Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP.

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.

Knocking Out Chloroplastic Aldolases/Rubisco Lysine Methyltransferase Enhances Biomass Accumulation in Nannochloropsis oceanica under High-Light Stress.

Rubisco large-subunit methyltransferase (LSMT), a SET-domain protein lysine methyltransferase, catalyzes the formation of trimethyl-lysine in the large subunit of Rubisco or in fructose-1,6-bisphosphate aldolases (FBAs). Rubisco and FBAs are both vital proteins involved in CO2 fixation in chloroplasts; however, the physiological effect of their trimethylation remains unknown. In Nannochloropsis oceanica, a homolog of LSMT (NoLSMT) is found. Phylogenetic analysis indicates that NoLSMT and other algae LSMTs are clustered in a basal position, suggesting that algal species are the origin of LSMT. As NoLSMT lacks the His-Ala/ProTrp triad, it is predicted to have FBAs as its substrate instead of Rubisco. The 18-20% reduced abundance of FBA methylation in NoLSMT-defective mutants further confirms this observation. Moreover, this gene (nolsmt) can be induced by low-CO2 conditions. Intriguingly, NoLSMT-knockout N. oceanica mutants exhibit a 9.7-13.8% increase in dry weight and enhanced growth, which is attributed to the alleviation of photoinhibition under high-light stress. This suggests that the elimination of FBA trimethylation facilitates carbon fixation under high-light stress conditions. These findings have implications in engineering carbon fixation to improve microalgae biomass production.

Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

RuBisCO Depletion and Subcellular Fractionation for Enhanced Florigen Detection in Arabidopsis.

In plants, complex signaling networks monitor and respond to environmental cues to determine the optimal time for the transition from the vegetative to reproductive phase. Understanding these networks requires robust tools to examine the levels and subcellular localization of key factors. The florigen FLOWERING LOCUS T (FT) is a crucial regulator of flowering time and occurs in soluble and membrane-bound forms. At low ambient temperatures, the ratio of these forms of FT undergoes a significant shift, which leads to a delay in the onset of flowering. To investigate these changes in FT localization, epitope-tagged FT protein can be isolated from plants by subcellular fractionation and its localization examined by immunoblot analysis of the resulting fractions. However, the highly abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) can interfere with methods to detect and characterize low-abundance proteins such as FT. In this chapter, we present a method for analyzing the ratio of HA-tagged FT (HA:FT) in different subcellular fractions while mitigating the interference from RuBisCO by using protamine sulfate (PS) to deplete RuBisCO during protein purification, thereby enhancing HA:FT detection in fractionated samples.

Rubisco is evolving for improved catalytic efficiency and CO2 assimilation in plants.

Rubisco is the primary entry point for carbon into the biosphere. However, rubisco is widely regarded as inefficient leading many to question whether the enzyme can adapt to become a better catalyst. Through a phylogenetic investigation of the molecular and kinetic evolution of Form I rubisco we uncover the evolutionary trajectory of rubisco kinetic evolution in angiosperms. We show that rbcL is among the 1% of slowest-evolving genes and enzymes on Earth, accumulating one nucleotide substitution every 0.9 My and one amino acid mutation every 7.2 My. Despite this, rubisco catalysis has been continually evolving toward improved CO2/O2 specificity, carboxylase turnover, and carboxylation efficiency. Consistent with this kinetic adaptation, increased rubisco evolution has led to a concomitant improvement in leaf-level CO2 assimilation. Thus, rubisco has been slowly but continually evolving toward improved catalytic efficiency and CO2 assimilation in plants.

Molecular characterization and expression pattern of Rubisco activase gene GhRCAß2 in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Rubisco activase (RCA) is a pivotal enzyme that can catalyse the activation of Rubisco in carbon assimilation pathway. Many studies have shown that RCA may be a potential target for genetic manipulation aimed at enhancing photosynthetic efficiency and crop yield. OBJECTIVE: To understand the biological function of the GhRCAß2 gene in upland cotton, we cloned the coding sequence (CDS) of the GhRCAß2 gene and investigated its sequence features, evolutionary relationship, subcellular localization, promoter sequence and expression pattern. METHODS: The bioinformatics tools were used to analyze the sequence features of GhRCAß2 protein. Transient transformation of Arabidopsis mesophyll protoplasts was performed to determine the subcellular localization of the GhRCAß2 protein. The expression pattern of the GhRCAß2 gene was examined by analyzing transcriptome data and using the quantitative real-time PCR (qRT-PCR). RESULTS: The full-length CDS of GhRCAß2 was 1317 bp, and it encoded a protein with a chloroplast transit peptide. The GhRCAß2 had two conserved ATP-binding domains, and did not have the C-terminal extension (CTE) domain that was unique to the RCA α-isoform in plants. Evolutionarily, GhRCAß2 was clustered in Group A, and had a close evolutionary relationship with the soybean RCA. Western blot analysis demonstrated that GhRCAß2 was immunoreactive to the RCA antibody displaying a molecular weight similar to that of the RCA ß-isoform. The GhRCAß2 protein was found in chloroplast, aligning with its role as a vital enzyme in the process of photosynthesis. The GhRCAß2 gene had a leaf tissue-specific expression pattern, and the yellow-green leaf mutant exhibited a decreased expression of GhRCAß2 in comparison to the wild-type cotton plants. The GhRCAß2 promoter contained several cis-acting elements that respond to light, phytohormones and stress, suggesting that the expression of GhRCAß2 may be regulated by these factors. An additional examination of stress response indicated that GhRCAß2 expression was influenced by cold, heat, salt, and drought stress. Notably, diverse expression pattern was observed across different stress conditions. Additionally, low phosphorus and low potassium stress may result in a notable reduction in the expression of GhRCAß2 gene. CONCLUSION: Our findings will establish a basis for further understanding the function of the GhRCAß2 gene, as well as providing valuable genetic knowledge to improve cotton photosynthetic efficiency and yield under challenging environmental circumstances.

Pyrenoid proteomics reveals independent evolution of the CO2-concentrating organelle in chlorarachniophytes.

Pyrenoids are microcompartments that are universally found in the photosynthetic plastids of various eukaryotic algae. They contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and play a pivotal role in facilitating CO2 assimilation via CO2-concentrating mechanisms (CCMs). Recent investigations involving model algae have revealed that pyrenoid-associated proteins participate in pyrenoid biogenesis and CCMs. However, these organisms represent only a small part of algal lineages, which limits our comprehensive understanding of the diversity and evolution of pyrenoid-based CCMs. Here we report a pyrenoid proteome of the chlorarachniophyte alga Amorphochlora amoebiformis, which possesses complex plastids acquired through secondary endosymbiosis with green algae. Proteomic analysis using mass spectrometry resulted in the identification of 154 potential pyrenoid components. Subsequent localization experiments demonstrated the specific targeting of eight proteins to pyrenoids. These included a putative Rubisco-binding linker, carbonic anhydrase, membrane transporter, and uncharacterized GTPase proteins. Notably, most of these proteins were unique to this algal lineage. We suggest a plausible scenario in which pyrenoids in chlorarachniophytes have evolved independently, as their components are not inherited from green algal pyrenoids.

A carboxysome-based CO2 concentrating mechanism for C3 crop chloroplasts: advances and the road ahead.

The introduction of the carboxysome-based CO2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields.

Insights on the enhancement of chilling tolerance in Rice through over-expression and knock-out studies of OsRBCS3.

Chilling stress is an important environmental factor that affects rice (Oryza sativa L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on OsRBCS3, a rice gene related to chilling tolerance at the booting stage, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit in photosynthesis. The aim of this study was to elucidate the role and mechanism of OsRBCS3 in rice chilling tolerance at the booting stage. The expression levels of OsRBCS3 under chilling stress were compared in two japonica rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between OsRBCS3 expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of OsRBCS3 were constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerance was evaluated at the seedling and booting stages. The results showed that OE lines exhibited higher chilling tolerance than wild-type (WT) lines at both seedling and booting stages, while KO lines showed lower chilling tolerance than WT lines. Furthermore, the antioxidant enzyme activities, malondialdehyde (MDA) content and Rubisco activity of four rice lines under chilling stress were measured, and it was found that OE lines had stronger antioxidant and photosynthetic capacities, while KO lines had the opposite effects. This study validated that OsRBCS3 plays an important role in rice chilling tolerance at the booting stage, providing new molecular tools and a theoretical basis for rice chilling tolerance breeding.

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts.

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology.

Perspectives on improving crop Rubisco by directed evolution.

Rubisco catalyses the entry of almost all CO2 into the biosphere and is often the rate-limiting step in plant photosynthesis and growth. Its notoriety as the most abundant protein on Earth stems from the slow and error-prone catalytic properties that require plants, cyanobacteria, algae and photosynthetic bacteria to produce it in high amounts. Efforts to improve the CO2-fixing properties of plant Rubisco has been spurred on by the discovery of more effective isoforms in some algae with the potential to significantly improve crop productivity. Incompatibilities between the protein folding machinery of leaf and algae chloroplasts have, so far, prevented efforts to transplant these more effective Rubisco variants into plants. There is therefore increasing interest in improving Rubisco catalysis by directed (laboratory) evolution. Here we review the advances being made in, and the ongoing challenges with, improving the solubility and/or carboxylation activity of differing non-plant Rubisco lineages. We provide perspectives on new opportunities for the directed evolution of crop Rubiscos and the existing plant transformation capabilities available to evaluate the extent to which Rubisco activity improvements can benefit agricultural productivity.

Evolution: Unearthing missing links in the origin of our planet's primary carbon-fixing enzyme.

Form I rubisco underpins life on Earth, but the origins of its anomalous and highly complex multimeric structure have long proven elusive. New research helps to unravel this enigma by uncovering key missing links in the enzyme's evolutionary history.

Deep-branching evolutionary intermediates reveal structural origins of form I rubisco.

The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme-found in plants, algae, and cyanobacteria-forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I') has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I' rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I'' rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I'' rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution.

Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield.

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.

Response to Tcherkez and Farquhar: Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off.

Rubisco is the primary entry point for carbon into the biosphere. It has been widely proposed that rubisco is highly constrained by catalytic trade-offs due to correlations between the enzyme's kinetic traits across species. In previous work, we have shown that the strength of these correlations, and thus the strength of catalytic trade-offs, have been overestimated due to the presence of phylogenetic signal in the kinetic trait data (Bouvier et al., 2021). We demonstrated that only the trade-offs between the Michaelis constant for CO2 and carboxylase turnover, and between the Michaelis constants for CO2 and O2 were robust to phylogenetic effects. We further demonstrated that phylogenetic constraints have limited rubisco adaptation to a greater extent than the combined action of catalytic trade-offs. Recently, however, our claims have been contested by Tcherkez and Farquhar (2021), who have argued that the phylogenetic signal we detect in rubisco kinetic traits is an artefact of species sampling, the use of rbcL-based trees for phylogenetic inference, laboratory-to-laboratory variability in kinetic measurements, and homoplasy of the C4 trait. In the present article, we respond to these criticisms on a point-by-point basis and conclusively show that all are unfounded. As such, we stand by our original conclusions. Namely, although rubisco kinetic evolution has been limited by biochemical trade-offs, these are not absolute and have been previously overestimated due to phylogenetic biases. Instead, rubisco adaptation has in fact been more limited by phylogenetic constraint.

The challenge of engineering Rubisco for improving photosynthesis.

Photosynthesis uses the energy of sunlight to convert water and atmospheric CO2 into sugars, providing food and oxygen for life. The fixation of atmospheric CO2 in this crucial biological process is mediated by the enzyme Rubisco. The inefficiencies of Rubisco have inspired researchers for decades to explore ways to improve its function with the goal of increasing crop yields [1-4], and more recently to combat global warming [5]. In this graphical review we highlight the challenges involved in engineering plant Rubisco, with a focus on the extensive chaperone requirement for its biogenesis. We discuss strategies for engineering the catalytic properties of Rubisco and for sequestering the enzyme in membraneless compartments to increase CO2 fixation.

A linker protein from a red-type pyrenoid phase separates with Rubisco via oligomerizing sticker motifs.

The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum. PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.

An ancient metabolite damage-repair system sustains photosynthesis in plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major catalyst in the conversion of carbon dioxide into organic compounds in photosynthetic organisms. However, its activity is impaired by binding of inhibitory sugars such as xylulose-1,5-bisphosphate (XuBP), which must be detached from the active sites by Rubisco activase. Here, we show that loss of two phosphatases in Arabidopsis thaliana has detrimental effects on plant growth and photosynthesis and that this effect could be reversed by introducing the XuBP phosphatase from Rhodobacter sphaeroides. Biochemical analyses revealed that the plant enzymes specifically dephosphorylate XuBP, thus allowing xylulose-5-phosphate to enter the Calvin-Benson-Bassham cycle. Our findings demonstrate the physiological importance of an ancient metabolite damage-repair system in degradation of by-products of Rubisco, and will impact efforts to optimize carbon fixation in photosynthetic organisms.

Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.

Computational and biochemical methods to measure the activity of carboxysomes and protein organelles in vivo.

Cyanobacteria are photosynthetic microorganisms that play important ecological roles as major contributors to global nutrient cycles. Cyanobacteria are highly efficient in carrying out oxygenic photosynthesis because they possess carboxysomes, a class of bacterial microcompartments (BMC) in which a polyhedral protein shell encapsulates the enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase and functions as the key component of the cyanobacterial CO2-concentrating mechanism (CCM). Elevated CO2 levels within the carboxysome shell as a result of carbonic anhydrase activity increase the efficiency of RuBisCO. Yet, there remain many questions regarding the flux or exclusion of metabolites across the shell and how the activity of BMCs varies over time. These questions have been difficult to address using traditional ensemble techniques due to the heterogeneity of BMCs extracted from their native hosts or with heterologous expression. In this chapter, we describe a method to film and extract quantitative information about carboxysome activity using molecular biology and live cell, timelapse microscopy. In our method, the production of carboxysomes is first controlled by deleting the native genes required for carboxysome assembly and then re-introducing them under the control of an inducible promoter. This system enables carboxysomes to be tracked through multiple generations of cells and provides a way to quantify the total biomass accumulation attributed to a single carboxysome. While the method presented here was developed specifically for carboxysomes, it could be modified to track and quantify the activity of bacterial microcompartments in general.