- Open Access
Engineering Cupriavidus necator H16 for enhanced lithoautotrophic poly(3-hydroxybutyrate) production from CO2
Microbial Cell Factories volume 21, Article number: 231 (2022)
A representative hydrogen-oxidizing bacterium Cupriavidus necator H16 has attracted much attention as hosts to recycle carbon dioxide (CO2) into a biodegradable polymer, poly(R)-3-hydroxybutyrate (PHB). Although C. necator H16 has been used as a model PHB producer, the PHB production rate from CO2 is still too low for commercialization.
Here, we engineer the carbon fixation metabolism to improve CO2 utilization and increase PHB production. We explore the possibilities to enhance the lithoautotrophic cell growth and PHB production by introducing additional copies of transcriptional regulators involved in Calvin Benson Bassham (CBB) cycle. Both cbbR and regA-overexpressing strains showed the positive phenotypes for 11% increased biomass accumulation and 28% increased PHB production. The transcriptional changes of key genes involved in CO2—fixing metabolism and PHB production were investigated.
The global transcriptional regulator RegA plays an important role in the regulation of carbon fixation and shows the possibility to improve autotrophic cell growth and PHB accumulation by increasing its expression level. This work represents another step forward in better understanding and improving the lithoautotrophic PHB production by C. necator H16.
With increasing global CO2 emissions at their highest in recent years, carbon neutrality by 2050 is the most significant mission in the world to alleviate climate change. To this end, the modern industry has reformed the current production systems to minimize greenhouse gas emissions and promote the conversion of CO2 into value added products . As industrial biotechnology has grown, the use of sustainable and industrial exhaust gas feedstocks including CO2, CO, and CH4 from various sources (e.g., steel mills, ethanol production plants, and biogases) shows to be a promising trend towards net-zero-carbon commodities [2, 3]. In order to further promote this trend, it is necessary to increase the capacity of natural organisms (e.g., plants, algae, cyanobacteria, other photo- and chemoautotrophic bacteria) for enhanced CO2 fixation. The development of synthetic biology has enabled promoting the microbial CO2 conversion into value added chemicals by engineering CO2-fixation pathways and energy-harvesting systems [3,4,5].
The Calvin-Benson-Bassham (CBB) cycle, utilizing one of the most abundant proteins on earth, the CO2 fixation enzyme ribulose-1,5- bisphosphate carboxylase/oxygenase (RuBisCO), is the most prevalent CO2 assimilation pathway widely distributed in higher plants, algae, and cyanobacteria [6, 7]. Despite its central role, RuBisCO is a notoriously inefficient enzyme that makes improving its efficiency a highly promising approach for the enhancement of CO2 fixation [7, 8]. There has been extensive effort on improving the catalytic activity of this key carbon fixation enzyme, but have resulted in limited success in cases of photoautotrophic cyanobacteria and algae [6, 9]. As well as improving the activity of RuBisCO itself, the CO2-fixation has been reinforced by regulating the expression of RuBisCO and carbon flux control enzymes (e.g., aldolase, fructose-1,6-sedoheptulose-1,7-bisphosphatase and transketolase) involved in the CBB cycle [5, 7, 10]. The regulation of cellular carbon flux can also be harnessed to reinforce CO2 assimilation by boosting product synthesis pathway and controlling transcriptional factors . Although it is difficult to identify the effective transcriptional factor, the metabolic networks can be fine-tuned at various levels by engineering and regulating transcriptional factors. In one example, the lipid production was doubled by modulating a transcriptional factor ZnCys expression of industrial microalgae Nannochloropsis gaditana while retaining its autotrophic cell growth .
Cupriavidus necator H16 (formerly known as Ralstonia eutropha) utilizing CBB cycle for CO2 fixation is a hydrogen-oxidizing chemolithotrophic bacterium capable of synthesizing poly(R)-3-hydroxybutyrate (PHB), which is used as a biodegradable plastic. This bacterium can utilize CO2 by using hydrogen and oxygen as electron donor and acceptor, respectively . C. necator H16 has been metabolically engineered to convert CO2 to PHB, ethanol, isopropanol, fatty acid, terpene, and lycopene. This suggests that the bacteria has great potential as a promising autotrophic platform strain with expanded synthetic biology tools [3, 13,14,15,16,17,18]. Assimilation of CO2 by C. necator H16 proceeds via the CBB cycle. The enzymes of the CBB cycle including CbbR, CbbL, CbbS, CbbX, CbbY, and others are encoded on two different replicons of its genome, the chromosome and mega plasmid. The polycistronic CBB expression cassette is mainly activated by the LysR-type transcriptional regulator CbbR located immediately upstream of cbb operon [19, 20]. Next to CbbR, the global transcriptional system RegA/RegB also plays a crucial role in the cbb promoter regulation . Previously, the autotrophic cell growth and PHB production improvement have been investigated by engineering RuBisCO, carbonic anhydrase, and hydrogenases [8, 21, 22]. However, studies providing a basis for metabolic engineering of C. necator H16 for enhancing CO2 fixation efficiency are still limited.
In this work, the lithoautotrophic bacterium C. necator H16 was engineered to enhance the autotrophic cell growth and PHB production by overexpressing the heterologous RuBisCO. Since the control of cbb operon expression is influenced by the regulatory network, the transcriptional regulators cbbR and regA were also overexpressed in C. necator H16. The results of the transcriptional changes of genes associated with the cellular metabolism in the recombinant C. necator H16 were investigated. Insights gained from transcriptome analysis enhances the overall understanding of autotrophic metabolism of C. necator H16 and can be used for the further strain engineering in order to improve the production efficiency of PHB and other commodities from CO2.
Results and discussion
Enhanced autotrophic cell growth of engineered C. necator strains
The CBB cycle is often limited by its low catalytic rate and some of its enzymes are more critical than others towards enhancing the efficiency of CO2 fixation. In this study, the key enzyme of CBB cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and transcriptional regulatory proteins (CbbR and RegA) were selected to be overexpressed in C. necator H16 to improve the lithoautotrophic cell growth. Overexpression plasmids with the heterologous RuBisCO genes (rbcL, rbcS and rbcX) derived from Synechocystis sp. PCC6803 or the native regulatory genes including cbbR and regA were constructed based on the pBBR1-MCS2 multiple-copy vector as listed in Fig. 1 and Table 1. The recombinant strains were subjected for autotrophic gas fermentation in minimal medium supplemented with the gas mixture (O2:H2:CO2 = 10:80:10).
As the ultimate limiting step in the CBB cycle, RuBisCO catalyzes the carboxylation reaction of ribulose-1,5-diphosphate (RuBP) and CO2 to generate 3-phosphoglycerate (3-PGA) for fixing CO2 into organic compounds, but its catalytic rate and efficiency are low . Despite its abundancy in nature, the evolution of RuBisCO into a more efficient enzyme has been constrained due to the trade-off between CO2 affinity and carboxylation rate [7, 23]. Unlike most RuBisCOs, the C. necator RubisCO seems to be evolved to retain optimal carboxylation rate in aerobic conditions with abundant competing O2 . However, its catalytic activity of carboxylation is known to be lower than those of cyanobacterial RuBisCOs . We therefore intended to improve the autotrophic cell growth of C. necator H16 by overexpressing alternative RuBisCO enzymes with more favorable kinetic parameters at first. Among the cyanobacterial RuBisCOs exhibiting high catalytic carboxylation activities, genes derived from Synechocystis sp. PCC6803, a well-studied and widely used model cyanobacterium, were chosen to be overexpressed. When RuBisCO genes (rbcL and rbcS) derived from Synechocystis sp. PCC6803 were overexpressed with the assembling chaperone gene rbcX, a higher cell growth rate was maintained compared to the control throughout 120 h (Fig. 2A). This is in line with previously reported findings that show the heterologous expression of rbcL and rbcS enhances the production of enzymatically active RuBisCO upon coexpression with rbcX . In Synechocystis sp. PCC6803, RubisCO is encoded by an operon in the order of rbcL-rbcX-rbcS. RuBisCO folding and assembly are complex processes involving chaperons that vary between species, thus making the heterologous RuBisCO expression strategy highly restricted [27, 28]. This findings of this study indicated that the assembly of a functional cyanobacterial RuBisCO in C. necator was successfully achieved with the aid of cyanobacterial rbcX. However, the overexpression of endogenous cbbLS with groES/EL chaperonin did not enhance the autotrophic cell growth (data not shown). Li et al.  also showed the feasibility of improving autotrophic cell growth rate of C. necator by overexpressing the heterologous RuBisCO enzyme derived from S. PCC7002 with assistance of endogenous GroES/EL chaperones although no significant enhancement of cell growth was observed after 96 h. Previous studies have shown that heterologous or hybrid overexpression of RuBisCOs to be positive for the enhanced carbon assimilation in many microorganisms, which include photosynthetic bacteria [6, 29,30,31], lithoautotrophic bacteria (e.g., Cupriavidus necator) , E. coli , and higher plants (e.g., tobacco) [33, 34]. Since RuBisCOs from cyanobacteria tend to have a faster carboxylation rate compared to other sources, many experiments have been conducted to improve photosynthesis of higher plants and bacteria by overexpressing cyanobacterial RuBisCOs despite its complex regulatory system. This makes the homologous/heterologous expression challenging .
While improving carbon fixation has mostly focused on enhancing the CO2 fixing enzyme RuBisCO, another promising strategy that was employed was overexpressing the transcriptional regulators including cbbR and/or regA (Fig. 2B). However, the overexpression of cbbR, the master regulator of the cbb operons, did not confer the significant improvement of autotrophic cell growth. Due to the heavy energy demands on the cell during CO2 assimilation, the expression levels of cbb gene clusters may be up- or down-regulated depending on the carbon-state of the cell [20, 35, 36]. While the overexpression of cbbR caused a minor improvement in cell growth, both regA only and cbbR/regA overexpressions significantly benefited the autotrophic cell growth of C. necator. These data demonstrated that RegA plays an important role in the regulation of carbon fixation and shows the possibility to increase autotrophic cell growth and biomass accumulation by increasing the expression level of RegA.
Increased PHB production by co-overexpressing the transcriptional regulators RegA and CbbR
The above studies demonstrated that overexpressing the cyanobacterial RuBisCO or transcriptional regulator regA resulted in the increased autotrophic cell growth. To further investigate their potential, the engineered strains were cultured under nitrogen-limited condition, allowing the question whether the increased CO2 fixation resulting from RuBisCO or regA overexpression can be utilized to enhance the accumulation of target product PHB to be addressed (Fig. 3). The PHB content of the cbbR/regA overexpressed strain increased from 6.3 (at 24 h) to 27.3% (at 168 h) when the cbbR/regA was induced by adding arabinose at 24 h (Additional file 1: Fig. S1). Compared to the control, the cbbR/regA and regA overexpressed strains exhibited comparable cell growth, but this was not correlated to the improved PHB production when the initial OD600 was 0.2 (Fig. 3A and B). Moreover, the PHB content of rbcLSX-overexpressing strain decreased by 42%. The level of nitrogen limitation may not have been optimal for boosting PHB accumulation at the initial OD600 of 0.2. For this reason, a higher initial cell density (OD600 of 2) was used in the following autotrophic fermentation experiments which allowed nitrogen deprivation to be accelerated. As shown in Fig. 3B and D, both regA and cbbR/regA-overexpressing strains with the initial OD600 of 2 showed the positive phenotypes for biomass accumulation as well as PHB production. Cells grown under this condition showed about 11% and 28% increased biomass accumulation and PHB titer, respectively. The PHB content (% of dry cell weight) also increased from 49 to 58%. The enhanced PHB production might be resulted from the re-distribution of carbon flux towards PHB accumulation rather than cell growth under nitrogen-deficient conditions.
However, this phenomenon did not appear to be true for rbcLXS recombinant strain. The strain with the heterologous RuBisCO overexpression was found to produce 16% less PHB than the control strain, implying that an increase in cell growth did not lead to a corresponding increase in carbon-based product formation. When the carbon fixation was enhanced by improving RuBisCO, the strain might channel more carbon fluxes towards cell growth, instead of towards PHB production. This result was in contrast to the previous studies that showed faster cell growth with increased production of target chemicals in many photosynthetic cyanobacteria overexpressing RuBisCO. For example, the increased isobutyraldehyde and fatty acid in cyanobacterial hosts with additional RuBisCO genes were reported [37, 38]. Regarding PHB production using C. necator H16, overexpressing regA showed to benefit both autotrophic cell growth and PHB production in the present study. Along with CbbR, the transcription of cbb operons additionally involves global transcription regulation system composed of RegA and RegB . While the cbb operon transcription regulation by CbbR is influenced by the carbon state of the cell (e.g., phosphoenolpyruvate (PEP)), RegA interacts with CbbR and finely tunes transcriptional control scenario in response to the redox state of the cell . The RegA/RegB system has an important role in the transcription of proteins involved in the control of energy-utilizing and energy-generating processes such as carbon fixation, nitrogen fixation, hydrogen utilization, respiration, electron transport and denitrification . The RegA/RegB two-component system imposes additional layers of redox control over the energetically costly process of CO2 assimilation and its involvement in the cbbR and cbb operon control is well identified in Rhodobacter capsulatus and R. sphaeroides [39, 40]. To further investigate the effect of cbbR and regA co-overexpression on the autotrophic cell metabolism of C. necator H16, a global transcriptional analysis was performed in the next section.
Global transcriptional profiling of cbbR/regA overexpressed C. necator H16
Genome-wide transcriptional analysis was performed using RNA sequencing to identify genes that are differentially expressed in response to the transcriptional regulatory gene (cbbR and regA) overexpression. We analyzed transcripts of the mid-exponential phase of the autotrophically grown cells under nitrogen-limited condition with the initial OD of 2. A total of 951 genes exhibited > twofold changes in expression (p < 0.05) in the engineered strain when compared to those of the control strain. Of these 951 genes, roughly half were observed to be upregulated.
Table 2 and Fig. 4 show the differentially expressed genes categorized according to CBB, tricarboxylic acid (TCA), and Entner-Doudoroff (ED) pathways. Since regA and cbbR were overexpressed in the engineered strain, those genes were significantly upregulated. Although regB as the component of regulatory system regA/regB was not overexpressed, a 4.3-fold increase of its expression level was also detected. Since RegB, a membrane associated histidine sensor kinase, phosphorylates its cognate response regulator RegA to stimulate the binding of CbbR to the cbb promoter region to regulate the transcription of cbb operon, its expression might be enhanced as well [20, 41]. By overexpressing the regulatory proteins, genes involved in CBB and TCA cycle were observed to be generally up-regulated in the engineered strain. Most of the enzymes required for the CBB cycle are encoded in the cbb operon, present in both the chromosomal and plasmid-borne clusters in C. necator H16 while the regulatory cbbR gene forms a monocistronic operon within the chromosomal cbb cluster . As shown in Table 1, large and small subunits of RuBisCO (cbbL and cbbS) of the chromosomal and plasmid-borne clusters were > twofold up-regulated while the RuBisCO accessory protein cbbX and cbbY located immediately downstream of the chromosomal RuBisCO gene cbbLS increased by 3.6-fold. The upregulated genes involved in the CBB cycle might be affected by RegA/RegB in combination with CbbR which play a crucial role in the transcriptional controls of both chromosomal and plasmid-borne cbb promoters. Among the TCA cycle related genes, citrate synthase encoding genes were highly up-regulated by 22–58.3 folds. Pyruvate kinase gene, pyk3, catalyzing the conversion of phosphoenolpyruvate (PEP) to pyruvate with the production of ATP was also upregulated 18-fold.
Since pyruvate and acetyl-CoA are key to the PHB synthesis from carbon dioxide in C. necator H16, genes related to those metabolisms were therefore analyzed. As shown in Fig. 2 and Table 3, the main PHB production gene cluster phaCAB except phaC1 did appear to exhibit changes in their expression levels. Although the expression level of phaC2 was 1.6-fold up-regulated, it might not directly affect the PHB synthesis since it has no activity. In particular, ß-ketothiolase (phaA1) involved in the first step of PHB synthesis condensing two moles of acetyl-CoA to acetoacetyl-CoA, was 2.2-fold up-regulated. Furthermore, phosphate acetyltransferase encoding genes converting acetyl-CoA to acetate such as pta1 (H16_B1631), ackA (H16_B1630) and ackA2 (H16_A0670) were observed to be down-regulated. These findings indicate that there is a change in the flux of acetyl-CoA in the cell, redirecting the acetyl-CoA into the cell growth and PHB synthesis, since the precursor of PHB is produced using two molecules of acetyl-CoA. A number of studies have revealed that the PHB production is highly dependent on the intracellular availability of acetyl-CoA [42,43,44]. Also, PHB biosynthesis in C. necator H16 could possibly be enhanced through the up-regulation of NADPH generation-related zwf gene encoding glucose-6-phosphate dehydrogenase (G6PDH) .
The expression level of carbonic anhydrase, can, which catalyzes the interconversion between carbon dioxide and bicarbonate (CO2 + H2O ↔ HCO3− + H+) was found to be increased 1.8-fold (Additional file 1: Table S1). In a previous study, the can gene-overexpressed C. necator strain revealed a 1.5-fold increase in PHB accumulation . While cyanobacteria use carboxysome as a unique CO2 concentrating mechanism to enhance its fixation efficiency, C. necator lacking this system expresses four carbonic anhydrases [21, 22]. In addition to the key CBB cycle enzymes, carbonic anhydrase is of great importance to maximize CO2 concentration near RuBisCO in autotrophic metabolism . Since C. necator H16 is able to fix CO2 through CBB cycle using hydrogen as the energy source, the transcriptional changes of hydrogenases were also investigated. However, the expression levels of genes encoding membrane-bound [NiFe] hydrogenases (hoxG and hoxK) and soluble hydrogenases (hoxF, hoxU, hoxY, and hoxI) except hoxH (2.5-fold down-regulated), were not significantly changed.
The transcription levels of majority genes encoding flagella were also down-regulated (Additional file 1: Table S1). The gene fliC, the main structural protein of bacterial flagella, was downregulated 5 folds and the changes in flagellations at each growth phase have been reported. Flagellation of cells is stagnated in the stationary phase allowing PHB accumulation while cells appear to be strongly flagellated in the early exponential phase . In the present study, the down-regulation of flagella gene clusters in the engineered strain may contribute to enhancing the PHB accumulation. Previous studies have shown that disrupting gene clusters relevant to outer membrane including flagella and pili benefited the PHB accumulation [47, 48]. Since the biosynthesis and assembly of various flagella and pili components requires high energetic cost, their biosynthesis might create a substantial metabolic burden . The decrease in flagella biosynthesis could help in saving energy and improve autotrophic cell growth and PHB accumulation in C. necator H16. Disrupting gene clusters relevant to flagella or pili may be an efficient strategy to improve cell performance to accumulate PHB. Altogether, these findings provide a reference for the construction of metabolic engineering C. necator H16 towards high efficiency for PHB production under lithoautotrophic cultivation conditions.
In this study, the overexpression of cbbR and regA in C. necator H16 enabled the improvement in its autotrophic cell growth and PHB accumulation. The global transcriptional regulator RegA seems to play an important role in the regulation of carbon fixation and shows possibility of enhancing the industrial applicability of C. necator H16. The comparative transcriptome provides references for the enhancement of PHB synthesis using carbon dioxide under autotrophic conditions. In summary, this study represents another step forward to better understanding the lithoautotrophic PHB production by C. necator H16.
Strains and plasmids
Cupriavidus necator H16 (KCTC 22469; Korean Collection for Type Cultures, Daejeon, Korea) was used in this study. All strains used in this study are listed in Table 1.
Cupriavidus necator H16 was routinely cultivated at 30 °C and 200 rpm. To grow C. necator strains autotrophically, the glycerol stock was first inoculated in a rich Luria–Bertani (LB) broth for 24 h and then pre-cultured in a 250 ml flask containing a minimal media with 10 g/L fructose for 24 h. Cultures were centrifuged at 4,200 rpm for 10 min, and the harvested cell was washed twice with the minimal medium. The final composition of the minimal medium was 6.74 g/L Na2HPO4·7H2O, 1.5 g/L KH2PO4, 1.0 g/L (NH4)2SO4, 80 mg/L MgSO4·7H2O, 1 mg/L CaSO4·2H2O, 0.56 mg/L NiSO4·7H2O, 0.4 mg/L ferric citrate, and 200 mg/L NaHCO3.
For gas fermentation, cultures were diluted into 20 mL minimal medium in the serum bottle to an OD600 of ~ 0.2, and then a total 150 kPa of mixture gas [(O2:H2:CO2 = 10:80:10); Airkorea corporation, South Korea] was pressurized into the headspace of the serum bottle (20 mL of minimal medium in 157 mL of serum bottle) every 24 h. For the growth of engineered strain, 200 μg/mL kanamycin was included in the plates or media to maintain the pBBR1 plasmid. Escherichia coli DH10β employed for DNA manipulation was cultured at 37 °C in LB medium supplemented with 50 μg/mL kanamycin.
All plasmids used in this study are listed in Table 1. The vector pBBR1MCS2 was a gift from Kenneth Peterson (Addgene plasmid #85168) . The homologous genes (regA and cbbR) were amplified from the wild type C. necator H16 genome. The heterologous genes from Synechocystis sp. PCC6803 (rbcL, rbcS, rbcX) were codon-optimized according to the codon usage of C. necator H16 using Gene designer (Atum, California, USA) and synthesized by IDT KOREA. All designated primers used in this study are listed in Additional file 1: Table S2. All plasmid constructions were performed in E. coli DH10β and transformed into C. necator H16 using the electroporation method. Plasmid isolation and DNA purification were carried out using Mini exprep plasmid SV (Geneall, Korea) and QIAquick gel extraction kit (Qiagen, Germany), respectively.
Quantification of PHB
Quantification of PHB was performed according to the modified method of Law and Slepecky (1961) . The cultures were centrifuged for 10 min at 4200 rpm and washed twice with deionized water. Next, the pellets were frozen in – 80 °C and dried using a freeze dryer (Operon, Gimpo, Korea). After transferring to a 1.5 mL microcentrifuge tube, 0.5 mL of 95% H2SO4 (Junsei Chemical, Tokyo, Japan) was added to the pellet and vortexed. The tubes were incubated at 95 °C for 1 h. Finally, the samples were diluted to 20–50 times and analyzed by high pressure liquid chromatography (Agilent technology 1260 Infinity, CA, USA) with HIPLEX-H column (300 × 7.7 mm, Agilent technology, CA, USA) using UV/Vis detector and the 5 mM H2SO4 was used as mobile phase at 0.6 mL/min.
For transcriptomic comparison of the control and engineered strains, RNA‐sequencing analyses were performed using tools from the commercial RNA‐Seq service Ebiogen, Inc. After 72 h of gas fermentation under nitrogen-limited conditions with the initial optical density of 2, the bacterial cells were harvested by centrifugation at 4200 rpm and 4 °C for 10 min. Total RNA was isolated with Trizol reagent (Invitrogen) according to the manufacturer's instructions. The purity and integrity of each total RNA sample were assessed according to the 28S/18S ratio and RNA integrity number measured on the 2100 Bioanalyzer system (Agilent Technologies). The cDNA library was generated using the Clontech SMARTer Stranded RNA‐Seq kit (Clontech). High‐throughput sequencing was performed on an Illumina HiSeq 2500 system (Illumina, Inc).
Availability of data and materials
All data for this study are included in this published article and its additional file.
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This research was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean government (MSIT) (No. RS-2022-00156236). The authors also appreciate the additional support provided by the Korea Institute of Science and Technology (KIST) Institutional Programs (2E31853 and 2E31833).
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Additional file 1
: Table S1. Transcriptional changes of genes involved in metabolism of cbbR and regA-overexpressing strain. Table S2. Primers used in this work. Figure S1. PHB accumulation of the control and cbbR/regA overexpressed strains with the different initial optical densities of 0.2 and 2 under nitrogen-limited conditions (0.2 g/L of (NH4)2SO4) at 24 h and 168 h during the autotrophic culture. The gene of interest was induced by adding 0.2% (w/v) of l-arabinose at 24 h of the autotrophic culture. The C. necator H16 strain harboring pBAD empty vector was used as a control. The data represent the means of duplicate or triplicate experiments.
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Kim, S., Jang, Y.J., Gong, G. et al. Engineering Cupriavidus necator H16 for enhanced lithoautotrophic poly(3-hydroxybutyrate) production from CO2. Microb Cell Fact 21, 231 (2022). https://doi.org/10.1186/s12934-022-01962-7
- Cupriavidus necator H16
- Calvin Benson Bassham (CBB) cycle
- Transcriptional regulator
- Carbon dioxide fixation
- Lithoautotrophic culture
- Polyhydroxybutyrate (PHB)