A phycocyanin-deletion mutant of Synechocystis (cyanobacteria) was generated upon replacement of the CPC-operon with a kanamycin resistance cassette. The Δcpc transformant strains (Δcpc) exhibited a green phenotype, compared to the blue-green of the wild type (WT), lacked the distinct phycocyanin absorbance at 625nm, and had a lower Chl per cell content and a lower PSI/PSII reaction center ratio compared to the WT. Molecular and genetic analyses showed replacement of all WT copies of the Synechocystis DNA with the transgenic version, thereby achieving genomic DNA homoplasmy. Biochemical analyses showed the absence of the phycocyanin α- and β-subunits, and the overexpression of the kanamycin resistance NPTI protein in the Δcpc. Physiological analyses revealed a higher, by a factor of about 2, intensity for the saturation of photosynthesis in the Δcpc compared to the WT. Under limiting intensities of illumination, growth of the Δcpc was slower than that of the WT. This difference in the rate of cell duplication diminished gradually as growth irradiance increased. Identical rates of cell duplication of about 13h for both WT and Δcpc were observed at about 800μmolphotonsm(-2)s(-1) or greater. Culture productivity analyses under simulated bright sunlight and high cell-density conditions showed that biomass accumulation by the Δcpc was 1.57-times greater than that achieved by the WT. Thus, the work provides first-time direct evidence of the applicability of the Truncated Light-harvesting Antenna (TLA)-concept in cyanobacteria, entailing substantial improvements in the photosynthetic efficiency and productivity of mass cultures upon minimizing the phycobilisome light-harvesting antenna size.

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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.

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial application. However, the slow catalytic activity of terpene synthases (k = 4 s or slower) makes them noncompetitive for the pool of available substrate, thereby limiting the rate and yield of product generation. Work in this paper applied transformation technologies in Synechocystis for the heterologous production of β-phellandrene (monoterpene) hydrocarbons. Conditions were defined whereby expression of the β-phellandrene synthase (PHLS), as a CpcB·PHLS fusion protein with the β-subunit of phycocyanin, accounted for up to 20 % of total cellular protein. Moreover, CpcB·PHLS was heterologously co-expressed with enzymes of the mevalonic acid (MVA) pathway and geranyl-diphosphate synthase, increasing carbon flux toward the terpenoid biosynthetic pathway and enhancing substrate availability. These improvements enabled yields of 10 mg of β-phellandrene per g of dry cell weight generated in the course of a 48-h incubation period, or the equivalent of 1 % β-phellandrene:biomass (w:w) carbon-partitioning ratio. The work helped to identify prerequisites for the efficient heterologous production of terpene hydrocarbons in cyanobacteria: (i) requirement for overexpression of the heterologous terpene synthase, so as to compensate for the slow catalytic turnover of the enzyme, and (ii) enhanced endogenous carbon partitioning toward the terpenoid biosynthetic pathway, e.g., upon heterologous co-expression of the MVA pathway, thereby supplementing the native metabolic flux toward the universal isopentenyl-diphosphate and dimethylallyl-diphosphate terpenoid precursors. The two prerequisites are shown to be critical determinants of yield in the photosynthetic CO to terpene hydrocarbons conversion process.

GUN4 is a regulatory subunit of Mg-chelatase involved in the control of tetrapyrrole synthesis in plants and cyanobacteria. Here, we report the first characterization of a gun4 insertion mutant of the unicellular green alga Chlamydomonas reinhardtii. The mutant contains 50% of chlorophyll as compared to wild-type and accumulates ProtoIX. In contrast to the increase in LHC transcription, the accumulation of most LHC proteins is drastically diminished, implying posttranscriptional down-regulation in the absence of transcriptional coordination. We found that 803 genes change their expression level in gun4 as compared to wild-type, by RNA-Seq, and this wide-ranging effect on transcription is apparent under physiological conditions. Besides LHCs, we identified transcripts encoding enzymes of the tetrapyrrole pathway and factors involved in signal transduction, transcription, and chromatin remodeling. Moreover, we observe perturbations in electron transport with a strongly decreased PSI-to-PSII ratio. This is accompanied by an enhanced activity of the plastid terminal oxidase (PTOX) that could have a physiological role in decreasing photosystem II excitation pressure.