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Although engineered Saccharomyces cerevisiae fermenting cellobiose is useful for the production of biofuels from cellulosic biomass, cellodextrin accumulation is one of the main problems reducing ethanol yield and productivity in cellobiose fermentation with S. cerevisiae expressing cellodextrin transporter (CDT) and intracellular β-glucosidase (GH1-1). In this study, we investigated the reason for the cellodextrin accumulation and how to alleviate its formation during cellobiose fermentation using engineered S. cerevisiae fermenting cellobiose. From the series of cellobiose fermentation using S. cerevisiae expressing only GH1-1 under several culture conditions, it was discovered that small amounts of GH1-1 were secreted and cellodextrin was generated through trans-glycosylation activity of the secreted GH1-1. As GH1-1 does not have a secretion signal peptide, non-conventional protein secretion might facilitate the secretion of GH1-1. In cellobiose fermentations with S. cerevisiae expressing only GH1-1, knockout of TLG2 gene involved in non-conventional protein secretion pathway significantly delayed cellodextrin formation by reducing the secretion of GH1-1 by more than 50%. However, in cellobiose fermentations with S. cerevisiae expressing both GH1-1 and CDT-1, TLG2 knockout did not show a significant effect on cellodextrin formation, although secretion of GH1-1 was reduced by more than 40%. These results suggest that the development of new intracellular β-glucosidase, not influenced by non-conventional protein secretion, is required for better cellobiose fermentation performances of engineered S. cerevisiae fermenting cellobiose.

Processed lignocellulosic biomass is a source of mixed sugars that can be used for microbial fermentation into fuels or higher value products, like chemicals. Previously, the yeast Saccharomyces cerevisiae was engineered to utilize its cellodextrins through the heterologous expression of sugar transporters together with an intracellular expressed β-glucosidase. In this study, we screened a selection of eight (putative) cellodextrin transporters from different yeast and fungal hosts in order to extend the catalogue of available cellobiose transporters for cellobiose fermentation in S. cerevisiae. We confirmed that several in silico predicted cellodextrin transporters from Aspergillus niger were capable of transporting cellobiose with low affinity. In addition, we found a novel cellobiose transporter from the yeast Lipomyces starkeyi, encoded by the gene Ls120451. This transporter allowed efficient growth on cellobiose, while it also grew on glucose and lactose, but not cellotriose nor cellotetraose. We characterized the transporter more in-depth together with the transporter CdtG from Penicillium oxalicum. CdtG showed to be slightly more efficient in cellobiose consumption than Ls120451 at concentrations below 1.0 g/L. Ls120451 was more efficient in cellobiose consumption at higher concentrations and strains expressing this transporter grew slightly slower, but produced up to 30% more ethanol than CdtG., (© FEMS 2020.)

To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat /K m ) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.

Objectives: To elucidate the mechanism of cellulase signal transduction in filamentous fungi including the components of the cellulase induction pathway., Results: Neurospora crassa ncw-1 encodes a non-anchored cell wall protein. The absence of ncw-1 increased cellulase gene expression and this is not due to relieving carbon catabolite repression mediated by the cre-1 pathway. A mutant lacking genes encoding both three major β-glucosidase enzymes and NCW-1 (Δ3βGΔncw-1) was constructed. Transcriptome analysis of the quadruple mutant demonstrated enhanced expression of cellodextrin transporters after ncw-1 deletion, indicating that ncw-1 affects cellulase expression and production by inhibiting the uptake of the cellodextrin., Conclusions: NCW-1 is a novel component that plays a critical role in the cellulase induction signaling pathway.

Yarrowia lipolytica is a promising production host for a wide range of molecules, but limited sugar consumption abilities prevent utilization of an abundant source of renewable feedstocks. In this study we created a Y. lipolytica strain capable of utilizing cellobiose as a sole carbon source by using endogenous promoters to express the cellodextrin transporter cdt-1 and intracellular β-glucosidase gh1-1 from Neurospora crassa. The engineered strain was also capable of simultaneous co-consumption of glucose and cellobiose. Although cellobiose was consumed slower than glucose when engineered strains were cultured with excess nitrogen, culturing with limited nitrogen led to cellobiose consumption rates comparable to those of glucose. Under limited nitrogen conditions, the engineered strain produced citric acid as a major product and we observed greater citric acid yields from cellobiose (0.37 g/g) than glucose (0.28 g/g). Culturing with a sole carbon source of either glucose or cellobiose induced additional differences on cell physiology and metabolism and a link is suggested to evasion of glucose-sensing mechanisms through intracellular creation and consumption of glucose. We ultimately applied this cellobiose-utilization system to produce citric acid from bioconversion of crystalline cellulose through simultaneous saccharification and fermentation (SSF)., (© 2014 Wiley Periodicals, Inc.)

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 β-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.

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 β-glucosidase enzymes (Δ3βG) lacks β-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 β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) 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.

Production of ethanol and xylitol from lignocellulosic hydrolysates is an alternative to the traditional production of ethanol in utilizing biomass. However, the conversion efficiency of xylose to xylitol is restricted by glucose repression, causing a low xylitol titer. To this end, we cloned genes CDT-1 (encoding a cellodextrin transporter) and gh1-1 (encoding an intracellular β-glucosidase) from Neurospora crassa and XYL1 (encoding a xylose reductase that converts xylose into xylitol) from Scheffersomyces stipitis into Saccharomyces cerevisiae, enabling simultaneous production of ethanol and xylitol from a mixture of cellobiose and xylose (main components of lignocellulosic hydrolysates). We further optimized the expression levels of CDT-1 and XYL1 by manipulating their promoters and copy-numbers, and constructed an engineered S. cerevisiae strain (carrying one copy of PGK1p-CDT1 and two copies of TDH3p-XYL1), which showed an 85.7% increase in xylitol production from the mixture of cellobiose and xylose than that from the mixture of glucose and xylose. Thus, we achieved a balanced co-fermentation of cellobiose (0.165 g/L/h) and xylose (0.162 g/L/h) at similar rates to co-produce ethanol (0.36 g/g) and xylitol (1.00 g/g).

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 β-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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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 β-glucosidase (gh1-1) from Neurospora crassa. Simultaneous fermentation of cellobiose and galactose can be applied to producing biofuels from hydrolysates of marine plant biomass.

β-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for β-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus ) showed 10 times greater β-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial β-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis-Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3 U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4 °C for Novozym 188. The significant thermostability of strain AP β-glucosidases was further confirmed when compared with Cellic CTec. The β-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for β-glucosidase activity.

The crystal structure of the carbohydrate-binding module (CBM) 4 Ig fused domain from the cellulosomal cellulase cellobiohydrolase A (CbhA) of Clostridium thermocellum was solved in complex with cellobiose at 2.11 A resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved noncellulosomal soluble oligosaccharide-binding CBM4 structures. However, this new structure possesses a significant feature-a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common to all cellulosomal clostridial CBM4 modules. Our results indicate that C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and, possibly, the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues Trp68 and Tyr110 play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose, and soluble oligodextrins. Binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose "whiskers" of BMCC. We propose that clostridial CBM4s have possibly evolved to bind the free-chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Rates of phosphorolytic cleavage of beta-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60 degrees C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35 degrees C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by > or = 20-fold for both cellobiose and cellopentaose over a 10-fold range of beta-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of beta-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V(max) values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K(m) for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).

Ruminococcus albus is an important fibrolytic bacterium in the rumen. Cellobiose is metabolized by this organism via hydrolytic and well as phosphorylytic enzymes, but the relative contributions of each pathway were not clear. The cellobiose consumption rate by exponentially growing cells was less than that of crude extracts (75 versus 243 nmol/min/mg protein). Cellobiose phosphorolytic cleavage was much greater than hydrolytic activity (179 versus 19 nmol/min/mg protein) indicating that phosphorylases were key enzymes in the initial metabolism of the soluble products of cellulose degradation. Cellodextrin phosphorylase appeared to be active against substrates as large as cellohexaose. Phosphorylase activities were cytoplasmic, but hydrolytic activities were associated with both the membrane and cytoplasmic fractions. Free glucose was phosphorylated with a GTP-dependent glucokinase, and this enzyme showed 20-fold higher activity with GTP or ITP (>324 nmol/min/mg protein) than with ATP, UTP, CTP, GDP, or PEP. The activity was decreased at least 57% when mannose, 2-deoxyglucose, or fructose was used as substrate compared with glucose. The Kms for glucose and GTP were 321 and 247 microM, respectively. Since phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, it is likely that such pathways provide for more efficient growth of R. albus in substrate-limiting conditions like those found in the rumen.

In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P(i)) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (> 92 nmol of P(i)/min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen.