolase and β-carotene hydroxylase genes. Successful integration of these genes enabled astaxanthin biosynthesis in this cyanobacterium under photoautotrophic conditions. It was demonstrated that astaxanthin accumulation was constitutive in the engineered strain and not triggered by nutrient starvation, distinguishing this strain from the typical stress-induced pigment production occurring in other microorganisms. Furthermore, high light intensities were well supported. In just 4 days, a productivity of 40 mg/L of astaxanthin was achieved, which exceeded that for Haematococcus lacustris in similar conditions. Astaxanthin production was successfully scaled up to a 330 L bioreactor system despite challenges, including limiting CO2 supply and insufficient light intensity. In collaboration with Algae for Future (A4F), a concentrated algal sludge was obtained, and it was used to evaluate various extraction methods to maximize astaxanthin yield. Initially, a chemical treatment using different simple organic solvents was tested to evaluate the highest extraction yield. Additionally, physical treatments were applied to the biomass, and different extraction times were evaluated. Finally, the optimal extraction method was used to enrich astaxanthin in sunflower oil, offering a potential application for this pigment in the feed industry. Eventually, a modified protocol for natural transformation of the cyanobacterium and the selection of fully segregated transformants using a continuous stirred tank reactor (CSTR) system was developed in collaboration with the University of Padova. This approach was implemented to enhance the selection and cultivation of homoplasmic transformants, wisely managing the high number of DNA copies present in Synechococcus sp. PCC 11901. These results highlight the potential of Synechococcus sp. PCC 11901 as a platform for sustainable astaxanthin production, with insights into a novel transformation method and optimization strategies for large-scale cultivation and recovery of the desired high added-value product.