Efficient biochemical production of acetoin from carbon dioxide using Cupriavidus necator H16
Biotechnology for Biofuels volume 12, Article number: 163 (2019)
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.
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%.
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.
Biotechnology can be the key to the advent of a bioeconomy as this technology can use renewable resources for sustainable production processes. In this study, we focused on the production of acetoin using Cupriavidus necator H16 as biocatalyst. C. necator is a model organism for both polyhydroxybutyrate (PHB) production and chemolithoautotrophic growth. Its status as a model organism is also due to the availability of its complete genome sequence [1, 2] and its genetic tractability. The organism grows heterotrophically with fructose, N-acetylglucosamine, gluconate, fatty acids, and other compounds as electron donors and carbon sources using oxygen, nitrate, nitrite, or dimethyl sulfoxide (DMSO) as electron acceptors. Under chemolithoautotrophic growth conditions, C. necator can use carbon dioxide (CO2) as a carbon source, hydrogen (H2) as an electron donor, and oxygen (O2) as an electron acceptor. Due to its metabolic versatility and accessible high cell densities, C. necator has often been suggested to be a good candidate for biotechnological processes. Consequently, multiple studies present strains that produce a large variety of chemicals ranging, for example, from α-humulene (over isopropanol, methyl ketones, and isobutanol) to 3-methyl-1-butanol [3,4,5,6,7]. However, studies evaluating the efficiency of autotrophic production processes for platform chemicals are rather sparse. Przybylski et al. , for instance, presented a strain for the autotrophic production of the methacrylate precursor 2-hydroxyisobutyric acid. The strain produced the C4 compound with an efficiency of 0.03 mol per mol of CO2 consumed. Krieg and colleagues  aimed for the production of the C15 compound, α-humulene. The complexity of this substance was most probably the reason why the carbon efficiency was 30-fold lower compared to hydroxyisobutyrate production .
Although the carbon efficiency might not be high enough at this point, it was suggested that knallgas bacteria, such as C. necator, could be the key for the production of more complex end products from syngas streams . Currently, acetogens are mostly used for syngas fermentation processes. The organisms are very efficient catalysts, especially due to Wood–Ljungdahl-based CO2 fixation and use of CO2 as a carbon source. Still, acetogens growth is rather energy limited compared to C. necator. The ΔG° value of an acetogen growing with hydrogen and CO2 and producing acetate is − 111 kJ mol−1. A knallgas bacterium using hydrogen as electron donor and oxygen as electron acceptor shows a ΔG° value of − 474 kJ mol−1. Hence, the possibilities to redirect this bacterium’s central metabolism toward valuable end products will be easier. Moreover, acetogens are often difficult to genetically manipulate, which hampers the development of strains with a metabolism tailored toward the production of the desired end products. A recent study suggested that heterologous expression of a CO reductase could render C. necator capable of using CO as electron carrier to a certain extent . The latter would allow to use a number of gas streams as substrates that originate, for instance, from steel production and contain CO in varying concentrations—a production niche that was exclusively exploited using acetogens to date. A benefit and challenge for the autotrophic production using C. necator are that the organism uses the Calvin cycle for CO2 fixation. The benefit is that the cycle funnels carbon as a C3 compound into glycolysis and/or gluconeogenesis, which allows the production of complex compound branching, for instance, from the central intermediate pyruvate. The disadvantage is that the unwanted oxygenase activity of the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) can divert a substantial amount of electrons toward the reduction in oxygen instead of CO2.
The C. necator wild-type strain is naturally producing the storage polymer PHB throughout all growth conditions and nutrition modes [11, 12]. The PHB concentration can be increased via limitation of nitrogen, phosphorus, magnesium, or sulfur and a surplus of the carbon source in the medium. Three enzymes are involved in PHB production. First, two molecules of acetyl-coenzyme A are converted to one molecule 3-acetoacetyl-CoA by the β-ketothiolase PhaA. The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent acetoacetyl-CoA reductase, PhaB, catalyzes the formation of 3-hydroxy-butyryl-CoA while the PHB synthase PhaC polymerizes this compound into PHB. The genome sequence of C. necator consists of multiple putative copies of genes encoding PhaA (38 genes), PhaB (15 genes), and PhaC (two genes) .
This study focused on the production of the platform chemical acetoin. Acetoin was proposed to be one of the top 30 value-added chemicals that can be produced from biomass . Acetoin has a wide spectrum of potential applications due to its character as a platform chemical  and is also a common additive in food industry due to its buttery flavor. On average, each person consumes 2 to 4 mg acetoin per day , which results in an annual global consumption of several thousand tons of acetoin. Acetoin is currently produced using fossil feedstocks and chemical processes . Bacteria produce acetoin via two steps: (1) the first step is an acetolactate synthase (alsS)-catalyzed condensation of two pyruvate molecules to acetolactate and the concomitant release of one CO2 molecule, and (2) in the second step, acetolactate decarboxylase (alsD) activity leads to acetoin formation by releasing a second CO2 molecule. Different bacterial strains naturally produce acetoin; the most prominent producer is Bacillus subtilis. An ultraviolet (UV)-mutagenized B. subtilis strain produced more than 50 g/l acetoin from 150 g/l glucose, which correlates to an acetoin yield of 73.5%, considering that a maximum of 1 mol of acetoin can be produced from 1 mol of glucose . The application of genetically manipulated bacterial strains can increase the production yield to up to 96% (mol/mol) of the theoretical maximum under oxic conditions . Under anoxic conditions, Förster et al.  and Bursac et al.  showed production yields in minimal medium with Escherichia coli and Shewanella oneidensis of up to 90% and 86% (mol/mol), respectively. To the best of our knowledge, no study has thus far elucidated the possibility of producing acetoin under chemolithoautotrophic conditions.
Consequently, this study aimed to develop a bacterial strain for the efficient production of acetoin from CO2. In order to reach this goal, we determined the optimal oxygen concentration for lithoautotrophic growth and studied the influence of different promoter systems and deletions of the PHB synthases on the acetoin production process. In the end, we engineered a strain with superior CO2 and electron efficiencies compared to the C. necator wild-type strain.
Materials and methods
Bacterial strains and growth conditions
All bacterial strains and plasmids used in this study are listed in Additional file 1: Table S1. C. necator strains were cultivated in lysogeny broth (LB) or in minimal media (MM) 81 (DSMZ) (Additional file 1: Table S2) at 30 °C. Minimal medium was supplemented for heterotrophic growth with 10 mM fructose or acetoin or for autotrophic growth with a gas mixture of 80% H2:5% CO2:15% O2. E. coli strains were cultivated in LB at 37 °C. If necessary, kanamycin (50 µg/ml in water) or tetracycline (15 µg/ml in DMSO) was added. For promoter induction, l-arabinose (1 µM up to 3 mM) or l-rhamnose (1 µM–1.5 mM) was added to the medium. E. coli WM3064 was grown in medium containing 0.3 mM diaminopimelic acid (DAP). If necessary, the medium was supplemented with 2% agar.
All experiments with explosive gas mixtures were conducted in fume hoods in order to assure a constant discharge of the explosive gas mixtures and with explosion protection devices.
Strain and plasmid construction
DNA was amplified with Hifi Polymerase (Nippon Genetics Europe, Dueren/Germany) in preparative polymerase chain reactions (PCRs) or with MangoMix (Bioline, Luckenwalde/Germany) in diagnostic PCRs. Gel purifications of DNA products were carried out using the Wizard® SV Gel and PCR Clean-Up System (Promega, Mannheim/Germany). The Wizard®Plus SV Minipreps DNA Purification System (Promega, Mannheim/Germany) was used for plasmid isolation. Restriction enzymes were purchased from New England Biolabs (Frankfurt/Germany). Primers were ordered from Sigma-Aldrich/Merck (Darmstadt/Germany). Sequencing of constructs was conducted by Eurofins Genomics (Ebersberg/Germany) and aligned with the expected sequences using the CLC main workbench (Version 8.0, Qiagen, Aarhus/Denmark).
The plasmids for gene deletions were constructed via amplification of approximately 500 bp upstream and downstream of the target gene (primers: ΔacoABC-up: 2596 and 2581, down: 2636 and 2597; ΔphaC1-up: 2682 and 2685, down: 2684 and 2683; ΔphaC2-up: 2687 and 2689, down: 2688 and 2690, see Additional file 1: Table S3). The PCR products had overlaps to the linearized pMQ150 plasmid (EcoRI and BamHI) and were assembled using the Gibson Assembly . Afterward, the dialyzed Gibson Assembly mix was transformed in E. coli WM3064. The strain carrying the correct plasmid was used as the donor strain for conjugation. The donor and recipient strains were streaked out on LB agar plates with diaminopimelic acid (DAP). After 24 h, the cell mixture was transferred to LB agar plates with antibiotic and without DAP. After an incubation time of 2 to 3 days, the colonies were tested for plasmid integration using diagnostic PCR. For the second cross, cells were first cultivated for 24 h in liquid LB without NaCl containing 25% sucrose and were then streaked onto agar plates containing the same medium. Verification was performed via diagnostic PCR and subsequent sequencing.
The alsSD genes from B. subtilis PY79 were selected for the expression experiment. The genes were codon optimized (see Additional file 2: Sequence alsS and Additional file 3: Sequence alsD) according to the codon usage of C. necator H16  and ordered as two strings (alsSD I and alsSD II). PCR products from the strings (primers: string alsSD I: 2588 and 2657; string alsSD II: 2589 and 2658) were assembled in a two-step PCR using primers 2657 and 2658 and cloned via Gibson Assembly in plasmid pKRrha-eGFP (cut by NdeI-HF and BsrGI-HF) resulting in plasmid pKRrha-alsSD.
For the construction of promoter exchange plasmids, the plasmid pKRrha-alsSD was amplified by two inverse PCRs without amplifying rhaR and rhaS (fragment I: primers 2701 and 188; fragment II: primers 2694 and 2693). The arabinose-inducible promoter and araC were amplified from the plasmid pBAD (using primers 2697 and 2698). A 2000 bp fragment upstream of the phaCAB operon in the genome of C. necator H16 wild type was amplified to gain the hypothetical promoter of the PHB-synthesis gene cluster phaCAB (primers 2695 and 2696). The vector fragments I and II were assembled with the ara promoter, and the hypothetical PHB promoter resulted in pKRara-alsSD and pKRphb-alsSD, respectively. The plasmids were conjugated into the recipient strains as previously described.
Quantification of substrate and products
Samples were taken at different time points and analyzed with regard to the optical density (OD600) as well as the fructose and acetoin concentration. The optical density was measured in a Genesys 20 spectrophotometer (Thermo Spectronic/Thermo Fisher Scientific, Darmstadt/Germany) or an Ultrospec 10 Cell Density Meter (Biochrom/Merck, Darmstadt/Germany).
In order to determine the fructose concentration, 150 µl samples were filtered through a 0.2 µm polytetrafluoroethylene (PTFE) membrane (VWR, Darmstadt/Germany) and mixed in a 96 well microtiter plate with 15 µl 0.5 M sulfuric acid and measured via high-performance liquid chromatography ([HPLC] Dionex UltiMate 3000 HPLC; Thermo Scientific, Waltham, MA) according to Bursac et al. .
Concentrations of H2, O2, N2, and CO2 gases were measured via gas chromatography (490 Micro GC, Agilent Technologies Deutschland, Waldbronn/Germany) and analyzed using the software Agilent OpenLAB CDS (EZChrom Edition) (Agilent Technologies Deutschland, Waldbronn/Germany). The method was: (1) 5 s stabilizing time; (2) 20 s sample time; (3) 110 °C sample line and injector temperatures; (4) 50 ms injector time; (5) first column: 10 m MS5A, argon as carrier gas, 70 °C column temperature, 150 kPa column pressure; and (6) second column: 10 m PPQ, helium as carrier gas, 45 °C column temperature, 150 kPa column pressure.
Optimal inductor concentration
In order to determine the optimal inductor concentration for the inducible promoters, cells were precultured in LB. Test tubes were filled with 5 ml MM 81 containing 10 mM fructose. The starting OD600 was adjusted to 0.6, and different inductor concentrations (arabinose: 0, 1, 5, 10, 50 µM, and 0.1, 0.5, 1, 1.5, 3, 5, 10 mM; rhamnose: 0, 1, 5, 10, 50 µM and 0.1, 0.5, 1, 1.5 mM) were added to the medium. OD600 values were measured, and samples were taken for the quantification of acetoin concentrations (Additional file 4: Figures S1 and S2).
Two methods were applied to analyze PHB production: (1) photometric measurement via Nile Red staining and (2) fluorescence microscopy. For the Nile Red staining, the different deletion mutants were cultivated for 20 h. One-hundred fifty microliters of an OD600 1.0 cell culture were mixed with 50 µl Nile Red (2 µg/ml in DMSO) and incubated in darkness for 30 min at room temperature. The samples’ cell densities were measured at 600 nm, while Nile Red was quantified using excitation and emission wavelengths of 530 and 575 nm (infinite M200 Pro, Tecan, Maennedorf/Switzerland).
For microscopic imaging, 1 ml culture was harvested and resuspended in 150 µl phosphate-buffered saline (PBS) consisting of 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.7 mM KH2PO4. After an incubation with 50 µl Nile Red (2 µg/ml in DMSO) for 30 min at room temperature in the dark, 40 µl 4′,6-diamidino-2-phenylindole (DAPI) at a concentration of 10 µg/ml in water was added, and the samples were incubated for another 10 min. The cells were washed with 150 µl PBS. Samples were viewed under a Leica DM 5500B microscope (objective lens 100×: HCX PL FLUOTAR, 1.4, oil immersion; eyepiece 10×: HC PLAN s  M), and images were taken with a Leica DFC 360 FX camera and analyzed with the corresponding Leica LAS AF Lite software.
Heterotrophic acetoin production
Cells were precultured in LB. Acetoin production was achieved in 100 ml MM 81 medium containing 10 mM fructose. The starting OD600 was 0.6. Samples were taken to measure OD600, acetoin, and fructose concentrations and to perform RNA isolation in addition to subsequent real-time qPCR.
qPCR reactions were conducted in order to determine alsS and alsD mRNA levels. The housekeeping gene, gyrB, served as positive control, while a non-transcribed area was used as the negative control (Additional file 1: Table S4). RNA was isolated from pooled triplicates corresponding to a cell number of an OD600 of 1.0 (Qiagen RNeasy Mini Kit, Hilden/Germany). DNA was hydrolyzed using the Ambion DNA-free DNase Treatment and Removal Reagents kit (Thermo Fisher Scientific, Darmstadt/Germany). Five microliters of DNA-free RNA samples (20–25 ng/µl) were transcribed into cDNA (iScript Select cDNA, Bio‐Rad Laboratories, Munich, Germany). 2 µl cDNA were used as template (SsoAdvanced™ Universal SYBR® Green Supermix, Bio‐Rad Laboratories, Munich, Germany). qPCR reactions were conducted and analyzed in triplicates using the CFX96™ Real‐Time PCR Detection System (Bio‐Rad Laboratories, Munich, Germany).
Determination of the optimal oxygen concentration
The optimal oxygen concentration for growth was determined using four Biostat A fermenters (Sartorius, Goettingen/Germany) with a continuous gas supply of H2, CO2, and O2 (Additional file 4: Figure S3). The composition of the gas mixture was adjusted using the gas mixer EL-FLOW (Bronkhorst, Ruurlo/Netherlands) and the software FlowDDE and FlowView (Bronkhorst, Ruurlo/Netherlands). Different gas mixtures and different gas flows were used (Additional file 1: Table S5). The fermenters were inoculated to a starting OD600 of 0.1. The stirring speed was adjusted to 400 rpm and the temperature to 30 °C. The OD600 was measured for 10 h after which the gas concentration was altered to increase or decrease the oxygen concentration. The cells then grew in the new gas mixture for 14 h. The experiments were performed with C. necator H16 wild-type cells in 1 l of MM 81 medium without any carbon source.
Autotrophic acetoin production
Cells were precultured in LB. One-liter anoxic Schott bottles with a gas-tight lid were flushed with the autotrophic gas mixture with a flow of 6 l/h for 2.5 h. Thereafter, the gas mixture in the bottle was measured with the Micro GC. Fifty microliters of MM 81 medium with a starting OD600 of 4.5 were injected. The gas pressure was balanced with air to a pressure of 1 bar. Samples were taken for OD600, and acetoin detection using a syringe and the gas mixture was measured by Micro GC analysis. The experiments were stopped at a CO2 concentration < 0.05%.
For acetoin production with a continuous gas flow, fermenters were inoculated to an OD600 of 2.0. Every other day, antibiotic was added to ensure that the plasmid was retained by the cells. The pH was adjusted to 7.0 with 1 M NaOH. Samples were taken to determine the OD600 and acetoin concentration.
Strain development toward heterotrophic acetoin production
The aim of this study was to develop efficient heterotrophic and autotrophic production of the platform chemical acetoin. An outline of the central carbon metabolism of C. necator H16 is given in Additional file 4: Figure S4. As depicted in this figure, the strain is able to metabolize acetoin to acetaldehyde and acetyl-CoA . Hence, the production of acetoin would potentially be hampered by its concurrent consumption. Therefore, the corresponding operon for acetoin consumption encoding the genes acoABC was deleted from the genome of the organism. In order to verify a successful deletion, both strains, C. necator H16 wild type and C. necator H16_ΔacoABC, were cultivated with 10 mM fructose or 10 mM acetoin as the sole carbon source (Fig. 1). Growth of both strains with 10 mM fructose did not show a significant difference. The wild-type strain grew with 10 mM acetoin, but the growth was slower than with fructose and reached a lower final optical density (OD600) of 1.2. As expected, the deletion strain could not thrive on acetoin as a carbon and electron source.
In order to achieve successful acetoin production, a plasmid with codon-optimized versions of the alsSD genes from B. subtilis PY79 was constructed (Additional file 4: Figure S4). The genes were placed under control of three different promoters: (1) plasmid, pKRrha-alsSD, had a rhamnose-inducible promoter; (2) pKRara-alsSD, an arabinose-inducible promoter; and (3) the last plasmid, pKRphb-alsSD, had the constitutively expressed promoter of the phaCAB operon, which is responsible for polyhydroxybutyrate (PHB) production [12, 25]. The optimal inductor concentrations for the inducible promoter systems (arabinose and rhamnose) were experimentally identified as 1.5 mM arabinose (Additional file 4: Figure S1) and 1 mM rhamnose (Additional file 4: Figure S2), respectively. All acetoin production experiments were inoculated to a starting OD600 of 0.6. The strains harboring the rhamnose- and arabinose-inducible plasmids grew to a maximal OD600 of 1.0 in medium containing the respective inductor (Fig. 2). The uninduced strains C. necator H16_ΔacoABC_pKRrha-alsSD and C. necator H16_ΔacoABC_pKRara-alsSD reached a maximum OD600 of 2.3 after 6 h. The strain with the constitutively expressed alsSD genes, C. necator H16_ΔacoABC_pKRphb-alsSD, had a final OD600 of 1.8 after 8 h. Acetoin production showed an inverse behavior with respect to growth. Acetoin production of 0.3 and 0.4 mM was measured for the uninduced ΔacoABC strains carrying the pKRrha-alsSD and pKRara-alsSD plasmids, respectively. The same strains cultivated with inducer reached a final acetoin concentration of up to 8.1 mM. The constitutive expression of the alsSD genes in C. necator H16_ΔacoABC_pKRphb-alsSD led to an acetoin production of 2.55 mM. In comparison with the consumed fructose, an acetoin yield of 89.9% and 87.1% of the theoretical maximum (mol/mol) was gained with the induced strains harboring plasmids pKRrha-alsSD and pKRara-alsSD, respectively. The acetoin yield of the strain containing the alsSD genes under phaC1 promoter control was with 26.2% (mol/mol) significantly lower.
Influence of PHB synthases on acetoin production
We hypothesized that it could be possible to increase the acetoin yield of the PHB promoter system, if the two PHB synthase genes (phaC1 and phaC2) were deleted from the genome as this would render the strain incapable of producing storage polymers . In order to verify the influence of the different deletions on the PHB production, C. necator H16 wild type, C. necator H16_ΔacoABC, C. necator H16_ΔacoABC_phaC1, C. necator H16_ΔacoABC_phaC2, and C. necator H16_ΔacoABC_phaC1_phaC2 were stained with Nile Red. The deletion of phaC1 had an impact on PHB production as the ΔphaC1 and ΔphaC1_ΔphaC2 both presented an approximately 2.5-fold lower Nile Red-based emission than the strains without a deletion of phaC1 (Fig. 3a). This result is corroborated by fluorescent microscopy experiments. Only strains containing the phaC1 gene produced PHB granula under the chosen growth conditions. These results are in agreement with a study by Peplinski and colleagues. The authors discovered that PhaC1 was the only active PHB synthase and that even a constitutive expression of phaC2 could not complement the mutation .
Although the phaC1 promoter-based expression led to rather low yields in the first experiments, we repeated the heterotrophic experiments using the phaC1 and phaC2 deletion strains as the presence of the phaC genes and their gene products might affect promoter activity. Therefore, the plasmid pKRphb-alsSD was introduced in the strains with either one or two deletions in the PHB synthases in order to direct the carbon flux toward acetoin production. The strain with the best growth was C. necator H16_ΔacoABC_ΔphaC1 followed by C. necator H16_ΔacoABC_ΔphaC2 (Fig. 4a). The triple deletion mutant (C. necator H16_ΔacoABC_ΔphaC1_ΔphaC2) showed the lowest increase in OD600. Acetoin production showed the opposite order, and the triple deletion mutant produced the highest acetoin concentration with 6.98 mM.
Quantitative reverse transcription PCR (qRT-PCR) was conducted on the alsSD genes in order to find a potential correlation between expression level and acetoin yield. The quantified expression levels were normalized to the housekeeping gene gyrB for each test strain. The highest transcription levels for alsS and alsD were measured for the ΔphaC1_ΔphaC2 double deletion mutant (Fig. 4b). alsS expression did not differ significantly between the ΔphaC1 and ΔphaC2 mutants, but alsD expression was lower in the ΔphaC1 compared to ΔphaC2 mutant. Hence, the expression values corresponded well to acetoin production. The acetoin yield from the strains with the best production with a deletion of either the ΔphaC2 gene alone or ΔphaC1 and ΔphaC2 was 64.6% and 63.0% (mol/mol).
Autotrophic acetoin production
Before testing the strains’ capability for autotrophic acetoin production, we aimed to elucidate the optimal oxygen concentration for lithoautotrophic growth. The experiments were conducted in 1 l fermenters with a continuous gas flow. The reactors were inoculated with C. necator H16 wild type to a starting OD600 of 0.1. The increase in O2 concentration from 3.8 to 20% had no significant influence on the growth rate, while concentrations > 25% seemed to inhibit growth. The boxplot indicates that the average growth rate was highest between 15 and 20% O2. Nevertheless, it appears that the standard deviation in most experiments was rather high (Fig. 5).
As a result of the experiments with different oxygen concentrations, we aimed for autotrophic acetoin production with a gas mixture of 75% H2:5% CO2:18% O2:2% N2. The experiments were repeated with the pKRphb-alsSD plasmid with the three different phaC deletion strains. The individual triplicate experiments were stopped when the CO2 concentration reached values < 0.05%. As expected, the highest acetoin levels were measured with the strain C. necator H16_ΔacoABC_ΔphaC2 (11.06 mM) and C. necator H16_ΔacoABC_ΔphaC1_ΔphaC2 (13.66 mM). Acetoin production corresponded to low or no growth, indicating low anabolic CO2 consumption in both strains. While H2 and O2 were consumed by all strains with similar rates, the phaC1 deletion mutant lacked behind the others with respect to CO2 consumption. The acetoin yields for C. necator H16_ΔacoABC and C. necator H16_ΔacoABC_ΔphaC1 were low with values of 6.82% and 12.03% (mol C/mol C), respectively. For the strains with the phaC2 deletion, the acetoin yield was determined to be 90.77% and 129.31% (C. necator H16_ΔacoABC_ΔphaC2 and C. necator H16_ΔacoABC_ΔphaC1_ΔphaC2, respectively) (mol C/mol C) (Fig. 6).
The best strain for acetoin production developed in this study was C. necator H16_ΔacoABC_ΔphaC1_ΔphaC2_pKRphb-alsSD, which was used in a fermenter with a continuous supply of the autotrophic gas mixture for 2 weeks. The starting OD600 was 2.0, and this strain grew up to an OD600 of around 5.0 (Fig. 7). The acetoin concentration increased to a final value of 44 mM (3.9 g/l).
The development of strains and processes for biotechnological production of platform chemicals from CO2 is a necessity for the realization of a bioeconomy. Knallgas bacteria, such as C. necator, offer the possibility to produce more complex chemicals from CO2 as the Calvin cycle leads to the production of C3 units, and aerobic respiratory metabolism delivers a sufficient amount of energy. Hence, this study does not only describe how efficient acetoin can be produced from CO2, but furthermore, it is a proof of principle for a multitude of different production processes branching from pyruvate.
Acetoin is a potential carbon source for C. necator, and the initially conducted experiments proved the necessity to delete the acoABC cluster if acetoin is chosen to be the final product of the process. The PHB promoter system led to rather inefficient acetoin production under heterotrophic conditions compared to the rhamnose- and arabinose-inducible promoters. This situation changed after deletion of the phaC2 gene although the gene product did not seem to be involved in PHB production under the study’ experimental conditions. Nevertheless, PhaC2 seems to negatively influence PHB promoter activity. This is also corroborated by the conducted quantitative analysis of alsSD expression, which revealed that the strain with the phaC2 deletion had higher alsD transcript abundance compared to the phaC1 deletion mutant. Still, the differences in the transcription levels between the three strains were moderate, and it seems unlikely that they alone can be the reason for the observed differences. It is also unlikely that PHB production funnels too much carbon away from acetoin synthesis as the phaC2 single deletion mutant produced similar amounts of PHB compared to the wild type. Hence, the influence of phaC2 and/or its gene product on the activity of the PHB promoter warrants further investigation in future research studies.
It was surprising to observe that the developed inducer-independent expression system led to very high carbon efficiencies in the phaC2 single and phaC1_phaC2 double deletion strains. The high efficiencies corresponded well with the observed lack of growth, which suggests that CO2 was used to a minor extent for anabolic purposes. Carbon recovery of > 100% mol C/mol C for the double mutant was most likely the result of catabolism as the optical density in the medium decreased slightly. In the long-term production experiment, a phase of 2 days in which growth was limited was followed by rapid growth of the organism for 3 days and a rather static cell density until the end of the experiment. The transition from exponential growth to the stationary phase marked the only period within the experiment in which the acetoin concentration remained rather stable. Hence, the developed constitutive PHB promoter system seems to be suitable for a stable long-term production of chemicals in C. necator.
Interestingly, not only the carbon but also hydrogen efficiencies were rather high. CO2 reduction to acetoin necessitates the addition of 20 mols of electrons per mol of acetoin produced. We observed a hydrogen consumption that was only 2.3-fold higher than the theoretical maximum (Table 1). As previously described, there are only a limited number of studies that would allow a comparison of efficiencies. Przybylski et al.  gained in their study a 2-hydroxyisobutyric acid production with a carbon recovery of 0.03 mol of 2-hydroxyisobutyric per mol of CO2. Similar efficiencies were gained in the constructed ΔacoABC strain containing the pKRphb-alsSD plasmid. It was the further deletion of the phaC2 gene in this strain that led to significantly increased carbon efficiency to values of 0.23 and 0.32 for the phaC2 single deletion and the phaC1_phaC2 double mutants, respectively.
The literature provides strategies for accelerating autotrophic growth of C. necator by adjusting the expression levels of the organism’s different carboanhydrases to an optimum level . Since this accelerated growth will most likely be due to a higher intracellular CO2 concentration, it seems likely that this will also lead to an increase in the RuBisCO efficiency. Hence, our next goal will be to advance hydrogen efficiency even further by adjusting the carboanhydrase expression according to the process conditions.
Overall, this study revealed that C. necator can be used for efficient bioproduction of platform chemicals although it uses the Calvin cycle and the high energy gain of the catalyzed knallgas reaction could lead to high anabolic rates. Moreover, the presented constitutive expression system will be a step toward a very cost efficient and long-term stable production system.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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The authors would like to thank the Nagelschneider Foundation for their generous financial support in the form of a stipend for Carina Windhorst.
Carina Windhorst was funded in form of a stipend from the Nagelschneider Foundation.
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Bacterial strains and plasmids used in this study. Table S2. Composition of the minimal media 81 (DSMZ). Table S3. Primers used in this study for cloning. Table S4. Primers used in this study for qPCR. Table S5. Gas mixtures and gas flows used in this study. The indicated values were used to determine the optimal oxygen concentration.
Codon optimization of the alsS gene from Bacillus subtilis to the codon usage of C. necator H16. Shown is an alignment of the developed gene sequence against the original B. subtilis gene.
Codon optimization of the alsD gene from Bacillus subtilis to the codon usage of C. necator H16. Shown is an alignment of the developed gene sequence against the original B. subtilis gene.
Optimal concentration of arabinose as inductor for acetoin production. C. necator H16_ΔacoABC_pKRara-alsSD cells were induced with 0, 1, 5, 10, 50 µM, 0.1, 0.5, 1, 1.5, 3, 5, 10 mM arabinose. A. OD measurement. B. acetoin concentration. Figure S2. Optimal concentration of rhamnose as inductor for acetoin production. C. necator H16_ΔacoABC_pKRrha-alsSD cells were induced with 0, 1, 5, 10, 50 µM, 0.1, 0.5, 1, 1.5 mM rhamnose. A. OD measurement. B. acetoin concentration. Figure S3. Schematic illustration of the fermenter for cultivation under autotrophic conditions. The gases H2 (red), O2 (blue) and CO2 (gray) were mixed according to the desired end concentrations in the gas mixer (green). The gas passed a 0.2-µm filter and was pumped into the fermenter. A sensor (orange) was installed to measure pH and temperature. Samples were taken through a septum (yellow). The gas mixer and the fermenter were installed in a fume cabinet. Figure S4. Central carbon metabolism of C. necator. Fructose and CO2 as carbon sources and acetoin as end product are highlighted in bold. Deleted or partially deleted genes are indicated in red (15) or orange (30) arrows, respectively. Introduced genes are shown in green (13 and 14). “Phospho” and “phosphate” are abbreviated by “P”. 1Fructokinase, 2Glucose-6-P isomerase, 3Glucose-6-P 1-dehydrogenase, 46-P-gluconolactanase, 5P-gluconate dehydrogenase, 62-keto-3-deoxy-6-P-gluconate aldolase, 7Glycerinaldehyde-3-P dehydrogenase, 8P-glycerate kinase, 9P-glycerate mutase, 10Enolase, 11Pyruvate kinase, 12Pyruvate dehydrogenase, 13Acetolactate synthase, 14Acetolactatede carboxylase, 15Acetoinoxido reductase, 16Ribulose-5-P kinase, 17Ribulose-1,5-bis-P carboxylase/oxygenase, 18Acetaldehyde dehydrogenase, 19Acetyl-CoA synthetase, 20Citrate synthase, 21Aconitase, 22Isocitrate dehydrogenase, 23Ketoglutarate dehydrogenase, 24Succinyl-CoA synthetase, 25Succinic dehydrogenase, 26Fumarase, 27Malate dehydrogenase, 28Ketothiolase, 29Acetoacetyl-CoA reduktase, 30Polyhydroxybutyryl synthase, 31Polyhydroxybutyryl depolymerase and 31β-Oxidation.
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Windhorst, C., Gescher, J. Efficient biochemical production of acetoin from carbon dioxide using Cupriavidus necator H16. Biotechnol Biofuels 12, 163 (2019). https://doi.org/10.1186/s13068-019-1512-x