Starch metabolism is a key determinant of durian (Durio zibethinus L.) fruit quality, as it influences texture and sweetness during ripening. However, the molecular basis of starch branching during its accumulation remains poorly understood. In this study, we characterized a durian starch branching enzyme, DzSBE1, which is highly expressed at the immature-to-mature stage. Transcriptome and phylogenetic analyses revealed that DzSBE1 belongs to the plant SBE1 subfamily and contains all of the conserved catalytic features of the α-amylase superfamily. Recombinant DzSBE1, expressed as a soluble protein in Escherichia coli and likely dimeric, exhibited clear α-1,6-branching activity toward both amylose and amylopectin, with a preference for amylose. The enzyme showed optimal activity at pH 6.0-7.0 and 30-35 °C and does not require metal ions for catalysis. Product structural analyses revealed that DzSBE1 converts linear glucans into a more highly branched structure with enhanced crystallinity. Subcellular localization and transient overexpression in Nicotiana benthamiana leaves confirmed that DzSBE1 functions within plastids to promote α-1,6-branching. Furthermore, promoter analysis of DzSBE1 identified MYB and bHLH1 binding motifs. Dual-luciferase assays confirmed that DzMYB61-like, DzMYBETC3-like, and DzbHLH1 act as transcriptional activators, whereas DzMYB1 functions as a repressor. Co-expression assays further demonstrated that specific MYB-bHLH1 pairs cooperatively regulate DzSBE1 promoter activity. These findings indicate that DzSBE1 may act as an important enzyme facilitating starch branching during durian fruit maturation and provide comprehensive insights into the regulatory mechanisms linking starch synthesis to quality development in this economically important tropical crop.

Levan-type fructooligosaccharides (LFOSs) possess valuable bioactivities but are produced in low yields by Gram-negative bacterial levansucrases. Here, we rationally engineered the levanbiose-binding site of Erwinia tasmaniensis levansucrase (EtLsc) to enhance LFOS synthesis. Eleven residues surrounding the levanbiose-binding site were substituted to disrupt levan chain binding or to modulate loop flexibility. The results indicate that D82A, R377A, and G379P do not synthesize levan polymers. In addition, R377A and G379P largely produced LFOS, converting 35% and 24% of sucrose into LFOS. Affinity PAGE confirmed the loss of the levan-binding ability of the R377A and G379P variants. MD simulations revealed that G379P induces a structural rearrangement of loop II, altering the orientation of the key residue R377. Prebiotic activity analysis demonstrated that LFOS synthesized by the R377A variant stimulated the growth of probiotic bacteria, including Limosilactobacillus fermentum, and Lacticaseibacillus casei, more effectively than inulin. Together, these results identify R377 and G379 as critical determinants of levan elongation and demonstrate a promising strategy for enhancing LFOS production with Gram-negative levansucrases.

Sialic acids - 9-carbon ulosonic acids - are implicated in many cell-cell and host-pathogen interactions due to their prevalent location at the non-reducing end of glycoconjugates. Sialic acids have recently been observed in microalgae, including the toxic bloom-forming Prymnesium parvum, which produces the deaminated sialic acid, ketodeoxynonulosonic acid (Kdn), through de novo biosynthesis. Here we report on the key CMP-sialic acid synthetase enzyme (CMAS), PpNeuA, which activates Kdn to its sugar nucleotide congener, CMP-Kdn. In the present study, the X-ray crystal structure of PpNeuA was determined to 1.8 Å resolution and shows that it adopts a similar overall fold to that of other sialic acid synthetase enzymes, with which it shares ca 30% amino acid sequence identity. PpNeuA specificity for Kdn is dependent upon Arg196, a hydrophilic residue that is only found in Kdn-specific sialic acid synthetases. R196L mutation switches the substrate preference of PpNeuA from Kdn to N-acetylneuraminic acid (Neu5Ac). Kinetic analysis shows that Arg196 plays both a role in substrate binding (impact on K M) and catalysis (impact on k cat). In the context of generating metabolic probes to identify the location and context (glycolipid vs glycoprotein) of Kdn in P. parvum, we also report on the ability of PpNeuA to accept both 5Az-Kdn and 9Az-Kdn as substrates.

Influenza viruses use haemagglutinins (HA) to target host sialic acids in the respiratory tract as do other pathogens, including coronaviruses, which engage using spike protein. The host adaptation of the HA protein, which leads to the accumulation of mutations, is a key descriptor of individual influenza strains, which aids zoonosis and is crucial in vaccine development. How each strain targets glycans is crucial to understanding function, designing new therapies, and optimizing candidates for vaccine development. Here, it is demonstrated that polymer-tethered plasmonic (gold) glyconanoparticles can be deployed for rapid evaluation of whole influenza virus binding, readable by simple UV-vis within a microwell plate as a low-tech alternative to printed microarrays. It is also demonstrated that the synthetic methodology is compatible with large branched glycans from chemoenzymatic synthesis, allowing a wider range of glycan structures to be probed. Particles are obtained by a modular capture and immobilisation process and used to interrogate the binding of five influenza strains as proof of concept. These results show that glycosylated nanoparticle probes are suitable for the rapid interrogation of live virus to map how glycan structure impacts binding and can enable at-bench, rapid virus/glycan binding readouts and aid the development of interventions for influenza and other viruses.

This study introduces a novel enzymatic cascade featuring five recombinant enzymes for the efficient synthesis of fucosylated glycosides, using sucrose exclusively as the sugar donor substrate. In our approach, we employed a sucrose synthase sourced from tomato to generate GDP-glucose from sucrose and GDP. By repurposing CDP-tyvelose 2-epimerase from Salmonella enterica, chosen for its catalytic efficiency from a panel of 27 CDP-tyvelose 2-epimerase candidates, it was possible to epimerize GDP-glucose into GDP-mannose. The subsequent transformation of GDP-d-mannose to GDP-l-fucose was achieved by GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose epimerase/reductase, also derived from S. enterica. In the final stage, Helicobacter pylori α1,3-fucosyltransferase was employed to fucosylate para-nitrophenyl β-lactoside, resulting in the production of para-nitrophenyl 3-fucosyllactoside with a conversion of more than 40%. Analysis of the synthesized compound by LC-MS and NMR analyses substantiated its structure. This investigation not only highlights the utility of this five-enzyme fucosylation cascade but also establishes a novel methodological paradigm for the biocatalytic production of α-l-fucosides for biochemical research and for biotechnological applications.

Levansucrase catalyzes the polymerization of fructose units from sucrose into a β-2,6-linked fructan called "Levan", which are versatile in many applications. While levansucrases from Gram-positive bacteria have well-characterized levan-binding track, the molecular basis for levan elongation by Gram-negative bacteria enzymes remains unclear. Here, we integrated rational mutagenesis, biochemical assays, and molecular dynamics (MD) simulations to elucidate the levan-binding architecture of levansucrease from the Gram-negative bacteria Erwinia tasmaniensis (EtLsc). Using surface topology and residue-frequency mapping, we identified three potential carbohydrate-binding tracks. Alanine scanning of 19 residues revealed that mutations in a distinct surface loop (track II) significantly decreased levan production, particularly F376A, which also abolished gel retention in levan-affinity PAGE. MD simulations proposed strong interactions between levan oligosaccharide and residues F376 and F349, and highlighted a conformational change within the 368-378 loop upon substrate binding. Interestingly, mutations in a second region (track III) selectively altered the product spectrum of β-2,1 fructooligosaccharides and promote levan biosynthesis, suggesting a dual-track model for fructan synthesis. Taken together, the results reveal a unique elongation mechanism in Gram-negative bacteria levansucrases that diverges from that of enzymes from Gram-positive bacteria. These findings provide a structural framework for engineering levansucrases with tunable product profiles for carbohydrate biotechnology applications.

Display technologies are used extensively in the discovery of peptides and antibodies towards the development of new medicines and diagnostic tools. Phage display technology enables the filtering of <1010 random peptides/antibodies down to and enriched pool of candidates with favourable binding affinities. In recent years, next-generation DNA sequencing technologies have increased the precision and accuracy with which the peptide sequences of phage display library clones are identified. Inaccuracies in DNA sequencing such as substitutions, insertions and deletions in the library oligonucleotide region have the potential to result in the identification of erroneous candidate sequences. Here, we describe a Python Pipeline for Phage Analysis through a Normative Unified Toolset (P3ANUT) which employs Levenshtein distance, k-mer approaches and a novel encoding scheme on paired-end sequencing outputs to correct sequencing errors from next-generation sequencing outputs of display library screens. We introduce an easy-to-use and highly customisable computational tool with graphical user- and command line interfaces to process entire datasets within a single input, as well as visualisation tools for candidate analysis and data generation. P3ANUT shows significant improvements in read recovery, overall read quality, and runtime compared to a previously published pipeline. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Medical Research Council, https://ror.org/03x94j517, MR/R015937/1 Biotechnology and Biological Sciences Research Council, BB/M011216/1 [1]: pending:yes

Levan is a versatile biomaterial because of its unique physicochemical properties and bioactivities. To synthesize levan efficiently, it is important to improve the thermostability of levansucrase. This study presents the first engineering study on Erwinia tasmaniensis levansucrase (EtLsc) employing a rational protein design approach. Molecular dynamics (MD) simulations were used to identify thermally sensitive regions of EtLsc, and thermostable variants were designed by using FireProt folding energy calculations. Among the designed candidates, the A197P and S239P mutants had largely higher melting temperatures (Tm) and half-life (t1/2) compared to the wild type. The double variant A197P/S239P exhibited a 7.9 °C increase in Tm and a 48-fold extension of t1/2 at 50 °C, which represents a more significant enhancement than previous studies. Kinetic and product analyses using HPSEC, HPAEC-PAD, and 1H NMR demonstrated that these mutations did not alter the catalytic efficiency or levan structure. The results demonstrate the potential of MD-aided energy-based engineering for thermostable EtLsc designs.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure–activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

We explore biocatalytic aldehyde generation under aqueous conditions, concomitantly delivering access to a one-pot Wittig reaction using stabilized phosphoranes and granting diverse alkene products. Using a recombinant choline oxidase mutant, we first undertake biocatalytic alcohol oxidation across a range of functional aliphatic primary alcohols, demonstrating a remarkable substrate tolerance for this enzyme, including chloride, bromide, azide, S-methyl, and alkynyl groups. Following this, we extend capability and deliver a practicable milligram-scale one-pot Wittig reaction in water.

Regio- and stereo-selective synthetic routes to 2-deoxy-2-fluoro-D-mannose D-mannose building blocks are often experimentally challenging when using Selectfluor with the corresponding glycal. We targeted a late-stage method to introduce fluorine in a stereospecific manner using inversion via a triflate. Accordingly, synthesis of a conventionally protected 2-deoxy-2-fluoro-D-mannose D-mannose beta-thioglycoside donor, directly applicable to oligosaccharide synthesis, was attempted using C2-triflate inversion of the corresponding D-glucoside with TBAF. Unexpectedly, an anomeric pyridinium salt was isolated when attempting to form the C2-triflate using Tf2O 2 O in pyridine. Indicatively, this proceeds via a 1 -> 2 S-migration delivering a 1,2- trans product with alpha-D-manno D-manno configuration and the anomeric pyridinium in a pseudo-equatorial position. The structure of this unexpected intermediate was confirmed in the solid-state using X-ray crystallography. Omission of the pyridine solvent led to dimer formation. Switching the aglycone to an O- para-methoxyphenyl enabled smooth C2 inversion to the desired 2-deoxy-2-fluoro D-mannose system, suitably equipped for further anomeric manipulation.

Correction for ‘Virtual screening, identification and in vitro validation of small molecule GDP-mannose dehydrogenase inhibitors’ by Jonathan P. Dolan et al., RSC Chem. Biol., 2023, 4, 865–870, https://doi.org/10.1039/D3CB00126A.

Cotton ovule in vitro cultures are a promising platform for exploring biofabrication of fibers with tailored properties. When the ovules' growth medium is supplemented with chemically synthesized cellulose precursors, it results in their integration into the developing fibers, thereby tailoring their end properties. Here, we report the feeding of synthetic glucosyl phosphate derivative, 6-deoxy-6-fluoro-glucose-1-phosphate (6F-Glc-1P) to cotton ovules growing in vitro, demonstrating the metabolic incorporation of 6F-Glc into the fibers with enhanced mechanical properties and moisture-retention capacity while emphasizing the role of molecular hierarchical architecture in defining functional characteristics and mechanical properties. This incorporation strategy bypasses the early steps of conventional metabolic pathways while broadening the range of functionalities that can be employed to customize fiber end properties. Our approach combines materials science, chemistry, and plant sciences to illustrate the innovation required to find alternative solutions for sustainable production of functional cotton fibers with enhanced and emergent properties.

In structural terms, the sialic acids are a large family of nine carbon sugars based around an alpha-keto acid core. They are widely spread in nature, where they are often found to be involved in molecular recognition processes, including in development, immunology, health and disease. The prominence of sialic acids in infection is a result of their exposure at the non-reducing terminus of glycans in diverse glycolipids and glycoproteins. Herein, we survey representative aspects of sialic acid structure, recognition and exploitation in relation to infectious diseases, their diagnosis and prevention or treatment. Examples covered span influenza virus and Covid-19, Leishmania and Trypanosoma, algal viruses, Campylobacter, Streptococci and Helicobacter, and commensal Ruminococci.

AbstractCellulose modification often employs chemical processes to tailor its properties and functionalities to fit the demands of a wide range of applications, maximizing its potential as a versatile and sustainable material. From both synthetic and environmental standpoints, one of the ultimate goals is to achieve significant modifications to enhance the end properties of the cellulose while minimizing the number of modified building blocks. The current study demonstrates that a synthetic glucose derivative, 6‐deoxy‐6‐fluoro‐glucose (6F‐Glc), fed into the fertilized cotton ovules, resulted in the accumulation of fluorine inside the cotton fibers with no apparent alteration to their morphology or development. These fibers exhibited a degree of substitution of 0.006, which is 170 times lower than that reported for chemical methods for cellulose modification. However, the physical characterization of the modified fibers showed a surprisingly large impact of this low‐level modification on the cellulose structure (e.g., hydrogen bonding network rearrangement) and a modest increase in the mechanical properties of the fibers. The obtained results exemplify the use of biological systems to introduce low quantities of new functionalities while maximizing the impact on fiber properties.

Upon undergoing mucoid conversion within the lungs of cystic fibrosis patients, the pathogenic bacterium Pseudomonas aeruginosa synthesises copious quantities of the virulence factor and exopolysaccharide alginate. The enzyme guanosine diphosphate mannose dehydrogenase (GMD) catalyses the rate-limiting step and irreversible formation of the alginate sugar nucleotide building block, guanosine diphosphate mannuronic acid. Since there is no corresponding enzyme in humans, strategies that could prevent its mechanism of action could open a pathway for new and selective inhibitors to disrupt bacterial alginate production. Using virtual screening, a library of 1447 compounds within the Known Drug Space parameters were evaluated against the GMD active site using the Glide, FRED and GOLD algorithms. Compound hit evaluation with recombinant GMD refined the panel of 40 potential hits to 6 compounds which reduced NADH production in a time-dependent manner; of which, an usnic acid derivative demonstrated inhibition six-fold stronger than a previously established sugar nucleotide inhibitor, with an IC50 value of 17 μM. Further analysis by covalent docking and mass spectrometry confirm a single site of GMD alkylation.

8-(1,2)-Mannan antigens incorporated into vaccines candidates for immunization studies, showed that antibodies raised against 8-(1,2)-mannotriose antigens can protect against disseminated candidiasis. Until recently, 8-(1,2)-mannans could only be obtained by isolation from microbial cultures, or by lengthy synthetic strategies involving protecting group manipulation. The discovery of two 8-(1,2)-mannoside phos-phorylases, Teth514_1788 and Teth514_1789, allowed efficient access to these compounds. In this work, Teth514_1788 was utilised to generate 8-(1,2)-mannan antigens, tri-and tetra-saccharides, decorated with a conjugation tether at the reducing end, suitable to be incorporated on a carrier en-route to novel vaccine can-didates, illustrated here by conjugation of the trisaccharide to BSA.

β-(1,2)-Mannan antigens incorporated into vaccines candidates for immunization studies, showed that antibodies raised against β-(1,2)-mannotriose antigens can protect against disseminated candidiasis. Until recently, β-(1,2)- mannans could only be obtained by isolation from microbial cultures, or by lengthy synthetic strategies involving protecting group manipulation. The discovery of two β-(1,2)-mannoside phosphorylases, Teth514_1788 and Teth514_1789, allowed efficient access to these compounds. In this work, Teth514_1788 was utilised to generate β-(1,2)-mannan antigens, tri- and tetra-saccharides, decorated with a conjugation tether at the reducing end, suitable to be incorporated on a carrier en-route to novel vaccine candidates, illustrated here by conjugation of the trisaccharide to BSA.

The (3-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixed- linkage glucans [MLG- (3-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Grampositive bacteria utilize MLG largely remains unknown. In this study, we used Blautia producta ATCC 27340 as a model organism to develop an understanding of MLG utilization. B. producta encodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16MLG), an ABC transporter, and a glycoside phosphorylase (BpGH94MLG) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)-encoding genes in this cluster when the organism is grown on MLG. We determined that recombinant BpGH16MLG cleaved various types of (3-glucan, generating oligosaccharides suitable for cellular uptake by B. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinant BpGH94MLG and (3-glucosidases (BpGH3-AR8MLG and BpGH3- X62MLG). Using targeted deletion, we demonstrated BpSBPMLG is essential for B. producta growth on barley (3-glucan. Furthermore, we revealed that beneficial bacteria, such as Roseburia faecis JCM 17581T, Bifidobacterium pseudocatenulatum JCM 1200T, Bifidobacterium adolescentis JCM 1275T, and Bifidobacterium bifidum JCM 1254, can also utilize oligosaccharides resulting from the action of BpGH16MLG. Disentangling the (3-glucan utilizing the capability of B. producta provides a rational basis on which to consider the probiotic potential of this class of organism.

Efficient and convenient access to short to medium chain length levan-type fructooligosaccharides (LFOS) is needed in order realise the nutritional potential of this class of bioactive oligosaccharides. While LFOS are synthesised by fructansucrase enzymes, these reactions are routinely associated with high molecular weight fructan polymer formation. Recent studies have shown that FOS production can be enhanced by the combination of levansucrase and inulosucrase in a one-pot reaction. In the present study, the novel mixed enzyme cross-linked enzyme aggregates (combi-CLEAs) based on levansucrase (Lev) and N543A variant inulosucrase (Inu) were prepared by ammonium sulfate precipitation, followed by glutaraldehyde cross-linking. The effect of Lev and Inu ratio on the activity of combi-CLEAs was explored. The results showed that >70% of total sucrase activity was recovered after immobilization and that the combi-CLEAs produced high amounts of LFOS (degree of polymerisation 3 to 21), while high molecular weight polysaccharide production was much reduced. Biochemical characterisation indicated that the optimum pH and temperature of combi-CLEAs (pH 5.5 and 50 degrees C, respectively) were comparable to those of free enzyme; however, the stability of the enzyme was improved. In addition, these combi-CLEAs have operational stability for several reaction cycles, which makes them very attractive for biotechnology applications.