Daniel C. Ducat

Synthetic trans-kingdom signaling systems with tunable transduction properties provide a unique avenue to explore how communication between organisms coordinate behaviors without the confounding complexity of natural systems. However, existing trans-kingdom signaling systems suffer from either toxic signal molecules, a need for rare precursors for signal generation, or transduction machinery which is either species-specific or cannot be easily tuned. This limits their capacity to be broadly applied across kingdoms and their utility as a model system to study trans-kingdom interactions. In this work we elucidate the design principles for a trans-kingdom signaling system based on the plant phytohormone, Indole 3-Acetic Acid (auxin), that overcomes these challenges. We demonstrate how auxin biosynthesis can be engineered into a model sender organism, the cyanobacteria Synechococcus elongatus PCC 7942, and how this signal can be tuned with low leak and high dynamic range. We also show these signals can be connected to gene expression using tunable synthetic auxin-responsive signaling systems across multiple kingdoms. We demonstrate that S. elongatus generated auxin signals can tune gene expression in co-cultured Saccharomyces cerevisiae via auxin-induced degradation. Additionally, we show that when the S. elongatus are associated with the roots of the model plant, Arabidopsis thaliana , the auxin they generate can control gene expression via auxin-responsive synthetic transcription factors expressed from the plant’s genome. Taken together, our results demonstrate the design principles for a tunable auxin-based trans-kingdom signaling system that can be broadly applied across species.

Sucrose metabolism is an important feature of the physiology of the green lineage of photosynthetic organisms and has therefore been the subject of considerable research on plants, algae, and cyanobacteria. Canonical sucrose biosynthesis pathways often involve the condensation of NDP-glucose and fructose-6-phosphate through the action of sucrose-phosphate synthase, then the dephosphorylation of sucrose 6-phosphate into sucrose via sucrose-phosphate phosphatase (SPP). However, many cyanobacterial genomes encode multiple homologs of SPP proteins (SPP-like), including variants that appear to lack key residues reported to be important for sucrose 6-phosphate binding. Herein, we examine these SPP-like proteins, focusing on the biochemical and physiological characterization of the SPP-like protein encoded by the cyanobacterial model, Synechococcus elongatus PCC 7942. Bioinformatic analysis suggests that the SPP-like family of proteins is highly conserved across cyanobacterial species and forms distinct phylogenetic clades that are more widely distributed than the canonical SPP proteins themselves. Biochemical analysis of the purified S. elongatus PCC 7942 SPP-like protein reveals that it not only retains the capacity to dephosphorylate sucrose 6-phosphate, but it also may have physiologically relevant phosphatase activity on 3-phosphoglycerate (3-PGA). We provide evidence that the SPP-like family of proteins represents a well-conserved group of phosphatases across cyanobacteria and suggest some enzymes in this family may have evolved a broader substrate specificity relative to the well-characterized SPP family. Our results have broader implications for cyanobacterial regulation of sucrose biosynthesis and other key steps of central carbon metabolism.

In cyanobacteria, carbohydrate products of the Calvin cycle can be utilized as precursors for cellular biosynthesis or stored as glucose polymers in glycogen granules. Cyanobacterial carbon fixation occurs in the carboxysome, while glycogen is synthesized in the cytosol via GlgC to support respiration and survival in the dark. Here, we identify a carboxysome-associated protein, C arboxysome I nteracting P rotein G (CipG), that appears to physically link these processes. CipG coassociates in cyanobacterial genomes with peripheral carboxysome structural proteins and localizes to carboxysomes in vivo via a process that requires conserved tryptophan residues. CipG coprecipitates with GlgC and is required for the recruitment of a subpopulation of GlgC to the surface of the carboxysome. CipG mutants display a glycogen deficiency, while CipG overexpression increases glycogen accumulation ~sixfold compared to wild type. Taken together, our results highlight a role for CipG in cyanobacterial energy metabolism and suggest an additional capacity for the carboxysome to scaffold key enzymes related to carbon partitioning in cyanobacteria.

Survival in dynamic environments requires photosynthetic organisms to rapidly sense and respond to stress. The stringent response, mediated by the signaling molecule guanosine-3',5'-bisdiphosphate (ppGpp), is crucial for acclimation to environmental changes such as darkness and nitrogen limitation. While it has been extensively characterized in heterotrophic bacteria such as Escherichia coli, the molecular mechanisms and regulatory targets of ppGpp in photosynthetic organisms remain less understood. Here, we report large-scale chemoproteomic identification of ppGpp-binding proteins across plant chloroplasts and cyanobacteria, revealing both conserved and novel targets compared to E. coli. In plants, we found that ppGpp regulates pyrimidine metabolism by inhibiting the chloroplastic enzyme aspartate transcarbamoylase (PyrB). In cyanobacteria, we found that ppGpp activates glucose-1-phosphate adenylyltransferase (GlgC) involved in glycogen synthesis, activates citrate synthase (GltA), and induces carboxysome aggregation. These findings expand the known ppGpp regulatory network in photosynthetic organisms and provide a foundation for understanding how ppGpp coordinates adaptation to nutrient and environmental stresses.

Gas exchange measurements are widely used in plant research to quantitatively resolve photosynthetic performance and CO2 assimilation rates, yet gas exchange techniques are less commonly applied to algae and cyanobacteria. Cyanobacteria have a well-characterized carbon-concentrating mechanism (CCM) that includes a proteinaceous microcompartment called the carboxysome. Carboxysome structural components have been extensively studied and consist of a nucleated core of rubisco encapsulated by a diverse family of structurally related shell proteins. In addition to the dominant shell proteins that compose the majority of carboxysome facets, accessory shell components are widely distributed throughout cyanobacterial phyla. Herein, we utilize gas exchange techniques to elucidate the photosynthetic impacts of carboxysome mutants in Synechoccocus elongatus PCC 7942, emphasizing analysis of three accessory shell proteins: CcmK3, CcmK4, and CcmP. We detail technical considerations important for increasing the throughput and accuracy of gas exchange measurements using a recently developed commercial aquatic chamber. We find distinct photosynthetic phenotypes associated with the roles of different accessory shell proteins, with context-specific photosynthetic impairment in ΔccmP mutants that contrast with the broader pleiotropic defects of ΔccmK3 ΔccmK4 strains under multiple environments. Our results have implications for understanding the role of accessory carboxysome shell components in facilitating carbon fixation and other carboxysomal functions.

Mycomaterials, materials made from filamentous fungi, have several advantages over traditional materials such as their genetic programmability and self-healing properties. However, their lack of mechanical strength and cost of production often constrain the applications in which they can be used in. In this work, we take inspiration from natural systems to overcome these challenges by elucidating design principles for mineralization-based enhancement of mechanical strength and synthetic lichen-based low-cost growth. We demonstrate that surface display of an enzyme from sea sponges, silicatein α, on the hyphae of the filamentous fungus Aspergillus niger enables mineralization of polysilicate and that this does not impact fungal growth. We also show that this strategy can be extended to other silicatein α variants and characterize how the degree of mineralization can be modulated. We then demonstrate that mineralization enhances the mechanical properties of the mycelium including its tensile strength, modulus, and toughness. Finally, we show how these reinforced mycelia can be grown without external carbon sources using a synthetic lichen-based coculture to facilitate low-cost biomanufacturing. Together, our results lay the groundwork for the sustainable production of mineralized mycomaterials and create a new model system to study how mineralization impacts growth and mechanical properties.

Non-biodegradable synthetic plastics are accumulating in the environment and current recycling methods are limited by harsh processing conditions and inferior quality of recycled products. While biological approaches to plastic degradation offer promise, available enzymes remain inefficient at degrading synthetic plastics due to their inherent physical and chemical properties. Inspired by nature's strategies for degrading resistant natural polymers like cellulose and chitin with enzyme complexes co-localized to polymer surfaces, we quantitatively evaluated a range of previously reported plastic-binding peptides (PBPs) for their capacity to anchor soluble proteins specifically to polystyrene and polypropylene. Among the PBPs tested, LCIM3 demonstrated better specificity when compared to other peptides, binding to polystyrene and polypropylene with an apparent dissociation constant (K_D) in the nanomolar range. By analogy to natural cellulosome complexes, we further investigated the rational design of scaffolds utilizing paired dockerin–cohesin protein binding domains and LCIM3 to enable recruitment of multiple user-defined proteins to plastics. Our study demonstrates that plastic binding peptides were marginally able to increase the localization of proteins and scaffolds onto plastic substrates. However, we also identify a significant non-specific adsorption of many untargeted proteins on plastic as a potential bottleneck for approaches to improve plastic degradation via rational engineering. This proof-of-concept study highlights both the opportunities and challenges in designing bio-inspired scaffolds to improve plastic degradation, laying a foundation for future advancements in sustainable plastic recycling and upcycling.

SUMMARY: Carboxysomes are bacterial microcompartments that encapsulate Rubisco and are a core component of the cyanobacterial carbon concentration mechanism (CCM). While carboxysome number, size, and spatial organization vary in different environmental conditions (CO₂, light availability, redox state, temperature, and light quality), the molecular mechanisms underlying this potentially adaptive process remain elusive. Herein, we observe that mutants of the circadian rhythm/metabolism factor, Regulator of Phycobilisome Association A (RpaA), exhibit a striking breakdown of carboxysomes under certain environmental conditions. We find that conditions leading to overreduction of the plastoquinone (PQ) pool (mixotrophic growth, high irradiance, or chemical inhibition of electron transfer from PQ to the cytochrome b₆f complex) are accompanied by an elevated generation of reactive oxygen species (ROS) and correlate with the loss of carboxysome integrity. Carboxysome breakdown is reversed by environmental conditions or chemical inhibitors that prevent PQ overreduction and accompanying ROS generation. Taken together, our data support a novel link between the redox status of the PQ pool and carboxysome integrity. Our results have implications for the fundamental understanding of cyanobacterial energy‐balancing pathways and may indicate new research directions for understanding how the carboxysome is remodeled in response to changing environments.

Mycomaterials, materials made from filamentous fungi, have several advantages over traditional materials, such as their genetic programmability and self-healing properties. However, their lack of mechanical strength and cost of production often constrain the applications they can be used in. In this work, we take inspiration from natural systems to overcome these challenges by elucidating design principles for mineralization-based enhancement of mechanical strength and synthetic lichen-based low-cost growth. We demonstrate that surface display of an enzyme from sea sponges, silicatein α, on the hyphae of the filamentous fungus Aspergillus niger enables mineralization of polysilicate and that this does not impact fungal growth. We also show that this strategy can be extended to other silicatein α variants and characterize how the degree of mineralization can be modulated. We then demonstrate that mineralization enhances the mechanical properties of the mycelium, including its tensile strength, modulus, and toughness. Finally, we show how these reinforced mycelia can be grown without external carbon sources using a synthetic lichen-based co-culture to facilitate low cost biomanufacturing. Together, our results lay the groundwork for the sustainable production of mineralized mycomaterials and create a new model system to study how mineralization impacts growth and mechanical properties. Significance Statement: Materials made from filamentous fungi, called mycomaterials, have several advantages over traditional materials, but their poor mechanical properties and relatively high production costs have limited their application. We elucidated design principles to enable tunable mineralization of fungal mycelium and have shown that it does not impact growth but significantly enhances mechanical strength. We have also shown that these reinforced mycelia can be grown without any external carbon in a synthetic lichen like consortia to minimize production costs. This work creates a novel experimental system to study how mineralization impacts growth and mechanical properties and will facilitate the broader application of mycomaterials in the future.