Fusion constructs enhance heterologous β-phellandrene production inSynechocystissp. PCC 6803

The impact of fusion genes on the overexpression of enzymes for the heterologous production of beta-phellandrene bySynechocystismutants 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 beta-phellandrene synthase (PHLS), along with the gene of geranyl diphosphate synthase (GPPS), were incorporated into the genomic DNA ofSynechocystissp. PCC 6803 following fusion with the highly expressed endogenouscpcBandcpcAgenes, encoding the phycocyanin beta- and alpha-subunits, respectively. Findings in this study indicated that the utilization of a strong promoter (cpc) in combination with thecpcBas a leader sequence was not by itself sufficient for cpcB.PHLS protein overexpression in the absence of the rest of thecpcoperon genes (cpcA,cpcC2,cpcC1,cpcD). Significantly higher expression of the CpcB.PHLS fusion protein was achieved only when allcpcoperon genes were present. In this case, the beta-phellandrene yield was substantially greater compared with strains that also expressed thecpcB.PHLSfusion gene in the absence of the remaindercpcoperon genes. Interestingly, when thecpcAwas used in the leader sequence position, the CpcA.PHLS fusion protein caused the heterologous production of a mixture of terpenoid isomers, instead of beta-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.