Beta-glucosidase Gene Research Articles

Long-insert sequence capture detects high copy numbers in a defence-related beta-glucosidase gene βglu-1 with large variations in white spruce but not Norway spruce

Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genes Ugt5/Ugt5b and βglu-1 may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm. This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number of βglu-1-like, Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40 P. glauca individuals from eight and nine provenances across Europe and North America respectively. We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity of βglu-1 and Ugt5 genes. We observed very large copy numbers of βglu-1, with up to 381 copies in a single P. glauca individual. We observed among-provenance CNV of βglu-1 in P. glauca but not P. abies. Ugt5b was predominantly single-copy in both species. This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences.

Genome-Wide Transcriptome Analysis Revealing the Genes Related to Sugar Metabolism in Kernels of Sweet Corn.

Sugar metabolism influences the quality of sweet corn (Zea mays var. saccharate Sturt) kernels, which is a major goal for maize breeding. In this study, the genome-wide transcriptomes from two supersweet corn cultivars (cv. Xuetian 7401 and Zhetian 11) with a nearly two-fold difference in kernel sugar content were carried out to explore the genes related to kernel sugar metabolism. In total, 45,748 differentially expressed genes (DEGs) in kernels and 596 DEGs in leaves were identified. PsbS, photosynthetic system II subunit S, showed two isoforms with different expression levels in leaf tissue between two cultivars, indicating that this gene might influence sugar accumulation in the kernel. On the other hand, hexokinases and beta-glucosidase genes involved in glycolysis, starch and sucrose metabolism were found in developing kernels with a genome-wide transcriptome analysis of developing kernels, which might contribute to the overaccumulation of water-soluble polysaccharides and an increase in the sweetness in the kernels of Xuetian 7401. These results indicated that kernel sugar accumulation in sweet corn might be influenced by both photosynthesis efficiency and the sugar metabolism rate. Our study supplied a new insight for breeding new cultivars with high sugar content and laid the foundation for exploring the regulatory mechanisms of kernel sugar content in corn.

A Novel Beta-Glucosidase Gene for Plant Type Was Identified by Genome-Wide Association Study and Gene Co-Expression Analysis in Widespread Bermudagrass.

Bermudagrass (Cynodon spp.) is one of the most widely distributed warm-season grasses globally. The growth habits and plant type of bermudagrass are strongly associated with the applied purpose of the landscape, livestock, and eco-remediation. Therefore, persistent efforts are made to investigate the genetic basis of plant type and growth habits of bermudagrass. Here, we dissect the genetic diversity of 91 wild bermudagrass resources by genome-wide association studies (GWAS) combined with weighted gene co-expression analysis (WGCNA). This work is based on the RNA-seq data and the genome of African bermudagrass (Cynodon transvaalensis Burtt Davy). Sixteen reliable single-nucleotide polymorphisms (SNPs) in transcribed regions were identified to be associated with the plant height and IAA content in diverse bermudagrass by GWAS. The integration of the results from WGCNA indicates that beta-glucosidase 31 (CdBGLU31) is a candidate gene underlying a G/A SNP signal. Furthermore, both qRT-PCR and correlation coefficient analyses indicate that CdBGLU31 might play a comprehensive role in plant height and IAA biosynthesis and signal. In addition, we observe lower plant height in Arabidopsis bglu11 mutants (homologs of CdBGLU31). It uncovers the breeding selection history of different plant types from diverse bermudagrass and provides new insights into the molecular function of CdBGLU31 both in plant types and in IAA biosynthetic pathways.

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test.

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.

Draft genome of the glucose tolerant β-glucosidase producing rare Aspergillus unguis reveals complete cellulolytic machinery with multiple beta-glucosidase genes

Draft genome sequence of the glucose tolerant beta glucosidase (GT-BGL) producing rare fungus Aspergillus unguis NII 08,123 was generated through Next Generation Sequencing (NGS). The genome size of the fungus was estimated to be 37.1 Mb. A total of 3116 contigs were assembled using SPades, and 15,161 proteins were predicted using AUGUSTUS 3.1. Among them, 13,850 proteins were annotated using UniProt. Distribution of CAZyme genes specifically those encoding lignocellulose degrading enzymes were analyzed and compared with those from the industrial cellulase producer Trichoderma reesei in view of the huge differences in detectable enzyme activities between the fungi, despite the ability of A. unguis to grow on lignocellulose as sole carbon source. Full length gene sequence of the inducible GT-BGL could be identified through tracing back from peptide mass fingerprint. A total of 403 CAZymes were predicted from the genome, which includes 232 glycoside hydrolases (GHs), 12 carbohydrate esterases (CEs), 109 glycosyl transferases (GTs), 15 polysaccharide lyases (PLs), and 35 genes with auxiliary activities (AAs). The high level of zinc finger motif containing transcription factors could possibly hint a tight regulation of the cellulolytic machinery, which may also explain the low cellulase activities even when a complete repertoire of cellulase degrading enzyme genes are present in the fungus.

Effects of Different Stress Parameters on Growth and on Oleuropein-Degrading Abilities of Lactiplantibacillus plantarum Strains Selected as Tailored Starter Cultures for Naturally Table Olives.

The use of β-glucosidase positive strains, as tailored-starter cultures for table olives fermentation, is a useful biotechnological tool applied to accelerate the debittering process. Nowadays, strains belonging to Lactiplantibacillus plantarum species are selected for their high versatility and tolerance to stress conditions. The present study investigated the effect of different stress factors (pH, temperature and NaCl) on growth and on oleuropein-degrading abilities of selected L. plantarum strains. In addition, the presence of the beta-glucosidase gene was investigated by applying a PCR based approach. Results revealed that, overall, the performances of the tested strains appeared to be robust toward the different stressors. However, the temperature of 16 °C significantly affected the growth performance of the strains both singularly and in combination with other stressing factors since it prolongs the latency phase and reduces the maximum growth rate of strains. Similarly, the oleuropein degradation was mainly affected by the low temperature, especially in presence of low salt content. Despite all strains displayed the ability to reduce the oleuropein content, the beta-glucosidase gene was detected in five out of the nine selected strains, demonstrating that the ability to hydrolyze the oleuropein is not closely related to the presence of beta-glucosidase. Data of the present study suggest that is extremely important to test the technological performances of strains at process conditions in order to achieve a good selection of tailored starter cultures for table olives.

A beta-glucosidase gene from Stevia rebaudiana may be involved in the steviol glycosides catabolic pathway.

We herein report the preparation of a full-length raucaffricine-O-beta-D-glucosidase gene of stevia rebaudiana Bertoni (named SrRG1, GenBank accession number MK920450). Sequence analysis indicated SrRG1 consists of a 1650bp open reading frame encoding a protein of 549 amino acids. Its deduced amino acid sequence showed a high identity of 82% with a raucaffricine-O-beta-D-glucosidase from H. annuus of glycoside hydrolase family 1. The expression pattern analyzed by real-time quantitative PCR showed no significant difference among different tissues, developmental stages, and cultivars under normal growth conditions. Furthermore, the genefunction of SrRG1 was preliminarily studied by agrobacterium-mediated transformation on instantaneous expression. In the test of agrobacterium-mediated transformation on instantaneous expression, it was observed that overexpression of SrRG1 increased the accumulation of steviol content and decreased the majorcomponents and total SGs contents. Such results demonstrated that SrRG1 may participate in the steviol glycosides catabolic pathway. However, the effect of silencing construct infiltration on steviol and SGs content wasnotsignificant and its expression pattern was constitutive, which most probably, attributed the hydrolysis of SGs to the secondary activity of SrRG1. This study firstly identified the bate-glucosidase in stevia and advances our understanding of steviol glycosides hydrolyzation.

Molecular cloning and bioinformatics analyses of a GH3 beta-glucosidase from Auricularia heimuer

The present study reports the isolation and analysis of a beta-glucosidase gene (accession number MN816859) from the fungus Auricularia heimuer. The Aa-bgl gene comprises 2583 bp and encodes a putative polypeptide of 860 amino acids. BlastP analysis revealed conserved glycoside hydrolase family 3 domains. Recombinant plasmid pET-32a-bgl was constructed to overexpress the target protein in Escherichia coli BL21, and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis revealed target bands between 100 kDa and 135 kDa, consistent with the expected molecular weight. Additionally, the transcription levels of Aa-bgl at different developmental stages were investigated, and the results showed that the expression levels increased with the maturation of the fruiting bodies. In mature fruiting bodies, the levels were 6.69-fold higher than in controls (mycelium at 6 days). We therefore speculate that Aa-bgl may be correlated with development in A. heimuer. The findings provide a theoretical basis for studying the function of this beta-glucosidase in future work.

Effects of Freezing Stress on the Expression of Fatty Acid Desaturase (FAD2, FAD6 and FAD7) and Beta-Glucosidase (BGLC) Genes in Tolerant and Sensitive Olive Cultivars

In this study, the effect of freezing stress on expression of fatty acid desaturases (FAD2-2, FAD6 and FAD7) and beta-glucosidase (BGLC) genes was investigated in freezing- tolerant (“Fishomi”) and sensitive (“Zard”) olive (Olea europaea L.) cultivars. The olive cultivars were exposed to freezing stress (–10°C) at three different times points (1, 4 and 8 h), followed by a recovery stage. The degree of cell membrane damage was determined by measurement of leaves ion leakage of two olive cultivars both at freezing and recovery condition. The results indicated that ion leakage of cv. “Fishomi” was significantly lower than cv. “Zard” when exposed to the freezing stress and recovery condition. Furthermore, BGLC gene expression level in cv. “Zard” significantly suppressed 8 h after freezing stress. At the recovery period, its expression increased about three-fold at cv. “Fishomi”. However, it was suppressed in cv. “Zard” at recovery period. FAD genes expression pattern in cv. “Fishomi” was different from that in cv. “Zard”. FAD2 expression level in cv. “Fishomi” initially increased at the first 1 h and 4 h of freezing stress time and thereafter, decreased to control level over 8 h. Following recovery period, FAD2 gene expression level in cv. “Fishomi” increased up 5-fold. While, no expression detected in “Zard” at any time point or recovery period. The expression of FAD6 and FAD7 genes showed similar patterns at both cultivars. FAD6 and FAD7 genes expression significantly up-regulated 5- and 4.5-fold, respectively, following recovery period in “Fishomi”. Overall, the results suggested that FADs and BGLC could be involved in the increasing of freezing tolerance in olive cultivars, particularly in plants recovery from freezing stress.

Gaucher disease: single gene molecular characterization of one-hundred Indian patients reveals novel variants and the most prevalent mutation

BackgroundGaucher disease is a rare pan-ethnic, lysosomal storage disorder resulting due to beta-Glucosidase (GBA1) gene defect. This leads to the glucocerebrosidase enzyme deficiency and an increased accumulation of undegraded glycolipid glucocerebroside inside the cells’ lysosomes. To date, nearly 460 mutations have been described in the GBA1 gene. With the aim to determine mutations spectrum and molecular pathology of Gaucher disease in India, the present study investigated one hundred unrelated patients (age range: 1 day to 31 years) having splenomegaly, with or without hepatomegaly, cytopenia and bone abnormality in some of the patients.MethodsThe biochemical investigation for the plasma chitotriosidase enzyme activity and β-Glucosidase enzyme activity confirmed the Gaucher disease. The mutations were identified by screening the patients’ whole GBA gene coding region using bidirectional Sanger sequencing.ResultsThe biochemical analysis revealed a significant reduction in the β-Glucosidase activity in all patients. Sanger sequencing established 71 patients with homozygous mutation and 22 patients with compound heterozygous mutation in GBA1 gene. Lack of identification of mutations in three patients suggests the possibility of either large deletion/duplication or deep intronic variations in the GBA1 gene. In four cases, where the proband died due to confirmed Gaucher disease, the parents were found to be a carrier. Overall, the study identified 33 mutations in 100 patients that also covers four missense mutations (p.Ser136Leu, p.Leu279Val, p.Gly383Asp, p.Gly399Arg) not previously reported in Gaucher disease patients. The mutation p.Leu483Pro was identified as the most commonly occurring Gaucher disease mutation in the study (62% patients). The second common mutations identified were p.Arg535Cys (7% patients) and RecNcil (7% patients). Another complex mutation Complex C was identified in a compound heterozygous status (3% patients). The homology modeling of the novel mutations suggested the destabilization of the GBA protein structure due to conformational changes.ConclusionsThe study reports four novel and 29 known mutations identified in the GBA1 gene in one-hundred Gaucher patients. The given study establishes p.Leu483Pro as the most prevalent mutation in the Indian patients with type 1 Gaucher disease that provide new insight into the molecular basis of Gaucher Disease in India.

Isolation of an algicidal bacterium and its effects against the harmful-algal- bloom dinoflagellate Prorocentrum donghaiense (Dinophyceae)

The relationship between algicidal bacteria and harmful-algal-bloom-forming dinoflagellates is understudied and their action modes are largely uncharacterized. In this study, an algicidal bacterium (FDHY-03) was isolated from a bloom of Prorocentrum donghaiense and the characteristics of its action against P. donghaiense was investigated at physiological, molecular, biochemical and cytological levels. 16S rDNA sequence analysis placed this strain in the genus of Alteromonas in the subclass of γ-proteobacteria. Algicidal activity was detected in the bacterial filtrate, suggesting a secreted algicidal principle from this bacterium. Strain FDHY-03 showed algicidal activity on a broad range of HAB-forming species, but the greatest effect was found on P. donghaiense, which showed 91.7% mortality in 24 h of challenge. Scanning electron microscopic analysis indicated that the megacytic growth zone of P. donghaiense cells was the major target of the algicidal action of FDHY-03. When treated with FDHY-03 culture filtrate, P. donghaiense cell wall polysaccharides decreased steadily, suggesting that the algicidal activity occurred through the digestion of cell wall polysaccharides. To verify this proposition, the expression profile of beta-glucosidase gene in FDHY-03 cultures with or without P. donghaiense cell addition was investigated using reverse-transcription quantitative PCR. The gene expression level increased in the presence of P. donghaiense cells, indicative of beta-glucosidase induction by P. donghaiense and the enzyme’s role in this dinoflagellate’s demise. This study has isolated a new bacterial strain with a strong algicidal capability, documented its action mode and biochemical mechanism, providing a potential source of bacterial agent to control P. donghaiense blooms.

Biochemical and molecular characterization of adult patients with type I Gaucher disease and carrier frequency analysis of Leu444Pro - a common Gaucher disease mutation in India

BackgroundGaucher disease is a rare pan-ethnic disorder which occurs due to an increased accumulation of undegraded glycolipid glucocerebroside inside the cells’ lysosomes. A beta-Glucosidase (GBA) gene defect results in glucocerebrosidase enzyme deficiency. Though the disease is mainly diagnosed in childhood, the adult manifestation is often missed or identified late due to the failure to recognize the heterogeneous clinical presentation. The present study includes seven unrelated Indian adult patients (age range: 20–40 years) having splenomegaly, with or without hepatomegaly, cytopenia and bone abnormality.MethodsThe biochemical investigation implicated measuring plasma chitotriosidase enzyme activity followed by confirmatory test of β-Glucosidase enzyme activity from the leukocytes. The molecular characterization involved patients’ initial screening for the common Gaucher mutation (Leu444Pro). Later, all patients were subjected to whole GBA gene coding region study using bidirectional Sanger sequencing. The population screening for common Gaucher disease mutation (Leu444Pro) was executed in 1200 unrelated and healthy Indian subjects by Restriction Fragment Length Polymorphism-Polymerase Chain Reaction technique. The allele frequency was calculated using Hardy-Weinberg formula.ResultsThe biochemical analysis revealed a significant reduction in the β-Glucosidase activity in all the patients. Also, an elevated level of plasma Chitotriosidase activity in five patients supported their diagnosis of Gaucher disease. Sanger sequencing established four patients with homozygous variation and three patients with compound heterozygous variation in GBA gene. This study uncovers two missense variants (Ala448Thr and Val17Gly) not previously reported in Gaucher disease patients. Also the known mutations like Leu444Pro, Arg329Cys, Asp315Asn, Ser125Arg, and Arg395Cys were identified in these patients. The homology modeling suggested the destabilization of the protein structure due to novel variants. The Leu444Pro mutation screening in the Indian population spotted two people as a carrier. This emerged the carrier frequency of 1:600 along with wild-type allele frequency 0.97113 and mutant allele frequency 0.02887.ConclusionsThe study reports novel and known variants identified in the GBA gene in seven adult patients. The given study is the first report on the carrier frequency of the Leu444Pro mutant allele in an Indian population which will help understanding the burden and susceptibility of Gaucher disease to affect next generation in India.

Purification and characterization of a novel GH1 beta-glucosidase from Jeotgalibacillus malaysiensis

Beta-glucosidase (BGL) is an important industrial enzyme for food, waste and biofuel processing. Jeotgalibacillus is an understudied halophilic genus, and no beta-glucosidase from this genus has been reported. A novel beta-glucosidase gene (1344 bp) from J. malaysiensis DSM 28777T was cloned and expressed in E. coli. The recombinant protein, referred to as BglD5, consists of a total 447 amino acids. BglD5 purified using a Ni-NTA column has an apparent molecular mass of 52 kDa. It achieved the highest activity at pH 7 and 65 °C. The activity and stability were increased when CaCl2 was supplemented to the enzyme. The enzyme efficiently hydrolyzed salicin and (1 → 4)-beta-glycosidic linkages such as in cellobiose, cellotriose, cellotetraose, cellopentose, and cellohexanose. Similar to many BGLs, BglD5 was not active towards polysaccharides such as Avicel, carboxymethyl cellulose, Sigmacell cellulose 101, alpha-cellulose and xylan. When BglD5 blended with Cellic® Ctec2, the total sugars saccharified from oil palm empty fruit bunches (OPEFB) was enhanced by 4.5%. Based on sequence signatures and tree analyses, BglD5 belongs to the Glycoside Hydrolase family 1. This enzyme is a novel beta-glucosidase attributable to its relatively low sequence similarity with currently known beta-glucosidases, where the closest characterized enzyme is the DT-Bgl from Anoxybacillus sp. DT3-1.

In vivo function of Pgβglu-1 in the release of acetophenones in white spruce.

Eastern spruce budworm (Choristoneura fumiferiana Clemens) (ESBW) is a major forest pest which feeds on young shoots of white spruce (Picea glauca) and can cause landscape level economic and ecological losses. Release of acetophenone metabolites, piceol and pungenol, from their corresponding glycosides, picein and pungenin, can confer natural resistance of spruce to ESBW. A beta-glucosidase gene, Pgβglu-1, was recently discovered and the encoded enzyme was characterized in vitro to function in the release of the defensive acetophenone aglycons. Here we describe overexpression of Pgβglu-1 in a white spruce genotype whose metabolome contains the glucosylated acetophenones, but no detectable amounts of the aglycons. Transgenic overexpression of Pgβglu-1 resulted in release of the acetophenone aglycons in planta. This work provides in vivo evidence for the function of Pgβglu-1.

Characterization of a new multifunctional beta-glucosidase from Musca domestica.

To engineer Pichia pastoris for heterologous production of cellulase from Musca domestica and explore its potential for industrial applications. A new beta-glucosidase gene (bg), encoding 562 amino acids, was cloned from M. domestica by using rapid amplification of cDNA ends. The gene bg was linked to pPICZαA and expressed in P. pastoris with a yield of 500mgl-1. The enzyme has the maximum activity with 27.6Umg-1 towards cellulose. The beta-glucosidase has stable activity from 20 to 70°C and can tolerate one-mole glucose. It has the maximum activities for salicin (25.9±1.8Umg-1), cellobiose (40.1±2.3Umg-1) and cellulose (27.6±3.5Umg-1). The wide-range substrate activities of the beta-glucosidase were further verified by matrix-assisted laser desorption/ionization mass spectra. Structural analysis shows that the beta-glucosidase belongs to glycoside hydrolase family Ι and possesses O-glycosylation sites. Thus, a multifunctional beta-glucosidase was expressed from M. domestica and provides a potential tool for industrial application of cellulose.

Transcriptional analysis of genes encoding β-glucosidase of Schizophyllum commune KUC9397 under optimal conditions.

The present study was conducted to determine the gene responsible for beta-glucosidase (BGL) production and to generate a full-length complementary DNA (cDNA) of one of the putative BGL genes, which showed a significant expression level when Schizophyllum commune KUC9397 was grown in optimized medium. The relative expression levels of seven genes encoding BGL of S. commune KUC9397 were determined with real-time quantitative reverse transcription PCR in cellulose-containing optimized medium (OM) compared to glucose-containing basal medium (BM). The most abundant transcript was bgl3a in OM. The transcript number of the bgl3a increased more than 57.60-fold when S. commune KUC9397 was grown on cellulose-containing OM compared to that on glucose-containing BM. The bgl3a was identified, and a deduced amino acid sequence of bgl3a shared homology (97%) with GH3 BGL of S. commune H4-8. This is the first report showing the transcription levels of genes encoding BGL and identification of full-length cDNA of glycoside hydrolase 3 (GH3) BGL from S. commune. Furthermore, this study is one of the steps for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

Co-expression of endoglucanase and β-glucosidase in Corynebacterium glutamicum DM1729 towards direct lysine fermentation from cellulose

The aim of the present study is the development of a consolidated bioprocess for the production of lysine with recombinant Corynebacterium glutamicum DM1729 strains expressing endoglucanase and β-glucosidase genes. Here, the endoglucanase genes from Xanthomonas campestris XCC3521 and XCC2387 and betaglucosidase gene from Saccharophagus degradans Sde1394 were cloned in C. glutamicum DM1729 and expressed either extracellularly or on cell surface. The highest β-glucosidase activity of 9±0.5U/OD600 of 1 and endoglucanase activity of 5.5±0.8U was obtained in C. glutamicum DM 1729 (pVWEx1-TATXCC2387) (pEKEx3-PorC-Sde1394) when cellobiose (20g/L) alone or in combination with carboxymethyl cellulose (20g/L) was used as the carbon sources respectively. The overall efforts resulted in a lysine titre of 5.9±0.5mM. The ability of the constructs to utilize carboxymethyl cellulose and cellobiose for growth and amino acid production proves the concept of utilization of C. glutamicum as a biocatalyst in the lignocellulosic biorefinery.

Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Beta-glucosidase (BG) is known as a multifunctional enzyme for social maintenance in terms of both cellulose digestion and social communication in termites. However, the expression profiles of each BG gene and their evolutionary history are not well understood. First, we cloned two types of BG homologs (RsBGI and RsBGII) from the termite Reticulitermes speratus (Kolbe). Gene expression analyses showed that RsBGI expression levels of primary queens and kings from 30 to 100days after colony foundation were high, but those of reproductives dropped after day 400. Extremely low gene expression levels of RsBGI were observed in eggs, whereas workers had significantly higher expression levels than those of soldiers and other colony members. Consequently, RsBGI gene expression levels changed among each developmental stage, and RsBGI was shown to be involved in cellulose digestion. On the other hand, the RsBGII gene was consistently expressed in all castes and developmental stages examined, and notable expression changes were not observed among them, including in eggs. It was indicated that RsBGII is a main component involved in social communication, for example, the egg-recognition pheromone shown in this species previously. Finally, we obtained partial gene homologs from other termite and cockroach species, including the woodroach (genus Cryptocercus), which is the sister group to termites, and performed molecular phylogenetic analyses. The results showed that the origin of the BG gene homologs preceded the divergence of termites and cockroaches, suggesting that the acquisition of multifunctionality of the BG gene also occurred in cockroach lineages.

RETRACTED ARTICLE: The effects of each beta-glucosidase gene deletion on cellulase gene regulation in Neurospora crassa (online publication: DOI 10.1007/s10482-013- 9972-7)

Takao Kasuga and Zhiliang Fan were listed as co-authors without their acknowledgement and would like to be removed from the authors list. This article is retracted on request of Weihua Wu due to inconsistent errors of intracellular and total beta-glucosidase activities assay and the mycelia weight measurement in this paper. These errors led to inaccurate results of total and intracellular beta-glucosidase activities, and the normalized endoglucanases and exoglucanases activities and therefore compromise the partial conclusions of this publication.

Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production

BackgroundMany microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed.ResultsTo engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-based gene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol.ConclusionSeven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host.