A SWEET surprise: Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast.

In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae.

Extremely Thermoacidophilic Metallosphaera Species Mediate Mobilization and Oxidation of Vanadium and Molybdenum Oxides.

Certain species from the extremely thermoacidophilic genus Metallosphaera directly oxidize Fe(II) to Fe(III), which in turn catalyzes abiotic solubilization of copper from chalcopyrite to facilitate recovery of this valuable metal. In this process, the redox status of copper does not change as it is mobilized. Metallosphaera species can also catalyze the release of metals from ores with a change in the metal's redox state. For example, Metallosphaera sedula catalyzes the mobilization of uranium from the solid oxide U3O8, concomitant with the generation of soluble U(VI). Here, the mobilization of metals from solid oxides (V2O3, Cu2O, FeO, MnO, CoO, SnO, MoO2, Cr2O3, Ti2O3, and Rh2O3) was examined for M. sedula and M. prunae at 70°C and pH 2.0. Of these oxides, only V and Mo were solubilized, a process accelerated in the presence of FeCl3 However, it was not clear whether the solubilization and oxidation of these metals could be attributed entirely to an Fe-mediated indirect mechanism. Transcriptomic analysis for growth on molybdenum and vanadium oxides revealed transcriptional patterns not previously observed for growth on other energetic substrates (i.e., iron, chalcopyrite, organic compounds, reduced sulfur compounds, and molecular hydrogen). Of particular interest was the upregulation of Msed_1191, which encodes a Rieske cytochrome b6 fusion protein (Rcbf, referred to here as V/MoxA) that was not transcriptomically responsive during iron biooxidation. These results suggest that direct oxidation of V and Mo occurs, in addition to Fe-mediated oxidation, such that both direct and indirect mechanisms are involved in the mobilization of redox-active metals by Metallosphaera species.IMPORTANCE In order to effectively leverage extremely thermoacidophilic archaea for the microbially based solubilization of solid-phase metal substrates (e.g., sulfides and oxides), understanding the mechanisms by which these archaea solubilize metals is important. Physiological analysis of Metallosphaera species growth in the presence of molybdenum and vanadium oxides revealed an indirect mode of metal mobilization, catalyzed by iron cycling. However, since the mobilized metals exist in more than one oxidation state, they could potentially serve directly as energetic substrates. Transcriptomic response to molybdenum and vanadium oxides provided evidence for new biomolecules participating in direct metal biooxidation. The findings expand the knowledge on the physiological versatility of these extremely thermoacidophilic archaea.

Identification of novel membrane proteins for improved lignocellulose conversion.

Lignocellulose processing yields a heterogeneous mixture of substances, which are poorly utilized by current industrial strains. For efficient valorization of recalcitrant biomass, it is critical to identify and engineer new membrane proteins that enable the broad uptake of hydrolyzed substrates. Whereas glucose consumption rarely presents a bottleneck for cell factories, there is also a lack of transporters that allow co-consumption of glucose with other abundant biomass sugars such as xylose. This review discusses recent efforts to bioinformatically identify membrane proteins of high biotech potential for lignocellulose conversion and metabolic engineering in both model and nonconventional organisms. Of particular interest are transporters sourced from anaerobic gut fungi resident to large herbivores, which produce Sugars Will Eventually be Exported Transporters (SWEETs) that enhance xylose transport in the yeast Saccharomyces cerevisiae and enable glucose and xylose co-utilization. Additionally, recently identified fungal cellodextrin transporters are valuable alternatives to mitigate glucose repression and transporter inhibition.

Harnessing Nature's Anaerobes for Biotechnology and Bioprocessing.

Industrial biotechnology has the potential to decrease our reliance on petroleum for fuel and bio-based chemical production and also enable valorization of waste streams. Anaerobic microorganisms thrive in resource-limited environments and offer an array of novel bioactivities in this regard that could revolutionize biomanufacturing. However, they have not been adopted for widespread industrial use owing to their strict growth requirements, limited number of available strains, difficulty in scale-up, and genetic intractability. This review provides an overview of current and future uses for anaerobes in biotechnology and bioprocessing in the postgenomic era. We focus on the recently characterized anaerobic fungi (Neocallimastigomycota) native to the digestive tract of large herbivores, which possess a trove of enzymes, pathways, transporters, and other biomolecules that can be harnessed for numerous biotechnological applications. Resolving current genetic intractability, scale-up, and cultivation challenges will unlock the potential of these lignocellulolytic fungi and other nonmodel micro-organisms to accelerate bio-based production.

Ionoprinted Multi-Responsive Hydrogel Actuators.

We report multi-responsive and double-folding bilayer hydrogel sheet actuators, whose directional bending response is tuned by modulating the solvent quality and temperature and where locally crosslinked regions, induced by ionoprinting, enable the actuators to invert their bending axis. The sheets are made multi-responsive by combining two stimuli responsive gels that incur opposing and complementary swelling and shrinking responses to the same stimulus. The lower critical solution temperature (LCST) can be tuned to specific temperatures depending on the EtOH concentration, enabling the actuators to change direction isothermally. Higher EtOH concentrations cause upper critical solution temperature (UCST) behavior in the poly(N-isopropylacrylamide) (pNIPAAm) gel networks, which can induce an amplifying effect during bilayer bending. External ionoprints reliably and repeatedly invert the gel bilayer bending axis between water and EtOH. Placing the ionoprint at the gel/gel interface can lead to opposite shape conformations, but with no clear trend in the bending behavior. We hypothesize that this is due to the ionoprint passing through the neutral axis of the bilayer during shrinking in hot water. Finally, we demonstrate the ability of the actuators to achieve shapes unique to the specific external conditions towards developing more responsive and adaptive soft actuator devices.