Ngk1 kinase-mediated N-acetylglucosamine metabolism promotes UDP-GlcNAc biosynthesis in Saccharomyces cerevisiae.

N-acetylglucosamine (GlcNAc) is an important structural component of the cell wall chitin, N-glycans, glycolipids, and GPI-anchors in eukaryotes. GlcNAc kinase phosphorylates GlcNAc into GlcNAc-6-phosphate, a precursor of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that serves as a substrate for glycan synthesis. Although GlcNAc kinase is found widely in organisms ranging from microorganisms to mammals, it has never been found in the model yeast Saccharomyces cerevisiae. Here, we demonstrate the presence of GlcNAc metabolism for UDP-GlcNAc biosynthesis in S. cerevisiae through Ngk1, a GlcNAc kinase we discovered previously. The overexpression or deletion of Ngk1 in the presence of GlcNAc affected the amount of both UDP-GlcNAc and chitin, suggesting that GlcNAc metabolism via Ngk1 promotes UDP-GlcNAc synthesis. Our data suggest that the Ngk1-mediated GlcNAc metabolism compensates for the hexosamine pathway, a known pathway for UDP-GlcNAc synthesis.

A C1/C4-Oxidizing AA10 Lytic Polysaccharide Monooxygenase from Paenibacillus xylaniclasticus Strain TW1.

Lytic polysaccharide monooxygenases (LPMO) are key enzymes for the efficient degradation of lignocellulose biomass with cellulases. A lignocellulose-degradative strain, Paenibacillus xylaniclasticus TW1, has LPMO-encoding PxAA10A gene. Neither the C1/C4-oxidizing selectivity nor the enzyme activity of PxAA10A has ever been characterized. In this study, the C1/C4-oxidizing selectivity of PxAA10A and the boosting effect for cellulose degradation with a cellulase cocktail were investigated. The full-length PxAA10A (rPxAA10A) and the catalytic domain (rPxAA10A-CD) were heterologously expressed in Escherichia coli and purified. To identify the C1/C4-oxidizing selectivity of PxAA10A, cellohexaose was used as a substrate with the use of rPxAA10A-CD, and the products were analyzed by MALDI-TOF/MS. As a result, aldonic acid cellotetraose and cellotetraose, the products from C1-oxidization and C4-oxidization, respectively, were detected. These results indicate that PxAA10A is a C1/C4-oxidizing LPMO. It was also found that the addition of rPxAA10A into a cellulase cocktail enhanced the cellulose-degradation efficiency.

Identification and biochemical characterization of a novel N-acetylglucosamine kinase in Saccharomyces cerevisiae.

N-acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae, a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The Km values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.

Characterization of a GH Family 43 ß-Xylosidase Having a Novel Carbohydrate-binding Module from Paenibacillus xylaniclasticus Strain TW1.

Paenibacillus xylaniclasticus strain TW1, a gram-positive facultative anaerobic bacterium, was isolated as a xylanolytic microorganism from the wastes of a pineapple processing factory. A gene encoding one of its xylanolytic enzymes, a ß-xylosidase, was cloned and sequenced. Sequence analysis revealed that this ß-xylosidase, named PxXyl43A, was composed of a glycoside hydrolase (GH) family 43 subfamily 12 catalytic module and an unknown function module (UM). The full-length PxXyl43A (PxXyl43A) was heterologously expressed in Escherichia coli and purified. Recombinant PxXyl43A exhibited hydrolysis activity against both p-nitrophenyl-ß-D-xylopyranoside (pNPX) and p-nitrophenyl-α-L-arabinofuranoside at specific activities of 250 and 310 mU/mg, respectively. The optimal reaction pH and temperature for pNPX hydrolysis were 7.1 and 54 ˚C, respectively. At pH 7.0 and 54 ˚C, the K m and k cat for pNPX were 1.2 mM and 2.8 ± 0.15 s-1, respectively. It was also discovered that the recombinant unknown function module of PxXyl43A (PxXyl43A-UM) could bind to insoluble xylans like birchwood xylan and oat spelt xylan, whereas it did not bind to cellulosic substrates such as ball-milled cellulose, carboxymethyl cellulose or lichenan. The PxXyl43A-UM's binding constant value K a for oat spelt xylan was 2.0 × 10-5 M-1. These results suggest that PxXyl43A possesses a novel carbohydrate-binding module, named as CBM91, specific for xylan-containing polysaccharides.

The expression of alternative sigma-I7 factor induces the transcription of cellulosomal genes in the cellulolytic bacterium Clostridium thermocellum.

The composition of cellulosomal carbohydrate-active enzymes (CAZymes) secreted from a cellulolytic bacterium Clostridium thermocellum varies depending on the cellulosic substrate used during cultivation. C. thermocellum detects the polysaccharides in cellulosic material via anti-sigma factors and expresses the appropriate CAZyme gene via alternative sigma factors, SigIs. Previous studies on the regulation of CAZyme gene expression via SigIs in C. thermocellum have been conducted in vitro or in a heterologous host, because of the limited genetic tools available for C. thermocellum. To characterize the in vivo function of SigIs, in the present study, we established a sigI7 gene expression strain of C. thermocellum. Transcriptome analysis of this strain revealed that SigI7 induced the expression of cellulosomal CAZyme genes and cellulosomal scaffold genes. However, there was a decrease in the degradation ability of the exoproteome from the sigI7 expression strain; the product of the downregulated gene, Clo1313_1002, rescued the activity of the C. thermocellum exoproteome from the sigI7 expression strain. In this study, we demonstrate the in vivo function of SigI7 and discuss the CAZymes that are important for cellulosic biomass degradation by C. thermocellum.

Metabolome Analysis of Constituents in Membrane Vesicles for Clostridium thermocellum Growth Stimulation.

The cultivation of the cellulolytic bacterium, Clostridium thermocellum, can have cost-effective cellulosic biomass utilizations, such as consolidated bioprocessing, simultaneous biological enzyme production and saccharification. However, these processes require a longer cultivation term of approximately 1 week. We demonstrate that constituents of the C. thermocellum membrane vesicle fraction significantly promoted the growth rate of C. thermocellum. Similarly, cell-free Bacillus subtilis broth was able to increase C. thermocellum growth rate, while several B. subtilis single-gene deletion mutants, e.g., yxeJ, yxeH, ahpC, yxdK, iolF, decreased the growth stimulation ability. Metabolome analysis revealed signal compounds for cell-cell communication in the C. thermocellum membrane vesicle fraction (ethyl 2-decenoate, ethyl 4-decenoate, and 2-dodecenoic acid) and B. subtilis broth (nicotinamide, indole-3-carboxaldehyde, urocanic acid, nopaline, and 6-paradol). These findings suggest that the constituents in membrane vesicles from C. thermocellum and B. subtilis could promote C. thermocellum growth, leading to improved efficiency of cellulosic biomass utilization.

The Emi2 Protein of Saccharomyces cerevisiae is a Hexokinase Expressed under Glucose Limitation.

Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg 2+ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.

Mitotic cyclin Clb4 is required for the intracellular adaptation to glucose starvation in Saccharomyces cerevisiae.

Cellular homeostasis in response to glucose availability is maintained through the tight coordination of various physiological processes, including cell proliferation, transcription, and metabolism. In this study, we use the budding yeast Saccharomyces cerevisiae to identify proteins implicated in carbon source-dependent modulation of physiological processes. We find that the mitotic cyclin Clb4 is required for optimal regulation of glucose-starvation-responsive pathways through the target of rapamycin complex 1. Cells lacking Clb4 are characterized by dysregulation of autophagy and impaired modulation of cell size. Notably, cell viability after prolonged glucose starvation is severely reduced by disruption of Clb4. We conclude that Clb4, in addition to its function in the cell cycle, plays a role in the intracellular adaptation to glucose starvation.

Cellulosomes localise on the surface of membrane vesicles from the cellulolytic bacterium Clostridium thermocellum.

Membrane vesicles released from bacteria contribute to cell-cell communication by carrying various cargos such as proteins, nucleic acids and signaling molecules. Cellulolytic bacteria have been isolated from many environments, yet the function of membrane vesicles for cellulolytic ability has been rarely described. Here, we show that a Gram-positive cellulolytic bacterium Clostridium thermocellum released membrane vesicles, each approximately 50-300 nm in diameter, into the broth. The observations with immunoelectron microscopy also revealed that cellulosomes, which are carbohydrate-active enzyme complexes that give C. thermocellum high cellulolytic activity, localized on the surface of the membrane vesicles. The membrane vesicles collected by ultracentrifugation maintained the cellulolytic activity. Supplementation with the biosurfactant surfactin or sonication treatment disrupted the membrane vesicles in the exoproteome of C. thermocellum and significantly decreased the degradation activity of the exoproteome for microcrystalline cellulose. However, these did not affect the degradation activity for soluble carboxymethyl cellulose. These results suggest a novel function of membrane vesicles: C. thermocellum releases cellulolytic enzymes on the surface of membrane vesicles to enhance the cellulolytic activity of C. thermocellum for crystalline cellulose.

Glucose production from cellulose through biological simultaneous enzyme production and saccharification using recombinant bacteria expressing the ß-glucosidase gene.

Efficient cellulosic biomass saccharification technologies are required to meet biorefinery standards. Biological simultaneous enzyme production and saccharification (BSES), which is glucose production from cellulosic biomass by Clostridium thermocellum, can be a reliable cellulose saccharification technology for biorefineries. However, the current BSES processes require purified ß-glucosidase supplementation. In this study, recombinant bacteria expressing the ß-glucosidase gene were developed and directly applied to BSES. The engineered Escherichia coli expressing the thermostable ß-glucosidase gene from Thermoanaerobacter brockii exhibited 0.5 U/ml of ß-glucosidase activities. The signal peptide sequence of lytF gene from Bacillus subtilis was the most appropriate for the ß-glucosidase secretion from Brevibacillus choshinensis, and the broth exhibited 0.74 U/ml of ß-glucosidase activities. The engineered E. coli and B. choshinensis expressing the thermostable ß-glucosidase gene produced 47.4 g/L glucose and 49.4 g/L glucose, respectively. Glucose was produced by the hydrolysis of 100 g/L Avicel cellulose for 10 days through BSES, and the product yield was similar to that obtained through BSES with purified ß-glucosidase supplementation. Our findings indicate that the direct supplementation of ß-glucosidase using bacterial cells expressing ß-glucosidase gene or their broth was applicable to BSES, suggesting the potential of this process as a cost-effective approach to cellulose saccharification.

Characterization of lignocellulose particles during lignocellulose solubilization by Clostridium thermocellum.

Clostridium thermocellum is a candidate bacterium for lignocellulose utilization due to its efficient lignocellulose solubilization ability. It has been reported that C. thermocellum efficiently degrades purified cellulose substrates, but cannot completely degrade milled lignocellulose powders. Evaluation of cellulose and hemicellulose contents in a lignocellulose residue after the cultivation of C. thermocellum indicated that C. thermocellum degraded cellulose and hemicellulose equally. Microscopic observations demonstrated that C. thermocellum significantly degraded small-sized lignocellulose particles, but it only partially degraded the larger sized particles. The lignin content of the large-sized particles was higher than that of the small particles. The remained large-sized particles included vascular tissues. These results suggest that the lignified structures such as vascular tissues in milled lignocellulose were less susceptible to bacterial lignocellulose solubilization.

Screening of yeast isolates from flowers for effective ethanol production.

The use of alternative substrates to produce biofuel is a striking option nowadays. This study aimed to screen bioethanolproducing yeast strains. From different flowers, 65 yeasts were isolated. Twelve isolates assimilated glucose by liberation of CO 2 anaerobically. Out of these, only 5 yeast isolates fermented glucose in medium consisting of 0.8 g/L Mg2+ ions to produce 2.05 ± 0.03% ethanol. The selected five strains were identified as members of the genera Metschnikowia or Meyerozyma based on molecular characterization. Selected yeast strains were used for conversion of rice into bioethanol following dilute acid hydrolysis and fermentation. Consistent ethanol production was 1.80 ± 0.05% at days 2-4 by Metschnikowia cibodasensis Y34 and 2.20 ± 0.21% by Meyerozyma caribica Y42 at days 4-6 with a gradual decrease at the time of experiment termination (day 10). Metschnikowia cibodasensis Y34 and Meyerozyma caribica Y42 produced the highest ethanol at pH 3, i.e. 1.75 ± 0.14% at days 3 and 5 and 2.05 ± 0.14% at days 1 and 3, respectively, upon incubation with different pH levels and 1% NaCl. Growth and ethanol production at pH 4 and 5 was close to that at pH 3, with a slight increase in production by Metschnikowia cibodasensis Y34 at pH 4 up to day 3.

Changes in nutrient composition and in vitro ruminal fermentation of total mixed ration silage stored at different temperatures and periods.

BACKGROUND: Total mixed ration (TMR) is widely used for dairy cattle and needs to be prepared daily because it deteriorates rapidly. Ensiling TMR allows preservation and saves labour at the farm; however, silage fermentation may influence various nutritional components. The objectives of this study were to evaluate nutritional changes and in vitro rumen fermentation of TMR silage that was stored at different temperatures and durations on a laboratory scale in comparison with those of typical TMR before ensiling. RESULTS: No distinct changes in crude protein (CP), neutral detergent fibre and non-fibrous carbohydrate contents were observed during silage fermentation. However, clear changes were observed in the soluble CP and soluble sugar fractions; solubilisation of the CP fraction in TMR silage was enhanced by prolonged storage and higher storage temperatures, and most soluble sugars were lost during ensiling. Short-chain fatty acid concentrations in the in vitro rumen from TMRs before and after ensiling were not significantly different; however, throughout incubation, NH3 -N concentrations from TMR silages were significantly higher than those from TMR before ensiling. CONCLUSION: A higher ruminal NH3 -N concentration from TMR silage may be a result of a shortage of fermentable sugars and enhanced deamination of CP. Feeding TMR ensiled under a high temperature must be investigated to balance proteins and carbohydrates for rumen fermentation.

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum.

Most cellulases contain carbohydrate-binding modules (CBMs) that largely contribute to their activity for insoluble substrates. Clostridium thermocellum Cel5E is an endoglucanase having xylanolytic activity. The Cel5E originally has a family 11 CBM preferentially binding to ß-1,4- and ß-1,3-1,4-mixed linkage glucans. In this study, we replaced the CBM with a different type of CBM, either a family 3 microcrystalline cellulose-directed CBM from Clostridium josui scaffoldin, or a family 6 xylan-directed CBM from Clostridium stercorarium xylanase 11A. Chimeric endoglucanases showed enhanced activity that was affected by CBM binding specificity. These chimeric enzymes could efficiently degrade milled lignocellulosic materials, such as corn hulls, because of heterologous components in the plant cell wall, indicating that diverse CBMs play roles in degradation of lignocellulosic materials.

Bacterial production and secretion of water-insoluble fuel compounds from cellulose without the supplementation of cellulases.

Achieving economic biofuel production from cellulosic biomass will require significant cost reductions. Enzymatic degradation of cellulosic biomass and distillation of water-soluble fuel compounds substantially increase the cost of biofuel production. Consolidated bioprocessing is a strategy to circumvent expensive biofuel production steps. Clostridium thermocellum is a promising bacterium for consolidated bioprocessing because it does not require the supplementation of lignocellulose-degrading enzymes. To produce water-insoluble fuel compounds, C. thermocellum was engineered to express a fatty acyl-acyl carrier protein reductase and an aldehyde-deformylating oxygenase from Synechococcus elongatus PCC 7942. Expression of the aldehyde-deformylating oxygenase gene was clearly detected, whereas only slight expression of the fatty acyl-acyl carrier protein reductase gene was detected. Cells expressing the fatty acyl-acyl carrier protein reductase and the aldehyde-deformylating oxygenase accumulated fatty aldehydes (higher alcohol precursors). After cultivation with cellulose, the higher alcohols, decanol and dodecanol, were detected in the organic solvent phase of the culture broth, indicating that the strain secreted the higher alcohols. These results suggest that the engineered C. thermocellum strain, expressing fatty acyl-acyl carrier protein reductase and aldehyde-deformylating oxygenase genes, directly produces and secretes higher alcohols from cellulose without the supplementation of cellulases. The higher alcohols can be collected by phase separation.

Cellulosomal carbohydrate-binding module from Clostridium josui binds to crystalline and non-crystalline cellulose, and soluble polysaccharides.

To understand the lignocellulose degradation activity of the Clostridium josui cellulosome, a carbohydrate-binding module of the scaffoldin CjCBM3 was characterized. CjCBM3 shows binding to crystalline cellulose, non-crystalline cellulose and soluble polysaccharides. The binding isotherm of CjCBM3 to acid-swollen cellulose is best fitted by the Langmuir two-site model, suggesting that there are two CjCBM3 binding sites on acid-swollen cellulose with different affinities. The second site shows lower affinity and larger binding capacity, suggesting that the cellulosome is directly targeted to the cellulose surface with high affinity, where larger amounts of the cellulosome bind to cellulose with low affinity.

Sequence analysis and heterologous expression of the wool cuticle-degrading enzyme encoding genes in Fusarium oxysporum 26-1.

Two protease-like proteins, KrtA and KrtC, were identified in Fusarium oxysporum 26-1. Genes coding these proteins, krtA and krtC, were isolated and characterized. Recombinant KrtA (rKrtA) and KrtC (rKrtC) were successfully expressed in Aspergillus oryzae and secreted. The combination of rKrtA and rKrtC completely removed the cuticle of wool fibers.

Characterization of an endo-processive-type xyloglucanase having a ß-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.

We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to ß-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.

Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase.

In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted ß-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of ß-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.

Surface carbohydrate analysis and bioethanol production of sugarcane bagasse pretreated with the white rot fungus, Ceriporiopsis subvermispora and microwave hydrothermolysis.

Effects of pretreatments with a white rot fungus, Ceriporiopsis subvermispora, and microwave hydrothermolysis of bagasse on enzymatic saccharification and fermentation were evaluated. The best sugar yield, 44.9 g per 100g of bagasse was obtained by fungal treatments followed by microwave hydrothermolysis at 180°C for 20 min. Fluorescent-labeled carbohydrate-binding modules which recognize crystalline cellulose (CjCBM3-GFP), non-crystalline cellulose (CjCBM28-GFP) and xylan (CtCBM22-GFP) were applied to characterize the exposed polysaccharides. The microwave pretreatments with and without the fungal cultivation resulted in similar levels of cellulose exposure, but the combined treatment caused more defibration and thinning of the plant tissues. Simultaneous saccharification and fermentation of the pulp fractions obtained by microwave hydrothermolysis with and without fungal treatment, gave ethanol yields of 35.8% and 27.0%, respectively, based on the holocellulose content in the pulp. These results suggest that C. subvermispora pretreatment could be beneficial part of the process to produce ethanol from bagasse.