Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli.

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.

The elucidation of phosphosugar stress response in Bacillus subtilis guides strain engineering for high N-acetylglucosamine production.

Bacillus subtilis is a preferred microbial host for the industrial production of nutraceuticals and a promising candidate for the synthesis of functional sugars, such as N-acetylglucosamine (GlcNAc). Previously, a GlcNAc-overproducer B. subtilis SFMI was constructed using glmS ribozyme dual-regulatory tool. Herein, we further engineered to enhance carbon flux from glucose towards GlcNAc synthesis. As a result, the increased flux towards GlcNAc synthesis triggered phosphosugar stress response, which caused abnormal cell growth. Unfortunately, the mechanism of phosphosugar stress response had not been elucidated in B. subtilis. To reveal the stress mechanism and overcome its negative effect in bioproduction, we performed comparative transcriptome analysis. The results indicate that cells slow glucose utilization by repression of glucose import and accelerate catabolic reactions of phosphosugar. To verify these results, we overexpressed the phosphatase YwpJ, which relieved phosphosugar stress and allowed us to identify the enzyme responsible for GlcNAc synthesis from GlcNAc 6-phosphate. In addition, the deletion of nagBB and murQ, responsible for GlcNAc precursor degradation, further improved GlcNAc synthesis. The best engineered strain, B. subtilis FMIP34, increased GlcNAc titer from 11.5 to 26.1 g/L in shake flasks and produced 87.5 g/L GlcNAc in 30-L fed-batch bioreactor. Our results not only elucidate, for the first time, the phosphosugar stress response mechanism in B. subtilis, but also demonstrate how the combination of rational metabolic engineering with novel insights into physiology and metabolism allows the construction of highly efficient microbial cell factories for the production of high-value chemicals.

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc.

Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N'-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.

Assembly of pathway enzymes by engineering functional membrane microdomain components for improved N-acetylglucosamine synthesis in Bacillus subtilis.

Enzyme clustering can improve catalytic efficiency by facilitating the processing of intermediates. Functional membrane microdomains (FMMs) in bacteria can provide a platform for enzyme clustering. However, the amount of FMMs at the cell basal level is still facing great challenges in multi-enzyme immobilization. Here, using the nutraceutical N-acetylglucosamine (GlcNAc) synthesis in Bacillus subtilis as a model, we engineered FMM components to improve the enzyme assembly in FMMs. First, by overexpression of the SPFH (stomatin-prohibitin-flotillin-HflC/K) domain and YisP protein, an enzyme involved in the synthesis of squalene-derived polyisoprenoid, the membrane order of cells was increased, as verified using di-4-ANEPPDHQ staining. Then, two heterologous enzymes, GlcNAc-6-phosphate N-acetyltransferase (GNA1) and haloacid dehalogenase-like phosphatases (YqaB), required for GlcNAc synthesis were assembled into FMMs, and the GlcNAc titer in flask was increased to 8.30 ± 0.57 g/L, which was almost three times that of the control strains. Notably, FMM component modification can maintain the OD600 in stationary phase and reduce cell lysis in the later stage of fermentation. These results reveal that the improved plasma membrane ordering achieved by the engineering FMM components could not only promote the enzyme assembly into FMMs, but also improve the cell fitness.

Synergetic engineering of central carbon and nitrogen metabolism for the production of N-acetylglucosamine in Bacillus subtilis.

N-acetylglucosamine (GlcNAc) is a nitrogen-containing compound, which is widely used as a nutraceutical and pharmaceutical. In our previous work, we constructed a recombinant Bacillus subtilis strain for the biosynthesis of GlcNAc by engineering the central carbon metabolism. However, nitrogen is also required for the synthesis of GlcNAc. Hence, it is necessary to simultaneously coordinate the carbon and nitrogen metabolism to improve production of GlcNAc. In this work, we attempted to enhance GlcNAc production in B. subtilis by increasing supply of precursors N-acetylglucosamine 6-phosphate (GlcNAc6P) and glutamate. The expression of a key enzyme, GlcNAc6P N-acetyltransferase (GNA1), was enhanced by engineering the promoter and ribosome binding site to enhance the production of GlcNAc6P. Next, we examined the effect of different nitrogen sources on GlcNAc synthesis. We observed that urea can promote nitrogen assimilation for GlcNAc synthesis. The glutamate synthesis was improved by deleting the two endogenous glutamate dehydrogenase genes (rocG and gudB) and by integrating one exogenous glutamate dehydrogenase gene (gdh). This strategy enhanced the intracellular glutamate and glutamine by 69.8% and 46.9%, respectively. The synergetic engineering of central carbon and nitrogen metabolisms increased the GlcNAc titer from 14.0 to 22.2 g/L in the shaker flask. Hence, our study demonstrated the importance of carbon and nitrogen metabolism coordination in the production of nitrogen-containing compounds.

CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N-Acetylglucosamine Production.

Lactobacillus plantarum is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of Lactobacillus plantarum without introducing exogenous genes or plasmids is of great importance. In this study, CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering was established in L. plantarum WCFS1 to seamlessly edit the genome, including gene knockouts, insertions, and point mutations. To optimize our editing method, phosphorothioate modification was used to improve the dsDNA insertion, and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer L. plantarum WCFS1 toward producing N-acetylglucosamine (GlcNAc). nagB was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in glmS1 were introduced to relieve feedback repression. The resulting strain produced 797.3 mg/liter GlcNAc without introducing exogenous genes or plasmids. This strategy may contribute to the available methods for precise and diverse genetic engineering in lactic acid bacteria and boost strain engineering for more applications.IMPORTANCE CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3 mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future.

Bioproduction of N-acetyl-glucosamine from colloidal α-chitin using an enzyme cocktail produced by Aeromonas caviae CHZ306.

N-acetyl-D-glucosamine (GlcNAc) is an important amino-monosaccharide with great potential for biotechnological applications. It has traditionally been produced by the chemical hydrolysis of chitin, despite certain industrial and environmental drawbacks, including acidic wastes, low yields and high costs. Therefore, enzymatic production has gained attention as a promising environmentally-friendly alternative to the chemical processes. In this study we demonstrate the GlcNAc bioproduction from colloidal α-chitin using an enzyme cocktail containing endochitinases and exochitinases (chitobiosidases and N-acetyl-glucosaminidases). The enzyme cocktail was extracted after fermentation in a bioreactor by Aeromonas caviae CHZ306, a chitinolytic marine bacterium with great potential for chitinase production. Hydrolysis parameters were studied in terms of temperature, pH, enzyme and substrate concentration, and reaction time, achieving over 90% GlcNAc yield within 6 h. The use of colloidal α-chitin as substrate showed a substantial improvement of GlcNAc yields, when compared with ß-chitin and α-chitin polymorphs. Such result is directly related to a significant decrease in crystallinity and viscosity from natural α-chitin, providing the chitinase with greater accessibility to the depolymerized chains. This study provides valuable information on the GlcNAc bioproduction from chitin using an enzymatic approach, addressing the key points for its production, including the enzyme cocktail composition and the substrate structures.

Bittersweet tumor development and progression: Emerging roles of epithelial plasticity glycosylations.

Altered metabolism is one of the hallmarks of cancer. The best-known cancer metabolic anomaly is an increase in aerobic glycolysis, which generates ATP and other basic building blocks, such as nucleotides, lipids, and proteins to support tumor cell growth and survival. Epithelial plasticity (EP) programs such as the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are evolutionarily conserved processes that are essential for embryonic development. EP also plays an important role during tumor progression toward metastasis and treatment resistance, and new roles in the acceleration of tumorigenesis have been found. Recent evidence has linked EMT-related transcriptomic alterations with metabolic reprogramming in cancer cells, which include increased aerobic glycolysis. More recent studies have revealed a novel connection between EMT and altered glycosylation in tumor cells, in which EMT drives an increase in glucose uptake and flux into the hexosamine biosynthetic pathway (HBP). The HBP is a side-branch pathway from glycolysis which generates the end product uridine-5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc). A key downstream utilization of UDP-GlcNAc is for the post-translational modification O-GlcNAcylation which involves the attachment of the GlcNAc moiety to Ser/Thr/Asn residues of proteins. Global changes in protein O-GlcNAcylation are emerging as a general characteristic of cancer cells. In our recent study, we demonstrated that the EMT-HBP-O-GlcNAcylation axis drives the O-GlcNAcylation of key proteins such as c-Myc, which previous studies have shown to suppress oncogene-induced senescence (OIS) and contribute to accelerated tumorigenesis. Here, we review the HBP and O-GlcNAcylation and their putative roles in driving EMT-related cancer processes with examples to illuminate potential new therapeutic targets for cancer.

Biochemical characterization of a ß-N-acetylhexosaminidase from Streptomyces alfalfae and its application in the production of N-acetyl-d-glucosamine.

N-Acetyl-d-glucosamine (GlcNAc) is a valuable monosaccharide widely used in the medical, agricultural, biofuel, and food industries. Its efficient and environment-friendly production depends on the binary system of ß-N-acetylhexosaminidase (HEX) and chitinase. In the present study, a HEX of glycoside hydrolasefamily 20 was identified in Streptomyces alfalfae ACCC40021, and was overexpressed in Escherichia coli. The purified recombinant SaHEX showed maximal activities at 60°C and pH 5.5, and retained stable up to 45°C. The enzyme not only exhibited broad substrate specificity including p-nitrophenyl ß-N-acetylglucosaminide, p-nitrophenyl ß-N-acetylgalactosaminide, chitooligosaccharides and colloidal chitin, but also had higher specific activities (up to 1149.7 ± 72.6 U/mg) towards natural and synthetic substrates. When combined with a commercial chitinase, it achieved a conversion rate of 93.7% from 1% of colloidal chitin to GlcNAc in 6 h, with the product purity of >98%. These excellent properties make SaHEX a potential enzyme candidate for the chitin conversion for various industrial purposes.

Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis.

One of the primary goals of microbial metabolic engineering is to achieve high titer, yield and productivity (TYP) of engineered strains. This TYP index requires optimized carbon flux toward desired molecule with minimal by-product formation. De novo redesign of central carbon and redox metabolism holds great promise to alleviate pathway bottleneck and improve carbon and energy utilization efficiency. The engineered strain, with the overexpression or deletion of multiple genes, typically can't meet the TYP index, due to overflow of central carbon and redox metabolism that compromise the final yield, despite a high titer or productivity might be achieved. To solve this challenge, we reprogramed the central carbon and redox metabolism of Bacillus subtilis and achieved high TYP production of N-acetylglucosamine. Specifically, a "push-pull-promote" approach efficiently reduced the overflown acetyl-CoA flux and eliminated byproduct formation. Four synthetic NAD(P)-independent metabolic routes were introduced to rewire the redox metabolism to minimize energy loss. Implementation of these genetic strategies led us to obtain a B. subtilis strain with superior TYP index. GlcNAc titer in shake flask was increased from 6.6 g L-1 to 24.5 g L-1, the yield was improved from 0.115 to 0.468 g GlcNAc g-1 glucose, and the productivity was increased from 0.274 to 0.437 g L-1 h-1. These titer and yield are the highest levels ever reported and, the yield reached 98% of the theoretical pathway yield (0.478 g g-1 glucose). The synthetic redesign of carbon metabolism and redox metabolism represent a novel and general metabolic engineering strategy to improve the performance of microbial cell factories.

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells.

In prokaryotic cells, 3'-5' exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3'-5' untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base "N." Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3'-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3'-terminus of the mRNA by blocking the processing action of the 3'-5' exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.

N-Acetyl-D-glucosamine Production by a Chitinase of Marine Fungal Origin: a Case Study of Potential Industrial Significance for Valorization of Waste Chitins.

Chitin is a linear homo-polymer of N-acetyl-D-glucosamine (GlcNAc) and the second most abundant biopolymer after cellulose. Several industries rely on the bioprocesses for waste chitin recycle and hydrolysis by chitinase (EC 3.2.1.14) for potential healthcare applications through the production of its monomeric subunit, GlcNAc. In the present study, a chitinase-producing fungus (named as MFSRK-S42) was isolated from the marine water sample of North Bay of the Andaman and Nicobar Islands. It was identified as Aspergillus terreus by morphological and molecular characterization methods leveraging the internal transcribed spacer between 18S rRNA and 5.8S rRNA. Chitinase that was isolated from the fermentation broth of marine Aspergillus terreus was used to carry out biotransformation of chitineaceous wastes. Prior to the enzymatic hydrolysis step, chitins from different sources were characterized for the presence of characteristic functional groups, grain size distribution, and surface morphology. Enzymatic hydrolysis of 50 mg/ml substrate with six units of enzyme incubated for 5 days revealed 15, 36.5, 40, and 46 mg/ml GlcNAc production from ground prawn shell, chitin flakes, colloidal prawn shell, and swollen chitin respectively under standardized conditions, as determined by HPLC. In this study, 30, 73, 80, and 92% GlcNAc yields were observed from ground prawn shell, chitin flakes, colloidal prawn shell, and swollen chitin conversion respectively. The HPLC-eluted product was confirmed as GlcNAc by the presence of characteristic functional groups in FTIR and 244 Da molecular weight peak in HRMS analyses.

CRISPRi allows optimal temporal control of N-acetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis.

Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ±â€¯0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ±â€¯0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ±â€¯0.85 g/L GlcNAc with a yield of 0.46 ±â€¯0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ±â€¯2.11 g/L and 1.17 ±â€¯0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories.

mTORC2 modulates the amplitude and duration of GFAT1 Ser-243 phosphorylation to maintain flux through the hexosamine pathway during starvation.

The mechanistic target of rapamycin (mTOR) controls metabolic pathways in response to nutrients. Recently, we have shown that mTOR complex 2 (mTORC2) modulates the hexosamine biosynthetic pathway (HBP) by promoting the expression of the key enzyme of the HBP, glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1). Here, we found that GFAT1 Ser-243 phosphorylation is also modulated in an mTORC2-dependent manner. In response to glutamine limitation, active mTORC2 prolongs the duration of Ser-243 phosphorylation, albeit at lower amplitude. Blocking glycolysis using 2-deoxyglucose robustly enhances Ser-243 phosphorylation, correlating with heightened mTORC2 activation, increased AMPK activity, and O-GlcNAcylation. However, when 2-deoxyglucose is combined with glutamine deprivation, GFAT1 Ser-243 phosphorylation and mTORC2 activation remain elevated, whereas AMPK activation and O-GlcNAcylation diminish. Phosphorylation at Ser-243 promotes GFAT1 expression and production of GFAT1-generated metabolites including ample production of the HBP end-product, UDP-GlcNAc, despite nutrient starvation. Hence, we propose that the mTORC2-mediated increase in GFAT1 Ser-243 phosphorylation promotes flux through the HBP to maintain production of UDP-GlcNAc when nutrients are limiting. Our findings provide insights on how the HBP is reprogrammed via mTORC2 in nutrient-addicted cancer cells.

Characterising N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12), the enzyme that catalyses the second step of GPI biosynthesis in Candida albicans.

Candida albicans N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12) recognises N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) from Saccharomyces cerevisiae and is able to complement ScGPI12 function. Both N- and C-terminal ends of CaGpi12 are important for its function. CaGpi12 was biochemically characterised using rough endoplasmic reticulum microsomes prepared from BWP17 strain of C. albicans. CaGpi12 is optimally active at 30°C and pH 7.5. It is a metal-dependent enzyme that is stimulated by divalent cations but shows no preference for Zn2+ unlike the mammalian homologue. It irreversibly loses activity upon incubation with a metal chelator. Two conserved motifs, HPDDE and HXXH, are both important for its function in the cell. CaGPI12 is essential for growth and viability of C. albicans. Its loss causes reduction of GlcNAc-PI de-N-acetylase activity, cell wall defects and filamentation defects. The filamentation defects could be specifically correlated to an upregulation of the HOG1 pathway.

ß-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin.

Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable ß-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55 °C (144 h, 75% activity), 60 °C (48 h, 85% activity; half-life 82 h), and 70 °C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2-5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1-3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MthNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.

Neural functions of bisecting GlcNAc.

Bisecting GlcNAc, a branch structure in N-glycan, has unique functions and is involved in several diseases including Alzheimer's disease (AD). In this review, we provide an overview of the biosynthesis of bisecting GlcNAc and its physiological and pathological functions, particularly in the nervous system where bisecting GlcNAc is most highly expressed. The biosynthetic enzyme of bisecting GlcNAc is N-acetylglucosaminyltransferase-III (GnT-III). Overexpression, knockdown, and knockout of GnT-III have so far revealed various functions of bisecting GlcNAc, which are mediated by regulating the functions of key carrier proteins. GnT-III-deficient AD model mice showed reduced amyloid-ß (Aß) accumulation in the brain by suppressing the function of a key Aß-generating enzyme, ß-site APP-cleaving enzyme-1 (BACE1), and greatly improved AD pathology. Altered BACE1 subcellular localization in GnT-III-deficient cells, from early endosomes to lysosomes, suggests that bisecting GlcNAc serves as a trafficking tag for the movement of modified proteins to an endosomal compartment. For therapeutic application, we have employed high-throughput screening to search for GnT-III inhibitors. These findings highlight the importance of bisecting GlcNAc modification in the nervous system.

Production of chitinase from Escherichia fergusonii, chitosanase from Chryseobacterium indologenes, Comamonas koreensis and its application in N-acetylglucosamine production.

The important platform polysaccharide N-acetylglucosamine (GlcNAc) has great potential to be used in the fields of food, cosmetics, agricultural, pharmaceutical, medicine and biotechnology. This GlcNAc is being produced by traditional methods of environment-unfriendly chemical digestion with strong acids. Therefore, researchers have been paying more attention to enzymatic hydrolysis process for the production of GlcNAc. Hence, in this study, we isolated novel chitinase (Escherichia fergusonii) and chitosanase (Chryseobacterium indologenes, Comamonas koreensis) producing strains from Korean native calves feces, and developed the potential of an eco-friendly microbial progression for GlcNAc production from swollen chitin and chitosan by enzymatic degradation. Maximum chitinase (7.24±0.07U/ml) and chitosanase (8.42±0.09, 8.51±0.25U/ml) enzyme activity were reached in submerged fermentation at an optimal pH of 7.0 and 30°C. In this study, sucrose, yeast extract, (NH4)2SO4, and NaCl were found to be the potential enhancers of exo-chitinase activity and glucose, corn flour, yeast extract, soybean flour, (NH4)2SO4, NH4Cl and K2HPO4 were found to be the potential activator for exo-chitosanase activity. Optimum concentrations of the carbon sources for enhanced chitinase activity were 9.91, 3.21, 9.86, 1.66U/ml and chitosanase activity were 1.63, 1.13, 2.28, 3.71, 9.02, 4.93, and 2.14U/ml. These enzymes efficiently hydrolyzed swollen chitin and chitosan to N-acetylglucosamine were characterized by thin layer chromatography and were further confirmed by high-pressure liquid chromatography. From a commercial perspective, we isolated, optimized and characterized exochitinase from Escherichia fergusonii (HANDI 110) and chitosanase from Chryseobacterium indologenes (HANYOO), and Comamonas koreensis (HANWOO) for the large-scale production of GlcNAc facilitating its potential use in industrial applications.

Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches.

Autosomal recessive loss-of-function mutations in N-glycanase 1 (NGLY1) cause NGLY1 deficiency, the only known human disease of deglycosylation. Patients present with developmental delay, movement disorder, seizures, liver dysfunction and alacrima. NGLY1 is a conserved cytoplasmic component of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. ERAD clears misfolded proteins from the ER lumen. However, it is unclear how loss of NGLY1 function impacts ERAD and other cellular processes and results in the constellation of problems associated with NGLY1 deficiency. To understand how loss of NGLY1 contributes to disease, we developed a Drosophila model of NGLY1 deficiency. Loss of NGLY1 function resulted in developmental delay and lethality. We used RNAseq to determine which processes are misregulated in the absence of NGLY1. Transcriptome analysis showed no evidence of ER stress upon NGLY1 knockdown. However, loss of NGLY1 resulted in a strong signature of NRF1 dysfunction among downregulated genes, as evidenced by an enrichment of genes encoding proteasome components and proteins involved in oxidation-reduction. A number of transcriptome changes also suggested potential therapeutic interventions, including dysregulation of GlcNAc synthesis and upregulation of the heat shock response. We show that increasing the function of both pathways rescues lethality. Together, transcriptome analysis in a Drosophila model of NGLY1 deficiency provides insight into potential therapeutic approaches.

Synthesis of N-acetyl-d-quinovosamine in Rhizobium etli CE3 is completed after its 4-keto-precursor is linked to a carrier lipid.

Bacterial O-antigens are synthesized on lipid carriers before being transferred to lipopolysaccharide core structures. Rhizobium etli CE3 lipopolysaccharide is a model for understanding O-antigen biological function. CE3 O-antigen structure and genetics are known. However, proposed enzymology for CE3 O-antigen synthesis has been examined very little in vitro, and even the sugar added to begin the synthesis is uncertain. A model based on mutagenesis studies predicts that 2-acetamido-2,6-dideoxy-d-glucose (QuiNAc) is the first O-antigen sugar and that genes wreV, wreQ and wreU direct QuiNAc synthesis and O-antigen initiation. Previously, synthesis of UDP-QuiNAc was shown to occur in vitro with a WreV orthologue (4,6-hexose dehydratase) and WreQ (4-reductase), but the WreQ catalysis in this conventional deoxyhexose-synthesis pathway was very slow. This seeming deficiency was explained in the present study after WreU transferase activity was examined in vitro. Results fit the prediction that WreU transfers sugar-1-phosphate to bactoprenyl phosphate (BpP) to initiate O-antigen synthesis. Interestingly, WreU demonstrated much higher activity using the product of the WreV catalysis [UDP-4-keto-6-deoxy-GlcNAc (UDP-KdgNAc)] as the sugar-phosphate donor than using UDP-QuiNAc. Furthermore, the WreQ catalysis with WreU-generated BpPP-KdgNAc as the substrate was orders of magnitude faster than with UDP-KdgNAc. The inferred product BpPP-QuiNAc reacted as an acceptor substrate in an in vitro assay for addition of the second O-antigen sugar, mannose. These results imply a novel pathway for 6-deoxyhexose synthesis that may be commonly utilized by bacteria when QuiNAc is the first sugar of a polysaccharide or oligosaccharide repeat unit: UDP-GlcNAc → UDP-KdgNAc → BpPP-KdgNAc → BpPP-QuiNAc.