RESUMEN
Photosynthetic organisms need to balance the rate of photosynthesis with the utilization of photosynthetic products by downstream reactions. While such "source/sink" pathways are well-interrogated in plants, analogous regulatory systems are unknown or poorly studied in single-celled algal and cyanobacterial species. Towards the identification of energy/sugar sensors in cyanobacteria, we utilized an engineered strain of Synechococcus elongatus PCC 7942 that allows experimental manipulation of carbon status. We conducted a screening of all two-component systems (TCS) and serine/threonine kinases (STKs) encoded in S. elongatus PCC 7942 by analyzing phenotypes consistent with sucrose-induced relaxation of sink inhibition. We narrowed the candidate sensor proteins by analyzing changes observed after sucrose feeding. We show that a clustered TCS network containing RpaA, CikB, ManS and NblS are involved in the regulation of genes related to photosynthesis, pigment synthesis, and Rubisco concentration in response to sucrose. Altogether, these results highlight a regulatory TCS group that may play under-appreciated functions in carbon partitioning and energy balancing in cyanobacteria.
Asunto(s)
Carbono , Synechococcus , Carbono/metabolismo , Fotosíntesis , Synechococcus/genética , Synechococcus/metabolismo , Sacarosa/metabolismoRESUMEN
Microbial communities have vital roles in systems essential to human health and agriculture, such as gut and soil microbiomes, and there is growing interest in engineering designer consortia for applications in biotechnology (e.g., personalized probiotics, bioproduction of high-value products, biosensing). The capacity to monitor and model metabolite exchange in dynamic microbial consortia can provide foundational information important to understand the community level behaviors that emerge, a requirement for building novel consortia. Where experimental approaches for monitoring metabolic exchange are technologically challenging, computational tools can enable greater access to the fate of both chemicals and microbes within a consortium. In this study, we developed an in-silico model of a synthetic microbial consortia of sucrose-secreting Synechococcus elongatus PCC 7942 and Escherichia coli W. Our model was built on the NUFEB framework for Individual-based Modeling (IbM) and optimized for biological accuracy using experimental data. We showed that the relative level of sucrose secretion regulates not only the steady-state support for heterotrophic biomass, but also the temporal dynamics of consortia growth. In order to determine the importance of spatial organization within the consortium, we fit a regression model to spatial data and used it to accurately predict colony fitness. We found that some of the critical parameters for fitness prediction were inter-colony distance, initial biomass, induction level, and distance from the center of the simulation volume. We anticipate that the synergy between experimental and computational approaches will improve our ability to design consortia with novel function.
Asunto(s)
Microbiota , Humanos , Consorcios Microbianos , Escherichia coli/metabolismo , Simulación por Computador , BiotecnologíaRESUMEN
Cyanobacteria must prevent imbalances between absorbed light energy (source) and the metabolic capacity (sink) to utilize it to protect their photosynthetic apparatus against damage. A number of photoprotective mechanisms assist in dissipating excess absorbed energy, including respiratory terminal oxidases and flavodiiron proteins, but inherently reduce photosynthetic efficiency. Recently, it has been hypothesized that some engineered metabolic pathways may improve photosynthetic performance by correcting source/sink imbalances. In the context of this subject, we explored the interconnectivity between endogenous electron valves, and the activation of one or more heterologous metabolic sinks. We coexpressed two heterologous metabolic pathways that have been previously shown to positively impact photosynthetic activity in cyanobacteria, a sucrose production pathway (consuming ATP and reductant) and a reductant-only consuming cytochrome P450. Sucrose export was associated with improved quantum yield of phtotosystem II (PSII) and enhanced electron transport chain flux, especially at lower illumination levels, while cytochrome P450 activity led to photosynthetic enhancements primarily observed under high light. Moreover, coexpression of these two heterologous sinks showed additive impacts on photosynthesis, indicating that neither sink alone was capable of utilizing the full "overcapacity" of the electron transport chain. We find that heterologous sinks may partially compensate for the loss of photosystem I (PSI) oxidizing mechanisms even under rapid illumination changes, although this compensation is incomplete. Our results provide support for the theory that heterologous metabolism can act as a photosynthetic sink and exhibit some overlapping functionality with photoprotective mechanisms, while potentially conserving energy within useful metabolic products that might otherwise be "lost."
Asunto(s)
Cianobacterias/metabolismo , Ingeniería Metabólica , Fotosíntesis , Complejo de Proteína del Fotosistema I/metabolismo , Cianobacterias/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Transporte de Electrón , Luz , Redes y Vías Metabólicas/genética , Oxidación-Reducción , Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/metabolismo , Sacarosa/metabolismo , Synechococcus/genética , Synechococcus/metabolismoRESUMEN
Photosynthetic organisms possess a variety of mechanisms to achieve balance between absorbed light (source) and the capacity to metabolically utilize or dissipate this energy (sink). While regulatory processes that detect changes in metabolic status/balance are relatively well studied in plants, analogous pathways remain poorly characterized in photosynthetic microbes. Here, we explored systemic changes that result from alterations in carbon availability in the model cyanobacterium Synechococcus elongatus PCC 7942 by taking advantage of an engineered strain where influx/efflux of a central carbon metabolite, sucrose, can be regulated experimentally. We observed that induction of a high-flux sucrose export pathway leads to depletion of internal carbon storage pools (glycogen) and concurrent increases in estimates of photosynthetic activity. Further, a proteome-wide analysis and fluorescence reporter-based analysis revealed that upregulated factors following the activation of the metabolic sink are concentrated on ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and auxiliary modules involved in Rubisco maturation. Carboxysome number and Rubisco activity also increased following engagement of sucrose secretion. Conversely, reversing the flux of sucrose by feeding exogenous sucrose through the heterologous transporter resulted in increased glycogen pools, decreased Rubisco abundance, and carboxysome reorganization. Our data suggest that Rubisco activity and organization are key variables connected to regulatory pathways involved in metabolic balancing in cyanobacteria.
Asunto(s)
Ribulosa-Bifosfato Carboxilasa , Synechococcus , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Glucógeno/metabolismo , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Sacarosa/metabolismo , Synechococcus/genética , Synechococcus/metabolismoRESUMEN
Carboxysomes are a class of bacterial microcompartments that form proteinaceous organelles within the cytoplasm of cyanobacteria and play a central role in photosynthetic metabolism by defining a cellular microenvironment permissive to CO2 fixation. Critical aspects of the assembly of the carboxysomes remain relatively unknown, especially with regard to the dynamics of this microcompartment. Progress in understanding carboxysome dynamics is impeded in part because analysis of the subtle changes in carboxysome morphology with microscopy remains a low-throughput and subjective process. Here we use deep learning techniques, specifically a Rotationally Invariant Variational Autoencoder (rVAE), to analyze fluorescence microscopy images of cyanobacteria bearing a carboxysome reporter and quantitatively evaluate how carboxysome shell remodelling impacts subtle trends in the morphology of the microcompartment over time. Toward this goal, we use a recently developed tool to control endogenous protein levels, including carboxysomal components, in the model cyanobacterium Synechococcous elongatus PCC 7942. By utilization of this system, proteins that compose the carboxysome can be tuned in real time as a method to examine carboxysome dynamics. We find that rVAEs are able to assist in the quantitative evaluation of changes in carboxysome numbers, shape, and size over time. We propose that rVAEs may be a useful tool to accelerate the analysis of carboxysome assembly and dynamics in response to genetic or environmental perturbation and may be more generally useful to probe regulatory processes involving a broader array of bacterial microcompartments.
Asunto(s)
Synechococcus , Proteínas Bacterianas/metabolismo , Dióxido de Carbono , Microscopía Fluorescente , Orgánulos/metabolismo , Fotosíntesis , Synechococcus/genética , Synechococcus/metabolismoRESUMEN
Enzymes of natural biochemical pathways are routinely subcellularly organized in space and time in order to improve pathway efficacy and control. Designer scaffolding platforms are under development to confer similar benefits upon engineered pathways. Herein, we evaluate bacterial microcompartment shell (pfam0936-domain) proteins as modules for constructing well-defined nanometer scale scaffolds in vivo. We use a suite of visualization techniques to evaluate scaffold assembly and dynamics. We demonstrate recruitment of target cargo molecules onto assembled scaffolds by appending reciprocally interacting adaptor domains. These interactions can be refined by fine-tuning the scaffold expression level. Real-time observation of this system reveals a nucleation-limited step where multiple scaffolds initially form within a cell. Over time, nucleated scaffolds reorganize into a single intracellular assembly, likely due to interscaffold competition for protein subunits. Our results suggest design considerations for using self-assembling proteins as building blocks to construct nanoscaffolds, while also providing a platform to visualize scaffold-cargo dynamics in vivo.
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Bacterias/química , Nanoestructuras/química , Bacterias/ultraestructura , Nanoestructuras/ultraestructuraRESUMEN
Rapid and directed electron transfer (ET) is essential for biological processes. While the rates of ET over 1-2 nm in proteins can largely be described by simplified nonadiabatic theory, it is not known how these processes scale to microscopic distances. We generated crystalline lattices of Small Tetraheme Cytochromes (STC) forming well-defined, three-dimensional networks of closely spaced redox centers that appear to be nearly ideal for multistep ET. Electrons were injected into specific locations in the STC crystals by direct photoreduction, and their redistribution was monitored by imaging. The results demonstrate ET over mesoscopic to microscopic (â¼100 µm) distances through sequential hopping in a biologically based heme network. We estimate that a hypothetical "nanowire" composed of crystalline STC with a cross-section of about 100 cytochromes could support the anaerobic respiration of a Shewanella cell. The crystalline lattice insulates mobile electrons from oxidation by O2, as compared to those in cytochromes in solution, potentially allowing for efficient delivery of current without production of reactive oxygen species. The platform allows direct tests of whether the assumptions based on short-range ET hold for sequential ET over mesoscopic distances. We estimate that the interprotein ET across 6 Å between hemes in adjacent proteins was about 105 s-1, about 100-fold slower than expectations based on simplified theory. More detailed analyses implied that additional factors, possibly contributed by the crystal lattice, may strongly impact mesoscale ET mainly by increasing the reorganizational energy of interprotein ET, which suggests design strategies for engineering improved nanowires suitable for future bioelectronic materials.
Asunto(s)
Citocromos/metabolismo , Cristalografía por Rayos X , Citocromos/química , Transporte de Electrón , Modelos Moleculares , Shewanella/química , Shewanella/citologíaRESUMEN
The cytoskeletal Filamenting temperature-sensitive Z (FtsZ) ring is critical for cell division in bacteria and chloroplast division in photosynthetic eukaryotes. While bacterial FtsZ rings are composed of a single FtsZ, except in the basal glaucophytes, chloroplast division involves two heteropolymer-forming FtsZ isoforms: FtsZ1 and FtsZ2 in the green lineage and FtsZA and FtsZB in red algae. FtsZ1 and FtsZB probably arose by duplication of the more ancestral FtsZ2 and FtsZA, respectively. We expressed fluorescent fusions of FtsZ from diverse photosynthetic organisms in a heterologous system to compare their intrinsic assembly and dynamic properties. FtsZ2 and FtsZA filaments were morphologically distinct from FtsZ1 and FtsZB filaments. When coexpressed, FtsZ pairs from plants and algae colocalized, consistent with heteropolymerization. Fluorescence recovery after photobleaching experiments demonstrated that subunit exchange was greater from FtsZ1 and FtsZB filaments than from FtsZ2 and FtsZA filaments and that FtsZ1 and FtsZB increased turnover of FtsZ2 and FtsZA, respectively, from heteropolymers. GTPase activity was essential only for turnover of FtsZ2 and FtsZA filaments. Cyanobacterial and glaucophyte FtsZ properties mostly resembled those of FtsZ2 and FtsZA, though the glaucophyte protein exhibited some hybrid features. Our results demonstrate that the more ancestral FtsZ2 and FtsZA have retained functional attributes of their common FtsZ ancestor, while eukaryotic-specific FtsZ1 and FtsZB acquired new but similar dynamic properties, possibly through convergent evolution. Our findings suggest that the evolution of a second FtsZ that could copolymerize with the more ancestral form to enhance FtsZ-ring dynamics may have been essential for plastid evolution in the green and red photosynthetic lineages.
Asunto(s)
Cloroplastos/metabolismo , Secuencia Conservada , Citoesqueleto/metabolismo , Fotosíntesis , Filogenia , Proteínas de Plantas/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , GTP Fosfohidrolasas/metabolismo , Transporte de ProteínasRESUMEN
The oscillatory Min system of Escherichia coli defines the cell division plane by regulating the site of FtsZ-ring formation and represents one of the best-understood examples of emergent protein self-organization in nature. The oscillatory patterns of the Min-system proteins MinC, MinD and MinE (MinCDE) are strongly dependent on the geometry of membranes they bind. Complex internal membranes within cyanobacteria could disrupt this self-organization by sterically occluding or sequestering MinCDE from the plasma membrane. Here, it was shown that the Min system in the cyanobacterium Synechococcus elongatus PCC 7942 oscillates from pole-to-pole despite the potential spatial constraints imposed by their extensive thylakoid network. Moreover, reaction-diffusion simulations predict robust oscillations in modeled cyanobacterial cells provided that thylakoid network permeability is maintained to facilitate diffusion, and suggest that Min proteins require preferential affinity for the plasma membrane over thylakoids to correctly position the FtsZ ring. Interestingly, in addition to oscillating, MinC exhibits a midcell localization dependent on MinD and the DivIVA-like protein Cdv3, indicating that two distinct pools of MinC are coordinated in S. elongatus. Our results provide the first direct evidence for Min oscillation outside of E. coli and have broader implications for Min-system function in bacteria and organelles with internal membrane systems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Synechococcus/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/metabolismo , División Celular , Membrana Celular/metabolismo , Simulación por Computador , Cianobacterias/metabolismo , Proteínas del Citoesqueleto/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Relación Estructura-Actividad , Synechococcus/fisiología , Tilacoides/fisiologíaRESUMEN
We previously reported that Synechococcus elongatus PCC 7942, engineered with the sucrose transporter CscB, can export up to 85% of its photosynthetically-fixed carbon as sucrose and shows considerable promise as an alternative carbohydrate source. One approach to effectively utilize this cyanobacterium is to generate synthetic, light-driven consortia in which sucrose-metabolizing heterotrophs catalyze the conversion of the low-value carbohydrate into higher-value compounds in co-culture. Here, we report an improved synthetic photoautotroph/chemoheterotroph consortial design in which sucrose secreted by S. elongatus CscB directly supports the bacterium Halomonas boliviensis, a natural producer of the bioplastic precursor, PHB. We show that alginate encapsulation of S. elongatus CscB enhances sucrose-export rates ~2-fold within 66h, to ~290mg sucrose L-1d-1 OD750-1 and enhances the co-culture stability. Consortial H. boliviensis accumulate up to 31% of their dry-weight as PHB, reaching productivities up to 28.3mg PHB L-1d-1. This light-driven, alginate-partitioned co-culture platform achieves PHB productivities that match or exceed those of traditionally engineered cyanobacterial monocultures. Importantly, S. elongatus CscB/H. boliviensis co-cultures were continuously productive for over 5 months and resisted invasive microbial species without the application of antibiotics or other chemical selection agents.
Asunto(s)
Halomonas , Luz , Consorcios Microbianos , Polímeros/metabolismo , Synechococcus , Halomonas/genética , Halomonas/crecimiento & desarrollo , Synechococcus/genética , Synechococcus/crecimiento & desarrolloRESUMEN
Cyanobacteria are emerging as alternative crop species for the production of fuels, chemicals, and biomass. Yet, the success of these microbes depends on the development of cost-effective technologies that permit scaled cultivation and cell harvesting. Here, we investigate the feasibility of engineering cell morphology to improve biomass recovery and decrease energetic costs associated with lysing cyanobacterial cells. Specifically, we modify the levels of Min system proteins in Synechococcus elongatus PCC 7942. The Min system has established functions in controlling cell division by regulating the assembly of FtsZ, a tubulin-like protein required for defining the bacterial division plane. We show that altering the expression of two FtsZ-regulatory proteins, MinC and Cdv3, enables control over cell morphology by disrupting FtsZ localization and cell division without preventing continued cell growth. By varying the expression of these proteins, we can tune the lengths of cyanobacterial cells across a broad dynamic range, anywhere from an â¼20% increased length (relative to the wild type) to near-millimeter lengths. Highly elongated cells exhibit increased rates of sedimentation under low centrifugal forces or by gravity-assisted settling. Furthermore, hyperelongated cells are also more susceptible to lysis through the application of mild physical stress. Collectively, these results demonstrate a novel approach toward decreasing harvesting and processing costs associated with mass cyanobacterial cultivation by altering morphology at the cellular level.IMPORTANCE We show that the cell length of a model cyanobacterial species can be programmed by rationally manipulating the expression of protein factors that suppress cell division. In some instances, we can increase the size of these cells to near-millimeter lengths with this approach. The resulting elongated cells have favorable properties with regard to cell harvesting and lysis. Furthermore, cells treated in this manner continue to grow rapidly at time scales similar to those of uninduced controls. To our knowledge, this is the first reported example of engineering the cell morphology of cyanobacteria or algae to make them more compatible with downstream processing steps that present economic barriers to their use as alternative crop species. Therefore, our results are a promising proof-of-principle for the use of morphology engineering to increase the cost-effectiveness of the mass cultivation of cyanobacteria for various sustainability initiatives.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ingeniería Genética , Synechococcus/citología , Synechococcus/crecimiento & desarrollo , Bacteriólisis , Biomasa , Centrifugación , Synechococcus/genéticaRESUMEN
Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.
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Proteínas Hierro-Azufre/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cisteína/química , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/química , Oxidación-ReducciónRESUMEN
In plants, a limited capacity to utilize or export the end-products of the Calvin-Benson cycle (CB) from photosynthetically active source cells to non-photosynthetic sink cells can result in reduced carbon capture and photosynthetic electron transport (PET), and lowered photochemical efficiency. The down-regulation of photosynthesis caused by reduced capacity to utilize photosynthate has been termed 'sink limitation'. Recently, several cyanobacterial and algal strains engineered to overproduce target metabolites have exhibited increased photochemistry, suggesting that possible source-sink regulatory mechanisms may be involved. We directly examined photochemical properties following induction of a heterologous sucrose 'sink' in the unicellular cyanobacterium Synechococcus elongatus PCC 7942. We show that total photochemistry increases proportionally to the experimentally controlled rate of sucrose export. Importantly, the quantum yield of PSII (ΦII) increases in response to sucrose export while the PET chain becomes more oxidized from less PSI acceptor-side limitation, suggesting increased CB activity and a decrease in sink limitation. Enhanced photosynthetic activity and linear electron flow are detectable within hours of induction of the heterologous sink and are independent of pigmentation alterations or the ionic/osmotic effects of the induction system. These observations provide direct evidence that secretion of heterologous carbon bioproducts can be used as an alternative approach to improve photosynthetic efficiency, presumably by by-passing sink limitation. Our results also suggest that engineered microalgal production strains are valuable alternative models for examining photosynthetic sink limitation because they enable greater control and monitoring of metabolite fluxes relative to plants.
Asunto(s)
Carbono/metabolismo , Metabolismo Energético , Regulación de la Expresión Génica de las Plantas , Ingeniería Metabólica , Sacarosa/metabolismo , Synechococcus/fisiología , Clorofila/metabolismo , Regulación hacia Abajo , Transporte de Electrón , Luz , Oxidación-Reducción , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Synechococcus/efectos de la radiaciónRESUMEN
Eukaryotic sugar transporters of the MFS and SWEET superfamilies consist of 12 and 7 α-helical transmembrane domains (TMs), respectively. Structural analyses indicate that MFS transporters evolved from a series of tandem duplications of an ancestral 3-TM unit. SWEETs are heptahelical proteins carrying a tandem repeat of 3-TM separated by a single TM. Here, we show that prokaryotes have ancestral SWEET homologs with only 3-TM and that the Bradyrhizobium japonicum SemiSWEET1, like Arabidopsis SWEET11, mediates sucrose transport. Eukaryotic SWEETs most likely evolved by internal duplication of the 3-TM, suggesting that SemiSWEETs form oligomers to create a functional pore. However, it remains elusive whether the 7-TM SWEETs are the functional unit or require oligomerization to form a pore sufficiently large to allow for sucrose passage. Split ubiquitin yeast two-hybrid and split GFP assays indicate that Arabidopsis SWEETs homo- and heterooligomerize. We examined mutant SWEET variants for negative dominance to test if oligomerization is necessary for function. Mutation of the conserved Y57 or G58 in SWEET1 led to loss of activity. Coexpression of the defective mutants with functional A. thaliana SWEET1 inhibited glucose transport, indicating that homooligomerization is necessary for function. Collectively, these data imply that the basic unit of SWEETs, similar to MFS sugar transporters, is a 3-TM unit and that a functional transporter contains at least four such domains. We hypothesize that the functional unit of the SWEET family of transporters possesses a structure resembling the 12-TM MFS structure, however, with a parallel orientation of the 3-TM unit.
Asunto(s)
Proteínas Bacterianas/metabolismo , Metabolismo de los Hidratos de Carbono , Proteínas de Transporte de Membrana/metabolismo , Familia de Multigenes , Proteínas de Plantas/metabolismo , Multimerización de Proteína , Sacarosa/metabolismo , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Proteínas Bacterianas/química , Transporte Biológico , Bradyrhizobium/metabolismo , Prueba de Complementación Genética , Glucosa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Transporte de Membrana/química , Modelos Biológicos , Filogenia , Proteínas de Plantas/química , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Carbohydrate feedstocks are at the root of bioindustrial production and are needed in greater quantities than ever due to increased prioritization of renewable fuels with reduced carbon footprints. Cyanobacteria possess a number of features that make them well suited as an alternative feedstock crop in comparison to traditional terrestrial plant species. Recent advances in genetic engineering, as well as promising preliminary investigations of cyanobacteria in a number of distinct production regimes have illustrated the potential of these aquatic phototrophs as biosynthetic chassis. Further improvements in strain productivities and design, along with enhanced understanding of photosynthetic metabolism in cyanobacteria may pave the way to translate cyanobacterial theoretical potential into realized application.
Asunto(s)
Biocombustibles , Carbohidratos/biosíntesis , Cianobacterias/metabolismo , Ingeniería Genética/métodos , Fotosíntesis/fisiología , Biomasa , Cianobacterias/genéticaRESUMEN
Hydrogenases catalyze the reversible reaction 2H(+) + 2e(-) â H(2) with an equilibrium constant that is dependent on the reducing potential of electrons carried by their redox partner. To examine the possibility of increasing the photobiological production of hydrogen within cyanobacterial cultures, we expressed the [FeFe] hydrogenase, HydA, from Clostridium acetobutylicum in the non-nitrogen-fixing cyanobacterium Synechococcus elongatus sp. 7942. We demonstrate that the heterologously expressed hydrogenase is functional in vitro and in vivo, and that the in vivo hydrogenase activity is connected to the light-dependent reactions of the electron transport chain. Under anoxic conditions, HydA activity is capable of supporting light-dependent hydrogen evolution at a rate > 500-fold greater than that supported by the endogenous [NiFe] hydrogenase. Furthermore, HydA can support limited growth solely using H(2) and light as the source of reducing equivalents under conditions where Photosystem II is inactivated. Finally, we demonstrate that the addition of exogenous ferredoxins can modulate redox flux in the hydrogenase-expressing strain, allowing for greater hydrogen yields and for dark fermentation of internal energy stores into hydrogen gas.
Asunto(s)
Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Synechococcus/enzimología , Transporte de Electrón , Hidrógeno/metabolismo , Oxidación-Reducción , Synechococcus/crecimiento & desarrolloRESUMEN
Cyanobacteria have been proposed as a potential alternative carbohydrate feedstock and multiple species have been successfully engineered to secrete fermentable sugars. To date, the most productive cyanobacterial strains are those designed to secrete sucrose, yet there exist considerable differences in reported productivities across different model species and laboratories. In this study, we investigate how cultivation conditions (specifically, irradiance, CO2, and cultivator type) affect the productivity of sucrose-secreting Synechococcus elongatus PCC 7942. We find that S. elongatus produces the highest sucrose yield in irradiances far greater than what is often experimentally utilized, and that high light intensities are tolerated by S. elongatus, especially under higher density cultivation where turbidity may attenuate the effective light experienced in the culture. By increasing light and inorganic carbon availability, S. elongatus cscB/sps produced a total of 3.8 g L-1 of sucrose and the highest productivity within that period being 47.8 mg L-1 h-1. This study provides quantitative description of the impact of culture conditions on cyanobacteria-derived sucrose that may assist to standardize cross-laboratory comparisons and demonstrates a significant capacity to improve productivity via optimizing cultivation conditions.
RESUMEN
Surface display technologies have been primarily developed for heterotrophic microbes, leaving photosynthetic counterparts like cyanobacteria with limited molecular tools. Here, we expanded upon surface display systems in Synechococcus elongatus PCC 7942 by modifying two outer-membrane proteins, SomA and Intimin, to display tags ( e.g. , SpyTag) to mediate physical interactions of living cyanobacteria with other biotic and abiotic targets. While re-engineered SomA constructs successfully translocated to the cell surface and could bind to compatible ligands, the efficacy of the best-performing designs was limited by a poorly-understood heterogeneity in the accessibility of the tags in living cells, resulting in low attachment penetrance.
RESUMEN
Synthetic biology conceptualizes biological complexity as a network of biological parts, devices, and systems with predetermined functionalities and has had a revolutionary impact on fundamental and applied research. With the unprecedented ability to synthesize and transfer any DNA and RNA across organisms, the scope of synthetic biology is expanding and being recreated in previously unimaginable ways. The field has matured to a level where highly complex networks, such as artificial communities of synthetic organisms, can be constructed. In parallel, computational biology became an integral part of biological studies, with computational models aiding the unravelling of the escalating complexity and emerging properties of biological phenomena. However, there is still a vast untapped potential for the complete integration of modelling into the synthetic design process, presenting exciting opportunities for scientific advancements. Here, we first highlight the most recent advances in computer-aided design of microbial communities. Next, we propose that such a design can benefit from an organism-free modular modelling approach that places its emphasis on modules of organismal function towards the design of multispecies communities. We argue for a shift in perspective from single organism-centred approaches to emphasizing the functional contributions of organisms within the community. By assembling synthetic biological systems using modular computational models with mathematical descriptions of parts and circuits, we can tailor organisms to fulfil specific functional roles within the community. This approach aligns with synthetic biology strategies and presents exciting possibilities for the design of artificial communities. Graphical Abstract.