FLIP sensors have been developed for a wide range of ions, amino acids and mono- and disaccharides and have been used to address important biological problems, such as the release of glutamate from neurons [10, 11], the exchange of tryptophan against kynurenine in cancer cells  or the transport of glucose across the endoplasmic reticulum membrane . Here it has been shown that the nanosensor technology also has potential applications for fermentation processes in the food, pharmaceutical and biofuel industries, since the FRET sensors robustly and quantitatively report changes in intracellular ligand concentrations in microorganisms. In this study, both a novel arabinose FRET nanosensor as well as an improved maltose nanosensor were used to quantify intracellular ligand concentrations in bacterial cell cultures in a 96-well microplate fluorimeter. These results demonstrate that, after addition of sugar, the rate of uptake exceeds the rate of metabolism in E. coli. Moreover, by using an injection module attached to the fluorimeter, it was possible to determine accumulation rates after the addition of the metabolite. Finally, the new Gateway-based vector system used here to construct the arabinose sensor will help to accelerate the development of novel sensors for other relevant compounds including xylose, one of the major cell wall-derived pentoses.
The apparent cytosolic steady-state levels of both arabinose and maltose were 20- to 200-fold lower in the cytosol compared with the extracellular medium, indicating the presence of a highly active metabolic flux that exceeds uptake rates even at high external levels. It should be noted that the maltose sensor also recognizes maltooligosaccharides ; thus, the actual maltose levels in the cytosol may be even lower if maltose is rapidly converted into maltooligosaccharides. At low maltose concentrations, maltose is imported by the maltoporin LamB across the outer membrane, and then bound by the maltose PBP, which delivers maltose to the ATP-dependent ABC transporter . However, at high maltose levels, this system with an affinity of around 1 μM (LamB) is not essential since the growth of lamB mutants is unaffected at high maltose concentrations . Consistent with the responses observed here, low-affinity/high-capacity uptake systems predominate at 1 to 100 mM maltose (stationary BL21-Gold(DE3) cells, kept in LB at 4°C overnight to allow full folding of the fluorophores, then washed and incubated in M9 medium for 1 hour to ensure low endogenous maltose levels). Given the successful use of the sensors in other systems, it will be possible to use physiological conditions; moreover, this system can be used to identify the nature of the low-affinity transport system.
The carbohydrate polymer loading in lignocellulosic biomass feedstock saccharification is typically at least 10%, resulting in monosaccharide concentrations up to several 100 mM, which is higher than encountered by fermentative organisms in nature and might result in increased carbon flux into undesired pathways . In addition, different organisms have a preference for fermenting one monosaccharide over another resulting in inefficient use of the feedstock substrate . Nanosensors might aid in identifying conditions of metabolite accumulation as a result of obstruction in, or repression of, metabolic pathways. An absence of, or decrease in, intracellular metabolite levels would indicate a lack of external supplies caused for instance, by the inhibition of uptake systems.
In this study, a sensor for monitoring intracellular levels of the pentose arabinose has been described, which was constructed by sandwiching the arabinose-binding protein gene of E. coli between the fluorophore genes in the Gateway destination vector pGWF1. The other main pentose present in cell walls of biofuel feedstocks such as corn fiber is xylose . Given the success in generating FRET sensors for ribose, arabinose, glucose, maltose and sucrose using this concept [9, 19–21], analogously, xylose sensors may be constructed using identified xylose-binding proteins as sensing domains [22, 23]. The developed Gateway-based cloning system enables rapid fusion of new recognition elements into a vector containing flanking fluorescent proteins. In addition, the linker deletion strategy used for optimization of the maltose sensor can be applied to metabolite sensors with high signal-to-noise ratios such as those shown here for the arabinose and unmodified maltose sensors. Given the wide spectrum of naturally available scaffolds that can be used for constructing additional FRET sensors relevant for metabolite analysis in relation to biofuels, the Gateway system and linker optimization strategy promise the rapid development of a suite of efficient FRET sensors for applications in bioengineering.
Furthermore, the high dynamic range of the responses, specifically shown in Figure 5A for maltose accumulation in E. coli make them suitable for use in high-throughput screens in, for instance, strain optimization. The technology is simple and fast to use and complements flux analysis by alternative methods, such as isotope pulse labeling combined with mass-spectroscopy . The data shown here demonstrate that FRET nanosensors can be used in prokaryotes and as such, have potential applications for the development of strains for the production of biofuels. The analysis of metabolite flux using an array of sensors will be valuable for ongoing efforts in kinetic modeling of carbohydrate fermentation in biofuel-producing organisms such as Zymomonas mobilis .