Exploring the Potential of the Model Cyanobacteria Synechococcus PCC 7002 and PCC 7942 for the Photoproduction of High-Value Terpenes: A Comparison with Synechocystis PCC 6803.

We have performed the first comparative analysis of the potential of two physiologically-diverse model cyanobacteria, Synechococcus PCC 7002 (S.7002) and Synechococcus elongatus PCC 7942 (S.7942), for the photosynthetic production of four chemically-different high-value terpenes: two monoterpenes limonene and pinene, and two sesquiterpenes bisabolene and farnesene. We showed, for the first time, that S.7002 and S.7942 can produce farnesene and bisabolene, respectively. Both cyanobacteria produced farnesene (S.7942 produced more efficiently than S.7002) more efficiently than the other tested terpenes (especially pinene, the weakest produced terpene). S.7002 produced limonene more efficiently than bisabolene, whereas S.7942 produced bisabolene more efficiently than limonene. These findings suggest that S.7942 is better suited to produce sesquiterpenes than monoterpenes. Interestingly, higher levels of terpenes were produced by S.7942 and S.7002 expressing a terpene-synthase gene from both an RSF1010-derived replicating plasmid and a neutral chromosomal site, as compared to either the plasmid alone or the chromosome alone. These results suggest that in both cyanobacteria, the production of terpenes is more limited by the activity of terpene synthases than the abundance of terpene precursors. Finally, higher levels of terpenes were produced by S.7002 growing on urea (a frequent pollutant) as compared to nitrate or ammonium, the standard nitrogen sources for cyanobacteria.

Exploring the potential of the model cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes.

BACKGROUND: The robust model cyanobacterium Synechocystis PCC 6803 is increasingly explored for its potential to use solar energy, water and atmospheric CO2 for the carbon-neutral production of terpenes, the high-value chemicals that can be used for the production of drugs, flavors, fragrances and biofuels. However, as terpenes are chemically diverse, it is extremely difficult to predict whether Synechocystis is a suitable chassis for the photosynthetic production of various terpenes or only a few of them. RESULTS: We have performed the first-time engineering and comparative analysis of the best-studied cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of five chemically diverse high-value terpenes: two monoterpenes (C10H16) limonene (cyclic molecule) and pinene (bicyclic), and three sesquiterpenes (C15H24) bisabolene (cyclic), farnesene (linear) and santalene (cyclic). All terpene producers appeared to grow well and to be genetically stable, as shown by the absence of changes in their production levels during the 5-9-month periods of their sub-cultivation under photoautotrophic conditions). We also found that Synechocystis PCC 6803 can efficiently and stably produce farnesene and santalene, which had never been produced before by this model organism or any other cyanobacteria, respectively. Similar production levels were observed for cells growing on nitrate (the standard nitrogen source for cyanobacteria) or urea (cheaper than nitrate). Furthermore, higher levels of farnesene were produced by cloning the heterologous farnesene synthase gene in a RSF1010-derived replicating plasmid as compared to the well-used slr0168 neutral cloning site of the chromosome. CONCLUSIONS:  Altogether, the present results indicate that Synechocystis PCC 6803 is better suited to produce sesquiterpenes (particularly farnesene, the most highly produced terpene of this study) than monoterpenes (especially pinene).

Iron Deficiency Promotes the Lack of Photosynthetic Cytochrome c550 and Affects the Binding of the Luminal Extrinsic Subunits to Photosystem II in the Diatom Phaeodactylum tricornutum.

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced QB and the S2/S3 states of the water-splitting machinery. However, electron transfer from QA to QB, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c550 subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c550 under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c550, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.

A Genetic Toolbox for the New Model Cyanobacterium Cyanothece PCC 7425: A Case Study for the Photosynthetic Production of Limonene.

Cyanobacteria, the largest phylum of prokaryotes, perform oxygenic photosynthesis and are regarded as the ancestors of the plant chloroplast and the purveyors of the oxygen and biomass that shaped the biosphere. Nowadays, cyanobacteria are attracting a growing interest in being able to use solar energy, H2O, CO2 and minerals to produce biotechnologically interesting chemicals. This often requires the introduction and expression of heterologous genes encoding the enzymes that are not present in natural cyanobacteria. However, only a handful of model strains with a well-established genetic system are being studied so far, leaving the vast biodiversity of cyanobacteria poorly understood and exploited. In this study, we focused on the robust unicellular cyanobacterium Cyanothece PCC 7425 that has many interesting attributes, such as large cell size; capacity to fix atmospheric nitrogen (under anaerobiosis) and to grow not only on nitrate but also on urea (a frequent pollutant) as the sole nitrogen source; capacity to form CO2-sequestrating intracellular calcium carbonate granules and to produce various biotechnologically interesting products. We demonstrate for the first time that RSF1010-derived plasmid vectors can be used for promoter analysis, as well as constitutive or temperature-controlled overproduction of proteins and analysis of their sub-cellular localization in Cyanothece PCC 7425. These findings are important because no gene manipulation system had been developed for Cyanothece PCC 7425, yet, handicapping its potential to serve as a model host. Furthermore, using this toolbox, we engineered Cyanothece PCC 7425 to produce the high-value terpene, limonene which has applications in biofuels, bioplastics, cosmetics, food and pharmaceutical industries. This is the first report of the engineering of a Cyanothece strain for the production of a chemical and the first demonstration that terpene can be produced by an engineered cyanobacterium growing on urea as the sole nitrogen source.

Recent Advances in the Photoautotrophic Metabolism of Cyanobacteria: Biotechnological Implications.

Cyanobacteria constitute the only phylum of oxygen-evolving photosynthetic prokaryotes that shaped the oxygenic atmosphere of our planet. Over time, cyanobacteria have evolved as a widely diverse group of organisms that have colonized most aquatic and soil ecosystems of our planet and constitute a large proportion of the biomass that sustains the biosphere. Cyanobacteria synthesize a vast array of biologically active metabolites that are of great interest for human health and industry, and several model cyanobacteria can be genetically manipulated. Hence, cyanobacteria are regarded as promising microbial factories for the production of chemicals from highly abundant natural resources, e.g., solar energy, CO2, minerals, and waters, eventually coupled to wastewater treatment to save costs. In this review, we summarize new important discoveries on the plasticity of the photoautotrophic metabolism of cyanobacteria, emphasizing the coordinated partitioning of carbon and nitrogen towards growth or compound storage, and the importance of these processes for biotechnological perspectives. We also emphasize the importance of redox regulation (including glutathionylation) on these processes, a subject which has often been overlooked.

Using agro-industrial wastes for mixotrophic growth and lipids production by the green microalga Chlorella sorokiniana.

There has been growing interest in the use of microalgae for the production of biofuels, but production costs continue to be too high to compete with fossil fuel prices. One of the main limitations for photobioreactor productivity is light shielding, especially at high cell densities. The growth of the green microalga Chlorella sorokiniana, a robust industrial species, has been evaluated under different trophic conditions with traditional carbon sources, such as glucose and sucrose, and alternative low cost carbon sources, such as carob pod extract, industrial glycerol and acetate-rich oxidized wine waste lees. The mixotrophic cultivation of this microalga with wine waste lees alleviated the problems of light shielding observed in photoautotrophic cultures, improving specific growth rate (0.052 h-1) compared with the other organic sources. The fed-batch mixotrophic culture of Chlorella sorokiniana in a 2 L stirred tank reactor, with optimized nutritional conditions, 100 mM of acetate coming from the oxidized wine waste lees and 30 mM of ammonium, produced an algal biomass concentration of 11 g L-1 with a lipid content of 38 % (w/w). This fed-batch strategy has been found to be a very effective means to enhance the biomass and neutral lipid productivity.

Efficiency of different heterologous promoters in the unicellular microalga Chlamydomonas reinhardtii.

Despite the biotechnological interest of microalgae, no robust and stable methods for genetic transformation of most microalgal strains exist. The scanty and disperse data about the efficiency of heterologous promoters in microalgae and the use of different transformation methods, DNA quantities and reporter genes in the existing studies makes very difficult a real comparison of their efficiency. Using Chlamydomonas reinhardtii as a host, we have evaluated the efficiency of the heterologous promoters of cauliflower mosaic virus 35S (CaMV 35S) and Agrobacterium nopaline synthase (NOS) genes. These promoters were fused to the paromomycin conferring-resistance aminoglycoside 3'-phosphotransferase encoding gene (APHVIII), and C. reinhardtii was transformed by the glass beads agitation method. The transformation efficiency and the APHVIII transcript and protein levels were evaluated in a series of transformants for each promoter. The chimeric promoter HSP70A/RBCS2 and the promoter-less APHVIII marker gene were used for comparison. We found significantly higher transformation efficiencies and higher level of APHVIII expression in those transformants harboring the NOS promoter than in those transformed with CaMV 35S promoter. The NOS promoter, widely used for genetic manipulation of higher plants, has been very rarely used for the transformation of microalgae. The results shown here suggest the possibilities of this heterologous promoter as an efficient system for the genetic manipulation of microalgae.

Promoter trapping in microalgae using the antibiotic paromomycin as selective agent.

The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.