Functional genetic elements for controlling gene expression in Cupriavidus necator H16

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements such as constitutive and inducible promoters, as well as ribosome binding sites (RBSs), are required. In this study, we design, build and test promoters and RBSs for controlling gene expression in the model lithoautotroph Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within more than 700-fold dynamic range is compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-L-arabinose and RhaRS/PrhaBAD-L-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems are evaluated. By supplying different concentrations of inducers, over a 1000-fold range of gene expression levels is achieved. Application of inducible systems for controlling expression of isoprene synthase gene ispS leads to isoprene yields that exhibit a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements such as mRNA stem-loop structure and A/U- rich sequence on the gene expression is also evaluated. A second-order polynomial relationship is observed between the RBS activities and isoprene yields. This study presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necator

Functional genetic elements for controlling gene expression in Cupriavidus necator H16

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements such as constitutive and inducible promoters, as well as ribosome binding sites (RBSs), are required. In this study, we design, build and test promoters and RBSs for controlling gene expression in the model lithoautotroph Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within more than 700-fold dynamic range is compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-L-arabinose and RhaRS/PrhaBAD-L-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems are evaluated. By supplying different concentrations of inducers, over a 1000-fold range of gene expression levels is achieved. Application of inducible systems for controlling expression of isoprene synthase gene ispS leads to isoprene yields that exhibit a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements such as mRNA stem-loop structure and A/U- rich sequence on the gene expression is also evaluated. A second-order polynomial relationship is observed between the RBS activities and isoprene yields. This study presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necator

13C-assisted metabolic flux analysis to investigate heterotrophic and mixotrophic metabolism in Cupriavidus necator H16

Introduction. Cupriavidus necator H16 is a gram-negative bacterium, capable of lithoautotrophic growth by utilizing hydrogen as an energy source and fixing carbon dioxide (CO2) through Calvin-Benson-Bassham (CBB) cycle. The potential to utilize synthesis gas (Syngas) and the prospects of rerouting carbon from polyhydroxybutyrate synthesis to value-added compounds makes C. necator an excellent chassis for industrial application. Objectives. In the context of lack of sufficient quantitative information of the metabolic pathways and to advance in rational metabolic engineering for optimized product synthesis in C. necator H16, we carried out a metabolic flux analysis based on steady-state 13C-labelling. Methods. In this study, steady-state carbon labelling experiments, using either D-[1-13C]fructose or [1,2-13C]glycerol, were undertaken to investigate the carbon flux through the central carbon metabolism in C. necator H16 under heterotrophic and mixotrophic growth conditions, respectively. Results. We found that the CBB cycle is active even under heterotrophic condition, and growth is indeed mixotrophic. While Entner-Doudoroff (ED) pathway is shown to be the major route for sugar degradation, tricarboxylic acid (TCA) cycle is highly active in mixotrophic condition. Enhanced flux is observed in reductive pentose phosphate pathway (redPPP) under the mixotrophic condition to supplement the precursor requirement for CBB cycle. The flux distribution was compared to the mRNA abundance of genes encoding enzymes involved in key enzymatic reactions of the central carbon metabolism. Conclusion. This study leads the way to establishing 13C-based quantitative fluxomics for rational pathway engineering in C. necator H16

Diurnal rhythm of a unicellular diazotrophic cyanobacterium under mixotrophic conditions and elevated carbon dioxide

Mixotrophic cultivation of cyanobacteria in wastewaters with flue gas sparging has the potential to simultaneously sequester carbon content from gaseous and aqueous streams and convert to biomass and biofuels. Therefore, it was of interest to study the effect of mixotrophy and elevated CO2 on metabolism, morphology and rhythm of gene expression under diurnal cycles. We chose a diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 as a model, which is a known hydrogen producer with robust circadian rhythm. Cyanothece 51142 grows faster with nitrate and/or an additional carbon source in the growth medium and at 3 % CO2. Intracellular glycogen contents undergo diurnal oscillations with greater accumulation under mixotrophy. While glycogen is exhausted by midnight under autotrophic conditions, significant amounts remain unutilized accompanied by a prolonged upregulation of nifH gene under mixotrophy. This possibly supports nitrogen fixation for longer periods thereby leading to better growth. To gain insights into the influence of mixotrophy and elevated CO2 on circadian rhythm, transcription of core clock genes kaiA, kaiB1 and kaiC1, the input pathway, cikA, output pathway, rpaA and representatives of key metabolic pathways was analyzed. Clock genes’ transcripts were lower under mixotrophy suggesting a dampening effect exerted by an external carbon source such as glycerol. Nevertheless, the genes of the clock and important metabolic pathways show diurnal oscillations in expression under mixotrophic and autotrophic growth at ambient and elevated CO2, respectively. Taken together, the results indicate segregation of light and dark associated reactions even under mixotrophy and provide important insights for further applications

Rhythmic oscillations in KaiC1 phosphorylation and ATP/ADP ratio in nitrogen-fixing cyanobacterium <i>Cyanothece</i> sp. ATCC 51142

<p>Cyanobacterial circadian clock composed of the Kai oscillator has been unraveled in the model strain <i>Synechococcus elongatus</i> PCC 7942. Recent studies with nitrogen-fixing <i>Cyanothece</i> sp. ATCC 51142 show rhythmic oscillations in the cellular program even in continuous light albeit with a cycle time of ~11 h. In the present study, we investigate correlation between cellular rhythms, KaiC1 phosphorylation cycle, ATP/ADP ratio, and the redox state of plastoquinone pool in <i>Cyanothece</i>. KaiC1 phosphorylation cycle of <i>Cyanothece</i> was similar to that of <i>Synechococcus</i> under diurnal cycles. However, under continuous light, the cycle time was shorter (11 h), in agreement with physiological and gene expression studies. Interestingly, the ATP/ADP ratio also oscillates with an 11 h period, peaking concomitantly with the respiratory burst. We propose a mathematical model with C/N ratio as a probable signal regulating the clock in continuous light and emphasize the existence of a single timing mechanism regardless of the cycle time.</p

Functional genetic elements for controlling gene expression in Cupriavidus necator H16

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements such as constitutive and inducible promoters, as well as ribosome binding sites (RBSs), are required. In this study, we design, build and test promoters and RBSs for controlling gene expression in the model lithoautotroph Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within more than 700-fold dynamic range is compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-L-arabinose and RhaRS/PrhaBAD-L-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems are evaluated. By supplying different concentrations of inducers, over a 1000-fold range of gene expression levels is achieved. Application of inducible systems for controlling expression of isoprene synthase gene ispS leads to isoprene yields that exhibit a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements such as mRNA stem-loop structure and A/U- rich sequence on the gene expression is also evaluated. A second-order polynomial relationship is observed between the RBS activities and isoprene yields. This study presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necator