Bgl Genes Research Articles

Promotion of extracellular lignocellulolytic enzymes production by restraining the intracellular β-glucosidase in Penicillium decumbens

In this study, the functions of β-glucosidases in regulation of the lignocellulolytic enzymes production in Penicillium decumbens 114-2 were investigated. The major extracellular β-glucosidase gene bgl1 and the major intracellular β-glucosidase gene bgl2 were deleted in P. decumbens 114-2 respectively. In Δbgl2, the production of extracellular lignocellulolytic enzymes (including endoglucanases, cellobiohydrolases and xylanases) on insoluble cellulose was significantly promoted, while in Δbgl1 there was no any difference compared with that of 114-2. The enhancement of the production of lignocellulolytic enzymes in Δbgl2 was likely attributed to the accumulation of intracellular cellobiose. Induction experiment in Δbgl1Δbgl2 showed that cellobiose was an inducer of lignocellulolytic enzymes expression in P. decumbens 114-2, and the induction was unrelated to the formation, if any, of gentiobiose or sophorose from cellobiose.

Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. crassa sextuple beta-glucosidase (bgl) gene deletion strains when grown on Avicel without exogenous cellulase addition. The sextuple bgl deletion strains expressing one of the three basally transcribed bgl genes are the best cellobiose producers. For most sextuple strains, the multiple bgl gene deletion has no negative effect on the production of other cellulases. The induction of major endoglucanases and exoglucanases on Avicel in most of the sextuple bgl deletions strains was as fast as or faster than that of the wild type, except for strain F4. The best cellobiose producing strain, F5, produced 7.7g/L of cellobiose from 20g/L of Avicel in four days and utilized the Avicel as fast as did the wild type (even in the presence of high cellobiose concentration). The cellobiose yield from cellulose was about 48.3%.

Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A.saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A.saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50°C, and at 60°C it had a half-life of approximately 6h.

Quantitative Secretomic Analysis of Trichoderma reesei Strains Reveals Enzymatic Composition for Lignocellulosic Biomass Degradation

Trichoderma reesei is a mesophilic, filamentous fungus, and it is a major industrial source of cellulases, but its lignocellulolytic protein expressions on lignocellulosic biomass are poorly explored at present. The extracellular proteins secreted by T. reesei QM6a wild-type and hypercellulolytic mutant Rut C30 grown on natural lignocellulosic biomasses were explored using a quantitative proteomic approach with 8-plex high throughput isobaric tags for relative and absolute quantification (iTRAQ) and analyzed by liquid chromatography tandem mass spectrometry. We quantified 230 extracellular proteins, including cellulases, hemicellulases, lignin-degrading enzymes, proteases, protein-translocating transporter, and hypothetical proteins. Quantitative iTRAQ results suggested that the expressions and regulations of these lignocellulolytic proteins in the secretome of T. reesei wild-type and mutant Rut C30 were dependent on both nature and complexity of different lignocellulosic carbon sources. Therefore, we discuss here the essential lignocellulolytic proteins for designing an enzyme mixture for optimal lignocellulosic biomass hydrolysis.

Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose

Backgroundβ-Glucosidase is an important component of the cellulase enzyme system. It does not only participate in cellulose degradation, it also plays an important role in hydrolyzing cellulose to fermentable glucose by relieving the inhibition of exoglucanase and endoglucanase from cellobiose. Therefore, the glucose-tolerant β-glucosidase with high specific activity for cellobiose might be a potent candidate for industrial applications.ResultsThe β-glucosidase gene bgl that encodes a 443-amino-acid protein was cloned and over-expressed from Thermoanaerobacterium thermosaccharolyticum DSM 571 in Escherichia coli. The phylogenetic trees of β-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the BGL was a novel β-glucosidase. By replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of bgl was increased from 6.6 to 11.2 U/mg in LB medium. Recombinant BGL was purified by heat treatment followed by Ni-NTA affinity. The optimal activity was at pH 6.4 and 70°C. The purified enzyme was stable over pH range of 5.2–7.6 and had a 1 h half life at 68°C. The activity of BGL was significantly enhanced by Fe2+ and Mn2+. The Vmax of 64 U/mg and 120 U/mg were found for p-nitrophenyl-β-D-glucopyranoside (Km value of 0.62 mM) and cellobiose (Km value of 7.9 mM), respectively. It displayed high tolerance to glucose and cellobiose. The Kcat for cellobiose was 67.7 s-1 at 60°C and pH 6.4, when the concentration of cellobiose was 290 mM. It was activated by glucose at concentrations lower that 200 mM. With glucose further increasing, the enzyme activity of BGL was gradually inhibited, but remained 50% of the original value in even as high as 600 mM glucose.ConclusionsThe article provides a useful novel β-glucosidase which displayed favorable properties: high glucose and cellobiose tolerance, independence of metal ions, and high hydrolysis activity on cellobiose.

Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33

BackgroundRecently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and β-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful β-glucosides. Although the β-glucosidases of different sources have been investigated, the amount of β-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of β-glucosidases, which was thermostable and with high catalytic efficiency.ResultsIn this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus Aspergillus fumigatus Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, bgl3, was cloned based on the peptide sequences obtained from the LC-MS/MS results. bgl3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant β-glucosidase (rBgl3) was obtained by the functional expression of bgl3 in Pichia pastoris X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-β-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan.ConclusionsAn native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of A. fumigatus Z5. The gene bgl3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene bgl3 was expressed in Pichia pastoris X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.

PCR detection of enzyme-encoding genes in Leuconostoc mesenteroides strains of wine origin

Fifteen isolates of lactic acid bacteria originating from South African grape and wine samples were identified as Leuconostoc mesenteroides subsp. mesenteroides through the taxonomic analysis of their 16S rDNA gene sequences. These isolates were further tested for the presence of genes coding for enzymes of oenological relevance using PCR detection technique. A type strain of Leuc. mesenteroides (NCDO 529(T)) was also incorporated for comparative analysis. From the PCR detection results, the estA, prtP, alsD, alsS, metK, metC and metB genes were present in all the strains tested. The bgl and gshR genes encoding β-glucosidase and glutathione reductase, respectively, were not detected in some strains. On the other hand, none of the tested strains possessed the genes encoding phenolic acid decarboxylase (padA), citrate permease (citP), citrate lyase (citD, citE and citF) and arginine deiminase pathway enzymes (arcA, arcB and arcC). The verification of PCR-generated fragments was performed by sequencing. GenBank database was used to search for homologous DNA sequences. Neighbour-joining trees based on nucleotide sequences of alsS, estA, metK and mleA genes were also constructed in order to study the phylogenetic relationship between Leuc. mesenteroides strains and closely related species. The phylogenetic analyses revealed that there are genetic heterogeneities between strains of Leuc. mesenteroides species. In conclusion, this study has improved our knowledge on the genetics of oenological strains of Leuc. mesenteroides and their genetic potential to contribute to certain wine aroma compounds.

Expressing β-glucosidase from Saccharomycopsis fibuligera in industrial ethanol producing yeast and evaluation of the expressing sufficiency

Recombinant β-glucosidase-expressing strains were constructed by integrating different copies of the BGL1 gene from Saccharomycopsis fibuligera into the chromosome of an industrial ethanol-producing strain of Saccharomyces cerevisiae. The engineered strains were evaluated for ethanol fermentation using cellobiose as sole carbon source. The results showed that most of the recombinant β-glucosidase produced by S. cerevisiae was secreted to the cell periplasm, with only a relatively small percentage released into the culture supernatant. Successful and unsuccessful utilization of cellobiose by the recombinant strains was determined by the copy number of BGL1 incorporated into the genome, which was related to specific β-glucosidase activity. Recombinant strain containing at least four copies of BGL1 produced 0.45 U ml−1 extracellularly, combined with 0.96 U mg−1 dry cell weight (DCW) cell pellet of β-glucosidase activity that could hydrolyze cellobiose effectively. Anaerobic growth (0.16 h−1) up to 0.82 g l−1 DCW of the engineered yeast was observed in medium containing 20 g l−1 cellobiose as sole carbohydrate source with concomitant ethanol production of up to 9.7 g l−1. This strain thus represents a promising alternative for efficient and cost-effective ethanol production.

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates ( μ max) could reach 0.03 and 0.05 h −1 under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobiose L −1 and produced 2.3 g ethanol L −1 in 48 h, while S. cerevisiae secreting β-glucosidase into culture broth used 3.6 g cellobiose L −1 and produced 1.5 g ethanol L −1 over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11 h −1) on cellobiose.

β-Glucosidase Activity andbglGene Expression ofOenococcus oeniStrains in Model Media and Cabernet Sauvignon Wine

Strains of Oenococcus oeni performing malolactic fermentation might prove an interesting source of β-glucosidase enzymes, which can release aromatic volatile compounds from their flavorless glycoconjugate precursors present in wine. Many studies have been conducted using strains cultured in winelike media, but little is known about the activity of these strains in real wine. In this work, real Cabernet Sauvignon red wine was used as the culture medium to test β-glucosidase activity and bgl gene expression of four O. oeni strains. Assays in wine were compared with winelike medium and mixtures of wine and MRS broth medium. β-Glucosidase activity was detected in both whole-cell and supernatant fractions for all four strains tested, although results varied depending on the conditions. The highest activity was found in wine (4 U/mg) and the activity was inhibited in the presence of fructose. To measure bgl expression, the RNA of O. oeni was extracted directly from red wine for the first time. Using real-time qPCR, the relative expression of the bgl gene was evaluated. The expression of this gene varied depending on the strain and it was overexpressed in samples of wine diluted with MRS. These results could be of importance to the wine industry and in studies of strain adaptation to wine for the selection of starters.

Translation efficiency of antiterminator proteins is a determinant for the difference in glucose repression of two β-glucoside phosphotransferase system gene clusters in Corynebacterium glutamicum R.

Corynebacterium glutamicum R has two β-glucoside phosphoenolpyruvate, carbohydrate phosphotransferase systems (PTS) encoded by bglF and bglF2 located in the respective clusters, bglF-bglA-bglG and bglF2-bglA2-bglG2. Previously, we reported that whereas β-glucoside-dependent induction of bglF is strongly repressed by glucose, glucose repression of bglF2 is very weak. Here, we reveal the mechanism behind the different effects of glucose on the two bgl genes. Deletion of the ribonucleic antiterminator sequence and transcriptional terminator located upstream of the translation initiation codon of bglF markedly relieved the glucose repression of a bglF-lacZ fusion, indicating that glucose affects the antitermination mechanism that is responsible for the β-glucoside-dependent induction of the bglF cluster. The glucose repression of bglF mRNA was also relieved by introducing a multicopy plasmid carrying the bglG gene encoding an antiterminator of the bglF cluster. Moreover, replacement of the GUG translation initiation codon of bglG with AUG was effective in relieving the glucose repression of bglF and bglG. Inversely, expression of bglF2 and bglG2 was subject to strict glucose repression in a mutant strain in which the AUG translation initiation codon of bglG2 encoding antiterminator of the bglF2 cluster was replaced with GUG. These results suggest that the translation initiation efficiency of the antiterminator proteins, at least in part, determines whether the target genes are subject to glucose repression. We also found that bglF expression was induced by glucose in the BglG-overexpressing strains, which may be explained by the ability of BglF to transport glucose.

Development of the cellulolytic fungus Trichoderma reesei strain with enhanced β-glucosidase and filter paper activity using strong artifical cellobiohydrolase 1 promoter

To improve the beta-glucosidase yield and total cellulase activity of Trichoderma reesei, extracellular beta-glucosidase (BGLI) was overexpressed under the control of the modified four-copy cbh1 promoter. Three transformants B2, B12 and B15 with successful integration of the bgl1 gene expression cassette were obtained, which exhibited 3.7-, 2.0- and 1.8-fold increase in beta-glucosidase activity than the parental strain RUT C30, respectively. The filter paper activities of the productive transformants B12 and B15 were improved by up to 130% and 55%, respectively. Saccharification of corncob residue with the crude enzyme dosages showed that the reducing sugar yields of B12 (5.59 mg/ml) and B15 (4.80 mg/ml) were 29% and 11% higher than that of RUT C30 (4.34 mg/ml), respectively. The present results proved that the modified four-copy cbh1 promoter was a useful tool for improving the cellulase activity of T. reesei, and the engineering strains developed in this study could be potentially used as promising cell factories for beta-glucosidase or cellulase production.

Revealing of Saccharomyces cerevisiae yeast cell wall proteins capable of binding thioflavin T, a fluorescent dye specifically interacting with amyloid fibrils

Proteins binding thioflavin T leading to its specific fluorescence were discovered in a fraction of noncovalently bound Saccharomyces cerevisiae yeast cell wall mannoproteins. Thioflavin-binding proteins display high resistance to trypsin digestion in solution. These data are the first experimental evidence for the presence of proteins whose properties are characteristic of amyloids in yeast cell wall, except for data on glucanotransferase Bgl2p that has amyloid properties. Our data suggest the anchoring of these proteins in the cell wall by a trypsin-sensitive part of the protein molecule. Experiments with a mutant strain devoid of the BGL2 gene suggest the compensation of absent amyloid-like protein Bgl2p by increase in contents of thioflavin-binding proteins in the cell wall.

Cloning, production, purification and preliminary crystallographic analysis of a glycosidase from the food lactic acid bacterium Lactobacillus plantarum CECT 748 T

In recent years, the exquisite stereoselectivity and high efficiency of carbohydrate-processing enzymes have been exploited for many biotechnological applications, including flavor enhancement in foods. In particular, much attention has been focused on the use of β-glucosidases for the enzymatic hydrolysis of flavorless glycoconjugates present in juices and wine beverages for the release aroma volatiles. With the aim to analyze a novel glycosidase with potential applications food industry we have produced and structurally characterized the Bgl glycosidase from the food lactic acid bacterium Lactobacillus plantarum. For that purpose, we have cloned and heterologously expressed the bgl gene (lp_3629) in Escherichia coli. The recombinant protein containing an amino terminal His 6 tag (Bgl) has been produced in a soluble form. Purified recombinant enzyme shows galactosidase activity against 4-nitrophenyl β- d-galactopyranoside but not glucosidase activity. Analytical size-exclusion gel filtration chromatography reveals that Bgl behaves in solution as a mixture of monomeric and a high-molecular weight assembly. Purified Bgl has been crystallized by the hanging-drop vapor-diffusion method at 18 °C. Diffraction data have been collected at ESRF to a resolution of 2.4 Å. The crystals belong to the space group C2 with unit-cell parameters a = 196.7, b = 191.7, c = 105.9, β = 102.7°. The structure refinement is in progress.

Application ofTrichoderma reeseiCellulase and Xylanase Promoters through Homologous Recombination for Enhanced Production of Extracellular β-Glucosidase I

One of the limiting factors for the application of Trichoderma reesei to degrade cellulosic biomass is its low beta-glucosidase activity, required to convert cellobiose to glucose. The egl3 and the xyn3 promoters were used to express beta-glucosidase 1 gene bgl1 through homologous recombination to improve the cellulose degradation ability of T. reesei. The recombinant strains expressing beta-glucosidase 1 (BGLI) under the control of either the egl3 or the xyn3 promoter had 4.0 and 7.5 fold higher beta-glucosidase activity than the native strain, which compares well to the finding that in wild-type T. reesei PC-3-7, the levels of egl3 and xyn3 mRNA expression were 6.0 and 12 fold higher respectively than that of bgl1. Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis of proteins secreted by the recombinant strains demonstrated that BGLI was overproduced. The increase in the transcription of bgl1 and the concomitant elevated level of BGLI in these recombinant strains were sufficient to degrade the cellobiose and cellotriose formed during the degradation of pretreated cedar powder.

Fate of the H-NS–Repressed bgl Operon in Evolution of Escherichia coli

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS–repressed locus is the bgl (aryl-β,D-glucoside) operon of E. coli. This locus is “cryptic,” as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli.

A novel mutant gene for (1-3, 1-4)-.BETA.-D-glucanless grain on barley (Hordeum vulgare L.) chromosome 7H

(1-3, 1-4)-β-D-glucan contained in barley (Hordeum vulgare L.) grains is a main component of endosperm cell walls and constitutes the cell wall matrix with arabinoxylan. A monofactorial recessive mutant gene for (1-3, 1-4)-β-D-glucanless grain was found and the new gene was designated as bgl (= (1-3, 1-4)-beta-D-glucanless grain). A linkage was found between the bgl gene and the naked caryopsis (nud) gene, and it was mapped to the centromeric region of chromosome 7H. Phenotypes in bgl cosegregated with the polymorphisms in HvCslF6, a member of cellulose synthase-like HvCslF gene family, indicating that bgl was caused by a mutation in the HvCslF6 locus. In order to clarify the characteristics of (1-3, 1-4)-β-D-glucanless barley grains, a near-isogenic line (NIL) was developed by backcrossing the Japanese two-rowed cultivar ‘Nishinohoshi’ as a recurrent parent. The NIL with bgl completely lacked (1-3, 1-4)-β-D-glucan in both the endosperm and aleurone layer cell walls. Microscopic analysis revealed that the NIL had thin endosperm cell walls. It also showed a softer grain texture and many more broken grains during the pearling process than the recurrent parent. The soft and friable grain texture of the NIL was probably caused by the thin endosperm cell walls.

Structural Mechanism of Signal Transduction between the RNA-binding Domain and the Phosphotransferase System Regulation Domain of the LicT Antiterminator

LicT belongs to a family of bacterial transcriptional antiterminators, which control the expression of sugar-metabolizing operons in response to phosphorylations by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Previous studies of LicT have revealed the structural basis of RNA recognition by the dimeric N-terminal co-antiterminator (CAT) domain on the one hand and the conformational changes undergone by the duplicated regulation domain (PRD1 and PRD2) upon activation on the other hand. To investigate the mechanism of signal transduction between the effector and regulation modules, we have undertaken the characterization of a fragment, including the CAT and PRD1 domains and the linker in-between. Comparative experiments, including RNA binding assays, NMR spectroscopy, limited proteolysis, analytical ultracentrifugation, and circular dichroism, were conducted on native CAT-PRD1 and on a constitutively active CAT-PRD1 mutant carrying a D99N substitution in PRD1. We show that in the native state, CAT-PRD1 behaves as a rather unstable RNA-binding deficient dimer, in which the CAT dimer interface is significantly altered and the linker region is folded as a trypsin-resistant helix. In the activated mutant form, the CAT-PRD1 linker becomes protease-sensitive, and the helix content decreases, and the CAT module adopts the same dimeric conformation as in isolated CAT, thereby restoring the affinity for RNA. From these results, we propose that a helix-to-coil transition in the linker acts as the structural relay triggered by the regulatory domain for remodeling the effector dimer interface. In essence, the structural mechanism modulating the LicT RNA antitermination activity is thus similar to that controlling the DNA binding activity of dimeric transcriptional regulators.

Lactic fermentation of cellobiose by a yeast strain displaying β-glucosidase on the cell surface

The Aspergillus aculeatus beta-glucosidase 1 (bgl1) gene was expressed in a lactic-acid-producing Saccharomyces cerevisiae strain to enable lactic fermentation with cellobiose. The recombinant beta-glucosidase enzyme was expressed on the yeast cell surface by fusing the mature protein to the C-terminal half region of the alpha-agglutinin. The beta-glucosidase expression plasmids were integrated into the genome. Three strong promoters of S. cerevisiae, the TDH3, PGK1, and PDC1 promoters, were used for beta-glucosidase expression. The specific beta-glucosidase activity varied with the promoter used and the copy number of the bgl1 gene. The highest activity was obtained with strain PB2 that possessed two copies of the bgl1 gene driven by the PDC1 promoter. PB2 could grow on cellobiose and glucose minimal medium at the same rate. Fermentation experiments were conducted in non-selective-rich media containing 95 g l(-1) cellobiose or 100 g l(-1) glucose as a carbon source under microaerobic conditions. The maximum rate of L-lactate production by PB2 on cellobiose (2.8 g l(-1) h(-1)) was similar to that on glucose (3.0 g l(-1) h(-1)). This indicates that efficient fermentation of cellobiose to L-lactate can be accomplished using a yeast strain expressing beta-glucosidase from a mitotically stable genomic integration plasmid.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells.

BackgroundContagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-β-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204.ResultsStrains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., β-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-β-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of β-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204.ConclusionOur results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of β-D-glucosides, thus contributing in some extent to mycoplasmaemia.