n-Butanol production by Rhodopseudomonas palustris TIE-1

Abstract

Anthropogenic carbon dioxide (CO₂) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO₂ to biofuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO₂. However, oxygen generation during oxygenic photosynthesis adversely affects biofuel production efficiency. To produce n-butanol (biofuel) from CO₂, here we introduce an n-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph, Rhodopseudomonas palustris TIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieve n-butanol production in wild-type TIE-1 and mutants lacking electron-consuming (nitrogen-fixing) or acetyl-CoA-consuming (polyhydroxybutyrate and glycogen synthesis) pathways. The mutant lacking the nitrogen-fixing pathway produce the highest n-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant produces n-butanol using CO₂, solar panel-generated electricity, and light with high electrical energy conversion efficiency. Overall, this approach showcases TIE-1 as an attractive microbial chassis for carbon-neutral n-butanol bioproduction using sustainable, renewable, and abundant resources.

Fig. 1. n-Butanol biosynthesis pathway, cassette design, and major metabolisms used for n-butanol production in Rhodopseudomonas palustris TIE-1 (TIE-1).

a n-butanol biosynthesis pathway involves five genes. The enzymes encoded by each gene and the reactions catalyzed by these enzymes are shown in dark blue. Two major byproducts (acetone and ethanol) are shown in dark red. NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate. b Cassette design. The 3-gene cassette relies on phaA and phaB on the genome of TIE-1 for the first two steps of n-butanol synthesis. Here, only 3-genes (phaJ, ter, and adhE2) were introduced on a plasmid under a constitutive promoter P_aphII. The 5-gene cassette has all five genes (phaA, phaB, phaJ, ter, and adhE2) on the plasmid under a constitutive promoter P_aphII. c Photoheterotrophy: TIE-1 uses organic acids as carbon and electron source, light as an energy source, and ammonium (NH₄⁺) or dinitrogen gas (N₂) as a nitrogen source. d Photoautotrophy: TIE-1 uses carbon dioxide as carbon source, hydrogen (H₂), ferrous iron [Fe(II)], or poised electrode as an electron source, light as an energy source, and NH₄⁺ or N₂ as a nitrogen source.

Fig. 2. The nitrogenase double mutant (Nif) produced the highest amount of n-butanol in the presence of 3-hydroxybutyrate.

The concentration of n-butanol in mg/L when TIE-1 was cultured with ammonium (NH₄⁺, red) or dinitrogen gas (N₂, blue) and a acetate (photoheterotrophy) b 3-hydroxybutyrate (photoheterotrophy) c hydrogen (H₂) (photoautotrophy) and d ferrous iron [Fe(II)] (photoautotrophy). CO₂ was present in all conditions. Data are from n = 3 of independent experiments. Boxes that only have two biological replicates are indicated by ‘*‘. WT-3: wild type with 3-gene cassette; WT-5: wild type with 5-gene cassette; Nif- 3: nitrogenase knockout t with 3-gene cassette; Nif - 5: nitrogenase knockout with 5-gene cassette; Gly- 3: glycogen synthase knockout with 3-gene cassette; Gly- 5: glycogen synthase knockout with 5-gene cassette; Phb- 3: hydroxybutyrate polymerase knockout with 3-gene cassette, n.d. (non-detectable).

Fig. 3. 3-Hydroxybutyrate resulted in the highest n-butanol productivity.

The n-butanol productivity when TIE-1 was cultured with ammonium (NH₄⁺, red) or dinitrogen gas (N₂, blue) and a acetate (photoheterotrophy) b 3-hydroxybutyrate (photoheterotrophy) c hydrogen (H₂) (photoautotrophy) and d ferrous iron [Fe(II)] (photoautotrophy). CO₂ was present in all conditions. Data are from n = 3 of independent experiments. Boxes with data from n = 2 independent experiments are indicated by ‘*’. WT-3: wild type with 3-gene cassette; WT-5: wild type with 5-gene cassette; Nif-3: nitrogenase knockout t with 3-gene cassette; Nif-5: nitrogenase knockout with 5-gene cassette; Gly-3: glycogen synthase knockout with 3-gene cassette; Gly-5: glycogen synthase knockout with 5-gene cassette; Phb-3: hydroxybutyrate polymerase knockout with 3-gene cassette, n.d. (non-detectable).

Fig. 4. High n-butanol production correlates to low acetone production amongst TIE-1 mutants.

The concentration of acetone in mg/L when TIE-1 was cultured with ammonium (NH₄⁺, red) or dinitrogen gas (N₂, blue) and a acetate (photoheterotrophy) b 3-hydroxybutyrate (photoheterotrophy) c hydrogen (H₂) (photoautotrophy) and d ferrous iron [Fe(II)] (photoautotrophy). CO₂ was present in all conditions. Data are from n = 3 of independent experiments. WT-3: wild type with 3-gene cassette; WT-5: wild type with 5-gene cassette; Nif-3: nitrogenase knockout with 3-gene cassette; Nif-5: nitrogenase knockout with 5-gene cassette; Gly-3: glycogen synthase knockout with 3-gene cassette; Gly-5: glycogen synthase knockout with 5-gene cassette; Phb-3: hydroxybutyrate polymerase knockout with 3-gene cassette, n.d. (non-detectable).

Fig. 5. The nitrogenase double mutant (Nif) converts carbon to n-butanol more efficiently.

a–d The CO₂ consumption (positive value)/production (negative value). e–h Carbon conversion efficiency to n-butanol. a, e: acetate (photoheterotrophy) b, f: 3-hydroxybutyrate (photoheterotrophy) c, g: hydrogen (H₂) (photoautotrophy) and d, h: ferrous iron [Fe(II)] (photoautotrophy). CO₂ was present in all conditions. Data are from n = 3 of independent experiments. WT-3: wild type with 3-gene cassette; WT-5: wild type with 5-gene cassette; Nif-3: nitrogenase knockout with 3-gene cassette; Nif-5: nitrogenase knockout with 5-gene cassette; Gly-3: glycogen synthase knockout with 3-gene cassette; Gly-5: glycogen synthase knockout with 5-gene cassette; Phb-3: hydroxybutyrate polymerase knockout with 3-gene cassette, n.d. non-detectable.

Fig. 6. The nitrogenase double mutant (Nif) converts electrons to n-butanol more efficiently.

The electron conversion efficiency towards n-butanol (%) when TIE-1 was cultured with ammonium (NH₄⁺, red) or dinitrogen gas (N₂, blue) and a acetate (photoheterotrophy) b (3-hydroxybutyrate (photoheterotrophy) c hydrogen (H₂) (photoautotrophy); and d ferrous iron [Fe(II)] (photoautotrophy). CO₂ was present in all conditions. Data are from n = 3 of independent experiments. Boxes that only have two biological replicates are indicated by ‘*’. WT-3: wild type with 3-gene cassette; WT-5: wild type with 5-gene cassette; Nif-3: nitrogenase knockout t with 3-gene cassette; Nif-5: nitrogenase knockout with 5-gene cassette; Gly-3: glycogen synthase knockout with 3-gene cassette; Gly-5: glycogen synthase knockout with 5-gene cassette; Phb-3: hydroxybutyrate polymerase knockout with 3-gene cassette, n.d. (non-detectable).

Fig. 7. Using Hydrogen as an electron donor resulted in low electron flow in byproduct generation.

Electron consumption of n-butanol/acetone/H₂/CO₂/biomass synthesis under various incubation conditions. a acetate + NH₄⁺ (photoheterotrophy) b acetate + N₂ (photoheterotrophy) c 3-hydroxybutyrate + NH₄⁺ (photoheterotrophy) d 3-hydroxybutyrate  + N₂ (photoheterotrophy) e hydrogen (H₂) + NH₄⁺ (photoautotrophy) f hydrogen (H₂) + N₂ (photoautotrophy) g ferrous iron [Fe(II)] + NH₄⁺ (photoautotrophy) h ferrous iron [Fe(II)] + N₂ (photoautotrophy) i electron consumption of n-butanol synthesis under various incubation conditions.CO₂ was present in all conditions. Data are from n = 3 of independent experiments. WT-3: wild type with 3-gene cassette; WT-5: wild type with 5-gene cassette; Nif-3: nitrogenase knockout t with 3-gene cassette; Nif-5: nitrogenase knockout with 5-gene cassette; Gly-3: glycogen synthase knockout with 3-gene cassette; Gly-5: glycogen synthase knockout with 5-gene cassette; Phb-3: hydroxybutyrate polymerase knockout with 3-gene cassette n.d. is non-detectable and n.a. is not available.

Fig. 8. Three-electrodes configured sealed type bioelectrochemical cell (BEC), n-butanol production, acetone production, carbon conversion efficiency, electron conversion efficiency, and electrical energy conversion efficiency (EECE) towards n-butanol by the nitrogenase double mutant with the 5-gene cassette under photoelectroautotrophy.

Under photoelectroautotrophic conditions, TIE-1 gains electrons from a poised electrode, using light as an energy source and carbon dioxide as a carbon source. For all the platforms, either ammonium (NH₄⁺) or dinitrogen gas (N₂) was supplied. a Schematic set up of BEC platform. platform set up: 1- electricity source 2-light source, 3- Purge inlet, 4- Reference electrode (Ag/AgCl in 3 M KCl), 5- Counter electrode (Pt foil, 5 cm²), 6- Working electrode (Graphite rod, 3.2 cm²), 7- electrical wire DAQ- Data acquisition); b n-butanol production; c acetone production; d carbon conversion efficiency (CCE) towards n-butanol; e electron conversion efficiency (ECE) towards n-butanol; f electrical energy conversion efficiency (EECE) towards n-butanol. PO potentiostat, IR infrared light, HA halogen light, SO solar panel. Data are from n = 2 of independent experiments.