Integrative analysis of large scale expression profiles reveals core transcriptional response and coordination between multiple cellular processes in a cyanobacterium

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria are the only known prokaryotes capable of oxygenic photosynthesis. They play significant roles in global biogeochemical cycles and carbon sequestration, and have recently been recognized as potential vehicles for production of renewable biofuels. <it>Synechocystis </it>sp. PCC 6803 has been extensively used as a model organism for cyanobacterial studies. DNA microarray studies in <it>Synechocystis </it>have shown varying degrees of transcriptome reprogramming under altered environmental conditions. However, it is not clear from published work how transcriptome reprogramming affects pre-existing networks of fine-tuned cellular processes.</p> <p>Results</p> <p>We have integrated 163 transcriptome data sets generated in response to numerous environmental and genetic perturbations in <it>Synechocystis</it>. Our analyses show that a large number of genes, defined as the core transcriptional response (CTR), are commonly regulated under most perturbations. The CTR contains nearly 12% of <it>Synechocystis </it>genes found on its chromosome. The majority of genes in the CTR are involved in photosynthesis, translation, energy metabolism and stress protection. Our results indicate that a large number of differentially regulated genes identified in most reported studies in <it>Synechocystis </it>under different perturbations are associated with the general stress response. We also find that a majority of genes in the CTR are coregulated with 25 regulatory genes. Some of these regulatory genes have been implicated in cellular responses to oxidative stress, suggesting that reactive oxygen species are involved in the regulation of the CTR. A Bayesian network, based on the regulation of various KEGG pathways determined from the expression patterns of their associated genes, has revealed new insights into the coordination between different cellular processes.</p> <p>Conclusion</p> <p>We provide here the first integrative analysis of transcriptome data sets generated in a cyanobacterium. This compilation of data sets is a valuable resource to researchers for all cyanobacterial gene expression related queries. Importantly, our analysis provides a global description of transcriptional reprogramming under different perturbations and a basic framework to understand the strategies of cellular adaptations in <it>Synechocystis</it>.</p

Redox Homeostasis in Cyanobacteria

Oxygenic photosynthetic organisms utilize high-energy electron transfer chains comprised of redox active intermediates and light harvesting complexes. While oxygen is a necessary byproduct of water oxidation and the source of photosynthetic electrons, its presence is also dangerous because leakage of electrons and excitation energy can interact with molecular oxygen to generate reactive oxygen species: ROS). Elaborate antioxidant networks and redox buffering systems have evolved to protect photosynthetic organisms from the threat of ROS. Glutathione: GSH) is a multifunctional molecule that is involved in core metabolism, detoxification of xenobiotics and in maintenance of cellular redox poise. The ubiquitous nature of glutathione and its importance to cellular metabolism has been observed in many organisms, however the specific roles of glutathione in photosynthetic organisms are not fully understood. To address these questions, we have generated several mutants in the glutathione biosynthesis and degradation pathways in the model organism Synechocystis sp. PCC 6803: Synechocystis 6803), an oxygenic photosynthetic cyanobacterium. We utilized targeted homologous recombination to generate deletion mutants of glutamate-cysteine ligase: GshA) and glutathione synthetase: GshB) in Synechocystis 6803. Our results indicate that GshA activity is essential for growth in cyanobacteria because we were unable to isolate a fully segregated ∆gshA deletion mutant. We did isolate a ∆gshB mutant strain that accumulates the biosynthetic intermediate γ-glutamylcysteine: γ-EC) instead of GSH. In this work, I have characterized the physiology of the ∆gshB mutant following environmental, genetic and redox perturbations. The results presented here also shed light on the dynamic nature of the low-molecular weight thiol pool in cyanobacteria. We quantified the levels of cellular thiols in Synechocystis 6803 during exposure to multiple environmental and redox perturbations and found that conditions promoting increased cellular metabolism and increased ROS production, including during high-light treatment and photomixotrophic growth, lead to higher cellular thiol levels. Furthermore, the intracellular pools of thiols decrease when the cell exhibits reduced metabolic capacity during conditions such as nutrient deprivation and dark incubation. Sulfate limitation results in dramatically decreased cellular thiol contents in a short period of time. We found that the ∆gshB strain is sensitive to sulfate limitation and exhibits delayed recovery upon sulfate repletion, indicating that GSH is important for acclimation to sulfate limiting conditions. To facilitate our understanding of GSH degradation in Synechocystis 6803 during sulfate limitation, we generated a mutant lacking γ-glutamyltranspeptidase: Ggt), an enzyme with GSH degradation activity. However, the ∆ggt mutant still exhibited GSH degradation during sulfate depletion, indicating the presence of an alternative system or mechanism. We did find increased levels of GSH in the growth media of the ∆ggt strain compared to the WT, which suggests a role in GSH uptake or prevention of leakage. Our results demonstrate that GSH is essential for protection from multiple environmental and redox perturbations in cyanobacteria. However, there are many pathways involved in maintenance of redox homeostasis in cyanobacteria. Therefore, we also aimed to determine whether these pathways function cooperatively to ameliorate damage from ROS. Several flavodiiron: Flv) proteins have been identified in Synechocystis 6803 that are involved in reduction of O2 to H2O without the formation of ROS intermediates. However, single ∆flv3 mutants do not exhibit severe growth defects under normal conditions. Therefore, we generated a ∆gshB/∆flv3 mutant to examine whether these systems cooperate to maintain redox homeostasis. Our results show that the ∆gshB/∆flv3 mutant exhibits reduced growth than either of the single mutants when grown on solid media, suggesting a degree of interaction between these pathways in cyanobacteria

A transcriptional-switch model for Slr1738-controlled gene expression in the cyanobacterium Synechocystis

<p>Abstract</p> <p>Background</p> <p>Protein-DNA interactions play a crucial role in the life of biological organisms in controlling transcription, regulation, as well as DNA recombination and repair. The deep understanding of these processes, which requires the atomic description of the interactions occurring between the proteins and their DNA partners is often limited by the absence of a 3D structure of such complexes.</p> <p>Results</p> <p>In this study, using a method combining sequence homology, structural analogy modeling and biochemical data, we first build the 3D structure of the complex between the poorly-characterized PerR-like regulator Slr1738 and its target DNA, which controls the defences against metal and oxidative stresses in <it>Synechocystis</it>. In a second step, we propose an expanded version of the Slr1738-DNA structure, which accommodates the DNA binding of Slr1738 multimers, a feature likely operating in the complex Slr1738-mediated regulation of stress responses. Finally, in agreement with experimental data we present a 3D-structure of the Slr1738-DNA complex resulting from the binding of multimers of the FUR-like regulator onto its target DNA that possesses internal repeats.</p> <p>Conclusion</p> <p>Using a combination of different types of data, we build and validate a relevant model of the tridimensional structure of a biologically important protein-DNA complex. Then, based on published observations, we propose more elaborated multimeric models that may be biologically important to understand molecular mechanisms.</p

The Transcriptional Landscape of the Photosynthetic Model Cyanobacterium Synechocystis sp. PCC6803.

Cyanobacteria exhibit a great capacity to adapt to different environmental conditions through changes in gene expression. Although this plasticity has been extensively studied in the model cyanobacterium Synechocystis sp. PCC 6803, a detailed analysis of the coordinated transcriptional adaption across varying conditions is lacking. Here, we report a meta-analysis of 756 individual microarray measurements conducted in 37 independent studies-the most comprehensive study of the Synechocystis transcriptome to date. Using stringent statistical evaluation, we characterized the coordinated adaptation of Synechocystis' gene expression on systems level. Evaluation of the data revealed that the photosynthetic apparatus is subjected to greater changes in expression than other cellular components. Nevertheless, network analyses indicated a significant degree of transcriptional coordination of photosynthesis and various metabolic processes, and revealed the tight co-regulation of components of photosystems I, II and phycobilisomes. Detailed inspection of the integrated data led to the discovery a variety of regulatory patterns and novel putative photosynthetic genes. Intriguingly, global clustering analyses suggested contrasting transcriptional response of metabolic and regulatory genes stress to conditions. The integrated Synechocystis transcriptome can be accessed and interactively analyzed via the CyanoEXpress website (http://cyanoexpress.sysbiolab.eu)

Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

<p>Abstract</p> <p>Background</p> <p>Unicellular cyanobacteria of the genus <it>Cyanothece </it>are recognized for their ability to execute nitrogen (N₂)-fixation in the dark and photosynthesis in the light. An understanding of these mechanistic processes in an integrated systems context should provide insights into how <it>Cyanothece </it>might be optimized for specialized environments and/or industrial purposes. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis should reveal fundamental insights into the control and regulation of these functions.</p> <p>Results</p> <p>To expand upon the current knowledge of protein expression patterns in <it>Cyanothece </it>ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Functional classification of labeled proteins suggested that proteins involved in respiration and glycogen metabolism showed increased expression in the dark cycle together with nitrogenase, suggesting that N₂-fixation is mediated by higher respiration and glycogen metabolism. Results indicated that <it>Cyanothece </it>ATCC51142 might utilize alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand.</p> <p>Conclusion</p> <p>This study provides a deeper systems level insight into how <it>Cyanothece </it>ATCC51142 modulates cellular functions to accommodate photosynthesis and N₂-fixation within the single cell.</p

Hydrocarbon Desaturation in Cyanobacterial Thylakoid Membranes Is Linked With Acclimation to Suboptimal Growth Temperatures

The ability to produce medium chain length aliphatic hydrocarbons is strictly conserved in all photosynthetic cyanobacteria, but the molecular function and biological significance of these compounds still remain poorly understood. This study gives a detailed view to the changes in intracellular hydrocarbon chain saturation in response to different growth temperatures and osmotic stress, and the associated physiological effects in the model cyanobacterium Synechocystis sp. PCC 6803. We show that the ratio between the representative hydrocarbons, saturated heptadecane and desaturated heptadecene, is reduced upon transition from 38°C toward 15°C, while the total content is not much altered. In parallel, it appears that in the hydrocarbon-deficient ∆ado (aldehyde deformylating oxygenase) mutant, phenotypic and metabolic changes become more evident under suboptimal temperatures. These include hindered growth, accumulation of polyhydroxybutyrate, altered pigment profile, restricted phycobilisome movement, and ultimately reduced CO2 uptake and oxygen evolution in the ∆ado strain as compared to Synechocystis wild type. The hydrocarbons are present in relatively low amounts and expected to interact with other nonpolar cellular components, including the hydrophobic part of the membrane lipids. We hypothesize that the function of the aliphatic chains is specifically associated with local fluidity effects of the thylakoid membrane, which may be required for the optimal movement of the integral components of the photosynthetic machinery. The findings support earlier studies and expand our understanding of the biological role of aliphatic hydrocarbons in acclimation to low temperature in cyanobacteria and link the proposed role in the thylakoid membrane to changes in photosynthetic performance, central carbon metabolism, and cell growth, which need to be effectively fine-tuned under alternating conditions in nature

Evaluating the possibilities and limitations of cyanobacterial hosts for future biotechnological applications

Carbon neutral solutions and circular economy are a relevant part of a sustainable future. Solutions based on photosynthetic cyanobacteria have potential due to several facts: i) Cyanobacteria can produce target compounds directly from CO2 and H2O using sunlight. ii) The expanding synthetic biology toolset allows the modifications of native pathways and even the introduction of entire heterologous pathways. iii) Many strains can tolerate harsh conditions and they do not compete for prime land areas with agriculture. Cyanobacteria are more complex biotechnological production platforms compared to traditional heterotrophic bacteria, and therefore a combination of basic research and synthetic biology is required. The work presented in this thesis combines basic research of native hydrocarbon metabolism to the applied utilization of cyanobacteria for heterologous ethylene production. In addition, the host cell properties of two different cyanobacterial strains were compared and, according to the principles of circular bioeconomy, a fermentative waste product was studied as an alternative carbon source for biomass production. The gained results highlight the unexpected importance of native hydrocarbons to cyanobacterial cells. Saturation and quantity of the aliphatic hydrocarbons is strictly controlled and membrane associated, while lack of hydrocarbons resulted in distorted growth and reduced photosynthetic activity, carbon fixation and the floating capacity of cells, which are important features for survival of photosynthetic organism in native aquatic environments. Ethylene, on the other hand, can be produced via the introduced ethylene forming enzyme. An alternative expression strategy enabled long-term ethylene production in two different host strains without the previously encountered loss of viability. Furthermore, both the original and modified gene sequence of efe enabled similar ethylene production in both hosts. This highlights the importance of taking into consideration strain-specific variations when designing production systems. Another important feature in production system design is the flexible use of alternative carbon sources which can function as a backup when light is limited and increase biomass accumulation. The potential for the utilization of acetate was studied as well, and the addition of an acetate transporter was observed to increase acetate intake. This was especially beneficial for cell growth under low light conditions, despite it being accompanied by alterations in photosynthetic activity and intracellular storage compounds.Hiilineutraalit ratkaisut ja kiertotalous ovat oleellinen osa kestävää kehitystä. Yhteyttäviin sinileviin pohjautuvalla bioteknologialla on useita etuja: i) Sinilevät pystyvät tuottamaan haluttuja yhdisteitä suoraan hiilidioksidista ja vedestä auringonvalon avulla. ii) Synteettisen biologian välineistön kehittyessä luontaisia aineenvaihdunta reitistöjä on mahdollista muokata ja uusia lisätä. iii) Monet kannat kestävät karujakin olosuhteita, eivätkä sinilevät kilpaile maatalouden kanssa hyvästä kasvualasta. Sinilevät ovat paljon monimutkaisempia organismeja kuin perinteiset, pieteetillä karakterisoidut, bakteereihin ja hiivoihin pohjautuvat tuottosysteemit, joten ne edellyttävät synteettisen biologian ja perustutkimuksen yhdistelemistä. Tämä väitöskirja käsittelee luontaisten hiilivetyjen merkityksen selvittämistä perustutkimuksen menetelmin ja solujen käyttöä solutehtaina lisätyn etyleenin tuottoreitin avulla. Lisäksi tutkimuksissa vertailtiin kahta sinileväkantaa isäntäsoluina ja sovellettiin kiertotalouden periaatteita käyttämällä käymistuotteena muodostuvaa asetaattia vaihtoehtoisena energian lähteenä biomassan lisäämiseksi. Tulokset osoittivat luontaisten alifaattisten hiilivetyjen merkityksen sinileville. Hiilivetyjen saturaatio ja määrä ovat tarkoin säädeltyjä ja yhteydessä solukalvoihin ja niiden puuttuminen vaikutti solujen kasvuominaisuuksiin sekä heikensi yhteyttämistä, hiilensidontaa ja solujen kellumista, jotka ovat tärkeitä ominaisuuksia yhteyttäville eliöille vesistöissä. Etyleenin tuotto puolestaan onnistuu lisätyn EFE–entsyymin avulla. Uudella tuottostrategialla pitkäaikainen etyleenin tuotto mahdollistettiin kahteen eri sinileväkantaan, ilman aikaisempaa isäntäsolun kärsimistä. Erilaisia efe-geenin variaatioita vertaamalla päästiin samoihin tuottomääriin ilman aikaisemmin käytössä olleita muokkauksia. Nämä tulokset korostivat sinileväkantojen välisiä eroja, joiden ottaminen huomioon on erityisen tärkeää tuotantostrategioita suunnitellessa. Suunnittelussa on tärkeää huomioida myös vaihtoehtoisten hiililähteiden joustava käyttö. Sekä biomassan lisäämisen että valon saatavuuden hetkellisten rajoitteiden vuoksi tutkittiin asetaatin käyttöä vaihtoehtoisena hiililähteenä. Asetaatti transportterin lisäys mahdollisti asetaatin tehokkaamman sisäänoton, joka lisäsi kasvua erityisesti vähäisessä valossa, vaikka aiheuttikin muutoksia myös solujen yhteyttämisaktiivisuuteen ja solun sisäisiin varastomolekyyleihin

Oxygen Photoreduction in Cyanobacteria

Cyanobacteria are well-known for their role in the global production of O2 via photosynthetic water oxidation. However, with the use of light energy, cyanobacteria can also reduce O2. In my thesis work, I have investigated the impact of O2 photoreduction on protection of the photosynthetic apparatus as well as the N2-fixing machinery. Photosynthetic light reactions produce intermediate radicals and reduced electron carriers, which can easily react with O2 to generate various reactive oxygen species. To avoid prolonged reduction of photosynthetic components, cyanobacteria use “electron valves” that dissipate excess electrons from the photosynthetic electron transfer chain in a harmless way. In Synechocystis sp. PCC 6803, flavodiiron proteins Flv1 and Flv3 comprise a powerful electron sink redirecting electrons from the acceptor side of Photosystem I to O2 and reducing it directly to water. In this work, I demonstrate that upon Ci-depletion Flv1/3 can dissipate up to 60% of the electrons delivered from Photosystem II. O2 photoreduction by Flv1/3 was shown to be vital for cyanobacteria in natural aquatic environments and deletion of Flv1/3 was lethal for both Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 under fluctuating light conditions. The lethal phenotype observed in the absence of Flv1/3 results from oxidative damage to Photosystem I, which appeared to be a primary target of reactive oxygen species produced upon sudden increases in light intensity. Importantly, cyanobacteria also possess other O2 photoreduction pathways which can protect the photosynthetic apparatus. This study demonstrates that respiratory terminal oxidases are also capable of initiating O2 photoreduction in mutant cells lacking the Flv1/3 proteins and grown under fluctuating light. Photoreduction of O2 by Rubisco was also shown in Ci-depleted cells of the mutants lacking Flv1/3, and thus provided the first evidence for active photorespiratory gas-exchange in cyanobacteria. Nevertheless, and despite the existence of other O2 photoreduction pathways, the Flv1/3 route appears to be the most robust and rapid system of photoprotection. Several groups of cyanobacteria are capable of N2 fixation. Filamentous heterocystous N2- fixing species, such as Anabaena sp. PCC 7120, are able to differentiate specialised cells called heterocysts for this purpose. In contrast to vegetative cells which perform oxygenic photosynthesis, heterocysts maintain a microoxic environment for the proper function of the nitrogenase enzyme, which is extremely sensitive to O2. The genome of Anabaena sp. PCC 7120 harbors two copies of genes encoding Flv1 and Flv3 proteins, designated as “A” and “B” forms. In this thesis work, I demonstrate that Flv1A and Flv3A are expressed only in the vegetative cells of filaments, whilst Flv1B and Flv3B are localized exclusively in heterocysts. I further revealed that the Flv3B protein is most responsible for the photoreduction of O2 in heterocysts, and that this reaction plays an important role in protection of the N2-fixing machinery and thus, the provision of filaments with fixed nitrogen. The function of the Flv1B protein remains to be elucidated; however the involvement of this protein in electron transfer reactions is feasible. Evidence provided in this thesis indicates the presence of a great diversity of O2 photoreduction reactions in cyanobacterial cells. These reactions appear to be crucial for the photoprotection of both photosynthesis and N2 fixation processes in an oxygenic environment.Syanobakteerien tiedetään osallistuvan maailmanlaajuiseen hapentuotantoon fotosynteesissä tapahtuvan veden hajotuksen, eli kemiallisesti veden hapetuksen, kautta. Valoenergian avulla syanobakteerit myös pelkistävät ilmakehän happea, jolloin lopputuotteena syntyy vettä. Väitöskirjatyössäni olen tutkinut hapen valopelkistyksen molekyylimekanismeja ja roolia syanobakteerien fotosynteesi- ja typensidontakoneistojen suojelemisessa. Fotosynteesin valoreaktioissa syntyy välituotteina monenlaisia radikaaleja ja pelkistyneitä elektroninsiirtäjiä, jotka reagoivat herkästi hapen kanssa synnyttäen erilaisia reaktiivisia happilajeja. Välttääkseen fotosynteesikoneiston pitkittynyttä pelkistymistilaa, syanobakteerit käyttävät ”elektroniventtiilejä”, joiden kautta ne purkavat fotosynteesikoneistoon kertyneet ylimääräiset elektronit vaarattomasti. Synechocystis sp. PCC 6803:n flavodiironproteiinit Flv1 ja Flv3 muodostavat tehokkaan elektroninielun ohjaamalla elektroneja valoreaktio I:n pelkistävältä puolelta suoraan hapelle pelkistäen sen vedeksi. Tässä työssä osoitan, että kun syanobakteereja kasvatetaan matalassa hiilidioksidipitoisuudessa (tasapainossa ympäröivän ilman kanssa), Flv1/3 proteiinipari pystyy kuluttamaan jopa 60% valoreaktio II:n veden hajotuksesta johdetuista elektroneista. Flv1/3-välitteisen hapen valopelkistyksen näytettiin olevan elintärkeää syanobakteereille luonnollisessa vesiympäristössä ja Flv1/3 proteiinien poistaminen johti sekä Synechocystis sp. PCC 6803:n että Anabaena sp. PCC 7120:n kuolemaan vaihtelevan valon olosuhteissa. Flv1/3:n puuttumisen aiheuttaman letaalin ilmiasun osoitettiin johtuvan valoreaktio I:n foto-oksidatiivisesta vaurioitumisesta: äkillinen valointensiteetin nousu johtaa reaktiivisten happilajien muodostumiseen, ja valoreaktio I näytti olevan näiden happilajien pääasiallinen kohde. Flv1/3:n lisäksi syanobakteereilla on myös muita hapen valopelkistämisreittejä, jotka voivat toimia fotosynteesikoneiston suojaamisessa. Tämä tutkimus osoittaa, että hengitysreitin terminaaliset oksidaasit kykenevät myös hapen valopelkistykseen vaihtelevan valon olosuhteissa mutanttisoluissa, joilta puuttuvat Flv1 ja 3 proteiinit. Matalassa hiilidioksidissa näissä mutanttisoluissa voitiin mitata myös Rubisco-välitteistä hapen valopelkistymistä, osoittautuen ensimmäiseksi raportiksi syanobakteerien aktiivisesta fotorespiratorisesta kaasujen vaihdosta. Huolimatta muista hapen valopelkistysreiteistä, Flv1/3 välitteinen reitti näyttäisi olevan kuitenkin vahvin ja nopein fotosynteesikoneiston suojausmekanismi. Monet syanobakteeriryhmät kykenevät ilmakehän typen sidontaan. Rihmamaiset typpeä sitovat syanobakteerit, kuten Anabaena sp. PCC 7120, kykenevät muodostamaan typen sidontaan erilaistuneita soluja: heterokystejä. Toisin kuin yhteyttävissä vegetatiivisissa soluissa, heterokysteissä ylläpidetään lähes hapetonta ympäristöä, joka vaaditaan happiherkän nitrogenaasientsyymin toimintaan. Anabaena sp. PCC 7120:n genomissa on Flv1 että Flv3 proteiineja koodaavista geeneistä kaksi kopiota, niin kutsutut ”A” ja ”B” muodot. Väitöskirjatyössäni osoitan, että Flv1A ja Flv3A ilmentyvät vain syanobakteeririhmojen vegetatiivisissa soluissa, kun taas Flv1B ja Flv3B sijaitsevat ainoastaan heterokysteissä. Lisäksi näytän, että Flv3B on pääasiassa vastuussa hapen valopelkistyksestä heterokysteissä ja että tämä reaktio on tärkeä typpeä sitovan koneiston suojaamisessa ja siten takaa rihmojen typen saannin. Flv1B proteiinin tarkka rooli on vielä selvittämättä, mutta on mahdollista, että se osallistuu elektroninsiirtoreaktioihin. Tässä väitöskirjassa esitetty todistusaineisto viittaa siihen, että syanobakteereilla on useita hapen valopelkistysreittejä. Nämä reaktiot ja reitit ovat välttämättömiä sekä fotosynteettisen että typpeä sitovan koneiston suojelemiseksi hapelta, jota fotosynteesi jatkuvasti tuottaa veden hajotusreaktioissaan.Siirretty Doriast

Evaluating the possibilities and limitations of cyanobacterial hosts for future biotechnological applications

Carbon neutral solutions and circular economy are a relevant part of a sustainable future. Solutions based on photosynthetic cyanobacteria have potential due to several facts: i) Cyanobacteria can produce target compounds directly from CO2 and H2O using sunlight. ii) The expanding synthetic biology toolset allows the modifications of native pathways and even the introduction of entire heterologous pathways. iii) Many strains can tolerate harsh conditions and they do not compete for prime land areas with agriculture. Cyanobacteria are more complex biotechnological production platforms compared to traditional heterotrophic bacteria, and therefore a combination of basic research and synthetic biology is required. The work presented in this thesis combines basic research of native hydrocarbon metabolism to the applied utilization of cyanobacteria for heterologous ethylene production. In addition, the host cell properties of two different cyanobacterial strains were compared and, according to the principles of circular bioeconomy, a fermentative waste product was studied as an alternative carbon source for biomass production.The gained results highlight the unexpected importance of native hydrocarbons to cyanobacterial cells. Saturation and quantity of the aliphatic hydrocarbons is strictly controlled and membrane associated, while lack of hydrocarbons resulted in distorted growth and reduced photosynthetic activity, carbon fixation and the floating capacity of cells, which are important features for survival of photosynthetic organism in native aquatic environments. Ethylene, on the other hand, can be produced via the introduced ethylene forming enzyme. An alternative expression strategy enabled long-term ethylene production in two different host strains without the previously encountered loss of viability. Furthermore, both the original and modified gene sequence of efe enabled similar ethylene production in both hosts. This highlights the importance of taking into consideration strain-specific variations when designing production systems. Another important feature in production system design is the flexible use of alternative carbon sources which can function as a backup when light is limited and increase biomass accumulation. The potential for the utilization of acetate was studied as well, and the addition of an acetate transporter was observed to increase acetate intake. This was especially beneficial for cell growth under low light conditions, despite it being accompanied by alterations in photosynthetic activity and intracellular storage compounds. Hiilineutraalit ratkaisut ja kiertotalous ovat oleellinen osa kestävää kehitystä. Yhteyttäviin sinileviin pohjautuvalla bioteknologialla on useita etuja: i) Sinilevät pystyvät tuottamaan haluttuja yhdisteitä suoraan hiilidioksidista ja vedestä auringonvalon avulla. ii) Synteettisen biologian välineistön kehittyessä luontaisia aineenvaihdunta reitistöjä on mahdollista muokata ja uusia lisätä. iii) Monet kannat kestävät karujakin olosuhteita, eivätkä sinilevät kilpaile maatalouden kanssa hyvästä kasvualasta. Sinilevät ovat paljon monimutkaisempia organismeja kuin perinteiset, pieteetillä karakterisoidut, bakteereihin ja hiivoihin pohjautuvat tuottosysteemit, joten ne edellyttävät synteettisen biologian ja perustutkimuksen yhdistelemistä. Tämä väitöskirja käsittelee luontaisten hiilivetyjen merkityksen selvittämistä perustutkimuksen menetelmin ja solujen käyttöä solutehtaina lisätyn etyleenin tuottoreitin avulla. Lisäksi tutkimuksissa vertailtiin kahta sinileväkantaa isäntäsoluina ja sovellettiin kiertotalouden periaatteita käyttämällä käymistuotteena muodostuvaa asetaattia vaihtoehtoisena energian lähteenä biomassan lisäämiseksi.Tulokset osoittivat luontaisten alifaattisten hiilivetyjen merkityksen sinileville. Hiilivetyjen saturaatio ja määrä ovat tarkoin säädeltyjä ja yhteydessä solukalvoihin ja niiden puuttuminen vaikutti solujen kasvuominaisuuksiin sekä heikensi yhteyttämistä, hiilensidontaa ja solujen kellumista, jotka ovat tärkeitä ominaisuuksia yhteyttäville eliöille vesistöissä. Etyleenin tuotto puolestaan onnistuu lisätyn EFE–entsyymin avulla. Uudella tuottostrategialla pitkäaikainen etyleenin tuotto mahdollistettiin kahteen eri sinileväkantaan, ilman aikaisempaa isäntäsolun kärsimistä. Erilaisia efe-geenin variaatioita vertaamalla päästiin samoihin tuottomääriin ilman aikaisemmin käytössä olleita muokkauksia. Nämä tulokset korostivat sinileväkantojen välisiä eroja, joiden ottaminen huomioon on erityisen tärkeää tuotantostrategioita suunnitellessa. Suunnittelussa on tärkeää huomioida myös vaihtoehtoisten hiililähteiden joustava käyttö. Sekä biomassan lisäämisen että valon saatavuuden hetkellisten rajoitteiden vuoksi tutkittiin asetaatin käyttöä vaihtoehtoisena hiililähteenä. Asetaatti transportterin lisäys mahdollisti asetaatin tehokkaamman sisäänoton, joka lisäsi kasvua erityisesti vähäisessä valossa, vaikka aiheuttikin muutoksia myös solujen yhteyttämisaktiivisuuteen ja solun sisäisiin varastomolekyyleihin.</p

Use of Proteomics to Probe Dynamic Changes in Cyanobacteria

Cyanobacteria are unicellular photosynthetic microorganisms that capture and convert light energy to chemical energy, which is the precursor for feed, fuel, and food. These oxygenic phototrophs appear blue-green in color due to the blue bilin pigments in their phycobilisomes and green chlorophyll pigments in their photosystems. They also have diverse morphologies, and thrive in terrestrial, marine water, fresh water, as well as extreme environments. Cyanobacteria have developed a number of protective mechanisms and adaptive responses that allow the photosynthetic process to operate optimally under diverse and extreme conditions. Prolonged deprivation of essential nutrients, such as nitrogen and sulfur, commonly found in the natural environments cyanobacteria grow in, can disrupt crucial metabolic activities and promote the production of lethal reactive oxygen species. The dynamic remodeling of protein complexes and structures facilitates adaptation to environmental stresses, however, specific protein modifications are poorly understood. Synthetic and systems biology approaches have been used to study how photosynthetic microorganisms optimize their cellular metabolism in response to adverse environmental conditions. To gain insights on how cyanobacteria cope with environmental changes, we created a global proteomics map of redox-sensitive amino acid residues and examined the degradation of light harvesting apparatus in cyanobacteria. These studies offered significant insights into the broad redox regulation and protein degradation, advancing knowledge of how photosynthetic microbial cells dynamically rely on protective mechanisms to survive changing environmental conditions