The microbial degradation of plant-derived compounds under salinity stress remains largely underexplored. The pretreatment of lignocellulose material, which is often needed to improve the production of lignocellulose monomers, leads to high salt levels, generating a saline environment that raises technical considerations that influence subsequent downstream processes. Here, we constructed halotolerant lignocellulose degrading microbial consortia by enriching a salt marsh soil microbiome on a recalcitrant carbon and energy source, i.e., wheat straw. The consortia were obtained after six cycles of growth on fresh substrate (adaptation phase), which was followed by four cycles on pre-digested (highly-recalcitrant) substrate (stabilization phase). The data indicated that typical salt-tolerant bacteria made up a large part of the selected consortia. These were “trained” to progressively perform better on fresh substrate, but a shift was observed when highly recalcitrant substrate was used. The most dominant bacteria in the consortia were Joostella marina, Flavobacterium beibuense, Algoriphagus ratkowskyi, Pseudomonas putida, and Halomonas meridiana. Interestingly, fungi were sparsely present and negatively affected by the change in the substrate composition. Sarocladium strictum was the single fungal strain recovered at the end of the adaptation phase, whereas it was deselected by the presence of recalcitrant substrate. Consortia selected in the latter substrate presented higher cellulose and lignin degradation than consortia selected on fresh substrate, indicating a specialization in transforming the recalcitrant regions of the substrate. Moreover, our results indicate that bacteria have a prime role in the degradation of recalcitrant lignocellulose under saline conditions, as compared to fungi. The final consortia constitute an interesting source of lignocellulolytic haloenzymes that can be used to increase the efficiency of the degradation process, while decreasing the associated costs.Electronic supplementary materialThe online version of this article (10.1007/s00253-017-8714-6) contains supplementary material, which is available to authorized users.

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single‐cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell‐intrinsic correlation between glycolytic flux and levels of fructose‐1,6‐bisphosphate (FBP), we transplanted the B. subtilis FBP‐binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

Metabolic heterogeneity between individual cells of a population harbors offers significantchallenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence requires tools to zoom into metabolism of individual cells.While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e. the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells, by drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), and by transplanting the B. subtilis FBP-binding transcription factor CggR into yeast. As proof of principle, using fluorescence microscopy, we applied the sensor to identify metabolic subpopulations in yeast cultures. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations. perform flux-dependent regulation 29,30 . Biosensors for such metabolites, such as recently accomplished for E. coli 31 , would in principle allow for measurement of metabolic fluxes in single cells, in combination with microscopy or flow cytometry.Here, drawing on glycolytic flux-signalling metabolite fructose-1,6-bisphosphate (FBP) levels in yeast 30 and using the B. subtilis FBP-binding transcription factor CggR 32,33 , we developed a biosensor, which allows to measure glycolytic flux in single yeast cells. To this end, we used computational protein design, biochemical, proteome and metabolome analyses, for (i) the development of a synthetic yeast promoter regulated by the bacterial transcriptional factor CggR, (ii) the engineering of the transcription factors' FBP binding site towards increasing the sensor's dynamic range, and (iii) the establishment of growthindependent CggR expression levels. We demonstrate the applicability of the biosensor for flow cytometry and time-lapse fluorescence microscopy. We envision that the biosensor will open new avenues for both fundamental and applied metabolic research, not only for monitoring glycolytic flux, but also for engineering control circuits with glycolytic flux as input variable. Results Design of biosensor conceptFor our biosensor, we exploited the fact that the level of the glycolytic intermediate fructose-1,6-biphosphate (FBP) in yeast 30,34 , similar to other organisms 27 , strongly correlates with the glycolysis flux 28,34,35 , and that changing FBP levels exert fluxdependent regulation. In B. subtilis, for instance, FBP binds to the transcription factor (TF) CggR 33 , which when bound to its target DNA forms a tetrameric assembly of two dimers, through which transcription gets inhibited 36 . Upon binding of FBP to the CggR-DNA complex, the dimer-dimer contacts of CggR are disrupted, and the promoter is derepressed 37 .Here, we aimed to transplant the B...