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.

The impact of fusion genes on the overexpression of enzymes for the heterologous production of β-phellandrene by Synechocystis mutants was investigated. The concept of overexpression of fusion genes was used in order to overcome the low expression level of these enzymes. Various constructs of the codon-optimized gene of β-phellandrene synthase (PHLS), along with the gene of geranyl diphosphate synthase (GPPS), were incorporated into the genomic DNA of Synechocystis sp. PCC 6803 following fusion with the highly expressed endogenous cpcB and cpcA genes, encoding the phycocyanin β- and α-subunits, respectively. Findings in this study indicated that the utilization of a strong promoter (cpc) in combination with the cpcB as a leader sequence was not by itself sufficient for cpcB.PHLS protein overexpression in the absence of the rest of the cpc operon genes (cpcA, cpcC2, cpcC1, cpcD). Significantly higher expression of the CpcB.PHLS fusion protein was achieved only when all cpc operon genes were present. In this case, the β-phellandrene yield was substantially greater compared with strains that also expressed the cpcB.PHLS fusion gene in the absence of the remainder cpc operon genes. Interestingly, when the cpcA was used in the leader sequence position, the CpcA.PHLS fusion protein caused the heterologous production of a mixture of terpenoid isomers, instead of β-phellandrene. This study extends previous findings in the field and provides new insights into the use of the fusion construct technology as a heterologous protein overexpression strategy for enzymes with slow catalytic activity.