Heterologous β-phellandrene production by alginate immobilized Synechocystis sp. PCC 6803

The aim of the present work was the investigation of various cultivation conditions in order to provide a foundation for a sustainable, financially viable, and environmentally friendly cultivation system for the heterologous β-phellandrene production by Synechocystis sp. PCC 6803. Synechocystis cells were able to grow and form distinct colonies both at the internal and the external surfaces of calcium alginate beads and maintained their ability to produce β-phellandrene, in considerably higher amounts compared to suspension cultures, with maximum production after 6 days. Both immobilized and suspended Synechocystis cells exhibited a continuous and long-term ability to produce β-phellandrene, only by CO2 addition, without renewal of the nutrients or the growth medium. However, photoheterotrophic growth of Synechocystis, with glucose as an alternative carbon source, had a negative impact on the heterologous production β-phellandrene. Despite the fact that cell growth and biomass accumulation were pronounced under photoheterotrophic growth conditions, β-phellandrene production was substantially decreased, indicating that this growth condition is not recommended for scale-up applications. Finally, combination of alkaline (pH 10.5) and saline (600 mM NaCl), i.e., extremophilic for Synechocystis growth conditions, proved to be amenable to cell growth and β-phellandrene production, albeit yields were a bit lower. The results provide new approaches for the development of larger scale, environmentally friendly, and financially viable cultivation systems for sustainable heterologous production of terpenoids by Synechocystis.