Unveiling the importance of the C-terminus in the sugar acid dehydratase of the IlvD/EDD superfamily.

Microbial non-phosphorylative oxidative pathways present promising potential in the biosynthesis of platform chemicals from the hemicellulosic fraction of lignocellulose. An L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii catalyzes the rate-limiting step in the non-phosphorylative oxidative pathways, that is, converts sugar acid to 2-dehydro-3-deoxy sugar acid. We have shown earlier that the enzyme forms a dimer of dimers, in which the C-terminal histidine residue from one monomer participates in the formation of the active site of an adjacent monomer. The histidine appears to be conserved across the sequences of sugar acid dehydratases. To study the role of the C-terminus, five variants (H579A, H579F, H579L, H579Q, and H579W) were produced. All variants showed decreased activity for the tested sugar acid substrates, except the variant H579L on D-fuconate, which showed about 20% increase in activity. The reaction kinetic data showed that the substrate preference was slightly modified in H579L compared to the wild-type enzyme, demonstrating that the alternation of the substrate preference of sugar acid dehydratases is possible. In addition, a crystal structure of H579L was determined at 2.4 Å with a product analog 2-oxobutyrate. This is the first enzyme-ligand complex structure from an IlvD/EDD superfamily enzyme. The binding of 2-oxobutyrate suggests how the substrate would bind into the active site in the orientation, which could lead to the dehydration reaction. KEY POINTS: • Mutation of the last histidine at the C-terminus changed the catalytic activity of L-arabinonate dehydratase from R. leguminosarum bv. trifolii against various C5/C6 sugar acids. • The variant H579L of L-arabinonate dehydratase showed an alteration of substrate preferences compared with the wild type. • The first enzyme-ligand complex crystal structure of an IlvD/EDD superfamily enzyme was solved.

Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras.

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).

Engineering Gluconbacter oxydans with efficient co-utilization of glucose and sorbitol for one-step biosynthesis of 2-keto-L-gulonic.

As the highest-demand vitamin, the development of a one-step vitamin C synthesis process has been slow for a long time. In previous research, a Gluconobacter oxydans strain (GKLG9) was constructed that can directly synthesize 2-keto-L-gulonic acid (2-KLG) from glucose, but carbon source utilization remained low. Therefore, this study first identified the gene 4kas (4-keto-D-arabate synthase) to reduce the loss of extracellular carbon and inhibit the browning of fermentation broth. Then, promoter engineering was conducted to enhance the intracellular glucose transport pathway and concentrate intracellular glucose metabolism on the pentose phosphate pathway to provide more reducing power. Finally, by introducing the D-sorbitol pathway, the titer of 2-KLG was increased to 38.6 g/L within 60 h in a 5-L bioreactor, with a glucose-to-2-KLG conversion rate of about 46 %. This study is an important step in the development of single-bacterial one-step fermentation to produce 2-KLG.

Catabolism of 2-keto-3-deoxy-galactonate and the production of its enantiomers.

2-Keto-3-deoxy-galactonate (KDGal) serves as a pivotal metabolic intermediate within both the fungal D-galacturonate pathway, which is integral to pectin catabolism, and the bacterial DeLey-Doudoroff pathway for D-galactose catabolism. The presence of KDGal enantiomers, L-KDGal and D-KDGal, varies across these pathways. Fungal pathways generate L-KDGal through the reduction and dehydration of D-galacturonate, whereas bacterial pathways produce D-KDGal through the oxidation and dehydration of D-galactose. Two distinct catabolic routes further metabolize KDGal: a nonphosphorolytic pathway that employs aldolase and a phosphorolytic pathway involving kinase and aldolase. Recent findings have revealed that L-KDGal, identified in the bacterial catabolism of 3,6-anhydro-L-galactose, a major component of red seaweeds, is also catabolized by Escherichia coli, which is traditionally known to be catabolized by specific fungal species, such as Trichoderma reesei. Furthermore, the potential industrial applications of KDGal and its derivatives, such as pyruvate and D- and L-glyceraldehyde, are underscored by their significant biological functions. This review comprehensively outlines the catabolism of L-KDGal and D-KDGal across different biological systems, highlights stereospecific methods for discriminating between enantiomers, and explores industrial application prospects for producing KDGal enantiomers. KEY POINTS: • KDGal is a metabolic intermediate in fungal and bacterial pathways • Stereospecific enzymes can be used to identify the enantiomeric nature of KDGal • KDGal can be used to induce pectin catabolism or produce functional materials.

Chemical Synthesis of an Orthogonally Protected 5,7-Diamino-3,5,7,9-tetradeoxy-d-glycero-l-gluco-2-nonulosonic Acid from N-Acetylneuraminic Acid.

Bacterial nonulosonic acids (NulOs), which feature a nine-carbon backbone, are associated with the biological functions of bacterial glycans. Here, an orthogonally protected 5-amino-7-azido-3,5,7,9-tetradeoxy-d-glycero-l-gluco-2-nonulosonic acid related to Fusobacterium nucleatum ATCC 23726 NulO was synthesized from N-acetylneuraminic acid with sequential performance of C5,7 azidation, C9 deoxygenation, C4 epimerization, and N5,7 differentiation. The C5 azido group in the obtained 5,7-diazido-NulO can be regioselectively reduced to differentiate the two amino groups.

Rhodotorula mucilaginosa A8, a potential helper strain in a vitamin C microbial fermentation process.

In the vitamin C microbial fermentation system, oxidative stress limits the growth and 2-keto-l-gulonic acid (2-KLG, the precursor of vitamin C) production of Ketogulonicigenium vulgare. Most Bacillus strains, as helper strains, have been reported to release key biomolecules to reduce oxidative stress and promote the growth and 2-KLG production of K. vulgare. To understand the specific mechanism by which the helper strain and K. vulgare interact to reduce oxidative stress, a novel helper strain, Rhodotorula mucilaginosa A8, was used to construct a consortium in the co-culture fermentation system. Based on the activities of the antioxidant enzymes and quantitative polymerase chain reaction (qPCR) analysis, R. mucilaginosa A8 could reduce oxidative stress and increase 2-KLG production in K. vulgare by upregulating antioxidant enzyme activities and related gene-expression levels. In addition, the carotenoids of R. mucilaginosa promoted 2-KLG production in K. vulgare. Coculture of R. mucilaginosa with K. vulgare increased the yield of carotenoids. This study suggested that helper strains with the ability to reduce oxidative stress in K. vulgare would likely act as potential helper strains for facilitating 2-KLG biosynthesis. This work could provide a theoretical basis for the search for potential helper strains for vitamin C microbial fermentation and for the construction of synthetic microbial communities to produce valuable products.

Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004.

The titer of the microbial fermentation products can be increased by enzyme engineering. l-Sorbosone dehydrogenase (SNDH) is a key enzyme in the production of 2-keto-l-gulonic acid (2-KLG), which is the precursor of vitamin C. Enhancing the activity of SNDH may have a positive impact on 2-KLG production. In this study, a computer-aided semirational design of SNDH was conducted. Based on the analysis of SNDH's substrate pocket and multiple sequence alignment, three modification strategies were established: (1) expanding the entrance of SNDH's substrate pocket, (2) engineering the residues within the substrate pocket, and (3) enhancing the electron transfer of SNDH. Finally, mutants S453A, L460V, and E471D were obtained, whose specific activity was increased by 20, 100, and 10%, respectively. In addition, the ability of Gluconobacter oxidans WSH-004 to synthesize 2-KLG was improved by eliminating H2O2. This study provides mutant enzymes and metabolic engineering strategies for the microbial-fermentation-based production of 2-KLG.

KpsS1 Mediates the Glycosylation of Pseudaminic Acid in Acinetobacter Baumannii.

Pseudaminic acid (Pse) is found in the polysaccharide structures of the cell surface of various Gram-negative pathogenic bacteria including Acinetobacter baumannii and considered as an important component of cell surface glycans including oligosaccharides and glycoproteins. However, the glycosyltransferase that is responsible for the Pse glycosylation in A. baumannii remains unknown yet. In this study, through comparative genomics analysis of Pse-positive and negative A. baumannii clinical isolates, we identified a potential glycosyltransferase, KpsS1, located right downstream of the Pse biosynthesis genetic locus. Deletion of this gene in an Pse-positive A. baumannii strain, Ab8, impaired the glycosylation of Pse to the surface CPS and proteins, while the gene knockout strain, Ab8ΔkpsS1, could still produce Pse with 2.86 folds higher amount than that of Ab8. Furthermore, impairment of Pse glycosylation affected the morphology and virulence potential of A. baumannii, suggesting the important role of this protein. This study will provide insights into the further understanding of Pse in bacterial physiology and pathogenesis.

[Dental caries - the most common noncommunicable disease and its impact on general health and disease]. / Karies ­ världens vanligaste icke smittsamma sjukdom.

More than 2.8 billion individuals worldwide suffer from untreated caries. Over ninety-five percent of all 50-year-olds in Sweden have caries experience. Caries is the most common cause of dental restorations and tooth loss. Tooth loss is associated with cardiovascular diseases, dementia, and death. Periapical tooth infections caused by caries can spread and cause severe infection, however rarely with lethal outcome. Sugars are a common risk factor for caries and other noncommunicable diseases such as cardiovascular diseases, diabetes, and obesity.  Caries is a consequence of sugar-provoked acid production and dysbiosis in the tooth biofilm (dental plaque). There are several conditions which may increase the risk for dental caries, such as different medical conditions and medications which may cause dry mouth. Treatment costs for caries are high.

Establishment of efficient 5-hydroxyvaleric acid production system by regenerating alpha-ketoglutaric acid and its application in poly(5-hydroxyvaleric acid) production.

5-hydroxyvaleric acid (5-HV) is a versatile C5 intermediate of bio-based high-value chemical synthesis pathways. However, 5-HV production faces a few shortcomings involving the supply of cofactors, especially α-ketoglutaric acid (α-KG). Herein, we established a two-cell biotransformation system by introducing L-glutamate oxidase (GOX) to regenerate α-KG. Additionally, the catalase KatE was adapted to inhibit α-KG degradation by the H2O2 produced during GOX reaction. We searched for the best combination of genes and vectors and optimized the biotransformation conditions to maximize GOX effectiveness. Under the optimized conditions, 5-HV pathway with GOX showed 1.60-fold higher productivity than that of without GOX, showing 11.3 g/L titer. Further, the two-cell system with GOX and KatE was expanded to produce poly(5-hydroxyvaleric acid) (P(5HV)), and it reached at 412 mg/L of P(5HV) production and 20.5% PHA contents when using the biotransformation supernatant. Thus, the two-cell biotransformation system with GOX can potentially give the practical and economic alternative of 5-HV production using bio-based methods. We also propose direct utilization of 5-HV from bioconversion for P(5HV) production.

Bacterial pseudaminic acid binding to Siglec-10 induces a macrophage interleukin-10 response and suppresses phagocytosis.

Pseudaminic acid (Pse) on pathogenic bacteria exopolysaccharide engages with the sialic acid-binding immunoglobulin-type lectin (Siglec)-10 receptor on macrophages via the critical 7-N-acetyl group. This binding stimulates macrophages to secrete interleukin 10 that suppresses phagocytosis against bacteria, but can be reverted by blocking Pse-Siglec-10 interaction with Pse-binding protein as a promising therapy.

Recent Advances in 2-Keto-l-gulonic Acid Production Using Mixed-Culture Fermentation and Future Prospects.

Vitamin C, also known as ascorbic acid, is an essential vitamin that cannot be synthesized by the human body and must be acquired through our diet. At present, the precursor of vitamin C, 2-keto-l-gulonic acid (2-KGA), is typically produced via a two-step fermentation process utilizing three bacterial strains. The second step of this traditional two-step fermentation method involves mixed-culture fermentation employing 2-KGA-producing bacteria (Ketogulonicigenium vulgare) along with associated bacteria. Because K. vulgare has defects in various metabolic pathways, associated bacteria are needed to provide key substances to promote K. vulgare growth and 2-KGA production. Unlike previous reviews where the main focus was the interaction between associated bacteria and K. vulgare, this Review presents the latest scientific research from the perspective of the metabolic pathways associated with 2-KGA production by K. vulgare and the mechanism underlying the interaction between K. vulgare and the associated bacteria. In addition, the dehydrogenases that are responsible for 2-KGA production, the 2-KGA synthesis pathway, strategies for simplifying 2-KGA production via a one-step fermentation route, and, finally, future prospects and research goals in vitamin C production are also presented.

Co-factor prosthesis facilitates biosynthesis of azido-pseudaminic acid probes for use as glycosyltransferase reporters.

Truncated thioester N,S-diacetylcysteamine (SNAc) was utilised as a co-factor mimic for PseH, an acetyl-coA dependent aminoglycoside N-acetyltransferase, in the biosynthesis of the bacterial sugar, pseudaminic acid. Additionally, an azido-SNAc analogue was used to smuggle N7-azide functionality into the pseudaminic acid backbone, facilitating its use as a reporter of pseudaminyltransferase activity.

The Retaining Pse5Ac7Ac Pseudaminyltransferase KpsS1 Defines a Previously Unreported glycosyltransferase family (GT118).

Cell surface sugar 5,7-diacetyl pseudaminic acid (Pse5Ac7Ac) is a bacterial analogue of the ubiquitous sialic acid, Neu5Ac, and contributes to the virulence of a number of multidrug resistant bacteria, including ESKAPE pathogens Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite its discovery in the surface glycans of bacteria over thirty years ago, to date no glycosyltransferase enzymes (GTs) dedicated to the synthesis of a pseudaminic acid glycosidic linkage have been unequivocally characterised in vitro. Herein we demonstrate that A. baumannii KpsS1 is a dedicated pseudaminyltransferase enzyme (PseT) which constructs a Pse5Ac7Ac-α(2,6)-Glcp linkage, and proceeds with retention of anomeric configuration. We utilise this PseT activity in tandem with the biosynthetic enzymes required for CMP-Pse5Ac7Ac assembly, in a two-pot, seven enzyme synthesis of an α-linked Pse5Ac7Ac glycoside. Due to its unique activity and protein sequence, we also assign KpsS1 as the prototypical member of a previously unreported GT family (GT118).

Discovery of a Novel Cellobiose Dehydrogenase from Cellulomonas palmilytica EW123 and Its Sugar Acids Production.

Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 (CpCDH) was cloned and characterized. The CpCDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in CpCDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of CpCDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of CpCDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The CpCDH displayed excellent enzymatic activity at low temperatures (below 30°C), unlike other CDHs. Moreover, CpCDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of CpCDH for cellobiose and lactose were 2.05 x 105 and 9.06 x 104 (M-1 s-1), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of CpCDH. This study establishes CpCDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.

Stereocontrolled Synthesis of α-3-Deoxy-d-manno-oct-2-ulosonic Acid (α-Kdo) Glycosides Using C3-p-Tolylthio-Substituted Kdo Donors: Access to Highly Branched Kdo Oligosaccharides.

3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an eight-carbon monosaccharide found widely in bacterial lipopolysaccharides (LPSs) and capsule polysaccharides (CPSs). We developed an indirect method for the stereoselective synthesis of α-Kdo glycosides with a C3-p-tolylthio-substituted Kdo phosphite donor. The presence of the p-tolylthio group enhanced the reactivity, suppressed the formation of elimination by-products (2,3-enes), and provided complete α-stereocontrol. A variety of Kdo α-glycosides were synthesized by our method in excellent yields (up to 98 %). After glycosylation, the p-tolylthio group can be efficiently removed by free-radical reduction. Subsequently, the orthogonality of the phosphite donor and thioglycoside donor was demonstrated by the one-pot synthesis of a trisaccharide in Helicobacter pylori and Neisseria meningitidis LPS. Moreover, an efficient total synthesis route to the challenging 4,5-branched Kdo trisaccharide in LPSs from several A. baumannii strains was highlighted. To demonstrate the high reactivity of our approach further, the highly crowded 4,5,7,8-branched Kdo pentasaccharide was synthesized as a model molecule for the first time. Additionally, the reaction mechanism was investigated by DFT calculations.

Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae.

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

Application of a novel chemical assay for the quantification of endotoxins in bacterial bioreactor samples.

A novel chemical assay, the so-called Kdo-DMB-liquid chromatography (LC) assay, was used for the accurate and cost-effective determination of the endotoxin content in supernatants of Gram-negative bacteria bioreactor samples. During mild acid hydrolysis, the endotoxin-specific sugar acid 3-deoxy-D-manno-oct-2-ulsonic acid (Kdo) is quantitatively released. Kdo is reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) to obtain the highly fluorescent derivate Kdo-DMB. It is separated from the reaction mixture by reversed phase-(U)HPLC and detected by fluorescence. From the Kdo content the endotoxin content of the sample is calculated. For three batch cultivations of Escherichia coli K12 and a fed-batch cultivation of Pseudomonas putida KT2440, the evolution of the endotoxin content in dependence on the cultivation time was monitored. Under optimal, constant cultivation conditions a linear correlation between the endotoxin content and the easy-to-access bioreactor parameters optical density at 600 nm and dry cell weight was found for both endotoxin kinds. Under stress cultivation conditions the E. coli K12 cultivation showed a stronger increase of the endotoxin content at harvest in comparison to optimal conditions. Optical density and dry cell weight may be used for production reactors as an economic real-time estimation tool to determine the endotoxin content at different cultivation time points and conditions. The optical density can further be used to establish straightforward sample dilution schemes for endotoxin quantification in samples of unknown endotoxin content. The endotoxin content [ng mL-1] measured by the Kdo-DMB-LC assay and the endotoxin activity [EU mL-1] obtained by the compendial Limulus Amoebocyte Lysate assay show a high correlation for the bacterial bioreactor samples tested.

Sialic acids Neu5Ac and KDN in adipose tissue samples from individuals following habitual vegetarian or non-vegetarian dietary patterns.

Sialic acids (Sias) are a class of sugar molecules with a parent nine-carbon neuraminic acid, generally present at the ends of carbohydrate chains, either attached to cellular surfaces or as secreted glycoconjugates. Given their position and structural diversity, Sias modulate a wide variety of biological processes. However, little is known about the role of Sias in human adipose tissue, or their implications for health and disease, particularly among individuals following different dietary patterns. The goal of this study was to measure N-Acetylneuraminic acid (Neu5Ac), N-Glycolylneuraminic acid (Neu5Gc), and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) concentrations in adipose tissue samples from participants in the Adventist Health Study-2 (AHS-2) and to compare the abundance of these Sias in individuals following habitual, long-term vegetarian or non-vegetarian dietary patterns. A method was successfully developed for the extraction and detection of Sias in adipose tissue. Sias levels were quantified in 52 vegans, 56 lacto-vegetarians, and 48 non-vegetarians using LC-MS/MS with Neu5Ac-D-1,2,3-13C3 as an internal standard. Dietary groups were compared using linear regression. Vegans and lacto-ovo-vegetarians had significantly higher concentrations of Neu5Ac relative to non-vegetarians. While KDN levels tended to be higher in vegans and lacto-ovo-vegetarians, these differences were not statistically significant. However, KDN levels were significantly inversely associated with body mass index. In contrast, Neu5Gc was not detected in human adipose samples. It is plausible that different Neu5Ac concentrations in adipose tissues of vegetarians, compared to those of non-vegetarians, reflect a difference in the baseline inflammatory status between the two groups. Epidemiologic studies examining levels of Sias in human adipose tissue and other biospecimens will help to further explore their roles in development and progression of inflammatory conditions and chronic diseases.

Efficient production of 2-keto-L-gulonic acid from D-glucose in Gluconobacter oxydans ATCC9937 by mining key enzyme and transporter.

Direct production of 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) from D-glucose through 2,5-diketo-D-gluconic acid (2,5-DKG) is a promising alternative route. To explore the pathway of producing 2-KLG from D-glucose, Gluconobacter oxydans ATCC9937 was selected as a chassis strain. It was found that the chassis strain naturally has the ability to synthesize 2-KLG from D-glucose, and a new 2,5-DKG reductase (DKGR) was found on its genome. Several major issues limiting production were identified, including the insufficient catalytic capacity of DKGR, poor transmembrane movement of 2,5-DKG and imbalanced D-glucose consumption flux inside and outside of the host strain cells. By identifying novel DKGR and 2,5-DKG transporter, the whole 2-KLG biosynthesis pathway was systematically enhanced by balancing intracellular and extracellular D-glucose metabolic flux. The engineered strain produced 30.5 g/L 2-KLG with a conversion ratio of 39.0%. The results pave the way for a more economical large-scale fermentation process for vitamin C.