Application of Dual Promoter Expression System for the Enhanced Heparosan Production in Bacillus megaterium.

Heparosan, a capsular polysaccharide synthesized by certain pathogenic bacteria, is a promising precursor for heparin production. Heparosan production is catalyzed by the formation of KfiC-KfiA complex and the subsequent action of KfiC and KfiA proteins. Polycistronic expression of kfiC and kfiA in Bacillus megaterium yielded an unbalanced expression of KfiC and KfiA proteins resulted in decreased heparosan production. In this study, dual promoter plasmid system was constructed to increase the expression levels of KfiC and KfiA proteins. Dual promoter plasmid system along with UDP-glucuronic acid pathway overexpression (CADuet-DB) increased the heparosan production to 203 mg/L in shake flask experiments. Batch fermentation of strain CADuet-DB under controlled conditions yielded a maximum heparosan concentration of 627 mg/L, which is 59% higher than strain CA-DB. A modified logistic model is applied to describe the kinetics of heparosan production and biomass growth. Fed batch fermentation resulted in 3-fold enhancement in heparosan concentration (1.96 g/L), compared to batch fermentation. Nuclear magnetic resonance analysis revealed that heparosan from strain CADuet-DB was similar to Escherichia coli K5 heparosan. These results suggested that dual promoter expression system is a promising alternative to polycistronic expression system to produce heparosan in B. megaterium.

Advance in glycosyltransferases, the important bioparts for production of diversified ginsenosides.

Ginsenosides are a series of glycosylated triterpenoids predominantly originated from Panax species with multiple pharmacological activities such as anti-aging, mediatory effect on the immune system and the nervous system. During the biosynthesis of ginsenosides, glycosyltransferases play essential roles by transferring various sugar moieties to the sapogenins in contributing to form structure and bioactivity diversified ginsenosides, which makes them important bioparts for synthetic biology-based production of these valuable ginsenosides. In this review, we summarized the functional elucidated glycosyltransferases responsible for ginsenoside biosynthesis, the advance in the protein engineering of UDP-glycosyltransferases (UGTs) and their application with the aim to provide in-depth understanding on ginsenoside-related UGTs for the production of rare ginsenosides applying synthetic biology-based microbial cell factories in the future.

Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells.

In mammalian cells, N-glycans may include multiple N-acetyllactosamine (poly-LacNAc) units that can play roles in various cellular functions and properties of therapeutic recombinant proteins. Previous studies indicated that ß-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß-1,4-galactotransferase 1 (B4GALT1) are two of the primary glycosyltransferases involved in generating LacNAc units. In the current study, knocking out sialyltransferase genes slightly enhanced the LacNAc content (≥4 repeats per glycan) on recombinant EPO protein. Next, the role of single and dual-overexpression of B3GNT2 and B4GALT1 was explored in recombinant EPO-expressing Chinese hamster ovary (CHO) cells. While overexpression of B4GALT1 slightly enhanced the levels of large glycans on recombinant EPO, overexpression of B3GNT2 in EPO-expressing CHO cells significantly decreased the recombinant EPO LacNAc content, resulting in N-glycans terminating primarily with GlcNAc structures, a limited number of Gals, and nearly undetectable sialylation, which was also observed in sialyltransferases knock-out-B3GNT2 overexpression cell lines. Considering the nature of the binding domain motifs present on B3GNT2, which evolved from ß1,3-galactosyltransferases, its overexpression may have competed and inhibited endogenous ß1,4-galactosyltransferases for exposed GlcNAc residues on the N-glycans, resulting in premature termination of many N-glycans at GlcNAc. Furthermore, B3GNT2 overexpression enhanced intracellular UDP-GlcNAc and CMP-Neu5Ac content while slightly lowering UDP-Gal content. The presence of a sink for UDP-GlcNAc in the form of B3GNT2 with no disposition may have also elevated the intracellular levels of this nucleotide as well as its downstream product, CMP-Neu5Ac. Furthermore, we were unable to overexpress B4GALT1 at either the transcriptional or translational levels following initial B3GNT2 expression. Expression of B3GNT2 following initial expression of B4GALT1 was also problematic in that transcriptional and translational analysis indicated the accumulation of truncated B3GNT2 missing a section of the B3GNT2 trans-Golgi lumen domain while transmembrane and cytoplasmic domains were present. Given that glycosylation is a very complex intra-network process, the addition of one or more recombinant glycosyltransferases may have an unexpected influence on the expression and activities of glycosyltransferases, which can disrupt the nucleotide sugar levels and lead to unexpected modifications of the resulting N-glycan patterns.

B3GNT5 is a novel marker correlated with stem-like phenotype and poor clinical outcome in human gliomas.

AIMS: Glioblastoma multiforme (GBM) is the most lethal tumor with a median patient survival of 14 to 15 months. Glioma stem cells (GSCs) play a critical role in tumor initiation and therapeutic resistance in GBM. B3GNT5 has been suggested as the key glycosyltransferase in the biosynthesis of the (neo-) lacto series of glycosphingolipid. In this study, we evaluated the B3GNT5 expression in GSCs as well as the correlation with clinical data in GBM. METHODS: The mRNA levels of B3GNT5 in normal astrocytes, four glioma cell lines, and four GSCs were evaluated using real-time PCR. Small interference RNAs (siRNAs) were used to inhibit B3GNT5 expression and analyze its ability to form neurospheres. Statistical analyses were conducted to determine the association with B3GNT5 expression and tumor grade and GBM subtypes as well as patient survival using public datasets. RESULTS: B3GNT5 expression was significantly elevated in GSCs compared with normal astrocytes, glioma cell lines, and their matched differentiated tumor cells. Knockdown of B3GNT5 in GSCs decreased the neurosphere formation. Patients with high B3GNT5 expression had a short overall survival. B3GNT5 is correlated with classical and mesenchymal GBM subtypes. CONCLUSION: The findings suggest the central role of B3GNT5 in regulating malignancy of GBM.

An Efficient Stevia rebaudiana Transformation System and In vitro Enzyme Assays Reveal Novel Insights into UGT76G1 Function.

Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1. By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E. coli, we show that two closely related UGT76G1 isoforms differ in their substrate specificities, providing new insights into mechanisms underlying the diversity of SG profiles that are observed across Stevia germplasm. Finally, we found evidence suggesting that alternative and/or aberrant splicing may serve to influence the ability of the plant to produce functional UGT76G1 transcripts, and possibly produce enzyme variants within the plant.

Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes.

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

GLT8D1 overexpression as a novel prognostic biomarker in human cutaneous melanoma.

Aberrant glycosylation plays a major role in the progression of melanoma, but little is known about glycosyltransferases. Glycosyltransferase 8 domain containing 1 (GLT8D1) is located in the Golgi apparatus and is related to transferase activity in mammals. However, its role in cancer remains unclear. The aim of this study was to investigate the expression of GLT8D1 in human melanoma and explore the relationship between GLT8D1 expression and the clinicopathological characteristics of melanoma patients via GEO data analysis combined with clinical patient data. The analysis of 45 malignant melanoma samples and 18 benign nevus samples from the GEO database was performed. Moreover, 67 patients with cutaneous melanoma and 38 patients with mucosal melanoma as well as 40 benign nevus samples were collected for our study. Immunohistochemistry analyses were implemented to evaluate GLT8D1 expression at protein level. The GEO data analysis exhibited that the GLT8D1 mRNA expression was upregulated in the melanoma samples compared with the benign nevus samples. Likewise, GLT8D1 protein expression in the cutaneous melanoma and mucosal melanoma samples was significantly higher than that in the benign nevus tissue samples (P = 0.001 and 0.046, respectively). Furthermore, the GLT8D1 protein expression in cutaneous melanoma was higher than that in mucosal melanoma (P = 0.001). The high GLT8D1 protein expression was remarkably correlated with Clark level (P = 0.027), AJCC stage (P = 0.003), ulceration status (P = 0.041), Ki-67 expression (P = 0.030) and especially with histopathological type (P = 0.001). The results of the Kaplan-Meier survival and Cox regression analyses revealed that cutaneous melanoma patients with high GLT8D1 expression (P = 0.036), Clark level (P = 0.018) and advanced AJCC stage (P = 0.003) encountered poor overall survival. Overall survival (P = 0.040) and progression-free survival (P = 0.019) were worse for the patients with high GLT8D1 expression than for the patients with low expression. These data implied that GLT8D1 could be an independent prognostic factor for an unfavorable prognosis in cutaneous malignant melanoma patients and that GLT8D1 overexpression might serve as a novel prognostic biomarker.

Comparison of uridine diphosphate-glycosyltransferase UGT76G1 genes from some varieties of Stevia rebaudiana Bertoni.

Stevia leaves contain various components, such as flavonoids, labdanes, chlorophylls, sterols, triterpenoids, mono-disaccharides, organic acids and inorganic salts. Stevia is known to accumulate diterpenoid steviol glycosides, which are approximately 300 times sweeter than regular sugar. Stevioside and rebaudioside A are the main diterpenic glycosides in stevia. Steviol glycosides are the secondary metabolites responsible for the sweetness of stevia. The main objectives of the present study were to determine the concentrations of diterpenic glycosides (stevioside and rebaudioside A) in three stevia varieties (Stevia rebaudiana) via the HPLC-UV technique and to amplify the UGT76G1 gene by PCR using gene-specific primers. The expression levels of the UGT76G1 gene were determined in the three stevia varieties. The PCR products were sequenced and analyzed, and the nucleotide sequences of the UGT76G1 gene were submitted to GenBank and assigned to the following three varieties: Egy1 (MH087463), China1 (MH087464) and Sponti (MH087465). Cluster analysis was used to separate the three varieties into two major clusters based on their phylogenetic relationship. In addition, chemical analysis was carried out to evaluate stevioside and rebaudioside A. The present study concluded that Egy1 and Sponti are closely related varieties as they fall in the same cluster, while China1 forms a separate cluster. Bioprospecting studies could be useful for selection of superior ecotypes of Stevia rebaudiana.

Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

Gentamicin B (GB), a valuable starting material for the preparation of the semisynthetic aminoglycoside antibiotic isepamicin, is produced in trace amounts by the wild-type Micromonospora echinospora. Though the biosynthetic pathway to GB has remained obscure for decades, we have now identified three hidden pathways to GB production via seven hitherto unknown intermediates in M. echinospora. The narrow substrate specificity of a key glycosyltransferase and the C6'-amination enzymes, in combination with the weak and unsynchronized gene expression of the 2'-deamination enzymes, limits GB production in M. echinospora. The crystal structure of the aminotransferase involved in C6'-amination explains its substrate specificity. Some of the new intermediates displayed similar premature termination codon readthrough activity but with reduced toxicity compared to the natural aminoglycoside G418. This work not only led to the discovery of unknown biosynthetic routes to GB, but also demonstrated the potential to mine new aminoglycosides from nature for drug discovery.

Optimization and purification of glucansucrase produced by Leuconostoc mesenteroides DRP2-19 isolated from Chinese Sauerkraut.

Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett-Burman (PB) experiment, sucrose, Ca2+, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87 g/L, Ca2+ 0.21 mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94 ± 0.43 U/mL in 24 hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56 U/mg protein.

Influence of the flow field on α-cyclodextrin glycosyltransferase production by Escherichia coli BL21.

The computational fluid dynamics (CFD) software package Fluent was utilized to simulate the flow field of Escherichia coli (E. coli) BL21 fermentation in a 50 L automatic bioreactor for producing α-cyclodextrin glycosyltransferase (α-CGTase) in this study. 4-down-pumping propeller (4DPP), 6-curved-blade disc turbine (6CBDT), and Rushton turbine (RT) were assembled to form eight impeller combinations (C1-C8). Through flow field simulating, four referential impeller combinations, in which C6, C7, and C8 were three layers stirring blades and C1 as a control, were selected to carry out batch fermentation experiments (TC1, TC6, TC7, and TC8) for validation. The correlation analysis between simulation results and experimental measurements indicated that TC6 (tank equipped with C6 impeller combination) exhibited lower enzymatic activity though it had the better mixing effect, fastest oxygen uptake rate (OUR), and maximum specific growth rate (µ) in the initial stage, which was just to the contrary in TC8. It was revealed by next fed-batch fermentation experiments in TC6 and TC8 that TC6 was considered as excellent flow field properties brought about the higher µ of E. coli BL21 and fast acetic acid (HAc) accumulation, which resulting in a serious inhibition on α-CGTase expression and this negative effect could not be removed. As a result, there should be a threshold of HAc accumulation rate which brought about a terrible inhibitory effect on α-CGTase expression. Moreover, the yield of α-CGTase activity reached 231.38 U mL- 1 in TC8, which elevated 31.74% compared to that obtained in TC1.

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases.

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 µg/ml for the single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

Exploring the functional significance of sterol glycosyltransferase enzymes.

Steroidal alkaloids (SAs) are widely synthesized and distributed in plants manifesting as natural produce endowed with potential for medicinal, pesticidal and other high-value usages. Glycosylation of these SAs raises complex and diverse glycosides in plant cells that indeed govern numerous functional aspects. During the glycosylation process of these valuable metabolites, the addition of carbohydrate molecule(s) is catalyzed by enzymes known as sterol glycosyltransferases (SGTs), commonly referred to as UGTs, leading to the production of steryl glycosides (SGs). The ratio of SGs and nonglyco-conjugated SAs are different in different plant species, however, their biosynthesis in the cell is controlled by different environmental factors. The aim of this review is to evaluate the current SGT enzyme research and the functional consequences of glycomodification of SAs on the physiology and plant development, which together are associated with the plant's primary processes. Pharmaceutical, industrial, and other potential uses of saponins have also been discussed and their use in therapeutics has been unveiled by in silico analysis. The field of biotransformation or conversion of nonglycosylated to glycosylated phytosterols by the activity of SGTs, making them soluble, available and more useful for humankind is the new field of interest towards drug therapy.

Characterization of UDP-glucosyltransferase from Indigofera tinctoria.

Indican is a secondary metabolite in Indigofera tinctoria; its synthesis from indoxyl and UDP-glucose is catalyzed by a UDP-glucosyltransferase (UGT). In this study, we partially purified UGT extracted from I. tinctoria leaves and analyzed the protein by peptide mass fingerprinting. We identified two fragments that were homologous to UGT after comparison with the transcriptomic data of I. tinctoria leaves. The fragments were named itUgt1 and itUgt2 and were amplified using rapid amplification of cDNA ends polymerase chain reaction to obtain full-length cDNAs. The resultant nucleotide sequences of itUgt1 and itUgt2 encoded peptides of 477 and 475 amino acids, respectively. The primary structure of itUGT1 was 89% identical to that of itUGT2 and contained an important plant secondary product glycosyltransferase (PSPG) box sequence and a UGT motif. The recombinant proteins expressed in Escherichia coli were found to possess high indican synthesis activity. Although the properties of the two proteins itUGT1 and itUGT2 were very similar, itUGT2 was more stable at high temperatures than itUGT1. Expression levels of itUGT mRNA and protein in plant tissues were examined by UGT assay, immunoblotting, and semi-quantitative reverse transcription polymerase chain reaction. So far, we presume that itUGT1, but not itUGT2, primarily catalyzes indican synthesis in I. tinctoria leaves.

Cell surface N-glycan alteration in HepAD38 cell lines expressing Hepatitis B virus.

Hepatitis B virus (HBV) is the smallest partially double-stranded DNA virus known to infect humans. Worldwide, more than 50% of hepatocellular carcinoma (HCC) cases are related to chronic Hepatitis B. Development of HCC from normal liver cells is characterized by changes in cell surface N-glycans, which can promote the invasive behavior of tumor cells, leading ultimately to the progression of cancer. However, little is understood about the cell surface N-glycans of HBV-infected liver cells. We try to address this by taking advantage of the HepAD38 cell line, which can replicate HBV in the absence of tetracycline [tet(-)] in growth medium. In the presence of tetracycline [tet(+)], this cell line is free from the virus due to the repression of pregenomic (pg) RNA synthesis. In culture medium without tetracycline, cells express viral pgRNA and start to secrete virions into the supernatant. Here we studied the expression of glycosyltransferases and the cell surface N-glycan composition of tet(+) and tet(-) HepAD38. Among the glycosyltransferases upregulated by the expression of HBV were GnT-II, GnT-IVa, ST6Gal1, and GnT-V, whereas GnT-I, GnT-III, ß4GalT1, and FUT8 were downregulated. About one-third of the total cell surface N-glycans found on tet(-)HepAD38 were sialylated. As for tet(+)HepAD38, sialylation was 6% lower compared to the tet(-) cells. Neither treatment changed the cell surface N-glycans expression of the total complex type or the total fucosylated type, which were about 50% or 60%, respectively. Our results showed that the expression of HBV triggers higher sialylation in HepAD38 cells. Altogether, the results show that HBV expression triggered the alteration of the cell surface N-glycosylation pattern and the expression levels of glycosyltransferases of HepAD38 cells.

N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae.

Recombinant proteins produced in insect cells and insects, unlike those produced in mammalian cells, have pauci-mannose-type N-glycans. In this study, we examined complex-type N-glycans on recombinant proteins via coexpression of human ß-1,2-N-acetylglucosaminyltransferase II (hGnT II) and human ß1,4-galactosyltransferase (hGalT I) in silkworm pupae, by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The actin A3 promoter from B. mori and the polyhedrin promoter from Autographa californica multiple nucleopolyhedroviruses (AcMNPVs) were used to coexpress hGnT II and hGalT I. These recombinant BmNPVs were coexpressed with human IgG (hIgG), hGnT II and hGalT I in silkworm pupae. When hIgG was coexpressed with hGnT II, approximately 15% of all N-glycans were biantennary, with both arms terminally modified with N-acetylglucosamine (GlcNAc). In contrast, when hIgG was coexpressed with both hGnT II and hGalT I under the control of the polyhedrin promoter, 27% of all N-glycans were biantennary and terminally modified with GlcNAc, with up to 5% carrying one galactose and 11% carrying two. The obtained N-glycan structure was dependent on the promoters used for coexpression of hGnT II or hGalT I. This is the first report of silkworm pupae producing a biantennary, terminally galactosylated N-glycan in a recombinant protein. These results suggest that silkworms can be used as alternatives to insect and mammalian hosts to produce recombinant glycoproteins with complex N-glycans.

Personalized genome sequencing coupled with iPSC technology identifies GTDC1 as a gene involved in neurodevelopmental disorders.

The cellular and molecular mechanisms underlying neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The ability to generate patient-specific induced pluripotent stem cells (iPSCs) now offers a novel strategy for modelling human diseases. Recent studies have reported the derivation of iPSCs from patients with neurological disorders. The key challenge remains the demonstration of disease-related phenotypes and the ability to model the disease. Here we report a case study with signs of neurodevelopmental disorders (NDDs) harbouring chromosomal rearrangements that were sequenced using long-insert DNA paired-end tag (DNA-PET) sequencing approach. We identified the disruption of a specific gene, GTDC1. By deriving iPSCs from this patient and differentiating them into neural progenitor cells (NPCs) and neurons we dissected the disease process at the cellular level and observed defects in both NPCs and neuronal cells. We also showed that disruption of GTDC1 expression in wild type human NPCs and neurons showed a similar phenotype as patient's iPSCs. Finally, we utilized a zebrafish model to demonstrate a role for GTDC1 in the development of the central nervous system. Our findings highlight the importance of combining sequencing technologies with the iPSC technology for NDDs modelling that could be applied for personalized medicine.

Catechol glucosides act as donor/acceptor substrates of glucansucrase enzymes of Lactobacillus reuteri.

Previously, we have shown that the glucansucrase GtfA-ΔN enzyme of Lactobacillus reuteri 121, incubated with sucrose, efficiently glucosylated catechol and we structurally characterized catechol glucosides with up to five glucosyl units attached (te Poele et al. in Bioconjug Chem 27:937-946, 2016). In the present study, we observed that upon prolonged incubation of GtfA-ΔN with 50 mM catechol and 1000 mM sucrose, all catechol had become completely glucosylated and then started to reappear. Following depletion of sucrose, this glucansucrase GtfA-ΔN used both α-D-Glcp-catechol and α-D-Glcp-(1→4)-α-D-Glcp-catechol as donor substrates and transferred a glucose unit to other catechol glycoside molecules or to sugar oligomers. In the absence of sucrose, GtfA-ΔN used α-D-Glcp-catechol both as donor and acceptor substrate to synthesize catechol glucosides with 2 to 10 glucose units attached and formed gluco-oligosaccharides up to a degree of polymerization of 4. Also two other glucansucrases tested, Gtf180-ΔN from L. reuteri 180 and GtfML1-ΔN from L. reuteri ML1, used α-D-Glcp-catechol and di-glucosyl-catechol as donor/acceptor substrate to synthesize both catechol glucosides and gluco-oligosaccharides. With sucrose as donor substrate, the three glucansucrase enzymes also efficiently glucosylated the phenolic compounds pyrogallol, resorcinol, and ethyl gallate; also these mono-glucosides were used as donor/acceptor substrates.

Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa.

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more ß-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of ß-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall.