Cyanobacterial conversion of carbon dioxide to 2,3-butanediol

Abstract

Conversion of CO(2) for the synthesis of chemicals by photosynthetic organisms is an attractive target for establishing independence from fossil reserves. However, synthetic pathway construction in cyanobacteria is still in its infancy compared with model fermentative organisms. Here we systematically developed the 2,3-butanediol (23BD) biosynthetic pathway in Synechococcus elongatus PCC7942 as a model system to establish design methods for efficient exogenous chemical production in cyanobacteria. We identified 23BD as a target chemical with low host toxicity, and designed an oxygen-insensitive, cofactor-matched biosynthetic pathway coupled with irreversible enzymatic steps to create a driving force toward the target. Production of 23BD from CO(2) reached 2.38 g/L, which is a significant increase for chemical production from exogenous pathways in cyanobacteria. This work demonstrates that developing strong design methods can continue to increase chemical production in cyanobacteria.

Fig. 1.

The pathway for acetoin and 23BD production in S. elongatus PCC7942. The acetoin/23BD production pathway contains three enzymatic steps from pyruvate.

Fig. 2.

Effect of acetoin and 23BD on growth. (A) Time course for the growth of S. elongatus in the absence of acetoin (●) or in the presence of 0.05 g/L (○), 0.1 g/L (◇), 0.2 g/L (□), and 1.0 g/L (△) acetoin. (B) Time course for the growth of S. elongatus in the absence of 23BD (●) or in the presence of 10 g/L (○), 15 g/L (◇), 30 g/L (□), and 50 g/L (△) 23BD. Cells were incubated in BG-11 containing 50 mM NaHCO₃ at 30 °C. Error bars are SD (n = 3).

Fig. 3.

Acetoin production in modified strains. (A) Schematic representation of recombination to integrate alsS and alsD into the S. elongatus chromosome. (B) Acetoin production in modified E. coli. Cells were grown for 16 h (hashed) and 40 h (blue). (C) Acetoin production in modified S. elongatus. Cells were grown for 72 h. alsS indicates inclusion (+) of alsS (B. subtilis) or absence (−) of the gene. alsD indicates the source organism for alsD (Table S3). A. h., A. hydrophila; B. l., B. licheniformis; B. s., B. subtilis; E. a., E. aerogenes; E. c., E. cloacae; G. x., G. xylinus.

Fig. 4.

23BD production in modified strains. (A) Schematic representation of recombination to integrate alsS, alsD, and adh into the S. elongatus chromosome. (B) Production in modified E. coli. Cells were grown for 40 h. Bars represent acetoin (light blue), (R,R)-23BD (red), and meso-23BD (dark blue). alsS indicates inclusion (+) of alsS (B. subtilis). alsD and adh rows indicate the source organism for the gene (Table S3). Activity is that of sADH expressed in E. coli and measured in cell extract (nmol NADPH min⁻¹⋅mg⁻¹). (C) 23BD production in modified S. elongatus. Cells were grown for 72 h. (D) Specific activities of ALS and sADH in cell extracts from modified S. elongatus strains. (E) Effect of IPTG in modified S. elongatus. Cells were grown for 72 h after induction with the specified concentration of IPTG. Values are normalized to those from uninduced cultures. Bars represent 23BD production (dark blue), activity of sADH protein measured with NADPH as cofactor (red), and activity of ALS protein (light blue). Error indicates SD (n = 3). A. h., A. hydrophila; B. l., B. licheniformis; B. s., B. subtilis; C. p., C. parapsilosis; C. b., C. beijerinckii; E. a., E. aerogenes; E. c., E. cloacae; G. x., G. xylinus; L. p., L. pseudomesenteroides; T. b., T. brockii.

Fig. 5.

Long-term 23BD production. (A–C) Summary of results for 23BD production in continuous cultures. Red: S. elongatus containing alsS (B. subtilis), alsD (A. hydrophila), and adh (C. beijerinckii). Blue: S. elongatus containing alsS (B. subtilis), alsD (A. hydrophila) and adh (T. brockii). Green: S. elongatus containing alsS (B. subtilis) and alsD (E. aerogenes). Purple: S. elongatus without alsS, alsD or adh. (A) Time courses for growth. (B) Total 23BD production. (C) Photosynthetic efficiency. (D) Total biomass production per day. Error bars indicate SD (n = 3).