Efficient biochemical production of acetoin from carbon dioxide using Cupriavidus necator H16

Abstract

Background: Cupriavidus necator is the best-studied knallgas (also termed hydrogen oxidizing) bacterium and provides a model organism for studying the production of the storage polymer polyhydroxybutyrate (PHB). Genetically engineered strains could be applied for the autotrophic production of valuable chemicals. Nevertheless, the efficiency of the catalyzed processes is generally believed to be lower than with acetogenic bacteria. Experimental data on the potential efficiency of autotrophic production with C. necator are sparse. Hence, this study aimed at developing a strain for the production of the bulk chemical acetoin from carbon dioxide and to analyze the carbon and electron yield in detail.

Results: We developed a constitutive promoter system based on the natural PHB promoter of this organism. Codon-optimized versions of the acetolactate dehydrogenase (alsS) and acetolactate decarboxylase (alsD) from Bacillus subtilis were cloned under control of the PHB promoter in order to produce acetoin from pyruvate. The production process's efficiency could be significantly increased by deleting the PHB synthase phaC2. Further deletion of the other PHB synthase encoded in the genome (phaC1) led to a strain that produced acetoin with > 100% carbon efficiency. This increase in efficiency is most probably due to a minor amount of cell lysis. Using a variation in hydrogen and oxygen gas mixtures, we observed that the optimal oxygen concentration for the process was between 15 and 20%.

Conclusion: To the best of our knowledge, this study describes for the first time a highly efficient process for the chemolithoautotrophic production of the platform chemical acetoin.

Keywords: Acetoin; Autotroph; Cupriavidus necator H16; Platform chemical; Polyhydroxybutyrate; Ralstonia eutropha H16.

Fig. 1

Growth of C. necator H16 wild type and C. necator H16_ΔacoABC with 10 mM fructose or acetoin as the sole carbon source

Fig. 2

Comparative acetoin production of the three different strains in the inductor’s presence and absence. The figures show growth (a) and acetoin production (b). C. necator H16_ΔacoABC_pKRphb-alsSD is depicted by a black line. The control experiments with an uninduced C. necator H16_ΔacoABC_pKRara-alsSD strain are shown in dark blue, while experiments with 1.5 mM arabinose are indicated in light blue. Experiments with uninduced C. necator H16_ΔacoABC_pKRrha-alsSD are indicated in red. Experiments under induction with 1 mM rhamnose are indicated in orange

Fig. 3

PHB production in different deletion strains. a Emission measurement at 575 nm after Nile Red staining and absorption of 530 nm. b Fluorescent microscopy pictures of C. necator H16 wild type after Nile Red (up) and DAPI straining (left), merged picture (right). c Fluorescent microscopy pictures of C. necator H16_ΔacoABC_ΔphaC1_ΔphaC2

Fig. 4

Influence of the phaC genes on acetoin production with the plasmid pKRphb-alsSD under heterotrophic growth conditions. The three different strains (C. necator H16_ΔacoABC_ΔphaC1, C necator H16_ΔacoABC_ΔphaC2, and C. necator H16_ΔacoABC_ΔphaC1_ΔphaC2) with the plasmid pKRphb-alsSD were characterized for growth, acetoin production, and alsSD expression levels. a Acetoin concentration (lines + up-facing triangles) and growth (dotted lines + squares). b Expression levels of alsD and alsS normalized to gyrB after 20 h

Fig. 5

Boxplot displaying the growth coefficient for different oxygen concentrations under autotrophic conditions. The small square in the middle of the boxes indicates the average growth rate while the horizontal line indicates the growth rate’s median

Fig. 6

Autotrophic acetoin production in batch experiments. The four different strains (C. necator H16_ΔacoABC, C. necator H16_ΔacoABC_ΔphaC1, C. necator H16_ΔacoABC_ΔphaC2, and C. necator H16_ΔacoABC_ΔphaC1_ΔphaC2) with the plasmid pKRphb-alsSD were characterized for growth and acetoin production in addition to CO₂ and H₂ consumption. a Growth (dotted lines + squares) and acetoin concentration (lines + up-facing triangles). b CO₂ concentration (dotted lines + squares) and H₂ concentration (lines + up-facing triangles)

Fig. 7

Autotrophic acetoin production under continuous growth conditions. C. necator H16_ΔacoABC_ΔphaC1_ΔphaC2_pKRphb-alsSD was inoculated to an OD₆₀₀ of 2.0. The gas mixture of 80% H₂:5% CO₂:15% O₂ was applied with a flow of 4 l/h for 2 weeks. Black squares: OD₆₀₀, red triangles: acetoin