Bgl Genes Research Articles

The role of intracellular β-glucosidase in cellulolytic response induction in filamentous fungus Penicillium verruculosum

In this study, CRISPR/Cas9 genome editing was used to knockout the bgl2 gene encoding intracellular β-glucosidase filamentous fungus Penicillium verruculosum. This resulted in a dramatic reduction of secretion of cellulolytic enzymes. The study of P. verruculosum Δbgl2 found that the transcription of the cbh1 gene, which encodes cellobiohydrolase 1, was impaired when induced by cellobiose and cellotriose. However, the transcription of the cbh1 gene remains at level of the host strain when induced by gentiobiose. This implies that gentiobiose is the true inducer of the cellulolytic response in P. verruculosum, in contrast to Neurospora crassa where cellobiose acts as an inducer.

A chromosome-scale Rhubarb (Rheum tanguticum) genome assembly provides insights into the evolution of anthraquinone biosynthesis

Rhubarb is the collective name for various perennial plants from the genus Rheum L. and the Polygonaceae family. They are one of the most ancient, commonly used, and important herbs in traditional Chinese medicine. Rhubarb is a major source of anthraquinones, but how they are synthesized remains largely unknown. Here, we generate a genome sequence assembly of one important medicinal rhubarb R. tanguticum at the chromosome level, with 2.76 Gb assembled into 11 chromosomes. The genome is shaped by two recent whole-genome duplication events and recent bursts of retrotransposons. Metabolic analyses show that the major anthraquinones are mainly synthesized in its roots. Transcriptomic analysis reveals a co-expression module with a high correlation to anthraquinone biosynthesis that includes key chalcone synthase genes. One CHS, four CYP450 and two BGL genes involved in secondary metabolism show significantly upregulated expression levels in roots compared with other tissues and clustered in the co-expression module, which implies that they may also act as candidate genes for anthraquinone biosynthesis. This study provides valuable insights into the genetic bases of anthraquinone biosynthesis that will facilitate improved breeding practices and agronomic properties for rhubarb in the future.

The effect of deletions of genes encoding Pho3p and Bgl2p on the polyphosphate level, stress adaptation and attachment of these proteins in <i>Saccharomyces cerevisiae</i> cell wall

Inorganic polyphosphates (polyP), according to literature data, are involved in the regulatory processes of molecular complex of the Saccharomyces cerevisiae cell wall (CW). The aim of the work was to reveal relationship between polyP, acid phosphatase Pho3p, and the major CW protein, glucanosyl transglycosylase Bgl2p, which is the main glucan-remodelling enzyme with amyloid properties. It has been shown that the yeast cells with deletion of the PHO3 gene contain more high molecular alkali-soluble polyP and are also more resistant to exposure to alkali and manganese ions compared to the wild type strain. This suggests that Pho3p is responsible for hydrolysis of the high molecular polyP on the surface of yeast cells, and these polyP belong to the stress resistance factors. The S. cerevisiae strain with deletion of the BGL2 gene is similar to the Δpho3 strain both in the level of high molecular alkali-soluble polyP and in the increased resistance to alkali and manganese. Comparative analysis of the CW proteins demonstrated correlation between the extractability of the acid phosphatase and Bgl2p, and also revealed a change in the mode of Bgl2p attachment to the CW of the strain lacking Pho3p. It has been suggested that Bgl2p and Pho3p are able to form a metabolon or its parts that connects biogenesis of the main structural polymer of the CW, glucan, and catabolism of an important regulatory polymer, polyphosphates.

Effect of Deletions of the Genes Encoding Pho3p and Bgl2p on Polyphosphate Level, Stress Adaptation, and Attachments of These Proteins to Saccharomyces cerevisiae Cell Wall.

Inorganic polyphosphates (polyP), according to literature data, are involved in the regulatory processes of molecular complex of the Saccharomyces cerevisiae cell wall (CW). The aim of the work was to reveal relationship between polyP, acid phosphatase Pho3p, and the major CW protein, glucanosyltransglycosylase Bgl2p, which is the main glucan-remodelling enzyme with amyloid properties. It has been shown that the yeast cells with deletion of the PHO3 gene contain more high molecular alkali-soluble polyP and are also more resistant to exposure to alkali and manganese ions compared to the wild type strain. This suggests that Pho3p is responsible for hydrolysis of the high molecular polyP on the surface of yeast cells, and these polyP belong to the stress resistance factors. The S. cerevisiae strain with deletion of the BGL2 gene is similar to the Δpho3 strain both in the level of high molecular alkali-soluble polyP and in the increased resistance to alkali and manganese. Comparative analysis of the CW proteins demonstrated correlation between the extractability of the acid phosphatase and Bgl2p, and also revealed a change in the mode of Bgl2p attachment to the CW of the strain lacking Pho3p. It has been suggested that Bgl2p and Pho3p are able to form a metabolon or its parts that connects biogenesis of the main structural polymer of the CW, glucan, and catabolism of an important regulatory polymer, polyphosphates.

Exogenous 24-epibrassinolide boosts plant growth under alkaline stress from physiological and transcriptomic perspectives: The case of broomcorn millet (Panicum miliaceum L.)

Land alkalization is an abiotic stress that affects global sustainable agricultural development and the balance of natural ecosystems. In this study, two broomcorn millet cultivars, T289 (alkaline-tolerant) and S223 (alkaline-sensitive), were selected to investigate the response of broomcorn millet to alkaline stress and the role of brassinolide (BR) in alkaline tolerance. Phenotypes, physiologies, and transcriptomes of T289 and S223 plants under only alkaline stress (AS) and alkaline stress with BR (AB) were compared. The results showed that alkaline stress inhibited growth, promoted the accumulation of soluble sugars and malondialdehyde, enhanced electrolyte leakage, and destroyed the integrity of broomcorn millet stomata. In contrast, BR lessened the negative effects of alkaline stress on plants. Transcriptome sequencing analysis showed that relative to control groups (CK, nutrient solution), in AS groups, 21,113 and 12,151 differentially expressed genes (DEGs) were identified in S223 and T289, respectively. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed various terms and pathways related to metabolism. Compared to S223, alkaline stress strongly activated the brassinosteroid biosynthesis pathway in T289. Conversely, ARF, TF, and TCH4, associated with cell growth and elongation, were inhibited by alkaline stress in S223. Moreover, alkaline stress induced the activation of the mitogen-activated protein kinase (MAPK) pathway, the abscisic acid signaling pathway that initiates stomatal closure, as well as the starch and sucrose metabolism. The EG and BGL genes, which are associated with cellulose degradation, were notably activated. BR enhanced alkaline tolerance, thereby alleviating the transcriptional responses of the two cultivars. Cultivar T289 is better in alkalized regions. Taken together, these results reveal how broomcorn millet responds to alkaline stress and BR mitigates alkaline stress, thus promoting agriculture in alkalized regions.

Selection of optimal culture conditions for expression of recombinant beta-glucosidase in Escherichia coli

Beta-glucosidase (BGL) is an enzyme involved in the hydrolysis of cellulose and plays an important role in many biological processes. This enzyme is widely available in animals, plants, and microorganisms. Expression of the recombinant enzyme in Escherichia coli is considered a suitable choice for high production via a fast growth rate and feasible manipulation. In this study, we optimised some conditions for the high soluble production of recombinant beta-glucosidase whose gene sequence was mined from the DNA metagenome of Cuc Phuong humus surrounding white rot fungi. The gene bgl was cloned in pET 22b(+) and expressed in the E. coli Rosetta 1 strain. Production of the recombinant BGL was examined at temperature ranges from 18 to 37oC. The recombinant BGL was obtained as a soluble form at low temperatures, and the optimal temperature was 25°C. In comparison to LB, TB, SB, PE media, mTB rich medium in presence of glucose produced the most BGL. Besides, the results assessing the inducer condition showed that the best IPTG concentration for producing the BGL was 0.3 mM IPTG. Furthermore, some fermentation conditions affecting the level of BGL production were assessed as the induction point and harvesting time. The BGL production was the most suitable when the cell at mid-log phase with an OD600 1 and harvesting time after 4 hours of induction. Importantly, the recombinant BGL had good activity on the esculin substrate plate. Based on the selected conditions, the recombinant BGL could be produced high amount to facilitate for future purification and characterization of the recombinant BGL.

Biochemical characterization of a novel glucose-tolerant GH3 β-glucosidase (Bgl1973) from Leifsonia sp. ZF2019.

Beta-glucosidase (Bgl) is an enzyme with considerable food, beverage, and biofuel processing potential. However, as many Bgls are inhibited by their reaction end product glucose, their industrial applications are greatly limited. In this study, a novel Bgl gene (Bgl1973) was cloned from Leifsonia sp. ZF2019 and heterologously expressed in E. coli. Sequence analysis and structure modeling revealed that Bgl1973 was 748 aa, giving it a molecular weight of 78kDa, and it showed high similarity with the glycoside hydrolase 3 (GH3) family Bgls with which its active site residues were conserved. By using pNPGlc (p-nitrophenyl-β-D-glucopyranoside) as substrate, the optimum temperature and pH of Bgl1973 were shown to be 50°C and 7.0, respectively. Bgl1973 was insensitive to most metal ions (12.5mM), 1% urea, and even 0.1% Tween-80. This enzyme maintained 60% of its original activity in the presence of 20% NaCl, demonstrating its excellent salt tolerance. Furthermore, it still had 83% residual activity in 1M of glucose, displaying its outstanding glucose tolerance. The Km, Vmax, and kcat of Bgl1973 were 0.22mM, 44.44μmol/min mg, and 57.78s-1, respectively. Bgl1973 had a high specific activity for pNPGlc (19.10 ± 0.59 U/mg) and salicin (20.43 ± 0.92 U/mg). Furthermore, molecular docking indicated that the glucose binding location and the narrow and deep active channel geometry might contribute to the glucose tolerance of Bgl1973. Our results lay a foundation for the studying of this glucose-tolerant β-glucosidase and its applications in many industrial settings. KEY POINTS: • A novel β-glucosidase from GH3 was obtained from Leifsonia sp. ZF2019. • Bgl1973 demonstrated excellent glucose tolerance. • The glucose tolerance of Bgl1973 was explained using molecular docking analysis.

Beta-Glucosidase 1 (bgl1) Gene Analysis in Mutant and Wild-type of Penicillium sp. ID10-T065

In the previous study, Penicillium sp. ID10-T065 has been mutated using ultraviolet (UV), ethyl methyl sulfonate (EMS) and the combination of UV-EMS to increase Beta-glucosidase (bgl) activity. There were three mutants selected, UV13 (UV mutant), EM31 (EMS mutant), and UM23 (UV-EMS mutant). This study examined the mutations in the bgl gene encoding (bgl1) as well as sequence differences between mutants and wild-type of Penicillium sp. ID10-T065. The gene analysis was performed by PCR amplification and sequencing of the bgl1 gene. The results of bgl1 gene sequences (600 bp) from mutants were aligned with the wild-type, it was discovered that there were base alterations from position 2025 to 2050. Mutant UV13 showed the highest base alterations (7 bases) which occurred at position 2027 (T→C); 2036 (T→G); 2040 (T→G); 2047 (G→C); and 2048-2050 (TTG→GGA). Mutant EM31 showed alterations in five bases at positions 2034 (G→A), 2036 (T→G), 2037 (G→C), 2044 (G→C), and 2047 (G→T). Mutant UM23 showed two base alterations at position 2025 (T→A) and 2037 (G→C). UV irradiation and EMS mutation resulted in transition and transversion DNA, whereas the combination of UV-EMS mutation resulted in transversion mutations. Base alterations in UV13 and EM31 mutants, causing missense and silent mutation, while in UM23 mutant only silent mutations occur. The bgl1 gene analysis showed that mutation using UV light was more effective than using EMS or a combination of UV-EMS in Penicillium sp. ID10-T065.

Morphological, molecular and phytochemical variations induced by colchicine and EMS chemical mutagens in Crocus sativus L.

Crocus sativus L., also known as saffron, is one of the most important medicinal and spice plants throughout the world. The plant is a rich source of apocarotenoids such as crocetin esters, picrocrocin, and safranal. The purpose of this study was to investigate the effect of colchicine and ethyl methanesulfonate (EMS) mutagens on possible inducing new variation in C. sativus. Accordingly, corms were exposed to EMS (0.1% and 0.2%) and colchicine (0.05% and 0.025%) for three incubation times. The lowest survival rate of corms was related to EMS treatments. The relative expression of ALDH, BGL, and CCD2 genes under 0.025% colchicine treatment for 12 h revealed a 2 fold increase compared with the control. The flow cytometric measurements (FCM) of the nuclear DNA content of the colchicine-treated plants did not reveal any significant changes in 2C DNA content. The results, manifest the potential of mutagens to create new variations in the plant.

Regulation Mechanism of Exogenous Brassinolide on Bulbil Formation and Development in Pinellia ternata.

The bulbil is the propagative organ of the P. ternata, which has a great effect on the yield of P. ternata. It is well known that plant hormones play important roles in bulbil formation and development. However, there is not clear about brassinolide (BR) regulation on bulbil formation and development. In this study, we revealed the effects of BR and BR biosynthesis inhibitors (propiconazole, Pcz) application on the histological observation, starch and sucrose metabolism, photosynthesis pathway, and hormone signaling pathway of P. ternata. The results showed that BR treatment reduced starch catabolism to maltodextrin and maltose in bulbil by decreasing BAM and ISA genes expression and increased cellulose catabolism to D-glucose in bulbil by enhancing edg and BGL genes expression. BR treatment enhanced the photosynthetic pigment content and potential maximum photosynthetic capacity and improved the photoprotection ability of P. ternata by increasing the dissipation of excess light energy to heat, thus reduced the photodamage in the PSII center. BR treatment increased the GA and BR content in bulbil of P. ternata, and decreased the ABA content in bulbil of P. ternata. Pcz treatment increased the level of GA, SL, ABA, and IAA in bulbil of P. ternata. BR regulated the signal transduction of BR, IAA, and ABA to regulate the formation and development of bulbil in P. ternata. These results provide molecular insight into BR regulation on bulbil formation and development.

The role of a β-1,3-1,4-glucanase derived from Bacillus amyloliquefaciens FS6 in the protection of ginseng against Botrytis cinerea and Alternaria panax

Gray mold and Alternaria blight, caused by Botrytis cinerea and Alternaria panax, respectively, are the most common diseases of ginseng (Panax ginseng), and cause significant yield losses and reduced quality. Previous field experiments revealed that treatment of ginseng with Bacillus amyloliquefaciens FS6 provided an excellent control against these two diseases, although its mechanism of action was unclear. In the present study, we demonstrated that the bacteria-free fermentation broth obtained from B. amyloliquefaciens FS6, a FS6-bgl strain of B. amyloliquefaciens overexpressing the bgl gene (encoding a β-1,3-1,4-glucanase), and purified recombinant β-1,3-1,4-glucanase can inhibit B. cinerea and A. panax both in vitro and in planta. The recombinant β-1,3-1,4-glucanase protein, BG-Ba, significantly inhibited the mycelial growth and spore germination of both A. panax and B. cinerea. Bacteria-free fermentation broth derived from FS6-bgl cultures overexpressing the bgl gene also inhibited mycelial growth and spore germination of A. panax and B. cinerea. The level of inhibition in the fermentation broth of the wild-type vs. the recombinant strain increased from 9 to 12% and 8 to 12% for the two pathogens, respectively. Inoculation assays on ginseng leaves indicated that the BG-Ba protein and the bacteria-free fermentation broth of the FS6-bgl strain provided significant protective effect against infection by A. panax and B. cinerea. The present study provides new information on the antimicrobial activity of FS6 and reveals that the use of β-1,3-1,4-glucanase alone has good potential for the management of ginseng diseases.

The combined effect of protein with amyloid properties Bgl2p and other components of the cell walls of yeast Saccharomyces cerevisiae on the condition of the skin and behavior of mice

The aim of the study was to carry out primary screening of changes occurring in the mice body as a result of intraperitoneal injections of a suspension of the cell walls (CW) of yeast Saccharomyces cerevisiae containing and not containing a protein with amyloid properties Bgl2p, and an extract from CW, in which the specified protein was present or removed. The studies were performed on CFW and C57BL/6 mouse lines using CW of wild-type strain of S.cerevisiae and a strain with the deletion of BGL2 gene encoding this protein. The presence of Bgl2p in the injection preparation was assessed according to immunoblotting data with antibodies to this protein. The ability to Bgl2p fibrillation was evaluated by electron microscopy and fluorescence analysis using Thioflavin T. It was shown that in all cases when Bgl2p protein was present in the injected drug, mice developed skin disorders of varying severity. In the case of CW injection obtained from wild-type yeast cells and treated with proteinase K, changes in animal behavior were observed, namely, a decrease in the parameters of their research activity. Data characterizing the ability of the Bgl2p protein to form fibrils of various morphologies after isolation from CW are presented. The conclusion is made about the Bgl2p toxic effect on the body of mice, the possibility of induction in animals of amyloidosis caused by contact with the Bgl2p protein is assumed. Other components of yeast CW toxic to animals have not been identified, however, their enhancing effect on the manifestation of toxic properties of Bgl2p is not excluded.

Engineering Trichoderma reesei for Hyperproduction of Cellulases on Glucose to Efficiently Saccharify Pretreated Corncobs.

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is widely used as a cellulase producer in the industry. Herein, we describe the rational engineering of the publicly available T. reesei QM9414 strain to achieve a remarkable high-level production of cellulase on glucose. Overexpression of the key cellulase regulator XYR1 by the copper-repressible promoter Ptcu1 was first implemented to achieve a full cellulase production in the context of catabolite repression (CCR) while eliminating the requirement of inducing sugars for enzyme production. The T. reesei bgl1 gene was further overexpressed to compensate for its low β-glucosidase activity on glucose. This overexpression resulted in a 102% increase in FPase activity compared with the CCR-released RUT-C30 strain cultured on Avicel. Moreover, the saccharification efficiency toward pretreated corncob residues by crude enzymes from the engineered strain on glucose increased by 85% compared with that treated by enzymes from RUT-C30 cultivated on Avicel. The engineered T. reesei strain thus shows great potential as a viable alternative to deliver commercial cellulases after further optimization for efficient saccharification of agricultural waste.

\u03b2-Glucosidase genes differentially expressed during composting

BackgroundCellulose degradation by cellulase is brought about by complex communities of interacting microorganisms, which significantly contribute to the cycling of carbon on a global scale. β-Glucosidase (BGL) is the rate-limiting enzyme in the cellulose degradation process. Thus, analyzing the expression of genes involved in cellulose degradation and regulation of BGL gene expression during composting will improve the understanding of the cellulose degradation mechanism. Based on our previous research, we hypothesized that BGL-producing microbial communities differentially regulate the expression of glucose-tolerant BGL and non-glucose-tolerant BGL to adapt to the changes in cellulose degradation conditions.ResultsTo confirm this hypothesis, the structure and function of functional microbial communities involved in cellulose degradation were investigated by metatranscriptomics and a DNA library search of the GH1 family of BGLs involved in natural and inoculated composting. Under normal conditions, the group of non-glucose-tolerant BGL genes exhibited higher sensitivity to regulation than the glucose-tolerant BGL genes, which was suppressed during the composting process. Compared with the expression of endoglucanase and exoglucanase, the functional microbial communities exhibited a different transcriptional regulation of BGL genes during the cooling phase of natural composting. BGL-producing microbial communities upregulated the expression of glucose-tolerant BGL under carbon catabolite repression due to the increased glucose concentration, whereas the expression of non-glucose-tolerant BGL was suppressed.ConclusionOur results support the hypothesis that the functional microbial communities use multiple strategies of varying effectiveness to regulate the expression of BGL genes to facilitate adaptation to environmental changes.

Assessment of β-D-glucosidase activity and bgl gene expression of Oenococcus oeni SD-2a.

Glycosidases enhance flavor during wine-making by mediating the enzymatic release of aroma molecules. In order to better understand the aroma enhancement potential of Oenococcus oeni SD-2a, β-D-glucosidase (βG) activities in the culture supernatant, whole cells, and disrupted cell lysate were assessed at mid log, late log and stationary growth phase. The enzymatic activity was also compared further from cell cultures with 5 different carbon sources (glucose, cellobiose, arbutin, glucose and cellobiose, glucose and arbutin) at late log phase. Correspondingly, expression levels of 3 bgl genes, OEOE-0224, OEOE-1210, and OEOE-1569 were investigated from cell cultures of the 3 growth phases, and the 5 cell cultures with different carbon sources. Finally, the volatile aroma compounds released by O. oeni SD-2a in synthetic wines with natural glycosides were evaluated by GC-MS. Results showed βG of O. oeni SD-2a was not extracellular enzyme, and the location of it didn’t change with the change of growth phase and carbon source studied. βG activities in the whole cells and disrupted cell lysate were similar and constant at the 3 growth phases. As for the carbon sources, βG activities of whole cells and disrupted lysate were positively affected by cellobiose. While arbutin displayed positive and negative effect on βG activity of whole cells and disrupted lysate, respectively. It is probably that bgl genes OEOE-0224 and OEOE-1210 were related to βG activity of SD-2a whole cells, while OEOE-1569 was responsible for βG activity of disrupted lysate. More kinds of volatile compounds and higher total concentration were released by SD-2a in synthetic wine compared with control. Thus, SD-2a showed a great potential for flavor enhancement under wine-like conditions. This study provides more information for further study of βG activity from O. oeni SD-2a.

Repetitive δ-integration of a cellulase-encoding gene into the chromosome of an industrial Angel Yeast-derived strain by URA3 recycling.

Genetic modification of industrial yeast strains often faces more difficulties than that of laboratory strains. Thus, new approaches are still required. In this research, the Angel Yeast-derived haploid strain Kα was genetically modified by multiple rounds of δ-integration, which was achieved via URA3 recycling. Three δ-integrative plasmids, pGδRU, pGδRU-BGL, and pGδRU-EG, were first constructed with two 167bp δ sequences and a repeat-URA3-repeat fragment. Then, the δ-integrative strains containing the bgl1 or egl2 gene were successfully obtained by one-time transformation of the linearized pGδRU-BGL or pGδRU-EG fragment, respectively. Their counterparts in which the URA3 gene was looped out were also easily isolated by selection for growth on 5´-fluoroorotic acid plates, although the ratio of colonies lacking URA3 to the total number of colonies decreased with increasing copy number of the corresponding integrated cellulase-encoding gene. Similar results were observed during the second round of δ-integration, in which the δ-integration strain Kα(δ::bgl1-repeat) obtained from the first round was transformed with a linearized pGδRU-EG fragment. After 10 rounds of cell growth and transfer to fresh medium, the doubling times and enzyme activities of Kα(δ::bgl1-repeat), Kα(δ::egl2-repeat), and Kα(δ::bgl1-repeat)(δ::egl2-repeat) showed no significant change and were stable. Further, their maximum ethanol concentrations during simultaneous saccharification and fermentation of pretreated corncob over a 7-day period were 46.35, 33.13, and 51.77g/L, respectively, which were all substantially higher than the parent Kα strain. Thus, repetitive δ-integration with URA3 recycling can be a feasible and valuable method for genetic engineering of Angel Yeast. These results also provide clues about some important issues related to δ-integration, such as the structural stability of δ-integrated genes and the effects of individual integration-site locations on gene expression. Further be elucidation of these issues should help to fully realize the potential of δ-integration-based methods in industrial yeast breeding.

Cloning and Expression of Bacillus pumilis Bg57 β-Glucanase Gene in Pichia pastoris: Purification and Characteristics of Recombinant Enzyme

The isolation, heterologous gene expression, and characteristics of a new β-glucanase from Bacillus pumilus is described. The bgl1 gene from the Bacillus pumilus Bg57 VKPM В-13195 strain, which consists of 729 nucleotides, encodes a secreted endo-β-l,3(4)-D-glucanase (EC 3.2.1.6) containing 214 amino acids and 28 residues of the putative signal peptide in the N-terminal area. The nucleotide sequence of bgl1 and amino acid sequence of the mature Bgl protein have the highest homology (89 and 95%, respectively) with the sequences for β-l,3-l,4-glucanase from Bacillus licheniformis. A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. The purified recombinant enzyme of Bgl was active towards barley β-glucan and lichenin. With barley β-glucan, the optimal pH and temperature were shown to be 6.0 and 50°C, respectively; Km and Vmax were 2.2 mg/mL and 3036.4 μmol/(min mg), respectively. The recombinant protein Bgl showed a high specific activity, thermal stability, and resistance to digestive enzymes. Other characteristics of the recombinant protein, including pH stability and sensitivity to metal ions and chemical reagents, were also determined.

Cloning, purification and study of recombinant GH3 family β-glucosidase from Penicillium verruculosum

A novel bgl1 gene, encoding GH3 family β-glucosidase from Penicillium verruculosum (PvBGL), was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain under the control of the strong xylA gene promoter. The recombinant rPvBGL was purified and their properties were studied in comparison with those of rAnBGL from Aspergillus niger expressed previously in the same fungal host. The rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data) and displayed the enzyme maximum activity at pH 4.6 and 65 °C. The enzyme half-life time at 60 °C was found to be 87 min. Unlike the rAnBGL, the rPvBGL was not adsorbed on microcrystalline cellulose, which gives the latter enzyme an advantage in cellulose conversion with a longer time of hydrolysis.

Construction of novel Trichoderma orientalis EU7-22, strain with improved β-glucosidase activity by cellobiohydrolase I promotor optimization from Trichoderma reesei

Abstract An upstream region of cellobiohydrolase I gene promoter was modified from Trichoderma reesei to enhance heterologous gene expression. The base pairs from −677 to −724 were deleted indicating glucose repressor binding sites. Trichoderma reesei is considered an important workplace for homologous/heterologous genes expressions under the modified cbh1 promoter. However, constitutive expression is desirable in some cases over inducible expression that leads to production of undesirable cellulase components as byproduct. Here we report that the reporter β-glucosidase gene (bgl1) from Aspergillus niger BE-2 was cloned and recombined with cbh1 promoter followed by terminator trpC cloned from fungal expression vector, pUR5750. The expression cassette was ligated to binary vector and transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT) using hygromycin B resistance gene as the screening marker. Four transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4 were screened. Compared with the host strain EU7-22, the enzyme activities of filter paper (FPA) and β-glucosidase (BG) of transformant Bgl-3 increased by 39% and 47.1% under repression condition respectively. The expression level of Bgl-3 was found 7.9 folds increase over host strain EU7-22. Our results indicate that cbh1 is a strong promoter and has successfully driven over-expression of bgl1 gene in EU7-22 under repression condition and has surprisingly improved the efficiency of heterologous expression of cellulase genes in EU7-22.

Selection-free markerless genome manipulations in the polyploid bacterium Thermus thermophilus.

A genome manipulation approach based on double-crossover homologous recombination was developed in the polyploid model organism Thermus thermophilus HB27 without the use of any selectable marker. The method was established and optimized by targeting the megaplasmid-encoded β-glucosidase gene bgl. When linear and supercoiled forms of marker-free suicide vector were used for transformations, the frequencies of obtaining apparent Bgl- mutant were 10- 5 and 10- 3, respectively; while the frequency could reach 10- 2 when transformation with concatemer form of the same vector. All randomly selected Bgl- colonies from the transformations were found to be true bgl knockout mutants. Thus, markerless gene deletion mutants could be constructed in T. thermophilus by the direct selection-free method. The functionality of this approach was further demonstrated by deletion of one chromosomal locus (TTC_0340-0341) as well as by generation of a reporter strain for the phytoene synthase promoter (PcrtB), homozygous mutants of the both targets could also be detected with a frequency of approximately 10- 2. During the genome modification process, heterozygous cells carrying two different alleles at a same locus (e.g., bgl and pyrE) could also be generated. However, in the absence of selection pressure, these strains could rapidly convert to homozygous strains containing only one of the two alleles. This indicated that allele segregation could occur in the heterozygous T. thermophilus cells, which probably explained the ease of obtaining homozygous gene deletion mutants with high frequency (10- 2) in the polyploid genomic background, as after the mutant allele had been introduced to the target region, allele segregation would lead to homozygous mutant cells. This marker-free genome manipulation approach does not require phenotype-based screens, and is applicable in gene deletion and tagging applications.