Evidence for transceptor function of cellodextrin transporters in Neurospora crassa.

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three ß-glucosidase enzymes (Δ3ßG) lacks ß-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the ß-glucosidase enzymes and cellodextrin transporters (Δ3ßGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as "transceptors" with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.

Analysis of cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation.

Saccharomyces cerevisiae can be engineered to ferment cellodextrins produced by cellulases as a product of cellulose hydrolysis. Direct fermentation of cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. To facilitate cellodextrin utilization by S. cerevisiae, a fungal cellodextrin-utilizing pathway from Neurospora crassa consisting of a cellodextrin transporter and a cellodextrin hydrolase has been introduced into S. cerevisiae. Two cellodextrin transporters (CDT-1 and CDT-2) were previously identified in N. crassa, but their kinetic properties and efficiency for cellobiose fermentation have not been studied in detail. In this study, CDT-1 and CDT-2, which are hypothesized to transport cellodextrin with distinct mechanisms, were introduced into S. cerevisiae along with an intracellular ß-glucosidase (GH1-1). Cellobiose transport assays with the resulting strains indicated that CDT-1 is a proton symporter while CDT-2 is a simple facilitator. A strain expressing CDT-1 and GH1-1 (DCDT-1G) showed faster cellobiose fermentation than the strain expressing CDT-2 and GH1-1 (DCDT-2G) under various culture conditions with different medium compositions and aeration levels. While CDT-2 is expected to have energetic benefits, the expression levels and kinetic properties of CDT-1 in S. cerevisiae appears to be optimum for cellobiose fermentation. These results suggest CDT-1 is a more effective cellobiose transporter than CDT-2 for engineering S. cerevisiae to ferment cellobiose.

Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters.

Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular ß-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6kJmol(-1)). A thermodynamic "push" from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae.

Cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain.

We demonstrate improved ethanol yield and productivity through cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain expressing genes coding for cellodextrin transporter (cdt-1) and intracellular ß-glucosidase (gh1-1) from Neurospora crassa. Simultaneous fermentation of cellobiose and galactose can be applied to producing biofuels from hydrolysates of marine plant biomass.

Microbial production of megadalton titin yields fibers with advantageous mechanical properties.

Manmade high-performance polymers are typically non-biodegradable and derived from petroleum feedstock through energy intensive processes involving toxic solvents and byproducts. While engineered microbes have been used for renewable production of many small molecules, direct microbial synthesis of high-performance polymeric materials remains a major challenge. Here we engineer microbial production of megadalton muscle titin polymers yielding high-performance fibers that not only recapture highly desirable properties of natural titin (i.e., high damping capacity and mechanical recovery) but also exhibit high strength, toughness, and damping energy - outperforming many synthetic and natural polymers. Structural analyses and molecular modeling suggest these properties derive from unique inter-chain crystallization of folded immunoglobulin-like domains that resists inter-chain slippage while permitting intra-chain unfolding. These fibers have potential applications in areas from biomedicine to textiles, and the developed approach, coupled with the structure-function insights, promises to accelerate further innovation in microbial production of high-performance materials.

Seeded Chain-Growth Polymerization of Proteins in Living Bacterial Cells.

Microbially produced protein-based materials (PBMs) are appealing due to use of renewable feedstock, low energy requirements, tunable side-chain chemistry, and biodegradability. However, high-strength PBMs typically have high molecular weights (HMW) and repetitive sequences that are difficult to microbially produce due to genetic instability and metabolic burden. We report the development of a biosynthetic strategy termed seeded chain-growth polymerization (SCP) for synthesis of HMW PBMs in living bacterial cells. SCP uses split intein (SI) chemistry to cotranslationally polymerize relatively small, genetically stable material protein subunits, effectively preventing intramolecular cyclization. We apply SCP to bioproduction of spider silk in Escherichia coli, generating HMW spider silk proteins (spidroins) up to 300 kDa, resulting in spidroin fibers of high strength, modulus, and toughness. SCP provides a modular strategy to synthesize HMW, repetitive material proteins, and may facilitate bioproduction of a variety of high-performance PBMs for broad applications.

A new diet for yeast to improve biofuel production.

In 2010, our group announced the discovery of two cellodextrin transporter families from the cellulolytic fungus, Neurospora crassa. Furthermore, we demonstrated the utility of these transporters in the production of lignocellulosic biofuels. This discovery was made possible by a decision to systematically study cell wall degradation by N. crassa. The identified transport pathway has opened up a new way of thinking about microbial fermentation of hexoses as well as pentoses derived from plant cell walls. Integrating this pathway with the endogenous metabolism and signaling networks of S. cerevisiae is now a major goal of our group.

Cellodextrin transport in yeast for improved biofuel production.

Fungal degradation of plant biomass may provide insights for improving cellulosic biofuel production. We show that the model cellulolytic fungus Neurospora crassa relies on a high-affinity cellodextrin transport system for rapid growth on cellulose. Reconstitution of the N. crassa cellodextrin transport system in Saccharomyces cerevisiae promotes efficient growth of this yeast on cellodextrins. In simultaneous saccharification and fermentation experiments, the engineered yeast strains more rapidly convert cellulose to ethanol when compared with yeast lacking this system.