Impact of Carbon Fixation, Distribution and Storage on the Production of Farnesene and Limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.

Development of a simple and versatile process for commercial and municipal lignocellulosic waste conversion into fermentable sugars.

Lignocellulosic wastes are valuable feedstock in biorefinery thanks to their high sugars content and low level of fibers intricacy. However, their massification is often a limiting parameter in the development of industrial projects. Hence, this study aims to develop an efficient process enabling the conversion of several waste streams within the same process line. Several pretreatment and enzymatic hydrolysis parameters were firstly evaluated with Old Corrugated Cardboards (OCC) as a model substrate. A chemical free pretreatment followed by an enzymatic hydrolysis (Cellic Ctec 3 enzymatic cocktail at 0.06 g of cocktail per g of Total Sugars (TS)) efficiently depolymerized OCC into monomeric sugars (0.50 g/gTS) consequently fermented into ethanol (0.24 g/gTS). Then, the suitability of this process was validated for sugars production from Pulp and Paper (P&P) sludge (0.48 g/gTS), sieved toilets papers (0.40 g/gTS), the Organic fraction of municipal solid waste (0.37 g/gTS) and Waste Wood B (0.08 g/gTS).

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.

Lactic acid production from food waste using a microbial consortium: Focus on key parameters for process upscaling and fermentation residues valorization.

In this study, the production of lactic acid from food waste in industrially relevant conditions was investigated. Laboratory assays were first performed in batch conditions to determine the suitable operational parameters for an efficient lactic acid production. The use of compost as inoculum, the regulation of temperature at 35 °C and pH at 5 enhanced the development of Lactobacillus sp. resulting in the production of 70 g/L of lactic acid with a selectivity of 89% over the other carboxylic acids. Those parameters were then applied at pilot scale in successive fed-batch fermentations. The subsequent high concentration (68 g/L), yield (0.38 g/gTS) and selectivity (77%) in lactic acid demonstrated the applicability of the process. To integrate the process into a complete value chain, fermentation residues were then converted into biogas through anaerobic digestion. Lastly, the experiment was successfully replicated using commercial and municipal waste collected in France.

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).

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.

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.