Figure 2. CCMB1 depends on rubisco carboxylation for growth on glycerol.

(A) Ribose-5-phosphate (Ri5P) is required for nucleotide biosynthesis. Deletion of ribose-phosphate isomerase (Δrpi) in CCMB1 blocks ribulose-5-phosphate (Ru5P) metabolism in the pentose phosphate (PP) pathway. Expression of rubisco (H. neapolitanus CbbLS) and phosphoribulokinase (S. elongatus PCC7942 prk) on the p1A plasmid (B) permits Ru5P metabolism, thus enabling growth on M9 glycerol media in 10% CO₂ (C). Mutating the rubisco active site (p1A CbbL^-) abrogates growth, as does mutating ATP-binding residues of Prk (p1A Prk^-). (D) CCMB1:p1A grows well under 10% CO₂, but fails to grow in ambient air. Cells were grown on M9 glycerol media throughout. The algorithmic design of CCMB1 is described in Figure 2—figure supplement 4 and Appendix 1. The mechanism of rubisco-dependence is diagrammed in Figure 2—figure supplement 3. Figure supplement 2 demonstrates growth of CCMB1:p1A on various media, Figure 2—figure supplement 5 demonstrates complementation by a variety of bacterial rubiscos and Figure 2—figure supplement 1 demonstrates anaerobic growth of CCMB1:p1A, establishing that oxygenation is not required for growth. Acronyms: ribulose 1, 5-bisphosphate (RuBP), 3-phosphoglycerate (3PG).

Figure 2—figure supplement 1. Expression of five kinetically and phylogenetically distinct rubiscos permits CCMB1 growth in glycerol minimal media with 5% CO₂.

(A) Expression of five diverse rubiscos in CCMB1 complemented growth in 5% CO₂ (colored lines) but not in ambient air (grey). Expressing the carboxysomal Form IA rubisco from H. neapolitanus along with prk on the p1A plasmid (CCMB1:p1A) permits growth in 5% CO2 (teal) but not in ambient air. A catalytically inactive variant (p1A CbbL K194M) failed to grow in both conditions, as expected and shown in Figure 2 and Figure 2—figure supplements 2, 5. The kinetic parameters of this rubisco have not been measured, but it is assumed to be relatively fast (k_cat,C ≈ 5–10 s⁻¹) and relatively non-specific towards CO₂ (S_C/O ≈ 30–50) like other carboxysome-localized Form IA rubiscos. Four additional rubiscos were expressed from an identical plasmid backbone and all of them permitted CCMB1 to grow in 5% CO₂ with varying kinetics. The non-carboxysomal Form IC rubisco from Ralstonia eutropha (also known as Cupriavidis necator) is in orange and is the most specific bacterial rubisco known, with k_cat,C ≈ 2–3 s⁻¹ and S_C/O ≈ 75–85. The Form IC rubisco from Rhodobacter sphaeroides (light blue) and has k_cat,C ≈ 1–2 s⁻¹ and S_C/O ≈ 55–60. The cyanobacterium S. elongatus PCC 6301 expresses a Form IB rubisco in the same family found in eukaryotic algae and land plants (pink). This enzyme is exceptionally fast for a Form I rubisco, with k_cat,C ≈ 10–14 s⁻¹ and S_C/O ≈ 40–50. Finally, the model Form II rubisco from R. rubrum (light green) also complemented CCMB1 for growth in 5% CO₂. Form II enzymes have relatively high k_cat,C ≈ 10 s⁻¹ and low S_C/O ≈ 10–20. Biological triplicate measurements were conducted for all rubiscos in panel A and were all consistent. Notably, none of these rubiscos permit growth in ambient air, even though they span a large fraction of the known diversity in maximum carboxylation rate (k_cat,C) and CO₂-specificity (S_C/O). For kinetic measurements of diverse rubiscos, see recent meta-analyses by Flamholz et al., 2019; Iñiguez et al., 2020. For recent measurements of the R. eutropha, R. rubrum, and S. elongatus enzymes, see Davidi et al., 2020; Occhialini et al., 2016; Satagopan and Tabita, 2016. (B) Growth rate and yield of CCMB1:p1A+vec depend on the CO₂ concentration, with higher CO₂ improving growth (‘vec’ denotes pFA-sfGFP). This result suggests that growth of CCMB1 is indeed coupled to the rate of carboxylation by rubisco, as predicted by the OptSlope algorithm described in Figure 2—figure supplement 4.

Figure 2—figure supplement 2. CCMB1 does not require oxygen for growth in minimal media.

(A) Titer plating assays were used to measure the viability of CCMB1:p1A grown on glycerol media under ambient air (≈0.04% CO₂, 21% O₂), 10% CO₂ (balance air), and an anoxic mix of 10% CO₂ and 90% N₂ (‘No O₂’). Since E. coli cannot ferment glycerol, 20 mM nitrate (NO₃^-) was provided as an alternate electron acceptor as marked. (B) CCMB1:p1A grows on glycerol media in the absence of O₂ so long as nitrate is provided. While CCMB1:p1A colonies are noticeably smaller than WT in panel (A), the colony count is indistinguishable, as quantified in panel (B). Experiments were conducted in biological duplicate (i.e. pre-cultures from distinct colonies) with at least two technical replicates (repeated spotting from the same preculture).

Figure 2—figure supplement 3. Proposed mechanisms of rubisco-dependent growth in CCMB1.

(A) CCMB1 depends on rubisco and prk for growth in glycerol, gluconate, and xylose minimal media. The common mechanism is an inability to metabolize ribulose-5-phosphate (Ru5P) due to the deletion of both ribose-phosphate isomerase genes (ΔrpiAB). When gluconate or xylose is the growth substrate, Ru5P must be produced in order to metabolize the carbon source. Although wild-type E. coli can metabolize gluconate via the ED pathway, the ED dehydratase knockout (Δedd) in CCMB1 blocks this route and forces 1:1 production of Ru5P from gluconate. Expression of prk and rubisco opens a new route of Ru5P metabolism, thus enabling CCMB1 to grow in gluconate or xylose media. Since extracellular glycerol is converted to glyceraldehyde 3-phosphate (GAP), it can be metabolized through lower glycolysis or through gluconeogenesis. The gluconeogenesis route produces hexoses that enter the pentose phosphate pathway, which is required to synthesize ribose 5-phosphate (Ri5P) for nucleotide and histidine biosynthesis. Depending on the growth rate, products of Ri5P make up 5–25% of E. coli biomass (Bremer and Dennis, 2008; Taymaz-Nikerel et al., 2010). As shown in (B), the pentose phosphate pathway forces co-production of Ri5P, Ru5P and xylulose 5-phosphate (Xu5P). In the absence of rpi activity, there is no pathway for metabolism of Xu5P or Ru5P. This defect is complemented by the expression of rubisco and prk. Notably, rubisco can also oxygenate RuBP, as shown in (C). E. coli can, in principle, recycle the oxygenation product 2-phosphoglycolate (2PG) through an ersatz salvage pathway via tartronate semialdehyde. This pathway is not the dominant mechanism of rubisco complementation because CCMB1:p1A cannot grow in ambient air, where O₂ is abundant (Figure 2D). Panel (D) describes the initial metabolism of extracellular glycerol, gluconate, and xylose in E. coli Extracellular carbon sources are marked with a grey background throughout. Abbreviations: 3-phosphoglycerate (3PG), 2-phosphoglycolate (2PG), glyceraldehyde 2-phosphate (GAP), dihydroxyacetone phosphate (DHAP), ribose 5-phosphate (Ri5P), ribulose 5-phosphate (Ru5P), xylulose 5-phosphate (Xu5P), ribulose 1, 5-bisphosphate (RuBP), 2-keto-3-deoxy-6-phosphogluconate (KDGP), fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (F-1,6-BP), phosphoenolpyruvate (PEP).

Figure 2—figure supplement 4. The OptSlope algorithm for designing rubisco-coupled E.colistrains.

Optslope searches for metabolic knockout mutants in which biomass production is coupled to flux through a reaction of choice (e.g. rubisco) at all growth rates. (A) shows the space of feasible biomass production and rubisco fluxes for wildtype (WT, grey) and a knockout mutant (green). For WT, biomass production and, therefore, growth rate, are independent of rubisco at all feasible growth rates (i.e. within the grey polygon). The mutant is ‘rubisco-coupled’ because maximal biomass production requires non-zero rubisco carboxylation flux and increasing biomass production demands increased carboxylation. The slope of this relationship is the ‘coupling slope.’ (B) We computationally generated pairs of E. coli central metabolic knockouts and calculated the coupling slope on nine carbon sources: glucose (gluc), fructose (fruc), gluconate (gnt), ribose (ribo), succinate (succ), xylose (xyl), glycerate (gly^ate), acetate (ace), and glycerol (gly^ol). Each double knockout is summarized as a 3 × 3 matrix of coupling slopes. Black denotes a rubisco-independent mutant and maroon a coupling slope of 0. The published mutant ∆gapA (Mueller-Cajar et al., 2007) has a coupling slope of 0 (left), while the ∆rpiAB ∆edd strain is rubisco-coupled on seven of the carbon sources (right). (C) Feasible phase space diagram for the ∆gapA strain shows that biomass production is not coupled to rubisco flux. (D) ∆rpiAB ∆edd has a positive coupling slope in glycerol, gluconate and xylose media.

Figure 2—figure supplement 5. CCMB1 depends on rubisco and prk for growth in minimal media.

(A) Expression of rubisco and prk complements CCMB1 growth on M9 glycerol and gluconate media under 10% CO₂, but not in ambient conditions (100 nM aTc induction in M9 plates). Mutations ablating rubisco (CbbL^-) or prk (Prk^-) activity abrogate growth in selective media but not in LB under 10% CO₂. Growth in LB is robust and rubisco-independent in 10% CO₂, but CCMB1 does not grow in ambient air even when supplied with rich media because it lacks CA genes (Merlin et al., 2003). Growth curves in (B) show the rubisco-dependence of CCMB1:p1A growth in glycerol (green) and gluconate (blue) media under 5% CO₂ in a gas controlled plate reader (Materials and methods). Negative controls (CCMB1:p1A CbbL^- in glycerol or gluconate media) and uninduced cultures failed to grow in these conditions (dashed grey lines). Experiments were conducted in technical sextuplicate and replicates were all consistent.