Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli

1Nearly all carbon in the biosphere enters by CO2 fixation in the Calvin-Benson-Bassham cycle. Ribulose Bisphosphate Carboxylase/Oxygenase - commonly known as rubisco - is the CO2 fixing enzyme in this cycle and likely the most abundant enzyme on Earth.

2As rubisco is abundant and central to biology, one might expect it to be an exceptional catalyst, but it is not. Photosynthetic rubiscos are modest enzymes, with carboxylation turnover numbers (kcat) ranging from 1 to 10 s−1. Moreover, all known rubiscos catalyze a competing oxygenation of the five-carbon organic substrate, ribulose 1, 5-bisphosphate. Rubisco oxygenation represents a ‘waste’ of cellular resources on two fronts: it fails to generate any new organic carbon and also produces a molecule (2-phosphoglycolate) that is not part of the Calvin cycle and therefore must be recycled through a salvage pathway to keep the cycle going.

3Rubisco arose >2.5 billion years ago, when Earth’s atmosphere contained little O2 and abundant CO2. In this environment, rubisco’s eponymous oxygenase activity could not have hindered carbon fixation or the growth of CO2-fixing organisms. Present-day atmosphere, however, poses a problem for plants and other autotrophs: their primary carbon source, CO2, is relatively scarce (≈0.04%) while a potent competing substrate, O2, is abundant (≈21%).

4CO2 concentrating mechanisms (CCMs) arose multiple times over the last 2 billion years and overcame rubisco’s limitations by concentrating CO2 near the enzyme (Figure 1A). In an elevated CO2 environment, most rubisco active sites will be occupied with CO2 and not O2. As such, high CO2 is expected to increase the rate of carboxylation and competitively inhibit oxygenation thereby improving overall carbon assimilation (Figure 1B). Today, at least four varieties of CCMs are found in plants, algae, and bacteria, organisms with CCMs are collectively responsible for ≈50% of global net photosynthesis, and some of the most productive human crops (e.g. maize and sugarcane) rely on CCMs.

5CCMs are particularly common among autotrophic bacteria: all Cyanobacteria and many Proteobacteria have CCM genes. Bacterial CCMs rely on two crucial features: (i) energy-coupled inorganic carbon uptake at the cell membrane and (ii) a 200+ MDa protein organelle called the carboxysome that encapsulates rubisco with a carbonic anhydrase enzyme. In the prevailing model of the carboxysome CCM, inorganic carbon uptake produces a high, above-equilibrium cytosolic HCO3- concentration (≈30 mM) that diffuses into the carboxysome, where carbonic anhydrase activity produces a high carboxysomal CO2 concentration that promotes efficient carboxylation by rubisco (Figure 1A–B).

6As CCMs accelerate CO2 fixation by rubisco, there is great interest in transplanting them into crops. Carboxysome-based CCMs are especially attractive because they natively function in single cells and appear to rely on a tractable number of genes. Modeling suggests that introducing bacterial CCM components could improve plant photosynthesis, especially if aspects of plant physiology can be modulated via genetic engineering. However, expressing bacterial rubiscos and carboxysome components has, so far, uniformly resulted in transgenic plants displaying impaired growth. More generally, as our understanding of the genes and proteins participating in the carboxysome CCM rests mostly on loss-of-function genetic experiments in native hosts, it is possible that some genetic, biochemical, and physiological aspects of CCM function remain unappreciated. We therefore sought to test whether current understanding is sufficient to reconstitute the bacterial CCM in a non-native bacterial host, namely Escherichia coli.

7Using a genome-wide screen in the CO2-fixing proteobacterium Halothiobacillus neapolitanus, we recently demonstrated that a 20-gene cluster encodes all activities required for the CCM, at least in principle. These genes are detailed in Supplementary file 1 and include rubisco large and small subunits, the carboxysomal carbonic anhydrase, seven structural proteins of the ɑ-carboxysome, an energy-coupled inorganic carbon transporter, three rubisco chaperones, and four genes of uncertain function (Figure 1C). We aimed to test whether these genes are sufficient to establish a functioning CCM in E. coli.

8As E. coli is a heterotroph, consuming organic carbon molecules to produce energy and biomass, it does not natively rely on rubisco. Therefore, in order to evaluate the effect of heterologous CCM expression, we first designed an E. coli strain that depends on rubisco carboxylation for growth. To grow on glycerol as the sole carbon source, E. coli must synthesize ribose 5-phosphate (Ri5P) for nucleic acids. Synthesis of Ri5P via the pentose phosphate pathway forces co-production of ribulose 5-phosphate (Ru5P). Deletion of ribose 5-phosphate isomerase (rpiAB genes, denoted Δrpi), however, makes Ru5P a metabolic ‘dead-end’ (Figure 2A). Expression of phosphoribulokinase (prk) and rubisco enables a ‘detour’ pathway converting Ru5P and CO2 into two units of the central metabolite 3-phosphoglycerate (3PG), enabling Ru5P metabolism and growth (Figure 2A). Additionally, cytosolic carbonic anhydrase activity is incompatible with the bacterial CCM. We therefore constructed a strain, named CCMB1 for ‘CCM Background 1’, lacking rpiAB and all endogenous carbonic anhydrases (Materials and methods, Appendix 1).

9As predicted, CCMB1 required rubisco and prk for growth on glycerol minimal media in 10% CO2 (Figure 2B–C). CCMB1:p1A failed to grow on glycerol media in ambient air, however, presumably due to insufficient carboxylation at low CO2 (Figure 2D). As such, CCMB1:p1A displays the ‘high-CO2 requiring’ phenotype that is the hallmark of CCM mutants. Four additional bacterial rubiscos were tested and displayed the same pattern, enabling CCMB1 to grow reproducibly in high CO2 but not in ambient air (Figure 2—figure supplement 1). When expressing rubisco and prk from the p1A plasmid, CCMB1 also grew reproducibly in an anoxic mix of 10:90 CO2:N2 (Figure 2—figure supplement 2) implying that carboxylation is sufficient for growth on glycerol media and rubisco-catalyzed oxygenation of RuBP is not required.

10We expected that expressing a functional CO2-concentrating mechanism would cure CCMB1 of its high-CO2 requirement and permit growth in ambient air. We therefore generated two plasmids, pCB and pCCM, that together express all 20 genes from the H. neapolitanus CCM cluster (Figure 1C). pCB encodes 10 carboxysome genes, including rubisco large and small subunits, along with prk. The remaining H. neapolitanus genes, including putative rubisco chaperones and an inorganic carbon transporter, were cloned into the second plasmid, pCCM.

11CCMB1 co-transformed with pCB and pCCM initially failed to grow on glycerol media. We therefore conducted selection experiments, described fully in Appendix 2, that ultimately resulted in the isolation of mutant plasmids conferring growth in ambient air. Briefly, CCMB1:pCB + pCCM cultures were grown to saturation in 10% CO2. These cultures were washed and plated on glycerol minimal media (Materials and methods). Colonies became visible after 20 days of incubation in ambient air, but only when induction and both plasmids were provided (Figure 3—figure supplement 1). Deep-sequencing of plasmid DNA revealed mutations in regulatory sequences (e.g. a promoter and transcriptional repressor) but none in sequences coding for CCM components (Supplementary file 1). Individual post-selection plasmids pCB’ and pCCM’ were reconstructed by PCR, resequenced, and transformed into naive CCMB1 (Materials and methods). As shown in Figure 3, pCB’ and pCCM’ together enabled reproducible growth of CCMB1 in ambient air, suggesting that the 20 genes expressed are sufficient to produce a heterologous CCM without any genomic mutations.

12To verify that growth in ambient air depends on the CCM, we generated plasmids carrying targeted mutations to known CCM components (Figure 4). An inactivating mutation to the carboxysomal rubisco (CbbL K194M) prohibited growth entirely. Mutations targeting the CCM, rather than rubisco itself, should ablate growth in ambient air while permitting growth in high CO2. Consistent with this understanding, an inactive mutant of the carboxysomal carbonic anhydrase (CsoSCA C173S) required high-CO2 for growth. Similarly, disruption of carboxysome formation by removal of the pentameric shell proteins or the N-terminal domain of CsoS2 also eliminated growth in ambient air. Removing the pentameric proteins CsoS4AB disrupts the permeability barrier at the carboxysome shell, while truncating CsoS2 prohibits carboxysome formation entirely. Finally, an inactivating mutation to the inorganic carbon transporter also eliminated growth in ambient air.

13These experiments demonstrate that pCB’ and pCCM’ enable CCMB1 to grow in ambient air in a manner that depends on the known components of the bacterial CCM. To confirm that these cells produce carboxysome structures, we performed thin section electron microscopy. Regular polyhedral inclusions of ≈100 nm diameter were visible in micrographs (Figure 5A), implying production of morphologically normal carboxysomes. Furthermore, we were able to purify carboxysome structures from CCMB1:pCB’+pCCM’ using established methods. Carboxysomes from CCMB1:pCB’+pCCM’ were similar in appearance to those from the native host, although more heterogeneous in size and shape (Figure 5B). The rubisco complex was visible inside isolated carboxysomes and confirmed to co-migrate with the structure via SDS-PAGE analysis (Figure 5—figure supplement 1).

14We next conducted isotopic labeling experiments to determine whether CCMB1:pCB’ + pCCM’ fixes CO2 from ambient air into biomass. Cells were grown in minimal media with 13C-labeled glycerol as the sole organic carbon source, such that CO2 from ambient air was the dominant source of 12C. The isotopic composition of amino acids in total biomass hydrolysate was analyzed via mass spectrometry (Materials and methods). Serine is a useful sentinel of rubisco activity because E. coli produces it from the rubisco product 3PG. 3PG is also an intermediate of lower glycolysis, and so the degree of 12C labeling on serine reports on the balance of fluxes through rubisco and lower glycolysis (Figure 6A). We therefore expected excess 12C labeling of serine when rubisco is active in CCMB1. Consistent with this expectation, serine from CCMB1:pCB’+pCCM’ cells contained roughly threefold more 12C than the rubisco-independent control (Figure 6B). We estimated the contribution of rubisco to 3PG synthesis in vivo by comparing labeling patterns between the rubisco-dependent experimental cultures and controls (Appendix 2). Based on these estimates, rubisco carboxylation was responsible for at least 10% of 3PG synthesis in all four biological replicates (Figure 6C, Materials and methods), confirming fixation of CO2 from ambient air. As such, this work represents the first functional reconstitution of any CCM.

15Reconstitution in E. coli enabled us to investigate which H. neapolitanus genes are necessary for CCM function in the absence of any regulation or genetic redundancy (i.e. genes with overlapping function) present in the native host. We focused on genes involved in rubisco proteostasis and generated plasmids lacking acRAF, a putative rubisco chaperone, or carrying targeted mutations to CbbQ, an ATPase involved in activating rubisco catalysis. Although acRAF deletion had a large negative effect in H. neapolitanus, neither acRAF nor CbbQ were strictly required for CCMB1 to grow in ambient air. Consistent with our screen in the native host; however, acRAF deletion produced a substantial growth defect (Figure 4—figure supplement 1, panel C), suggesting that the rate of rubisco complex assembly is an important determinant of carboxysome biogenesis.

16Today, CCMs catalyze about half of global photosynthesis, but this was not always so. Land plant CCMs, for example, arose only in the last 100 million years. Although all contemporary Cyanobacteria have CCM genes, these CCMs are found in two convergently evolved varieties, suggesting that the ancestor of present-day Cyanobacteria and chloroplasts did not have a CCM. So how did carboxysome CCMs come to dominate the cyanobacterial phylum?

17Here, we demonstrated that the ɑ-carboxysome CCM from H. neapolitanus can be readily transferred between species and confers a large growth benefit, suggesting that these CCMs became so widespread by horizontal transfer between bacteria. We constructed a functional bacterial CCM by expressing 20 genes in an E. coli strain, CCMB1, engineered to depend on rubisco carboxylation. In accordance with its role in native autotrophic hosts, the transplanted CCM required (i) ɑ-carboxysome structures containing both rubisco and carbonic anhydrase and (ii) inorganic carbon uptake at the cell membrane in order to enable CCMB1 to grow by fixing CO2 from ambient air (Figures 3–6). These results conclusively demonstrate that at most 20 gene products are required to produce a bacterial CCM. The ɑ-carboxysome CCM is apparently genetically compact and ‘portable’ between organisms. It is possible, therefore, that expressing bacterial CCMs in non-native autotrophic hosts will improve CO2 assimilation and growth. This is a promising approach to improving plant growth characteristics and also engineering enhanced microbial production of fuel, food products, and commodity chemicals from CO2.

18Reconstitution also enabled us to test, via simple genetic experiments, whether particular genes play a role in the CCM (Figure 4—figure supplement 1). These experiments demonstrated that the rubisco chaperones are strictly dispensable for producing a functional bacterial CCM, although removing acRAF produced a substantial growth defect that warrants further investigation. Further such experiments can use our reconstituted CCM to delineate a minimal reconstitution of the bacterial CCM suitable for plant expression, test hypotheses about carboxysome biogenesis, and probe the relationship between CCMs and host physiology. This last point deserves special emphasis as the growth physiologies of plants and bacteria are exceedingly different and it remains unclear whether microbial CCMs can function efficiently when expressed in macroscopic land plants.

19Our approach to studying CCMs by reconstitution in tractable non-native hosts can be also applied to other CCMs, including β-carboxysome CCMs, the algal pyrenoid, and plausible evolutionary ancestors thereof. Historical trends in atmospheric CO2 likely promoted the evolution of CCMs, so testing the growth of plausible ancestors of bacterial CCMs (e.g. carboxysomes lacking carbonic anhydrase activity) may provide insight into paths of CCM evolution and the composition of the ancient atmosphere at the time bacterial CCMs arose. In response to these same pressures, diverse eukaryotic algae evolved CCMs relying on micron-sized rubisco aggregates called the pyrenoids. Pyrenoid CCMs are collectively responsible for perhaps 70–80% of oceanic photosynthesis, yet many fundamental questions remain regarding the composition and operation of algal CCMs. Functional reconstitution of a pyrenoid CCM is a worthy goal which, once achieved, will indicate enormous progress in our collective understanding of the genetics, cell biology, biochemistry, and physical processes supporting the eukaryotic complement of oceanic photosynthesis. We hope such studies will further our principled understanding of, and capacity to engineer, the cell biology supporting CO2 fixation in diverse organisms.

20Unless otherwise noted, cells were grown on M9 minimal media supplemented with 0.4% v/v glycerol, 0.5 ppm thiamin (104 fold dilution of 0.5% w/v stock) and a trace element mix. The trace element mix components and their final concentrations in M9 media are: 50 mg/L EDTA, 31 mM FeCl3, 6.2 mM ZnCl2, 0.76 mM CuSO4·5H2O, 0.42 mM CoCl2·6H2O, 1.62 mM H3BO3, 81 nM MnCl2·4H2O. 100 nM anhydrotetracycline (aTc) was used in induced cultures. For routine cloning, 25 mg/L chloramphenicol and 60 mg/L kanamycin selection were used as appropriate. Antibiotics were reduced to half concentration (12.5 and 30 mg/L, respectively) for CCMB1 growth experiments and kanamycin was omitted when evaluating rubisco-dependence of growth as pF-derived plasmids carrying kanamycin resistance also express rubisco. Culture densities were measured at 600 nm in a table top spectrophotometer (Genesys 20, Thermo Scientific) and turbid cultures were measured in five- or tenfold dilution as appropriate in order to reach the linear regime of the spectrophotometer.

21Agar plates were incubated at 37°C in defined CO2 pressures in a CO2 controlled incubator (S41i, New Brunswick). For experiments in which a frozen bacterial stock was used to inoculate the culture, cells were first streaked on agar plates and incubated at 10% CO2 to facilitate fast growth. Pre-cultures derived from colonies were grown in 2–5 mL liquid M9 glycerol media under 10% CO2 with a matching 1 mL control in ambient air. Negative control strains unable to grow in minimal media (i.e. active site mutants of rubisco) were streaked on and pre-cultured in LB media under 10% CO2.

22Growth curves were obtained using two complementary methods: an eight-chamber bioreactor for large-volume cultivation (MC1000, PSI), and 96-well plates in a gas controlled plate reader plate (Spark, Tecan). For the 96-well format, cells were pre-cultured in the appropriate permissive media, M9 glycerol under 10% CO2 where possible. If rich media was used, for example for negative controls, stationary phase cells were washed in 2x the culture volume and resuspended in 1x culture volume of M9 media with no carbon source. Cultures were diluted to an OD of 1.0 (600 nm) and 250 μl cultures were inoculated by adding 5 μl of cells to 245 μl media. A humidity cassette (Tecan) was refilled daily with distilled water to mitigate evaporation during multi-day cultivation at 37 °C. Evaporation nonetheless produced irregular growth curves (e.g. Figure 3—figure supplement 2), which motivated larger volume cultivation in the bioreactor, which mixes by bubbling ambient air into each growth vessel. 80 mL bioreactor cultures were inoculated to a starting OD of 0.005 (600 nm) and grown at 37°C to saturation. Optical density was monitored continuously at 680 nm.

23Anaerobic cultivation of agar plates was accomplished using a BBL GasPak 150 jar (BD) flushed six times with an anoxic mix of 10% CO2 and 90% N2. Tenfold titers of biological duplicate cultures were plated on M9 glycerol media with and without 20 mM NaNO3 supplementation. Because E. coli cannot ferment glycerol, NO3- was supplied as an alternative electron acceptor. Plates without NO3- showed no growth (Figure 2—figure supplement 1), confirming the presence of an anaerobic atmosphere in the GasPak.

24To computationally design mutant strains in which growth is coupled to rubisco carboxylation flux, we used a variant of Flux Balance Analysis called ‘OptSlope’. Starting from a published model of E. coli central metabolism, the Core Escherichia coli Metabolic Model, we considered all pairs of central metabolic knockouts and ignored those that permit growth in silico in the absence of rubisco and phosphoribulokinase (Prk) activities. For the remaining knockouts, we evaluated the degree of coupling between rubisco flux and biomass production during growth in nine carbon sources: glucose, fructose, gluconate, ribose, succinate, xylose, glycerate, acetate, and glycerol. This approach highlighted several candidate rubisco-dependent knockout strains, including ΔrpiAB Δedd, which is the basis of the CCMB1 strain. Full discussion of our algorithmic approach to strain design is given in Appendix 1 along with detailed description of the proposed mechanisms of rubisco coupling in CCMB1 and a comparison to other rubisc-dependent E. coli strains. OptSlope source code is available at https://gitlab.com/elad.noor/optslope and calculations specific to CCMB1 can be found at https://github.com/flamholz/carboxecoli (; copy archived at swh:1:rev:76596e1e8614173d8ef64aa13e93674307cfa3de).

25Strains used in this study are documented in Supplementary file 1. To produce CCMB1, we first constructed a strain termed ‘Δrpi’. This strain has the genotype ΔrpiAB Δedd and was constructed in the E. coli BW25113 background by repeated rounds of P1 transduction from the KEIO collection followed by pCP20 curing of the kanamaycin selection marker. Deletion of edd removes the Entner-Doudoroff pathway, forcing rubisco-dependent metabolism of gluconate via the pentose phosphate pathway (Figure 2—figure supplement 3). CCMB1 has the genotype BW25113 ΔrpiAB Δedd ΔcynT Δcan and was constructed from ΔrpiAB by deleting both native carbonic anhydrases using the same methods, first transducing the KEIO ΔcynT and then Δcan from EDCM636, which was obtained from the Yale Coli Genetic Stock Center. Transformation was performed by electroporation (ECM 630, Harvard Biosciences) and electrocompetent stocks were prepared using standard protocols. Strain genotypes were routinely verified by PCR, as described below.

26pFE21 and pFA31 are compatible vectors derived from pZE21 and pZA31. These vectors use an anhydrotetracycline (aTc) inducible PLtetO-1 promoter to regulate gene expression. pF plasmids were modified from parent vectors to constitutively express the tet repressor (TetR) under the Pbla promoter so that expression is repressed by default. We found that an inducible system aids in cloning problematic genes like prk. We refer to these vectors as pFE and pFA, respectively. The p1A plasmid (Figure 2A) derives from pFE and expresses two additional genes: the Form IA rubisco from H. neapolitanus and a prk gene from Synechococcus elongatus PCC 7942. The pCB plasmid is properly called pFE-CB, while pCCM is pFA-CCM. The two CCM plasmids are diagrammed in Figure 3—figure supplement 1. Cloning was performed by Gibson and Golden-Gate approaches as appropriate. Large plasmids (e.g. pCB, pCCM) were verified by Illumina resequencing (Harvard MGH DNA Core plasmid sequencing service) and maps were updated manually after reviewing results compiled by breseq resequencing software. Plasmids used in this study are described in Supplementary file 1 and available on Addgene at https://www.addgene.org/David_Savage/.

27As CCMB1 is a relatively slow-growing knockout strain, we occasionally observed contaminants in growth experiments. We used two strategies to detect contamination by faster-growing organisms (e.g. wild-type E. coli). As most strains grew poorly or not at all in ambient air, pre-cultures grown in 10% CO2 were accompanied by a matching 1 mL negative control in ambient air. Pre-cultures showing growth in the negative control were discarded or verified by PCR genotyping in cases where air-growth was plausible.

28PCR genotyping was performed using primer sets documented in Supplementary file 1. Three primer pairs were used to probe a control locus (zwf) and two target loci (cynT and rpiA). The zwf locus is intact in all strains. cynT and rpiA probes test for the presence of the CCMB1 strain (genotype BW25113 ΔrpiAB Δedd ΔcynT Δcan). Notably, the CAfree strain (BW25113 ΔcynT Δcan) that we previously used to test the activity of DAB-type transporters is a cynT knockout but has a wild-type rpiA locus, so this primer set can distinguish between wild-type, CAfree, and CCMB1. This was useful for some experiments where CAfree was used as a control (e.g. Figures S7-8). Pooled colony PCRs were performed using Q5 polymerase (NEB), annealing at 65°C and with a 50 s extension time.

29CCMB1:pCB did not initially grow in M9 media supplemented with glycerol, which was unexpected because pCB carries rubisco and prk genes. We therefore performed a series of selection experiments to isolate plasmids conferring growth at elevated CO2 and then in ambient air. Here we describe the methodology; the full series of experiments is described in Appendix 2 and illustrated in Figure 3—figure supplement 1. CCMB1 cultures carrying appropriate plasmids were first grown to saturation in rich LB media in 10% CO2. Stationary phase cultures were pelleted by centrifugation for 10 min at 4000 x g, washed in 2x the culture volume, and resuspended in 1x culture volume of M9 media with no carbon source. After resuspension, multiple dilutions were plated on selective media (e.g. M9 glycerol media) and incubated in the desired conditions (e.g. in ambient air) with a positive control in 10% CO2 on appropriate media. When colonies formed in restrictive conditions, they were picked into permissive media, grown to saturation, washed and tested for re-growth in restrictive conditions by titer plating or streaking. Plasmid DNA was isolated from verified colonies and transformed into naive CCMB1 cells to test whether plasmid mutations confer improved growth (i.e. in the absence of genomic mutations).

30We first selected for CCMB1:pCB growth on M9 glycerol media in 10% CO2 and then in M9 gluconate media under 10% CO2. The resulting plasmid, pCB-gg for ‘gluconate grower,’ was isolated and deep sequenced (Harvard MGH DNA Core plasmid sequencing service). Plasmid maps were manyally updated based on results from the breseq software. Following this first round of selection, CCMB1 was co-transformed with pCB-gg and pCCM and selected for growth in ambient air. Washed stationary phase cultures of CCMB1:pCB-gg+pCCM were plated on M9 glycerol media in ambient CO2. Parallel negative control selections were plated on uninduced plates (no aTc) and using CCMB1:p1A+pCCM, which lacks carboxysome genes. Plates were incubated in a humidified incubator for 20 days until colonies became visible.

31Forty colonies were picked and tested for re-growth in ambient air by titer plating. Pooled plasmid DNA was extracted from verified colonies and electroporated into naive CCMB1 to test plasmid-linkage of growth. Colony #4 re-transformant #13 grew robustly was chosen due to replicable growth. Pooled plasmid DNA extracted from this strain was resequenced by a combination deep sequencing and targeted Sanger sequencing of the TetR locus and origins of replication, as these regions share sequence between pCB and pCCM. The individual post-selection plasmids, termed pCB’ and pCCM’, were reconstructed from pooled plasmid extract by PCR and Gibson cloning. These plasmids, termed pCB’ and pCCM’, were again verified by resequencing. Naive CCMB1 was transformed with the reconstructed post-selection plasmids pCB’ and pCCM’ and tested for growth in ambient air in plate reader (Spark, Tecan) and bioreactor (MC1000, PSI) assay formats.

32To verify that growth in ambient air depends on CCM components, we generated variants of pCB’ and pCCM’ carrying targeted null mutations. CCMB1 was then co-transformed with two plasmids: a mutant plasmid (of either pCB’ or pCCM’) and its cognate, unmodified plasmid. Mutant plasmids are listed here along with expected growth phenotypes, with full detail in Supplementary file 1. pCB’ CbbL K194M, or pCB’ cbbL-, contains an inactivating mutation to the large subunit of the carboxysomal Form 1A rubisco. This mutation was expected to abrogate rubisco-dependent growth entirely.

33Mutations targeting the CCM, rather than rubisco itself, are expected to ablate growth in ambient air but permit growth in high CO2. The following plasmid mutations were designed to specifically target essential components of the CCM. pCB’ CsoSCA C173S, or pCB’ CsoSCA-, carries a mutation to an active site cysteine residue responsible for coordinating the catalytic Zn2+ ion in β-carbonic anhydrases. pCB’ CsoS2 ΔNTD lacks the N-terminal domain of CsoS2, which is responsible for recruiting rubisco to the carboxysome during the biogenesis of the organelle. Similarly, pCB’ CbbL Y72R carries an arginine residue instead of the tyrosine responsible for mediating cation-π interactions between the rubisco large subunit and the N-termus of CsoS2. This mutation eliminates binding between the rubisco complex and the N-termus of CsoS2. pCB’ ΔcsoS4AB lacks both pentameric shell proteins, CsoS4AB, which was shown to disrupt the permeability barrier at the carboxysome shell. pCCM’ DabA1 C462A, D464A, or pCCM’ DabA1-, carries inactivating mutations to the putative active site of the inorganic carbon transporter component DabA1.

34Two more mutant plasmids were designed to test the roles of rubisco chaperones in producing a functional CCM. pCCM’ CbbQ K46A, E107Q, denoted pCCM’ CbbQ-, carries mutations that inactivate the ATPase activity of the CbbQ subunit of the CbbOQ rubisco activase complex. pCCM’ ΔacRAF lacks the putative rubisco chaperone acRAF. acRAF is homologous to a plant rubisco folding chaperone and likely involved in the folding of the H. neapolitanus Form IA rubisco. Experimental evaluation of growth phenotypes for the above-described mutants is detailed below and results are given in Figure 4—figure supplement 1.

35To interrogate the phenotypic effects of mutations to the CCM, we tested the growth of matched biological replicate cultures of CCM mutants (e.g. disruption of carboxysome components or transporter function) in M9 glycerol medium in 10% CO2 and ambient air (Figure 4A). Individual colonies were picked into a round-bottom tube with 4 mL of M9 glycerol media with full strength antibiotic and 100 nM aTc. 1 mL of culture was then transferred to a second tube. The 3 mL pre-culture was incubated in 10% CO2, while the 1 mL culture was incubated in ambient air as a negative control. Control strains unable to grow in minimal media (e.g. those expressing inactive rubisco mutants) were pre-cultured in LB media. High-CO2 pre-cultures were grown to saturation, after which optical density (OD600) was measured in five-fold dilution. Experimental cultures were inoculated to a starting OD600 of 0.01 in 3 mL of M9 glycerol media with 12.5 mg/L chloramphenicol and 100 nM aTc. Each pre-culture was used to inoculate a matched pair of experimental cultures, one incubated in 10% CO2 and another in ambient air (Figure 4A). The endpoint culture density was measured at 600 nm. All experiments were performed in biological quadruplicate. As a positive control we used a complemented double carbonic anhydrase knockout (CAfree:pFE-sfGFP+pFA-HCAII) as its growth in air depends on expression of the human carbonic anhydrase II.

36Roughly 1.2 L of CCMB1:pCB’+pCCM’ was grown in M9 glycerol media in ambient air in two identical bioreactors (MC1000, PSI) as described above. Sixteen distinct 80 mL cultures were grown, comprising eight technical replicates of two biological replicates deriving from distinct colonies. Cells were harvested before the onset of stationary phase, with optical densities ranging from ≈0.5 to ≈2.0 (600 nm, Genesys 20, Thermo Scientific) and pooled before subsequent purification steps. Wild type H. neapolitanus cells were grown in a 10 L bioreactor (Eppendorf BioFlo 115) modified to function as a chemostat. A continuous culture was grown in DSMZ-68 medium at a dilution rate of 0.03–0.05/hour. The culture was grown at 30°C, sparged with ambient air and the pH was held constant at 6.4 by addition of KOH. Chemostat effluent was collected in a 20 L glass flask and cells harvested every 2–3 day by centrifugation at 6000 x g for 15 min. A cell pellet of approximately 10 L of culture was used for subsequent purification.

37Cells were chemically lysed in B-PER II (Thermo Fischer) diluted to 1x with TEMB buffer (10 mM Tris pH 8.0, 10 mM MgCl2, 20 mM NaHCO3 and 1 mM EDTA) supplemented with 0.1 mg / mL lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 ul of benzonase/mL (Sigma-Aldrich). E. coli cells (CCMB1:pCB’+pCCM’) were lysed for 30 min under mild shaking while H. neapolitanus cells were stirred vigorously with a magnetic stirrer for 1 hr. Lysed cells were centrifuged 12,000 x g for 15 min to remove cell debris. The clarified lysate (supernatant) was centrifuged 40,000 x g for 30 min to pellet carboxysomes and obtained pellets were gently resuspended in 1.5 mL TEMB buffer. Resuspended pellets were loaded on top of a 25 mL 10–50% sucrose step gradient (10, 20, 30, 40% and 50% w/v sucrose, made in TEMB buffer) and ultracentrifuged at 105,000 x g for 35 min (SW 32 Ti Swinging-bucket, Beckman Coulter). Gradients were fractionated, analysed by SDS-PAGE and carboxysome containing fractions pooled. Due to the low concentration of carboxysomes in the CCMB1:pCB’+pCCM’ sample, fraction numbers corresponding to H. neapolitanus gradient were pooled. Pooled fractions were ultracentrifuged 100,000 x g for 90 min and pellets were gently resuspended in TEMB to obtain the final purified carboxysome sample. The co-migration of carboxysomes with rubisco confirmed by coomassie and silver stained SDS-page gels of the final sample. Purified carboxysomes were visualized by negative stain TEM. Sample was applied to glow discharged formvar/carbon coated copper grids. Grids were then washed with deionized water and stained with 2% aqueous uranyl acetate. Imaging was performed on a JEOL 1200 EX TEM (H. neapolitanus) or a Tecnai 12 TEM at 120 KV (FEI) (CCMB1:pCB’+pCCM’). Images were collected using UltraScan 1000 digital micrograph software (Gatan Inc).

38CCMB1:pCB’+pCCM’ was grown in ambient air in 3 mL of M9 glycerol medium and induced with 100 nM aTc. A carboxysome-negative control, CAfree:pFE-sfGFP+pFA-HCAII, was grown in the same conditions. Sample preparation and sectioning were performed by the University of California Berkeley Electron Microscope Laboratory. Cell pellets were fixed for 30 min at room temperature in 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4. Fixed cells were stabilized in 1% very low melting-point agarose and cut into small cubes. Cubed sample was then rinsed three times at room temperature for 10 min in 0.1 M sodium cacodylate buffer, pH 7.4 and then immersed in 1% osmium tetroxide with 1.6% potassium ferricyanide in 0.1 M cacodylate buffer for an hour in the dark on a rocker. Samples were later rinsed three times with a cacodylate buffer and then subjected to an ascending series of acetone for 10 min each (35%, 50%, 75%, 80%, 90%, 100%, 100%). Samples were progressively infiltrated with Epon resin (EMS, Hatfield, PA, USA) while rocking and later polymerized at 60°C for 24 hr. 70 nm thin sections were cut using an Ultracut E (Leica) and collected on 100 mesh formvar coated copper grids. The grids were further stained for 5 min with 2% aqueous uranyl acetate and 4 min with Reynold's lead citrate. The sections were imaged using a Tecnai 12 TEM at 120 KV (FEI) and images were collected using UltraScan 1000 digital micrograph software (Gatan Inc).

39Protein-bound amino acids were analyzed in total biomass hydrolysate of cultures grown in minimal media with 99% 13C glycerol (Cambridge Isotopes) as the sole organic carbon source. Biological quadruplicate cultures of the experimental strain, CCMB1:pCB’ + pCCM’, and the rubisco-independent control strain, CAfree:pFE-sfGFP + pFA-HCAII, were grown in 80 mL volumes in a bioreactor bubbling ambient air into each growth vessel (MC1000, PSI). After harvesting biomass, samples were prepared and analyzed as described in. Briefly, the OD600 was recorded and 2 OD x mL of sample were pelleted by centrifugation for 15 min at 4000 x g. The pellet was resuspended in 1 mL of 6 N HCl and incubated for 24 hr at 110°C. The acid was subsequently evaporated under a nitrogen stream using a custom gas manifold, resulting in a dry hydrolysate. Dry hydrolysates were resuspended in 0.6 mL of MilliQ water, centrifuged for 5 min at 14,000 x g, and supernatant was analyzed by liquid chromatography-mass spectrometry (LC-MS).

40Hydrolyzed amino acids were separated using ultra performance liquid chromatography (UPLC, Acquity, Waters) on a C-8 column (Zorbax Eclipse XBD, Agilent) at a flow rate of 0.6 mL/min, and eluted off the column using a hydrophobicity gradient. Buffers used were: (A) H2O + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid with the following gradient: 100% of A (0–3 min), 100% A to 100% B (3–9 min), 100% B (9–13 min), 100% B to 100% A (13–14 min), 100% A (14–20 min). The UPLC was coupled online to a triple quadrupole mass spectrometer (TQS, Waters). Data were acquired using MassLynx v4.1 (Waters). Amino acids and metabolites used for analysis were selected according to the following criteria: amino acids that had peaks at a distinct retention time and m/z values for all isotopologues and also showed correct 13C labeling fractions in control samples that contained protein hydrolyzates of WT cells grown with known ratios of uniformly 13C-labeled (U-13C) glucose to 12C-glucose. We further analyzed the serine M+2 isotopologue (parent ion in positive ionization mode with 108.1 m/z) using multiple reaction monitoring (MRM). This approach by selecting the channels of a daughter ion (fragment) with the formula [C2H6NO]+: (A) 61.1 m/z, where the undetected fragment contains a carboxylic acid carbon which is a 13C isotope and (B) 62.1 m/z, where the undetected fragment contains a carboxylic acid carbon which is 12C. We looked at the ratio of the peak integrals of A/B to infer the distribution of 13C/12C for that particular carboxyl carbon. Since the carboxylic acid on L-serine derives from the rubisco carboxylation product 3-phosphoglycerate, measuring the 13C/12C distribution at this position reports directly on carboxylation by rubisco in vivo (Figure 6 and supplements) and with lower background than the total mass measurement described above.

41The total 13C fraction of each metabolite was determined as the weighted average of the fractions of all the isotopologues for that metabolite:

42Here, N is the number of carbons in the compound (e.g. N = 3 for serine) and fi is the relative fraction of the i-th isotopologue, that is containing i 13C carbon atoms. Each metabolite’s total 12C fraction was calculated as. Our quantitative approach to inferring the rubisco carboxylation flux from these data is described fully in Appendix 3; source code and data are available at https://github.com/flamholz/carboxecoli.