Global metabolic rewiring for improved CO2 fixation and chemical production in cyanobacteria

Abstract

Cyanobacteria have attracted much attention as hosts to recycle CO₂ into valuable chemicals. Although cyanobacteria have been engineered to produce various compounds, production efficiencies are too low for commercialization. Here we engineer the carbon metabolism of Synechococcus elongatus PCC 7942 to improve glucose utilization, enhance CO₂ fixation and increase chemical production. We introduce modifications in glycolytic pathways and the Calvin Benson cycle to increase carbon flux and redirect it towards carbon fixation. The engineered strain efficiently uses both CO₂ and glucose, and produces 12.6 g l^-1 of 2,3-butanediol with a rate of 1.1 g l^-1 d^-1 under continuous light conditions. Removal of native regulation enables carbon fixation and 2,3-butanediol production in the absence of light. This represents a significant step towards industrial viability and an excellent example of carbon metabolism plasticity.

Figure 1. Rewiring of carbon metabolism in cyanobacteria.

(a) Photoautotrophic conversion of CO₂ to 23BD and biomass. (b) Coupling glucose metabolism with the CB cycle to enhance CO₂ fixation and 23BD production in both light and dark conditions. (c) Relative amounts of intracellular metabolites of Strain 3 (alsS-alsD-adh+galP) grown with (grey) and without (white) glucose in continuous light conditions for 72 h where n=3 biological replicates, and error bars represent s.d. Metabolites significantly elevated and decreased with the addition of glucose are labelled red and blue, respectively. ADH, alcohol dehydrogenase; ALDC, acetolactate decarboxylase; ALS, acetolactate synthase; CIT, citrate; DHAP, dihydroxyacetone phosphate; EDA, 2-keto-3-deoxygluconate-6-phosphate aldolase; EDD, 6PG dehydratase; E4P, erythrose-4-phosphate; FBP, fructose-1,6-bisphosphate; FUM, fumarate; F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; GalP, galactose-proton symporter; GND, 6PG dehydrogenase; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; ICIT, isocitrate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; MAL, malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PGI, phosphoglucose isomerase; PFK, phosphofructokinase; PRK, phophoribulokinase; PYR, pyruvate; AcCoA, acetyl-CoA; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; R5P, ribose-5-phosphate; R15P, ribulose-1,5-bisphosphate; Ru5P, ribulose-5-phosphate; SUC, succinate; S7P, sedoheptulose-7-phosphate; Xu5P, xylulose-5-phosphate; ZWF, G6P dehydrogenase; 2OG, 2-oxoglutarate; 23BD, 2,3-butanediol; 3PGA, 3-phosphoglycerate; and 6PG, 6-phosphogluconate.

Figure 2. Characterization and activation of glucose metabolism.

Cells were cultured in 10 ml of BG11 media containing 10 g l⁻¹ glucose and 20 mM NaHCO₃ in continuous light conditions. IPTG (0.1 mM) was added at 0 h. Cell growth (a) and 23BD concentration (b) profiles of Strains 3 (black), 4 (Δzwf, red), 5 (gnd/Δgnd, green), 6 (pgi/Δpgi, blue), 7 (Δpfk, purple) and 8 (eda/Δeda, orange). (c–e) Cell growth (c), 23BD concentration (d) and glucose concentration (e) profiles of Strains 3 (grey), 9 (galP-zwf-edd, green), 10 (galP-pgi, blue) and 11 (galP-zwf-gnd, red). N=3 biological replicates; error bars represent s.d.

Figure 3. Redirection of carbon flux towards CO₂ fixation in light and dark conditions.

(a–c) Cells were cultured in 10 ml of BG11 media containing 10 g l⁻¹ U-¹³C glucose and 20 mM unlabelled NaHCO₃ in continuous light conditions. ‘–', ‘+' and ‘+++' indicate that each gene in the corresponding row is deleted, natively expressed or overexpressed, respectively. (a) The percentage of 23BD produced from either glucose or CO₂ by each strain. Growth (b) and 23BD concentration (c) profiles of Strains 3 (grey), 12 (Δcp12, purple), 13 (Δcp12:: rbcLXS, green) and 14 (Δcp12:: prk-rbcLXS, orange). (d,e) Strains 3, 12 and 14 were cultured in 10 ml of BG11 media containing 10 g l⁻¹ unlabelled glucose and ¹³C-NaHCO₃ for 24 h under continuous dark conditions. ¹³C labelling ratio of intracellular 3PGA (d) and concentration of intracellular R15P normalized with that of Strain 12 (e). N=3 biological replicates; error bars represent s.d. N.D. indicates not detectable.

Figure 4. Coupling the OPP pathway and the CO₂ fixation pathway.

Cells were cultured in 10 ml of BG11 media containing 10 g l⁻¹ glucose and 20 mM NaHCO₃ in continuous light conditions (a–e) and dark conditions (f,g). OPP=with (+++) and without (+) overexpression of zwf and gnd; CP12=cp12 gene was intact (+) and deleted (–) with prk and rbcLXS. Metabolite abbreviations are the same as those used in Fig. 1. Growth (a), 23BD concentration (b) and glucose concentration (c) profiles of Strains 3 (grey), 11 (galP-zwf-gnd, blue), 14 (Δcp12:: prk-rbcLXS, brown) and 15 (11+Δcp12:: prk-rbcLXS, red). Intracellular concentrations of metabolites (d) of Strain 3, 11 and 15 at 48 h. Relative content of intracellular R15P of Strains 3, 11, 14 and 15 grown in continuous light (e) and dark (f) conditions for 24 h. Cell biomass and 23BD production (g) of the same strains grown in continuous dark conditions for 24 h. N=3 biological replicates; error bars represent s.d.' N.D. indicates not detectable.

Figure 5. Long-term production in continuous light and diurnal light conditions.

Strain 15 (1+galP-zwf-gnd+Δcp12:: prk-rbcLXS) was cultured in 10 ml BG11 media containing 15 g l⁻¹ glucose and 20 mM NaHCO₃ in continuous light conditions (a–c) and diurnal light conditions (d–f). Grey shades represent dark phases. Cells were induced with 1 mM IPTG on day 0. On days 3, 6 and 9, cells were collected by centrifugation and resuspended at an OD₇₃₀ of 5.0 in fresh production media. 23BD production (a,d), glucose consumption (b,e) and growth (c,f) profiles of Strain 15 where N=3 biological replicates, and error bars represent s.d.