Modeling bacterial microcompartment architectures for enhanced cyanobacterial carbon fixation.

The carboxysome is a bacterial microcompartment (BMC) which plays a central role in the cyanobacterial CO2-concentrating mechanism. These proteinaceous structures consist of an outer protein shell that partitions Rubisco and carbonic anhydrase from the rest of the cytosol, thereby providing a favorable microenvironment that enhances carbon fixation. The modular nature of carboxysomal architectures makes them attractive for a variety of biotechnological applications such as carbon capture and utilization. In silico approaches, such as molecular dynamics (MD) simulations, can support future carboxysome redesign efforts by providing new spatio-temporal insights on their structure and function beyond in vivo experimental limitations. However, specific computational studies on carboxysomes are limited. Fortunately, all BMC (including the carboxysome) are highly structurally conserved which allows for practical inferences to be made between classes. Here, we review simulations on BMC architectures which shed light on (1) permeation events through the shell and (2) assembly pathways. These models predict the biophysical properties surrounding the central pore in BMC-H shell subunits, which in turn dictate the efficiency of substrate diffusion. Meanwhile, simulations on BMC assembly demonstrate that assembly pathway is largely dictated kinetically by cargo interactions while final morphology is dependent on shell factors. Overall, these findings are contextualized within the wider experimental BMC literature and framed within the opportunities for carboxysome redesign for biomanufacturing and enhanced carbon fixation.

Toward a Predictive Understanding of Cyanobacterial Harmful Algal Blooms through AI Integration of Physical, Chemical, and Biological Data.

Freshwater cyanobacterial harmful algal blooms (cyanoHABs) are a worldwide problem resulting in substantial economic losses, due to harm to drinking water supplies, commercial fishing, wildlife, property values, recreation, and tourism. Moreover, toxins produced from some cyanoHABs threaten human and animal health. Climate warming can affect the distribution of cyanoHABs, where rising temperatures facilitate more intense blooms and a greater distribution of cyanoHABs in inland freshwater. Nutrient runoff from adjacent watersheds is also a major driver of cyanoHAB formation. While some of the physicochemical factors behind cyanoHAB dynamics are known, there are still major gaps in our understanding of the conditions that trigger and sustain cyanoHABs over time. In this perspective, we suggest that sufficient data sets, as well as machine learning (ML) and artificial intelligence (AI) tools, are available to build a comprehensive model of cyanoHAB dynamics based on integrated environmental/climate, nutrient/water chemistry, and cyanoHAB microbiome and 'omics data to identify key factors contributing to HAB formation, intensity, and toxicity. By taking a holistic approach to the analysis of all available data, including the rapidly growing number of biological data sets, we can provide the foundational knowledge needed to address the increasing threat of cyanoHABs to the security of our water resources.

Monatomic ions influence substrate permeation across bacterial microcompartment shells.

Bacterial microcompartments (BMCs) are protein organelles consisting of an inner enzymatic core encased within a selectively permeable shell. BMC shells are modular, tractable architectures that can be repurposed with new interior enzymes for biomanufacturing purposes. The permeability of BMC shells is function-specific and regulated by biophysical properties of the shell subunits, especially its pores. We hypothesized that ions may interact with pore residues in a manner that influences the substrate permeation process. In vitro activity comparisons between native and broken BMCs demonstrated that increasing NaCl negatively affects permeation rates. Molecular dynamics simulations of the dominant shell protein (BMC-H) revealed that chloride ions preferentially occupy the positive pore, hindering substrate permeation, while sodium cations remain excluded. Overall, these results demonstrate that shell properties influence ion permeability and leverages the integration of experimental and computational techniques to improve our understanding of BMC shells towards their repurposing for biotechnological applications.

Validation of an insertion-engineered isoprene synthase as a strategy to functionalize terpene synthases.

Terpene synthases are biotechnologically-relevant enzymes with a variety of applications. However, they are typically poor catalysts and have been difficult to engineer. Structurally, most terpene synthases share two conserved domains (α- and ß-domains). Some also contain a third domain containing a second active site (γ-domain). Based on the three-domain architecture, we hypothesized that αß terpene synthases could be engineered by insertion of a heterologous domain at the site of the γ-domain (an approach we term "Insertion-engineering terpene synthase"; Ie-TS). We demonstrate that by mimicking the domain architecture of αßγ terpene synthases, we can redesign isoprene synthase (ISPS), an αß terpene synthase, while preserving enzymatic activity. Insertion of GFP or a SpyCatcher domain within ISPS introduced new functionality while maintaining or increasing catalytic turnover. This insertion-engineering approach establishes that the γ-domain position is accessible for incorporation of additional sequence features and enables the rational engineering of terpene synthases for biotechnology.

Nuclear, Chloroplast, and Mitochondrial Genome Sequences of the Prospective Microalgal Biofuel Strain Picochlorum soloecismus.

Picochlorum soloecismus is a halotolerant, fast-growing, and moderate-lipid-producing microalga that is being evaluated as a renewable feedstock for biofuel production. Herein, we report on an improved high-quality draft assembly and annotation for the nuclear, chloroplast, and mitochondrial genomes of P. soloecismus DOE 101.

Carboxysomes: metabolic modules for CO2 fixation.

The carboxysome is a bacterial microcompartment encapsulating the enzymes carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase. As the site of CO2 fixation, it serves an essential role in the carbon dioxide concentrating mechanism of many chemoautotrophs and all cyanobacteria. Carboxysomes and other bacterial microcompartments self-assemble through specific protein-protein interactions that are typically mediated by conserved protein domains. In this review, we frame our current understanding of carboxysomes in the context of their component protein domains with their inherent function as the 'building blocks' of carboxysomes. These building blocks are organized in genetic modules (conserved chromosomal loci) that encode for carboxysomes and ancillary proteins essential for the integration of the organelle with the rest of cellular metabolism. This conceptual framework provides the foundation for 'plug-and-play' engineering of carboxysomes as CO2 fixation modules in a variety of biotechnological applications.

Bacterial microcompartments as metabolic modules for plant synthetic biology.

Bacterial microcompartments (BMCs) are megadalton-sized protein assemblies that enclose segments of metabolic pathways within cells. They increase the catalytic efficiency of the encapsulated enzymes while sequestering volatile or toxic intermediates from the bulk cytosol. The first BMCs discovered were the carboxysomes of cyanobacteria. Carboxysomes compartmentalize the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) with carbonic anhydrase. They enhance the carboxylase activity of RuBisCO by increasing the local concentration of CO2 in the vicinity of the enzyme's active site. As a metabolic module for carbon fixation, carboxysomes could be transferred to eukaryotic organisms (e.g. plants) to increase photosynthetic efficiency. Within the scope of synthetic biology, carboxysomes and other BMCs hold even greater potential when considered a source of building blocks for the development of nanoreactors or three-dimensional scaffolds to increase the efficiency of either native or heterologously expressed enzymes. The carboxysome serves as an ideal model system for testing approaches to engineering BMCs because their expression in cyanobacteria provides a sensitive screen for form (appearance of polyhedral bodies) and function (ability to grow on air). We recount recent progress in the re-engineering of the carboxysome shell and core to offer a conceptual framework for the development of BMC-based architectures for applications in plant synthetic biology.

Streamlined Construction of the Cyanobacterial CO2-Fixing Organelle via Protein Domain Fusions for Use in Plant Synthetic Biology.

Bacterial microcompartments (BMCs) are self-assembling organelles that sequester segments of biochemical pathways within a protein shell. Given their functional diversity, BMCs constitute a rich source of metabolic modules for applications in synthetic biology. The carboxysome, the cyanobacterial BMC for CO(2) fixation, has attracted significant attention as a target for installation into chloroplasts and serves as the foundation for introducing other types of BMCs into plants. Carboxysome assembly involves a series of protein-protein interactions among at least six gene products to form a metabolic core, around which the shell assembles. This complexity creates significant challenges for the transfer, regulation, and assembly of carboxysomes, or any of the myriad of functionally distinct BMCs, into heterologous systems. To overcome this bottleneck, we constructed a chimeric protein in the cyanobacterium Synechococcus elongatus that structurally and functionally replaces four gene products required for carboxysome formation. The protein was designed based on protein domain interactions in the carboxysome core. The resulting streamlined carboxysomes support photosynthesis. This strategy obviates the need to regulate multiple genes and decreases the genetic load required for carboxysome assembly in heterologous systems. More broadly, the reengineered carboxysomes represent a proof of concept for a domain fusion approach to building multifunctional enzymatic cores that should be generally applicable to the engineering of BMCs for new functions and cellular contexts.

ClpB1 overproduction in Synechocystis sp. strain PCC 6803 increases tolerance to rapid heat shock.

ClpB1 is a heat shock protein known to disaggregate large protein complexes. Constitutive, 16-fold ClpB1 overproduction in the cyanobacterium Synechocystis sp. strain PCC 6803 increased cell survival by 20-fold when cultures were heated quickly (1°C/s) to 50°C and delayed cell death by an average of 3 min during incubation at high temperatures (>46°C). Cooverexpression of ClpB1 and another heat shock protein, DnaK2, further increased cell survival. According to immunocytochemistry results, ClpB1 is dispersed throughout the cytoplasm but is concentrated in specific areas and is more prevalent near thylakoid membranes. However, ClpB1 overproduction does not lead to a change in the morphology, chlorophyll content, or photosystem ratio. Whereas electron microscopy demonstrated that apparent protein aggregation occurred after heat treatment in the control strain, protein aggregate size was maintained in the ClpB1 overexpresser. Constitutive ClpB1 overproduction allows an earlier response to heat shock and protects from rapid heating of cultures.