Vers la reprogrammation métabolique de la cyanobactérie modèle Synechocystis pour la production durable de biocarburants : structuration des flux du carbone par CP12 et implications sur l’équilibre bioénergétique, l’hydrogénase et l’intégrité génomique

Biotechnology is a powerful tool allowing exploitation of biological circuits to produce compounds with multiple uses (medicine, nutrition, industrial…). Cyanobacteria have valuable genetic and trophic properties which could reduce the costs and the environmental footprint of these processes (photosynthesis, CO₂ fixation, assimilation of diverse nitrogen sources…). They also naturally produce energetic molecules such as H₂ from which new and sustainable biofuels sectors may rise. However, a global and fine understanding of their physiology is required in order to design an efficient biological chassis with these organisms. They are genetically manipulable but also exhibit a strong versatility favoring fixation of mutations that can be either beneficial or harmful to their large-scale cultivation. Over the course of my PhD, I constructed and studied mutants of a CO₂ fixation regulator whose activation is linked to photosynthesis. I showed that the Calvin cycle activity synchronizes carbon fluxes and redox status in Synechocystis and that its deregulation affects the metabolism in a pleiotropic manner. I was specifically interested into the carbon/nitrogen balance in this species and its urea metabolism which is of prime interest in biotechnology. I demonstrated that the latter was in competition with the hydrogenase for the insertion of nickel into their respective catalytic centers. Scarcity of this metal leads to selection of mutants thriving upon prolonged exposure to urea that retained a high capacity of H₂ production in presence of this nitrogenic substrate. This work shows that the metabolism of Synechocystis can be altered in favor of other cellular processes. Omics approaches allow global identification of the physiological responses induced as well as the biological compensation mechanisms. These observations are discussed with regards to biotechnological implications of genetic instability and the need to strengthen our understanding of metabolic and genetic plasticity in cyanobacteria.Les biotechnologies sont un outil puissant permettant d’emprunter les circuits biologiques pour produire des composés aux applications multiples (médecine, alimentation, industries…). Les cyanobactéries possèdent des propriétés génétiques et trophiques précieuses pour réduire les coûts et l’empreinte environnementale de ces procédés (photosynthèse, fixation du CO₂, sources d’azote assimilables...). Elles produisent aussi naturellement certaines molécules énergétiques comme le H₂ dont pourraient émerger de nouvelles filières propres de biocarburants. Cependant, une compréhension globale et approfondie de leur physiologie est nécessaire pour concevoir un châssis biologique performant à partir de ces organismes. Elles sont aisément manipulables génétiquement mais présentent une versatilité favorisant la fixation de mutations bénéfiques mais aussi délétères pour leur exploitation à grande échelle. Au cours de ma thèse, j’ai construit et étudié des mutants d’un régulateur de l’assimilation du CO₂ dont l’activation est liée à la photosynthèse. J’ai montré que l’activité du cycle de Calvin synchronise les flux du carbone et le statut rédox de Synechocystis et que sa dérégulation se répercute de manière pléiotropique sur son métabolisme. Plus spécifiquement, je me suis intéressé au déséquilibre carbone/azote dans cette espèce et à son métabolisme de l’urée qui présente un intérêt biotechnologique considérable. J’ai démontré que ce dernier était en compétition avec l’hydrogénase pour l’insertion du nickel dans leurs centres catalytiques respectifs. L’insuffisance de ce métal a permis de sélectionner des mutants de l’uréase tolérant une exposition prolongée à l’urée et conservant une forte capacité de production de H₂ en présence de ce substrat azoté. L’ensemble de ces résultats montre que le métabolisme de Synechocystis peut être détourné au profit de certains processus cellulaires. Les approches « omiques » permettent d’identifier globalement les réponses physiologiques induites ainsi que les leviers biologiques de compensation. Ces travaux sont discutés au regard des implications biotechnologiques de l’instabilité génétique et de la nécessité de renforcer notre compréhension de la plasticité métabolique et génomique des cyanobactéries

Towards the metabolic reprogramming of the cyanobacterium Synechocystis for sustainable biofuels production : Structuration of carbon fluxes by CP12 and implications on the bioenergetic balance, hydrogenase and genomic integrity

Les biotechnologies sont un outil puissant permettant d’emprunter les circuits biologiques pour produire des composés aux applications multiples (médecine, alimentation, industries…). Les cyanobactéries possèdent des propriétés génétiques et trophiques précieuses pour réduire les coûts et l’empreinte environnementale de ces procédés (photosynthèse, fixation du CO₂, sources d’azote assimilables...). Elles produisent aussi naturellement certaines molécules énergétiques comme le H₂ dont pourraient émerger de nouvelles filières propres de biocarburants. Cependant, une compréhension globale et approfondie de leur physiologie est nécessaire pour concevoir un châssis biologique performant à partir de ces organismes. Elles sont aisément manipulables génétiquement mais présentent une versatilité favorisant la fixation de mutations bénéfiques mais aussi délétères pour leur exploitation à grande échelle. Au cours de ma thèse, j’ai construit et étudié des mutants d’un régulateur de l’assimilation du CO₂ dont l’activation est liée à la photosynthèse. J’ai montré que l’activité du cycle de Calvin synchronise les flux du carbone et le statut rédox de Synechocystis et que sa dérégulation se répercute de manière pléiotropique sur son métabolisme. Plus spécifiquement, je me suis intéressé au déséquilibre carbone/azote dans cette espèce et à son métabolisme de l’urée qui présente un intérêt biotechnologique considérable. J’ai démontré que ce dernier était en compétition avec l’hydrogénase pour l’insertion du nickel dans leurs centres catalytiques respectifs. L’insuffisance de ce métal a permis de sélectionner des mutants de l’uréase tolérant une exposition prolongée à l’urée et conservant une forte capacité de production de H₂ en présence de ce substrat azoté. L’ensemble de ces résultats montre que le métabolisme de Synechocystis peut être détourné au profit de certains processus cellulaires. Les approches « omiques » permettent d’identifier globalement les réponses physiologiques induites ainsi que les leviers biologiques de compensation. Ces travaux sont discutés au regard des implications biotechnologiques de l’instabilité génétique et de la nécessité de renforcer notre compréhension de la plasticité métabolique et génomique des cyanobactéries.Biotechnology is a powerful tool allowing exploitation of biological circuits to produce compounds with multiple uses (medicine, nutrition, industrial…). Cyanobacteria have valuable genetic and trophic properties which could reduce the costs and the environmental footprint of these processes (photosynthesis, CO₂ fixation, assimilation of diverse nitrogen sources…). They also naturally produce energetic molecules such as H₂ from which new and sustainable biofuels sectors may rise. However, a global and fine understanding of their physiology is required in order to design an efficient biological chassis with these organisms. They are genetically manipulable but also exhibit a strong versatility favoring fixation of mutations that can be either beneficial or harmful to their large-scale cultivation. Over the course of my PhD, I constructed and studied mutants of a CO₂ fixation regulator whose activation is linked to photosynthesis. I showed that the Calvin cycle activity synchronizes carbon fluxes and redox status in Synechocystis and that its deregulation affects the metabolism in a pleiotropic manner. I was specifically interested into the carbon/nitrogen balance in this species and its urea metabolism which is of prime interest in biotechnology. I demonstrated that the latter was in competition with the hydrogenase for the insertion of nickel into their respective catalytic centers. Scarcity of this metal leads to selection of mutants thriving upon prolonged exposure to urea that retained a high capacity of H₂ production in presence of this nitrogenic substrate. This work shows that the metabolism of Synechocystis can be altered in favor of other cellular processes. Omics approaches allow global identification of the physiological responses induced as well as the biological compensation mechanisms. These observations are discussed with regards to biotechnological implications of genetic instability and the need to strengthen our understanding of metabolic and genetic plasticity in cyanobacteria

Genomics of Urea Transport and Catabolism in Cyanobacteria: Biotechnological Implications

International audienceCyanobacteria are widely-diverse prokaryotes that colonize our planet. They use solar energy to assimilates huge amounts of atmospheric CO2 and produce a large part of the biomass and oxygen that sustain most life forms. Hence cyanobacteria are increasingly studied for basic research objective, as well as for the photosynthetic production of chemicals of industrial interests. One potential approach to reduce the cost of the future bioproduction processes will be to couple them with the treatment of wastewaters that are often polluted with urea, which in any case is cheaper than nitrate. As yet, however, research has mostly focused on a very small number of model cyanobacteria growing on nitrate. Thus, the genetic inventory of the cyanobacterial phylum is still insufficiently employed to meaningfully select the right host for the right purpose. This review reports what is known about urea transport and catabolism in cyanobacteria, and what can be inferred from the comparative analysis of the publicly available genome sequence of 308 cyanobacteria. We found that most cyanobacteria harbor the genes encoding the urea catabolytic enzymes urease (ureABCDEFG) mostly, but not systematically, together with the urea transport (urtABCDE). These findings are consistent with the capacity of the few tested cyanobacteria to grow on urea as the sole nitrogen source. They also indicate that urease is important for the detoxification of internally generated urea (re-cycling its carbon and nitrogen). In contrast, several cyanobacteria have urtABCDE but not ureABCDEFG, suggesting that urtABCDE could operate in the transport of not only urea but also of other nutrients. Only four cyanobacteria appeared to have the genes encoding the urea carboxylase (uc) and allophanate hydrolase (ah) enzymes that sequentially catabolize urea. Three of these cyanobacteria belongs to the genera Gloeobacter and Gloeomargarita that have likely diverged early from other cyanobacteria, suggesting that the urea carboxylase and allophanate hydrolase enzymes appeared in cyanobacteria before urease

Advances in the Function and Regulation of Hydrogenase in the Cyanobacterium Synechocystis PCC6803

In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism and identify bottlenecks limiting H2 production. Most of the recent advances in our understanding of the bidirectional [Ni-Fe] hydrogenase (Hox) came from investigations performed in the widely-used model cyanobacterium Synechocystis PCC6803 where Hox is the sole enzyme capable of combining electrons with protons to produce H2 under specific conditions. Recent findings suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. Furthermore, plasmid-encoded functions and glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) are proposed as possible new players in the function and regulation of hydrogen production

Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications.

International audienceCyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings indicate that not all cyanobacteria have an E. coli-type SOS system. Also interestingly, several cyanobacteria possess multiple copies of E. coli-like DNA repair genes, such as Acaryochloris marina MBIC11017 (2 alkB, 3 ogt, 7 recA, 3 recD, 2 ssb, 3 umuC, 4 umuD, and 8 xerC), Cyanothece ATCC51142 (2 lexA and 4 ruvC), and Nostoc PCC7120 (2 ssb and 3 xerC)

Overproduction of the cyanobacterial hydrogenase and selection of a mutant thriving on urea, as a possible step towards the future production of hydrogen coupled with water treatment.

Using a combination of various types of genetic manipulations (promoter replacement and gene cloning in replicating plasmid expression vector), we have overproduced the complex hydrogenase enzyme in the model cyanobacterium Synechocystis PCC6803. This new strain overproduces all twelve following proteins: HoxEFUYH (hydrogen production), HoxW (maturation of the HoxH subunit of hydrogenase) and HypABCDEF (assembly of the [NiFe] redox center of HoxHY hydrogenase). This strain when grown in the presence of a suitable quantities of nickel and iron used here exhibits a strong (25-fold) increase in hydrogenase activity, as compared to the WT strain growing in the standard medium. Hence, this strain can be very useful for future analyses of the cyanobacterial [NiFe] hydrogenase to determine its structure and, in turn, improve its tolerance to oxygen with the future goal of increasing hydrogen production. We also report the counterintuitive notion that lowering the activity of the Synechocystis urease can increase the photoproduction of biomass from urea-polluted waters, without decreasing hydrogenase activity. Such cyanobacterial factories with high hydrogenase activity and a healthy growth on urea constitute an important step towards the future development of an economical industrial processes coupling H2 production from solar energy and CO2, with wastewater treatment (urea depollution)

First in vivo analysis of the regulatory protein CP12 of the model cyanobacterium Synechocystis PCC 6803: Biotechnological implications

International audienceWe report the first in vivo analysis of a canonical CP12 regulatory protein, namely the unique CP12 of the model cyanobacterium Synechocystis PCC 6803, which has the advantage of being able to grow photoautotrophically, photomixotrophically, and photoheterotrophically. The data showed that CP12 is dispensable to cell growth under standard (continuous) light and light/dark cycle, whereas it is essential for the catabolism of exogenously added glucose that normally sustains cell growth in absence of photosynthesis. Furthermore, to be active in glucose catabolism, CP12 requires its three conserved features: its AWD_VEEL motif and its two pairs of cysteine residues. Also interestingly, CP12 was found to regulate the redox equilibrium of NADPH, an activity involving its AWD_VEEL motif and its C-ter cysteine residues, but not its N-ter cysteine residues. This finding is important because NADPH powers up the methylerythritol 4-phosphate (MEP) pathway that synthesizes the geranyl-diphosphate (GPP) and farnesyl-diphosphate (FPP) metabolites, which can be transformed into high-value terpenes by recombinant cyanobacteria producing plant terpene synthase enzymes. Therefore, we have introduced into the Δ cp12 mutant and the wild-type (control) strain our replicative plasmids directing the production of the monoterpene limonene and the sesquiterpene bisabolene. The photosynthetic production of both bisabolene and limonene appeared to be increased (more than two-fold) in the Δ cp12 mutant as compared to the WT strain. Furthermore, the level of bisabolene production was also higher to those previously reported for various strains of Synechocystis PCC 6803 growing under standard (non-optimized) photoautotrophic conditions. Hence, the presently described Δ cp12 strain with a healthy photoautotrophic growth and an increased capability to produce terpenes, is an attractive cell chassis for further gene manipulations aiming at engineering cyanobacteria for high-level photoproduction of terpenes

Recent Advances in the Photoautotrophic Metabolism of Cyanobacteria: Biotechnological Implications

International audienceCyanobacteria 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

Comparative analysis of the strains over-expressing the genes <i>hoxEFUYH</i> (CE1) or <i>hoxEFUYHW</i> (CE4; CE4u) alone, or together with the <i>hypABCDEF</i> genes (CE5; CE5u).

<p>All experiments were performed at least three times. (<b>A</b>) Typical growth of the wild type (WT; squares), CE-<i>hoxEFUYH</i> (CE1; white triangles) and CE-<i>hoxEFUYHW</i> (CE4; black squares) cells incubated under standard conditions. (<b>B</b>) Histogram plot representation of transcript abundance (measured by Real-time quantitative PCR) of the <i>hoxEFUYHW</i> genes in strains WT (small light-grey bars), CE1 (grey rectangles) and CE4 (hatched bars) mutants. (<b>C</b>) Western blot analysis of the abundance of the HoxF and HoxH proteins in WT, CE1, CE4 and CE5 cells growing in MM* medium (MM + 17 μM Fe). (<b>D</b>) Histogram plot representation of the transcript abundance (RT-qPCR) of the <i>hypABC-F</i> genes in the strains WT (light-grey rectangles), CE1 (grey) and CE5 (arrow-filled bars). (<b>E</b>) Histograms representation of the hydrogenase activities of WT (light grey), CE1 (grey), CE2 (dark grey) CE4 (light grey-hatched bars), CE5 (white arrow-filled bars), CE4u (dark grey-hatched bars) and CE5u cells (grey arrow-filled bars) growing in standard medium (MM) or MM* (MM + 17 μM Fe) supplemented with 2.5 μM NiSO₄.</p

Influence of urea on the growth of <i>Synechocystis</i> WT and mutants overexpressing the <i>hoxEFUYHW</i> genes alone (CE4) or in combination with the <i>hypABCDEF</i> genes (CE5).

<p>(<b>A</b>) Typical growth of WT cells cultivated on medium containing Ni (1 μM) and urea (2.5–20 mM) as the sole nitrogen source. (<b>B</b>) Typical growth on urea (5 mM as the sole nitrogen source) and Ni (2.5 μM) of the WT strain, and the CE4 and CE5 strain without or with (CE4u and CE5u) a mutation in <i>ureG</i>. Influence of prolonged growth on urea (5 mM as the sole nitrogen source) and Ni (2.5 μM) on the cell appearance <b>C</b>) and urease activity (<b>D</b>) of the studied strains. All experiments were performed at least three times.</p