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Abstract Synthetic biology conceptualises 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 synthesise 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 multi-species communities. We argue for a shift in perspective from single organism-centred approaches to emphasising 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. 

Abstract 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. 

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-secretingSynechococcus elongatusPCC 7942. We find thatS. elongatusproduces the highest sucrose yield in irradiances far greater than what is often experimentally utilized, and that high light intensities are tolerated byS. 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/spsproduced a total of 3.8 g L^-1of sucrose and the highest productivity within that period being 47.8 mg L^-1h^-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. 

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. 

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 anin-silicomodel of a synthetic microbial consortia of sucrose-secretingSynechococcus elongatusPCC 7942 andEscherichia coliW. 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. 

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.