Cinzia Formighieri

Maximizing photosynthetic efficiency and culture productivity in cyanobacteria upon minimizing the phycobilisome light-harvesting antenna size.

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.

A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria)

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms.

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

AbstractMain conclusionSuccessful 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 Synechocystiscpc 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.

Carbon partitioning to the terpenoid biosynthetic pathway enables heterologous β-phellandrene production in Escherichia coli cultures

Escherichia coli was used as a microbial system for the heterologous synthesis of β-phellandrene, a monoterpene of plant origin with several potential commercial applications. Expression of Lavandula angustifolia β-phellandrene synthase (PHLS), alone or in combination with Picea abies geranyl-diphosphate synthase in E. coli, resulted in no β-phellandrene accumulation, in sharp contrast to observations with PHLS-transformed cyanobacteria. Lack of β-phellandrene biosynthesis in E. coli was attributed to the limited endogenous carbon partitioning through the native 2-C-methylerythritol-4-phosphate (MEP) pathway. Heterologous co-expression of the mevalonic acid pathway, enhancing cellular carbon partitioning and flux toward the universal isoprenoid precursors, isopentenyl-diphosphate and dimethylallyl-diphosphate, was required to confer β-phellandrene production. Differences in endogenous carbon flux toward the synthesis of isoprenoids between photosynthetic (Synechocystis) and non-photosynthetic bacteria (E. coli) are discussed in terms of differences in the regulation of carbon partitioning through the MEP biosynthetic pathway in the two systems.

Cyanobacteria as a Platform for Direct Photosynthesis-to-Fuel Conversion

Expression of heterologous genes allows to introduce novel biosynthetic pathways in cyanobacteria, more prone to genetic transformation than eukaryotic microalgae. Cyanobacteria are consequently endowed with the biosynthesis of fuel molecules, such as alcohols, free fatty acids, and terpene hydrocarbons, from photosynthesis-associated metabolism.

Biodiesel from Microalgae

Microalgae have the almost unique ability among microorganisms to naturally store significant amounts of carbon as neutral lipids that can be converted to biodiesel. Accumulation of neutral lipids has mainly evolved as a response strategy to stress conditions, which on the other hand are detrimental for growth and biomass production. Genetic improvement would be required as part of the effort for algae domestication in order to meet industrial lipid production requirements.

Biofuels: An Emerging Industry

Dealing with the nonrenewable nature of fossil fuels, and with the effect of carbon dioxide emissions on global warming, requires transformation of the energy sector toward renewable, carbon neutral resources. This chapter introduces the biofuel industry, which relies on the energy derived from living organisms or from their metabolic products, in the contest of global energy supply.

Cyanobacterial production of plant essential oils

Main conclusionSynechocystis (a cyanobacterium) was employed as an alternative host for the production of plant essential oil constituents. β-Phellandrene synthase (PHLS) genes from different plants, when expressed in Synechocystis, enabled synthesis of variable monoterpene hydrocarbon blends, converting Synechocystis into a cell factory that photosynthesized and released useful products.Monoterpene synthases are secondary metabolism enzymes that catalyze the generation of essential oil constituents in terrestrial plants. Essential oils, including monoterpene hydrocarbons, are of interest for their commercial application and value. Therefore, heterologous expression of monoterpene synthases for high-capacity essential oil production in photosynthetic microorganism transformants is of current interest. In the present work, the cyanobacterium Synechocystsis PCC 6803 was employed as an alternative host for the production of plant essential oil constituents. As a case study, β-phellandrene synthase (PHLS) genes from different plants were heterologously expressed in Synechocystis. Genomic integration of individual PHLS-encoding sequences endowed Synechocystis with constitutive monoterpene hydrocarbons generation, occurring concomitant with photosynthesis and cell growth. Specifically, the β-phellandrene synthase from Lavandula angustifolia (lavender), Solanum lycopersicum (tomato), Pinus banksiana (pine), Picea sitchensis (Sitka spruce) and Abies grandis (grand fir) were active in Synechocystis transformants but, instead of a single product, they generated a blend of terpene hydrocarbons comprising β-phellandrene, α-phellandrene, β-myrcene, β-pinene, and δ-carene with variable percentage ratios ranging from < 10 to > 90% in different product combinations and proportions. Our results suggested that PHLS enzyme conformation and function depends on the cytosolic environment in which they reside, with the biochemical properties of the latter causing catalytic deviations from the products naturally observed in the corresponding gene-encoding plants, giving rise to the terpene hydrocarbon blends described in this work. These findings may have commercial application in the generation of designer essential oil blends and will further assist the development of heterologous cyanobacterial platforms for the generation of desired monoterpene hydrocarbon products.

Economic Viability of Algal Biodiesel

Production of biodiesel from oleaginous microalgae has been evaluated in terms of economic profitability, as an example that can be extended to other algal biofuels. Biodiesel has to reach cost parity with fossil fuels in order to be economically sustainable in the long term. Major costs for generation of algal biodiesel are not related to the feedstock, but they depend on construction and maintenance of the cultivation system, biomass harvesting and processing. The cost balance of the system can be improved by co-production of high value-added commodities and/or by substantial increase in biomass productivity and oil yield.

Light-Utilization Inefficiency of Wild-Type Algal Mass Cultures

Photosynthetic productivity of microalgae depends on photosynthate production and utilization by the single cell. At the same time, large light-harvesting antenna systems found in wild-type strains are responsible for inefficient light-utilization by the culture as a whole. Growth conditions in the natural habitat are opposite compared to mass cultivation, and configurations positively selected in the wild may become detrimental in photobioreactors.