Regulation of β-phellandrene synthase gene expression, recombinant protein accumulation, and monoterpene hydrocarbons production in Synechocystis transformants.

Successful application of the photosynthesis-to-fuels approach requires a high product-to-biomass carbon-partitioning ratio. The work points to the limiting amounts of heterologous terpene synthase in cyanobacteria as a potential barrier in the yield of terpene hydrocarbons via photosynthesis. Cyanobacteria like Synechocystis sp. can be exploited as platforms in a photosynthesis-to-fuels process for the generation of terpene hydrocarbons. Successful application of this concept requires maximizing photosynthesis and attaining a high endogenous carbon partitioning toward the desirable product. The work addressed the question of the regulation of β-phellandrene synthase transgene expression in relation to product yield from the terpenoid biosynthetic pathway of cyanobacteria. The choice of strong alternative transcriptional and translational cis-regulatory elements and the choice of the Synechocystis genomic DNA loci for transgene insertion were investigated. Specifically, the β-phellandrene synthase transgene was expressed under the control of the endogenous psbA2 promoter, or under the control of the Ptrc promoter from Escherichia coli with the translation initiation region of highly expressed gene 10 from bacteriophage T7. These heterologous elements allowed for constitutive transgene expression. In addition, the β-phellandrene synthase construct was directed to replace the Synechocystis cpc operon, encoding the peripheral phycocyanin rods of the phycobilisome antenna. Results showed that a 4-fold increase in the cellular content of the β-phellandrene synthase was accompanied by a 22-fold increase in β-phellandrene yield, suggesting limitations in rate and yield by the amount of the transgenic enzyme. The work points to the limiting amount of transgenic terpene synthases as a potential barrier in the heterologous generation of terpene products via the process of photosynthesis.