Clinical and biochemical evolution after partial dietary liberalization of two cases of galactosemia due to galactose mutarotase deficiency.

BACKGROUND: The recommended diet attitude in the recently described galactose mutarotase (GALM) deficiency is not yet established. We describe two 9-years twins who remain asymptomatic despite prolonged partial dietary liberalization from 18 months of age, after two periods of galactose-free diet. It represents the second report in Europe of GALM deficiency. CASE PRESENTATION: Two male monochorionic diamniotic twins were detected through newborn screening by galactosuria and increased total blood galactose. They started galactose dietary restriction with biochemical normalization. After exclusion of the three previously described types of galactosemia, a progressively galactose reintroduction was initiated. The clinical follow-up developed include neurological assessment and intelligence quotient, annual ophthalmological evaluation and biannual abdominal ultrasound; whereas the biochemical assessment comprises quarterly determinations of galactose 1-phosphate and galactosuria and annual determination of liver and renal function, 25-OH-vitamin D and calcium levels. Sanger sequencing of GALM gene was complemented by the study of gene dose using SNPs array and a protein modeling to study the conformational changes induced in GALM protein. In both siblings a novel and complete deletion of exon 4 in GALM gene was detected. Both remained asymptomatic, with normal growth and intellectual development, despite dietary liberalization. Evolutionarily, the biochemical profile in blood remained normal with intermittent galactosuria. CONCLUSIONS: The absence of clinical involvement after 7 years of dietary liberalization is interesting to expand the knowledge about the recommended dietary management in this pathology.

Computer-aided mining of a psychrophilic cellobiose 2-epimerase from the Qinghai-Tibet Plateau gene catalogue.

Cellobiose 2-epimerase (CE) catalyzes the conversion of the lactose into its high-value derivatives, epilactose and lactulose, which has great prospects in food applications. In this study, CE sequences from the Qinghai-Tibet Plateau gene catalogue, we screened these for structural flexibility through molecular dynamics simulation to identify potential psychrophilic CE candidates. One such psychrophilic CE we termed psyCE demonstrated exceptional epimerization activity, achieving an optimum activity of 122.2 ± 1.6 U/mg. Its kinetic parameters (Kcat and Km) for epimerization activity were 219.9 ± 5.6 s-1 and 261.9 ± 18.1 mM, respectively, representing the highest Kcat recorded among known cold-active CEs. Notably, this is the first report of a psychrophilic CE. The psyCE can effectively produce epilactose at 8 °C, converting 20.3 % of 200 mM lactose into epilactose within four hours. These findings suggest that psyCE is highly suitable for cryogenic food processing, and glaciers may serve as a valuable repository of psychrophilic enzymes.

Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor.

BACKGROUND: D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants. RESULTS: The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L-1 D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle. CONCLUSION: All these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.

Discovery of a Thermostable Tagatose 4-Epimerase Powered by Structure- and Sequence-Based Protein Clustering.

d-Tagatose is a highly promising functional sweetener known for its various physiological functions. In this study, a novel tagatose 4-epimerase from Thermoprotei archaeon (Thar-T4Ease), with the ability to convert d-fructose to d-tagatose, was discovered through a combination of structure similarity search and sequence-based protein clustering. The recombinant Thar-T4Ease exhibited optimal activity at pH 8.5 and 85 °C, in the presence of 1 mM Ni2+. Its kcat and kcat/Km values toward d-fructose were measured to be 248.5 min-1 and 2.117 mM-1·min-1, respectively. Notably, Thar-T4Ease exhibited remarkable thermostability, with a t1/2 value of 198 h at 80 °C. Moreover, it achieved a conversion ratio of 18.9% using 100 g/L d-fructose as the substrate. Finally, based on sequence and structure analysis, crucial residues for the catalytic activity of Thar-T4Ease were identified by molecular docking and site-directed mutagenesis. This research expands the repertoire of enzymes with C4-epimerization activity and opens up new possibilities for the cost-effective production of d-tagatose from d-fructose.

Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose.

In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition.

Cell-surface d-glucuronyl C5-epimerase binds to porcine deltacoronavirus spike protein facilitating viral entry.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. The coronavirus spike (S) glycoprotein, especially the S1 subunit, mediates viral entry by binding to cellular receptors. However, the functional receptor of PDCoV remains poorly understood. In this study, we used the soluble PDCoV S1 protein as bait to capture the S1-binding cellular transmembrane proteins in combined immunoprecipitation and mass spectrometry analyses. A single guide RNA screen identified d-glucuronyl C5-epimerase (GLCE), a heparan sulfate-modifying enzyme, as a proviral host factor for PDCoV infection. GLCE knockout significantly inhibited the attachment and internalization stages of PDCoV infection. We also demonstrated the interaction between GLCE and PDCoV S with coimmunoprecipitation in both an overexpression system and PDCoV-infected cells. GLCE could be localized to the cell membrane, and an anti-GLCE antibody suppressed PDCoV infection. Although GLCE expression alone did not render nonpermissive cells susceptible to PDCoV infection, GLCE promoted the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. In conclusion, our results demonstrate that GLCE is a novel cell-surface factor facilitating PDCoV entry and provide new insights into PDCoV infection. IMPORTANCE: The identification of viral receptors is of great significance, potentially extending our understanding of viral infection and pathogenesis. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus with the potential for cross-species transmission. However, the receptors or coreceptors of PDCoV are still poorly understood. The present study confirms that d-glucuronyl C5-epimerase (GLCE) is a positive regulator of PDCoV infection, promoting viral attachment and internalization. The anti-GLCE antibody suppressed PDCoV infection. Mechanically, GLCE interacts with PDCoV S and promotes the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. This work identifies GLCE as a novel cell-surface factor facilitating PDCoV entry and paves the way for further insights into the mechanisms of PDCoV infection.

The transcriptional regulator Fur modulates the expression of uge, a gene essential for the core lipopolysaccharide biosynthesis in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a Gram-negative pathogen that has become a threat to public health worldwide due to the emergence of hypervirulent and multidrug-resistant strains. Cell-surface components, such as polysaccharide capsules, fimbriae, and lipopolysaccharides (LPS), are among the major virulence factors for K. pneumoniae. One of the genes involved in LPS biosynthesis is the uge gene, which encodes the uridine diphosphate galacturonate 4-epimerase enzyme. Although essential for the LPS formation in K. pneumoniae, little is known about the mechanisms that regulate the expression of uge. Ferric uptake regulator (Fur) is an iron-responsive transcription factor that modulates the expression of capsular and fimbrial genes, but its role in LPS expression has not yet been identified. This work aimed to investigate the role of the Fur regulator in the expression of the K. pneumoniae uge gene and to determine whether the production of LPS by K. pneumoniae is modulated by the iron levels available to the bacterium. RESULTS: Using bioinformatic analyses, a Fur-binding site was identified on the promoter region of the uge gene; this binding site was validated experimentally through Fur Titration Assay (FURTA) and DNA Electrophoretic Mobility Shift Assay (EMSA) techniques. RT-qPCR analyses were used to evaluate the expression of uge according to the iron levels available to the bacterium. The iron-rich condition led to a down-regulation of uge, while the iron-restricted condition resulted in up-regulation. In addition, LPS was extracted and quantified on K. pneumoniae cells subjected to iron-replete and iron-limited conditions. The iron-limited condition increased the amount of LPS produced by K. pneumoniae. Finally, the expression levels of uge and the amount of the LPS were evaluated on a K. pneumoniae strain mutant for the fur gene. Compared to the wild-type, the strain with the fur gene knocked out presented a lower LPS amount and an unchanged expression of uge, regardless of the iron levels. CONCLUSIONS: Here, we show that iron deprivation led the K. pneumoniae cells to produce higher amount of LPS and that the Fur regulator modulates the expression of uge, a gene essential for LPS biosynthesis. Thus, our results indicate that iron availability modulates the LPS biosynthesis in K. pneumoniae through a Fur-dependent mechanism.

Production, purification, characterization, and safety evaluation of constructed recombinant D-psicose 3-epimerase.

BACKGROUND: D-psicose 3-epimerase (DPEase) is a potential catalytic enzyme for D-psicose production. D-psicose, also known as D-allulose, is a low-calorie sweetener that has gained considerable attention as a healthy alternative sweetener due to its notable physicochemical properties. This research focused on an in-depth investigation of the expression of the constructed DPEase gene from Agrobacterium tumefaciens in Escherichia coli for D-psicose synthesis. Experimentally, this research created the recombinant enzyme, explored the optimization of gene expression systems and protein purification strategies, investigated the enzymatic characterization, and then optimized the D-psicose production. Finally, the produced D-psicose syrup underwent acute toxicity evaluation to provide scientific evidence supporting its safety. RESULTS: The optimization of DPEase expression involved the utilization of Mn2+ as a cofactor, fine-tuning isopropyl ß-D-1-thiogalactopyranoside induction, and controlling the induction temperature. The purification process was strategically designed by a nickel column and an elution buffer containing 200 mM imidazole, resulting in purified DPEase with a notable 21.03-fold increase in specific activity compared to the crude extract. The optimum D-psicose conversion conditions were at pH 7.5 and 55 °C with a final concentration of 10 mM Mn2+ addition using purified DPEase to achieve the highest D-psicose concentration of 5.60% (w/v) using 25% (w/v) of fructose concentration with a conversion rate of 22.42%. Kinetic parameters of the purified DPEase were Vmax and Km values of 28.01 mM/min and 110 mM, respectively, which demonstrated the high substrate affinity and efficiency of DPEase conversion by the binding site of the fructose-DPEase-Mn2+ structure. Strategies for maintaining stability of DPEase activity were glycerol addition and storage at -20 °C. Based on the results from the acute toxicity study, there was no toxicity to rats, supporting the safety of the mixed D-fructose-D-psicose syrup produced using recombinant DPEase. CONCLUSIONS: These findings have direct and practical implications for the industrial-scale production of D-psicose, a valuable rare sugar with a broad range of applications in the food and pharmaceutical industries. This research should advance the understanding of DPEase biocatalysis and offers a roadmap for the successful scale-up production of rare sugars, opening new avenues for their utilization in various industrial processes.

Biochemical identification of D-mannose 2-epimerase from Cytophagaceae bacterium SJW1-29 for efficient bioconversion of D-glucose to D-mannose.

Enzymatic production of D-mannose attracts increasing attention because of the health effects and commercial values of D-mannose. Several kinds of epimerases or isomerases have been used for enzymatic production of D-mannose from D-glucose or D-fructose. D-Mannose epimerase (MEase), belonging to N-acyl-D-glucosamine 2-epimerase superfamily enzymes, catalyzes the C-2 epimerization between D-glucose and D-mannose. In this study, a novel MEase was identified from Cytophagaceae bacterium SJW1-29. Sequence and structure alignments indicate that it is highly conserved with the reported R. slithyformis MEase with the known crystal structure. It was a metal-independent enzyme, with an optimal pH of 8.0 and an optimal temperature of 40 °C. The specific activities on D-glucose and D-mannose were 2.90 and 2.96 U/mg, respectively. The Km, kcat, and kcat/Km on D-glucose were measured to be 194.9 mM, 2.72 s-1, and 0.014 mM-1 s-1, respectively. The purified enzyme produced 23.15 g/L of D-mannose from 100 g/L of D-glucose at pH 8.0 and 40 °C for 8 h, with a conversion rate of 23.15 %.

Production of lactulose from lactose using a novel cellobiose 2-epimerase from Clostridium disporicum.

Lactulose is a semisynthetic nondigestive sugar derived from lactose, with wide applications in the food and pharmaceutical industries. Its biological production routes which use cellobiose 2-epimerase (C2E) as the key enzyme have attracted widespread attention. In this study, a set of C2Es from different sources were overexpressed in Escherichia coli to produce lactulose. We obtained a novel and highly efficient C2E from Clostridium disporicum (CDC2E) to synthesize lactulose from lactose. The effects of different heat treatment conditions, reaction pH, reaction temperature, and substrate concentrations were investigated. Under the optimum biotransformation conditions, the final concentration of lactulose was up to 1.45 M (496.3 g/L), with a lactose conversion rate of 72.5 %. This study provides a novel C2E for the biosynthesis of lactulose from low-cost lactose.

The Formation of D-Allulose 3-Epimerase Hybrid Nanoflowers and Co-Immobilization on Resins for Improved Enzyme Activity, Stability, and Processability.

As a low-calorie sugar, D-allulose is produced from D-fructose catalyzed by D-allulose 3-epimerase (DAE). Here, to improve the catalytic activity, stability, and processability of DAE, we reported a novel method by forming organic-inorganic hybrid nanoflowers (NF-DAEs) and co-immobilizing them on resins to form composites (Re-NF-DAEs). NF-DAEs were prepared by combining DAE with metal ions (Co2+, Cu2+, Zn2+, Ca2+, Ni2+, Fe2+, and Fe3+) in PBS buffer, and were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and X-ray diffraction. All of the NF-DAEs showed higher catalytic activities than free DAE, and the NF-DAE with Ni2+ (NF-DAE-Ni) reached the highest relative activity of 218%. The NF-DAEs improved the thermal stability of DAE, and the longest half-life reached 228 min for NF-DAE-Co compared with 105 min for the free DAE at 55 °C. To further improve the recycling performance of the NF-DAEs in practical applications, we combined resins and NF-DAEs to form Re-NF-DAEs. Resins and NF-DAEs co-effected the performance of the composites, and ReA (LXTE-606 neutral hydrophobic epoxy-based polypropylene macroreticular resins)-based composites (ReA-NF-DAEs) exhibited outstanding relative activities, thermal stabilities, storage stabilities, and processabilities. The ReA-NF-DAEs were able to be reused to catalyze the conversion from D-fructose to D-allulose, and kept more than 60% of their activities after eight cycles.

Designable immobilization of D-allulose 3-epimerase on bimetallic organic frameworks based on metal ion compatibility for enhanced D-allulose production.

D-allulose, a low-calorie rare sugar catalyzed by D-allulose 3-epimerase (DAE), is highly sought after for its potential health benefits. However, poor reusability and stability of DAE limited its popularization in industrial applications. Although metal-organic frameworks (MOFs) offer a promising enzyme platform for enzyme immobilization, developing customized strategies for MOF immobilization of enzymes remains challenging. In this study, we introduce a designable strategy involving the construction of bimetal-organic frameworks (ZnCo-MOF) based on metal ions compatibility. The DAE@MOFs materials were prepared and characterized, and the immobilization of DAE and the enzymatic characteristics of the MOF-immobilized DAE were subsequently evaluated. Remarkably, DAE@ZnCo-MOF exhibited superior recyclability which could maintain 95 % relative activity after 8 consecutive cycles. The storage stability is significantly improved compared to the free form, with a relative activity of 116 % remaining after 30 days. Molecular docking was also employed to investigate the interaction between DAE and the components of MOFs synthesis. The results demonstrate that the DAE@ZnCo-MOF exhibited enhanced catalytic efficiency and increased stability. This study introduces a viable and adaptable MOF-based immobilization strategy for enzymes, which holds the potential to expand the implementation of enzyme biocatalysts in a multitude of disciplines.

Induced Muscle and Liver Absence of Gne in Postnatal Mice Does Not Result in Structural or Functional Muscle Impairment.

Background: GNE Myopathy is a unique recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, caused by mutations in the GNE gene which is a key enzyme in the biosynthesis of sialic acid. To date, the precise pathophysiology of the disease is not well understood and no reliable animal model is available. Gne KO is embryonically lethal in mice. Objective: To gain insights into GNE function in muscle, we have generated an inducible muscle Gne KO mouse. To minimize the contribution of the liver to the availability of sialic acid to muscle via the serum, we have also induced combined Gne KO in liver and muscle. Methods: A mouse carrying loxp sequences flanking Gne exon3 was generated by Crispr/Cas9 and bred with a human skeletal actin (HSA) promoter driven CreERT mouse. Gne muscle knock out was induced by tamoxifen injection of the resulting homozygote GneloxpEx3loxp/HSA Cre mouse. Liver Gne KO was induced by systemic injection of AAV8 vectors carrying the Cre gene driven by the hepatic specific promoter of the thyroxine binding globulin gene. Results: Characterization of these mice for a 12 months period showed no significant changes in their general behaviour, motor performance, muscle mass and structure in spite of a dramatic reduction in sialic acid content in both muscle and liver. Conclusions: We conclude that post weaning lack of Gne and sialic acid in muscle and liver have no pathologic effect in adult mice. These findings could reflect a strong interspecies versatility, but also raise questions about the loss of function hypothesis in Gne Myopathy. If these findings apply to humans they have a major impact on therapeutic strategies.

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis.

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ( Δ H ‡ = 54 kJ mol-1), significant loss in entropy ( - T Δ S ‡ = 20 kJ mol-1; 298 K) and negative activation heat capacity ( Δ C p ‡ = -0.64 kJ mol-1 K-1). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.

Enhancing the stability of a novel D-allulose 3-epimerase from Ruminococcus sp. CAG55 by interface interaction engineering and terminally attached a self-assembling peptide.

D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.

Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts.

Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%-86% of DS-epi1 activity at 10 µM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients.

A mutation in CsGME encoding GDP-mannose 3,5-epimerase results in little and wrinkled leaf in cucumber.

KEY MESSAGE: Map-based cloning revealed that a mutation in a highly conserved amino acid of the CsGME gene encoding GDP-mannose 3,5-epimerase, causes the phenotype of little and wrinkled leaves in cucumbers. Leaf size is a critical determinant of plant architecture in cucumbers, yet only a few genes associated with this trait have been mapped or cloned. Here, we identified and characterized a mutant with little and wrinkled leaves, named lwl-1. Genetic analysis revealed that the phenotype of the lwl-1 was controlled by a single recessive gene. Through map-based cloning, the lwl-1 locus was narrowed down to a 12.22-kb region exclusively containing one fully annotated gene CsGME (CsaV3_2G004170). CsGME encodes GDP-mannose 3,5-epimerase, which is involved in the synthesis of ascorbic acid (ASA) and one of the components of pectin, RG-II. Whole-length sequencing of the 12.22 kb DNA fragment revealed the presence of only a non-synonymous mutation located in the sixth exon of CsGME in lwl-1, resulting in an amino acid alteration from Pro363 to Leu363. This mutation was unique among 118 inbred lines from cucumber natural populations. CsGME expression significantly reduced in various organs of lwl-1, accompanied by a significant decrease in ASA and pectin content in leaves. Both CsGME and Csgme proteins were localized to the cytoplasm. The mutant phenotype exhibited partial recovery after the application of exogenous boric acid. Silencing CsGME in cucumber through VIGS confirmed its role as the causal gene for lwl-1. Transcriptome profiling revealed that CsGME greatly affected the expression of genes related to the cell division process and cell plate formation. This study represents the first report to characterize and clone the CsGME in cucumber, indicating its crucial role in regulating leaf size and development.

Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis.

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production.

Rational design improves both thermostability and activity of a new D-tagatose 3-epimerase from Kroppenstedtia eburnean to produce D-allulose.

D-allulose is a naturally occurring rare sugar and beneficial to human health. However, the efficient biosynthesis of D-allulose remains a challenge. Here, we mined a new D-tagatose 3-epimerase from Kroppenstedtia eburnean (KeDt3e) with high catalytic efficiency. Initially, crucial factors contributing to the low conversion of KeDt3e were identified through crystal structure analysis, density functional theory calculations (DFT), and molecular dynamics (MD) simulations. Subsequently, based on the mechanism, combining restructuring the flexible region, proline substitution based onconsensus sequence analysis, introducing disulfide bonds, and grafting properties, and reshaping the active center, the optimal mutant M5 of KeDt3e was obtained with enhanced thermostability and activity. The optimal mutant M5 exhibited an enzyme activity of 130.8 U/mg, representing a 1.2-fold increase; Tm value increased from 52.7 °C to 71.2 °C; and half-life at 55 °C extended to 273.7 min, representing a 58.2-fold improvement, and the detailed mechanism of performance improvement was analyzed. Finally, by screening the ribosome-binding site (RBS) of the optimal mutant M5 recombinant bacterium (G01), the engineered strain G08 with higher expression levels was obtained. The engineered strain G08 catalyzed 500 g/L D-fructose to produce 172.4 g/L D-allulose, with a conversion of 34.4% in 0.5 h and productivity of 344.8 g/L/h on a 1 L scale. This study presents a promising approach for industrial-scale production of D-allulose.

Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.

Among the epimerases specific to alginate, some of them in Azotobacter genera convert ß-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.