Nature-Inspired Strategies for Sustainable Degradation of Synthetic Plastics.

Synthetic plastics have become integral to our daily lives, yet their escalating production, limited biodegradability, and inadequate waste management contribute to environmental contamination. Biological plastic degradation is one promising strategy to address this pollution. The inherent chemical and physical properties of synthetic plastics, however, pose challenges for microbial enzymes, hindering the effective degradation and the development of a sustainable biological recycling process.

A new era of synthetic biology-microbial community design.

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.

Impact of irradiance and inorganic carbon availability on heterologous sucrose production in 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, CO, and cultivator type) affect the productivity of sucrose-secreting PCC 7942.

Population-level heterogeneity complicates utilization of 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 PCC 7942 by modifying two outer-membrane proteins, SomA and Intimin, to display tags ( , 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.

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.

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).

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.

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.

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 CO 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.

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.

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.

Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria.

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 (, 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 by adapting a method using degron tags from the transfer-mRNA (tmRNA) system.

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.

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 CO through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P.

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 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 .

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.

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.

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 PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown.

The Molecular Machinery of Chloroplast Division.

Recent studies advance understanding of the mechanisms, spatial control, and regulation of chloroplast division, but many questions remain.

A synthetic, light-driven consortium of cyanobacteria and heterotrophic bacteria enables stable polyhydroxybutyrate production.

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.

Engineering the Bacterial Microcompartment Domain for Molecular Scaffolding Applications.

As synthetic biology advances the intricacy of engineered biological systems, the importance of spatial organization within the cellular environment must not be marginalized. Increasingly, biological engineers are investigating means to control spatial organization within the cell, mimicking strategies used by natural pathways to increase flux and reduce cross-talk. A modular platform for constructing a diverse set of defined, programmable architectures would greatly assist in improving yields from introduced metabolic pathways and increasing insulation of other heterologous systems.

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.