Population-level heterogeneity complicates utilization of Synechococcus elongatus PCC 7942 surface display platforms.

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.

Impact of irradiance and inorganic carbon availability on heterologous sucrose production in Synechococcus elongatus PCC 7942.

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.

Coordination of carbon partitioning and photosynthesis by a two-component signaling network in Synechococcus elongatus PCC 7942.

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.

Predicting partner fitness based on spatial structuring in a light-driven microbial community.

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.

Cyanobacteria as cell factories for the photosynthetic production of sucrose.

Biofuels and other biologically manufactured sustainable goods are growing in popularity and demand. Carbohydrate feedstocks required for industrial fermentation processes have traditionally been supplied by plant biomass, but the large quantities required to produce replacement commodity products may prevent the long-term feasibility of this approach without alternative strategies to produce sugar feedstocks. Cyanobacteria are under consideration as potential candidates for sustainable production of carbohydrate feedstocks, with potentially lower land and water requirements relative to plants. Several cyanobacterial strains have been genetically engineered to export significant quantities of sugars, especially sucrose. Sucrose is not only naturally synthesized and accumulated by cyanobacteria as a compatible solute to tolerate high salt environments, but also an easily fermentable disaccharide used by many heterotrophic bacteria as a carbon source. In this review, we provide a comprehensive summary of the current knowledge of the endogenous cyanobacterial sucrose synthesis and degradation pathways. We also summarize genetic modifications that have been found to increase sucrose production and secretion. Finally, we consider the current state of synthetic microbial consortia that rely on sugar-secreting cyanobacterial strains, which are co-cultivated alongside heterotrophic microbes able to directly convert the sugars into higher-value compounds (e.g., polyhydroxybutyrates, 3-hydroxypropionic acid, or dyes) in a single-pot reaction. We summarize recent advances reported in such cyanobacteria/heterotroph co-cultivation strategies and provide a perspective on future developments that are likely required to realize their bioindustrial potential.

Developing Cyanobacterial Quorum Sensing Toolkits: Toward Interspecies Coordination in Mixed Autotroph/Heterotroph Communities.

There has been substantial recent interest in the promise of sustainable, light-driven bioproduction using cyanobacteria, including developing efforts for microbial bioproduction using mixed autotroph/heterotroph communities, which could provide useful properties, such as division of metabolic labor. However, building stable mixed-species communities of sufficient productivity remains a challenge, partly due to the lack of strategies for synchronizing and coordinating biological activities across different species. To address this obstacle, we developed an inter-species communication system using quorum sensing (QS) modules derived from well-studied pathways in heterotrophic microbes. In the model cyanobacterium, Synechococcus elongatus PCC 7942 (S. elongatus), we designed, integrated, and characterized genetic circuits that detect acyl-homoserine lactones (AHLs), diffusible signals utilized in many QS pathways. We showed that these receiver modules sense exogenously supplied AHL molecules and activate gene expression in a dose-dependent manner. We characterized these AHL receiver circuits in parallel with Escherichia coli W (E. coli W) to dissect species-specific properties, finding broad agreement, albeit with increased basal expression in S. elongatus. Our engineered "sender" E. coli strains accumulated biologically synthesized AHLs within the supernatant and activated receiver strains similarly to exogenous AHL activation. Our results will bolster the design of sophisticated genetic circuits in cyanobacterial/heterotroph consortia and the engineering of QS-like behaviors across cyanobacterial populations.

Investigating Carboxysome Morphology Dynamics with a Rotationally Invariant Variational Autoencoder.

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.

Rubisco regulation in response to altered carbon status in the cyanobacterium Synechococcus elongatus PCC 7942.

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.

Generation of Stable, Light-Driven Co-cultures of Cyanobacteria with Heterotrophic Microbes.

Co-cultivation of an autotrophic species with one or more heterotrophic microbes is a strategy for photobiological production of high-value compounds and is relatively underexplored in comparison to cyanobacterial or microalgal monocultures. Long-term stability of such consortia is required for useful collaboration between the partners, and this property can be increased by encapsulation of phototrophic partners within a hydrogel. Encapsulated cyanobacteria have advantages relative to planktonic cultures that may be useful to explore the potential for artificial microbial communities for targeted biomolecule synthesis, such as increased control over population sizes and reduced liquid handling requirements. In this chapter, we describe a method for encapsulation of genetically modified cyanobacterial strain (Synechococcus elongatus PCC 7942, CscB+) into a sodium alginate matrix, and the utilization of these encapsulated cells to construct stable, artificial autotroph/heterotroph co-cultures. This method has applications for the study of phototroph-based synthetic microbial consortia, and multi-species photobiological production.

Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria.

Synechococcus elongatus PCC 7942 is a model cyanobacterium for study of the circadian clock, photosynthesis, and bioproduction of chemicals, yet nearly 40% of its gene identities and functions remain unknown, in part due to limitations of the existing genetic toolkit. While classical techniques for the study of genes (e.g., deletion or mutagenesis) can yield valuable information about the absence of a gene and its associated protein, there are limits to these approaches, particularly in the study of essential genes. Herein, we developed a tool for inducible degradation of target proteins in S. elongatus by adapting a method using degron tags from the Mesoplasma florum transfer-mRNA (tmRNA) system. We observed that M. florum lon protease can rapidly degrade exogenous and native proteins tagged with the cognate sequence within hours of induction. We used this system to inducibly degrade the essential cell division factor, FtsZ, as well as shell protein components of the carboxysome. Our results have implications for carboxysome biogenesis and the rate of carboxysome turnover during cell growth. Lon protease control of proteins offers an alternative approach for the study of essential proteins and protein dynamics in cyanobacteria.

Improved photosynthetic capacity and photosystem I oxidation via heterologous metabolism engineering in cyanobacteria.

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."

Mesoscopic to Macroscopic Electron Transfer by Hopping in a Crystal Network of Cytochromes.

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 (∼100 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.

Biotransformation of 2,4-dinitrotoluene in a phototrophic co-culture of engineered Synechococcus elongatus and Pseudomonas putida.

In contrast to the current paradigm of using microbial mono-cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed-species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co-culture: cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4-dinitrotoluene (2,4-DNT). By encapsulating S. elongatus within a barium-alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4-DNT, enhancing the performance of the co-culture. The synthetic consortium was able to convert 2,4-DNT with light and CO2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose-dependent accumulation of polyhydroxyalkanoate, an added-value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4-DNT while simultaneously synthesizing biopolymers using light and CO2 as the primary inputs.

Visualizing in Vivo Dynamics of Designer Nanoscaffolds.

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.

New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering.

Cyanobacteria are promising microorganisms for sustainable biotechnologies, yet unlocking their potential requires radical re-engineering and application of cutting-edge synthetic biology techniques. In recent years, the available devices and strategies for modifying cyanobacteria have been increasing, including advances in the design of genetic promoters, ribosome binding sites, riboswitches, reporter proteins, modular vector systems, and markerless selection systems. Because of these new toolkits, cyanobacteria have been successfully engineered to express heterologous pathways for the production of a wide variety of valuable compounds. Cyanobacterial strains with the potential to be used in real-world applications will require the refinement of genetic circuits used to express the heterologous pathways and development of accurate models that predict how these pathways can be best integrated into the larger cellular metabolic network. Herein, we review advances that have been made to translate synthetic biology tools into cyanobacterial model organisms and summarize experimental and in silico strategies that have been employed to increase their bioproduction potential. Despite the advances in synthetic biology and metabolic engineering during the last years, it is clear that still further improvements are required if cyanobacteria are to be competitive with heterotrophic microorganisms for the bioproduction of added-value compounds.

Functionalization of Bacterial Microcompartment Shell Proteins With Covalently Attached Heme.

Heme is a versatile redox cofactor that has considerable potential for synthetic biology and bioelectronic applications. The capacity to functionalize non-heme-binding proteins with covalently bound heme moieties in vivo could expand the variety of bioelectronic materials, particularly if hemes could be attached at defined locations so as to facilitate position-sensitive processes like electron transfer. In this study, we utilized the cytochrome maturation system I to develop a simple approach that enables incorporation of hemes into the backbone of target proteins in vivo. We tested our methodology by targeting the self-assembling bacterial microcompartment shell proteins, and inserting functional hemes at multiple locations in the protein backbone. We found substitution of three amino acids on the target proteins promoted heme attachment with high occupancy. Spectroscopic measurements suggested these modified proteins covalently bind low-spin hemes, with relative low redox midpoint potentials (about -210 mV vs. SHE). Heme-modified shell proteins partially retained their self-assembly properties, including the capacity to hexamerize, and form inter-hexamer attachments. Heme-bound shell proteins demonstrated the capacity to integrate into higher-order shell assemblies, however, the structural features of these macromolecular complexes was sometimes altered. Altogether, we report a versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins, and provide design considerations on how heme incorporation may interface with native assembly properties in engineered proteins.

Protein gradients on the nucleoid position the carbon-fixing organelles of cyanobacteria.

Carboxysomes are protein-based bacterial organelles encapsulating key enzymes of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we identify a small protein, termed McdB that localizes to carboxysomes and drives emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly stimulates McdA ATPase activity and its release from DNA, driving carboxysome-dependent depletion of McdA locally on the nucleoid and promoting directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a previously unknown class of self-organizing proteins that utilize a Brownian-ratchet mechanism to position carboxysomes in cyanobacteria, rather than a cytoskeletal system. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria.

Conserved Dynamics of Chloroplast Cytoskeletal FtsZ Proteins Across Photosynthetic Lineages.

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.