AraC-Based Biosensor for the Detection of Isoprene in E. coli.

Isoprene is a valuable platform chemical, which is produced by engineered microorganisms, albeit in low quantities. The amount of isoprene produced is usually measured by gas chromatography, which can be time-consuming and expensive. Alternatively, biosensors have evolved as a powerful tool for real-time high-throughput screening and monitoring of product synthesis. The AraC-pBAD-inducible system has been widely studied, evolved, and engineered to develop biosensors for small molecules. In our preliminary studies, the AraC-pBAD system was mildly induced at higher isoprene concentrations when arabinose was also available. Hence, in the present study, we designed and constructed a synthetic biosensor based on the AraC-pBAD system, wherein the ligand-binding domain of AraC was replaced with IsoA. On introducing this chimeric AraC-IsoA (AcIa) transcription factor with the native PBAD promoter system regulating rfp gene expression, fluorescence output was observed only when wild-type Escherichia coli cells were induced with both isoprene and arabinose. The biosensor sensitivity and dynamic range were further enhanced by removing operator sequences and by substituting the native promoter (PAraC) with the strong tac promoter (Ptac). The chimeric sensor did not work in AraC knockout strains; however, functionality was restored by reintroducing AraC. Hence, AraC is essential for the functioning of our biosensor, while AcIa provides enhanced sensitivity and specificity for isoprene. However, insights into how AraC-AcIa interacts and the possible working mechanism remain to be explored. This study provides a prototype for developing chimeric AraC-based biosensors with proteins devoid of known dimerizing domains and opens a new avenue for further study and exploration.

Quantitative insight into the metabolism of isoprene-producing Synechocystis sp. PCC 6803 using steady state 13C-MFA.

Cyanobacteria are photosynthetic bacteria, widely studied for the conversion of atmospheric carbon dioxide to useful platform chemicals. Isoprene is one such industrially important chemical, primarily used for production of synthetic rubber and biofuels. Synechocystis sp. PCC 6803, a genetically amenable cyanobacterium, produces isoprene on heterologous expression of isoprene synthase gene, albeit in very low quantities. Rationalized metabolic engineering to re-route the carbon flux for enhanced isoprene production requires in-dept knowledge of the metabolic flux distribution in the cell. Hence, in the present study, we undertook steady state 13C-metabolic flux analysis of glucose-tolerant wild-type (GTN) and isoprene-producing recombinant (ISP) Synechocystis sp. to understand and compare the carbon flux distribution in the two strains. The R-values for amino acids, flux analysis predictions and gene expression profiles emphasized predominance of Calvin cycle and glycogen metabolism in GTN. Alternatively, flux analysis predicted higher activity of the anaplerotic pathway through phosphoenolpyruvate carboxylase and malic enzyme in ISP. The striking difference in the Calvin cycle, glycogen metabolism and anaplerotic pathway activity in GTN and ISP suggested a possible role of energy molecules (ATP and NADPH) in regulating the carbon flux distribution in GTN and ISP. This claim was further supported by the transcript level of selected genes of the electron transport chain. This study provides the first quantitative insight into the carbon flux distribution of isoprene-producing cyanobacterium, information critical for developing Synechocystis sp. as a single cell factory for isoprenoid/terpenoid production.

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 designed, built, and tested 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 a >700-fold dynamic range was compared to the native P phaC , with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/P araBAD -l-arabinose and RhaRS/P rhaBAD -l-rhamnose) and negatively (AcuR/P acuRI -acrylate and CymR/P cmt -cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase gene ispS led to isoprene yields that exhibited 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 gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necatorIMPORTANCE This report provides tools for robust and predictable control of gene expression in the model lithoautotroph C. necator H16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression in C. necator H16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/P araBAD -l-arabinose and RhaRS/P rhaBAD -l-rhamnose) and two negatively (AcuR/P acuRI -acrylate and CymR/P cmt -cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.

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.

Rhythmic and sustained oscillations in metabolism and gene expression of Cyanothece sp. ATCC 51142 under constant light.

Cyanobacteria, a group of photosynthetic prokaryotes, oscillate between day and night time metabolisms with concomitant oscillations in gene expression in response to light/dark cycles (LD). The oscillations in gene expression have been shown to sustain in constant light (LL) with a free running period of 24 h in a model cyanobacterium Synechococcus elongatus PCC 7942. However, equivalent oscillations in metabolism are not reported under LL in this non-nitrogen fixing cyanobacterium. Here we focus on Cyanothece sp. ATCC 51142, a unicellular, nitrogen-fixing cyanobacterium known to temporally separate the processes of oxygenic photosynthesis and oxygen-sensitive nitrogen fixation. In a recent report, metabolism of Cyanothece 51142 has been shown to oscillate between photosynthetic and respiratory phases under LL with free running periods that are temperature dependent but significantly shorter than the circadian period. Further, the oscillations shift to circadian pattern at moderate cell densities that are concomitant with slower growth rates. Here we take this understanding forward and demonstrate that the ultradian rhythm under LL sustains at much higher cell densities when grown under turbulent regimes that simulate flashing light effect. Our results suggest that the ultradian rhythm in metabolism may be needed to support higher carbon and nitrogen requirements of rapidly growing cells under LL. With a comprehensive Real time PCR based gene expression analysis we account for key regulatory interactions and demonstrate the interplay between clock genes and the genes of key metabolic pathways. Further, we observe that several genes that peak at dusk in Synechococcus peak at dawn in Cyanothece and vice versa. The circadian rhythm of this organism appears to be more robust with peaking of genes in anticipation of the ensuing photosynthetic and respiratory metabolic phases.

Model based optimization of high cell density cultivation of nitrogen-fixing cyanobacteria.

In the present study, fed-batch cultivation of Cyanothece sp. ATCC 51142, a known hydrogen producer, was optimized for maximizing biomass production. Decline in growth of this organism in dense cultures was attributed to increased substrate consumption for maintenance and respiration, and photolimitation due to self shading. A model incorporating these aspects was developed, and by using control vector parameterization (CVP), substrate feeding recipe was optimized to achieve 12-fold higher biomass concentration. The optimization results were verified experimentally on shake flask and bioreactor. The latter resulted in greater exponential growth rate possibly by overcoming photolimitation by simulating flashing light effect. Such a strategy can be readily applied for mixotrophic cultivation of cyanobacterial cultures in the first stage followed by photoautotrophic growth at the production stage.

Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions.

Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

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.