Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine.

As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.

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.

Efficient biosynthesis of D-allulose in Bacillus subtilis through D-psicose 3-epimerase translation modification.

The combined catalysis of glucose isomerase (GI) and D-psicose 3-epimerase (DPEase) provided a convenient route for the direct synthesis of D-allulose from d-glucose, whose cost is lower than d-fructose. In the present research, the weak activity of DPEase was the key rate-limiting step and resulted in the accumulation of d-fructose in engineered Bacillus subtilis. Then, the 5'-untranslated region (5'-UTR) structure of the mRNA translational initiation region was optimized for the precise control of DPEase expression. The manipulation of the 5'-UTR region promoted the accessibility to ribosome binding and the stability of mRNA, resulting in a maximum of 1.73- and 1.98-fold increase in DPEase activity and intracellular mRNA amount, respectively. Under the optimal catalytic conditions of 75 °C, pH 6.5, 110 g/L d-glucose, and 1 mmol/L Co2+, the reaction equilibrium time was reduced from 7.6 h to 6.1 h. We hope that our results could provide a facilitated strategy for large-scale production of D-allulose at low-cost.

Exo-inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production.

Aspergillus fumigatus was found to produce thermostable exo-inulinase (EC 3.8.1.80; 38 U/ml) on inulin-rich infusions. Exo-inulinase (14.6 U/mg) was immobilized on glutaraldehyde activated Ca-alginate beads for continuous generation of fructose by hydrolyzing sucrose, chicory, and dandelion substrates. Immobilization of enzyme was confirmed by microscopic and spectroscopic techniques. The exo-inulinase was purified using ion-exchange (1.30-folds) and size-exclusion chromatography (2.71-folds). The purified exo-inulinase showed 64 kDa band on gel and was optimally active at 60 °C and pH 6.0. Kinetic constants, Km and Vmax of purified exo-inulinase, were 5.88 mM and 1.66 µM/min, respectively, and its relative activity was found to be enhanced (125.8%) in the presence of calcium ion. Immobilized preparation was utilized for continuous generation of fructose from chicory juice (26 to 70%) and dandelion root extracts (16 to 24%) by recycling upto five cycles, respectively. In comparison to other sweeteners, such as sucrose, fructose is considered as a healthy alternative. The present study demonstrated the use of immobilized exo-inulinase in continuous generation of fructose from some underutilized plant sources that can be used in food industry. PRACTICAL APPLICATION: Thermostable exo-inulinase produced by A. fumigatus was immobilized on calcium alginate matrix and was employed for continuous hydrolysis of chicory juice and dandelion root extract for generation of fructose syrup.

D-Allulose 3-epimerase of Bacillus sp. origin manifests profuse heat-stability and noteworthy potential of D-fructose epimerization.

BACKGROUND: D-Allulose is an ultra-low calorie sugar of multifarious health benefits, including anti-diabetic and anti-obesity potential. D-Allulose 3-epimerase family enzymes catalyze biosynthesis of D-allulose via epimerization of D-fructose. RESULTS: A novel D-allulose 3-epimerase (DaeB) was cloned from a plant probiotic strain, Bacillus sp. KCTC 13219, and expressed in Bacillus subtilis cells. The purified protein exhibited substantial epimerization activity in a broad pH spectrum, 6.0-11.0. DaeB was able to catalyze D-fructose to D-allulose bioconversion at the temperature range of 35 °C to 70 °C, exhibiting at least 50 % activity. It displaced excessive heat stability, with the half-life of 25 days at 50 °C, and high turnover number (kcat 367 s- 1). The coupling of DaeB treatment and yeast fermentation of 700 g L- 1 D-fructose solution yielded approximately 200 g L- 1 D-allulose, and 214 g L- 1 ethanol. CONCLUSIONS: The novel D-allulose 3-epimerase of Bacillus sp. origin discerned a high magnitude of heat stability along with exorbitant epimerization ability. This biocatalyst has enormous potential for the large-scale production of D-allulose.

Prospective production of fructose and single cell protein from date palm waste

BACKGROUND: Fructose and single cell protein are important products for the food market. Abundant amounts of low-grade dates worldwide are annually wasted. In this study, highly concentrated fructose syrups and single cell protein were obtained through selective fermentation of date extracts by Saccharomyces cerevisiae. RESULTS: The effect of air flow (0.1, 0.5, 0.75, 1, 1.25 and 1.5 vvm) and pH (4.5, 4.8, 5, 5.3 and 5.6) was investigated. Higher air flow led to lower fructose yield. The optimum cell mass production of 10 g/L was achieved at air flow of 1.25 vvm with the fructose yield of 91%. Similar cell mass production was obtained in the range pH of 5.0­5.6, while less cell mass was obtained at pH less than 5. Controlling the pH at 4.5, 5.0 and 5.3 failed to improve the production of cell mass which were 5.6, 5.9 and 5.4 g/L respectively; however, better fructose yield was obtained. CONCLUSIONS: Extension of the modified Gompertz enabled excellent predictions of the cell mass, fructose production and fructose fraction. The proposed model was also successfully validated against data from literatures. Thus, the model will be useful for wide application of biological processes.

Immobilization of Agrobacterium tumefaciensd-psicose 3-epimerase onto titanium dioxide for bioconversion of rare sugar.

d-Psicose (d-ribo-2-hexulose or d-allulose) is the Carbon-3 epimer of d-fructose sugar and considered as an unnatural (rare) sugar found in low amount in nature. It has about 70% of the relative sweetness but 0.3% of the energy of sucrose, which is suggested as the most suitable sucrose substitute for food additives. Enzymatic biosynthesis using ketose 3-epimerases is a necessary procedure for the production of d-Psicose from d-fructose. However, significant drawbacks in the application of ketose 3-epimerases at industrial scale observe lower thermal stability as well as bioconversion efficiency, reusability and recovery of the enzyme. We have attempted immobilization of ketose 3-epimerases from Agrobacterium tumefaciens (agtu) d-psicose 3-epimerase (DPEase) on titanium dioxide. Further, Scanning electron microscopy (SEM), inverted microscopy, Fourier transform infrared spectroscopy (FTIR) and UV-vis spectroscopy showed that the enzyme was successfully immobilized on the titanium dioxide (TiO2) surface. Titanium dioxide immobilized agtu-DPEase (TiO2-agtu-DPEase) shows pH optima at 6.0 and 60 °C as a higher working temperature. TiO2-agtu-DPEase showed a half-life of 180 min at 60 °C, which is higher as compared to Agrobacterium tumefaciens (agtu) DPEase (3.99 min at 50 °C). At equilibrium, 36:64 (D-psicose: d-fructose), the bioconversion efficiency was accounted for titanium dioxide immobilized DPEase, which is higher than the agtu-DPEase. Titanium dioxide immobilized DPEase showed bioconversion efficiency up to 9 cycles of reusability.

5-Keto-D-fructose production from sugar alcohol by isolated wild strain Gluconobacter frateurii CHM 43.

GLUCONOBACTER FRATEURII: CHM 43 have D-mannitol dehydrogenase (quinoprotein glycerol dehydrogenase) and flavoprotein D-fructose dehydrogenase in the membranes. When the two enzymes are functional, D-mannitol is converted to 5-keto-D-fructose with 65% yield when cultivated on D-mannitol. 5-Keto-D-fructose production with almost 100% yield was realized with the resting cells. The method proposed here should give a smart strategy for 5-keto-D-fructose production.

Novel plasmid-free Gluconobacter oxydans strains for production of the natural sweetener 5-ketofructose.

BACKGROUND: 5-Ketofructose (5-KF) has recently been identified as a promising non-nutritive natural sweetener. Gluconobacter oxydans strains have been developed that allow efficient production of 5-KF from fructose by plasmid-based expression of the fructose dehydrogenase genes fdhSCL of Gluconobacter japonicus. As plasmid-free strains are preferred for industrial production of food additives, we aimed at the construction of efficient 5-KF production strains with the fdhSCL genes chromosomally integrated. RESULTS: For plasmid-free 5-KF production, we selected four sites in the genome of G. oxydans IK003.1 and inserted the fdhSCL genes under control of the strong P264 promoter into each of these sites. All four recombinant strains expressed fdhSCL and oxidized fructose to 5-KF, but site-specific differences were observed suggesting that the genomic vicinity influenced gene expression. For further improvement, a second copy of the fdhSCL genes under control of P264 was inserted into the second-best insertion site to obtain strain IK003.1::fdhSCL2. The 5-KF production rate and the 5-KF yield obtained with this double-integration strain were considerably higher than for the single integration strains and approached the values of IK003.1 with plasmid-based fdhSCL expression. CONCLUSION: We identified four sites in the genome of G. oxydans suitable for expression of heterologous genes and constructed a strain with two genomic copies of the fdhSCL genes enabling efficient plasmid-free 5-KF production. This strain will serve as basis for further metabolic engineering strategies aiming at the use of alternative carbon sources for 5-KF production and for bioprocess optimization.

High Conversion of D-Fructose into D-Allulose by Enzymes Coupling with an ATP Regeneration System.

D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM D-fructose, and 20 mM Mg2+ when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.

Redesign of a novel D-allulose 3-epimerase from Staphylococcus aureus for thermostability and efficient biocatalytic production of D-allulose.

BACKGROUND: A novel D-allulose 3-epimerase from Staphylococcus aureus (SaDAE) has been screened as a D-allulose 3-epimerase family enzyme based on its high specificity for D-allulose. It usually converts both D-fructose and D-tagatose to respectively D-allulose and D-sorbose. We targeted potential biocatalysts for the large-scale industrial production of rare sugars. RESULTS: SaDAE showed a high activity on D-allulose with an affinity of 41.5 mM and catalytic efficiency of 1.1 s-1 mM-1. Four residues, Glu146, Asp179, Gln205, and Glu240, constitute the catalytic tetrad of SaDAE. Glu146 and Glu240 formed unique interactions with substrates based on the structural model analysis. The redesigned SaDAE_V105A showed an improvement of relative activity toward D-fructose of 68%. The conversion rate of SaDAE_V105A reached 38.9% after 6 h. The triple mutant S191D/M193E/S213C showed higher thermostability than the wild-type enzyme, exhibiting a 50% loss of activity after incubation for 60 min at 74.2 °C compared with 67 °C for the wild type. CONCLUSIONS: We redesigned SaDAE for thermostability and biocatalytic production of D-allulose. The research will aid the development of industrial biocatalysts for D-allulose.

Microbial co-culturing strategies for fructo-oligosaccharide production.

Fructo-oligosaccharide (FOS) mixtures produced by fermentation contain large amounts of non-prebiotic sugars. Here we propose a mixed culture of Aureobasidium pullulans and Saccharomyces cerevisiae cells to produce FOS and consume the small saccharides simultaneously, thereby increasing FOS purity in the mixture. The use of immobilised A. pullulans in co-culture with encapsulated S. cerevisiae, inoculated after 10 h fermentation, enhanced FOS production in a 5 L bioreactor. Using this strategy, a maximal FOS concentration of 119 g L-1, and yield of 0.59 gFOS gsucrose-1, were obtained after 20 h fermentation, increasing FOS productivity from about 4.9 to 5.9 gFOS L-1 h-1 compared to a control fermentation of immobilized A. pullulans in monoculture. In addition, the encapsulated S. cerevisiae cells were able to decrease the glucose in the medium to about 7.6% (w/w) after 63 h fermentation. This provided a final fermentation mixture with 2.0% (w/w) sucrose and a FOS purity of over 67.0% (w/w). Moreover, a concentration of up to 58.0 g L-1 of ethanol was obtained through the enzymatic transformation of glucose. The resulting pre-purified FOS mixture could improve the separation and purification of FOS in downstream treatments, such as simulated moving bed chromatography.

Uric acid activates aldose reductase and the polyol pathway for endogenous fructose and fat production causing development of fatty liver in rats.

Dietary, fructose-containing sugars have been strongly associated with the development of nonalcoholic fatty liver disease (NAFLD). Recent studies suggest that fructose also can be produced via the polyol pathway in the liver, where it may induce hepatic fat accumulation. Moreover, fructose metabolism yields uric acid, which is highly associated with NAFLD. Here, using biochemical assays, reporter gene expression, and confocal fluorescence microscopy, we investigated whether uric acid regulates aldose reductase, a key enzyme in the polyol pathway. We evaluated whether soluble uric acid regulates aldose reductase expression both in cultured hepatocytes (HepG2 cells) and in the liver of hyperuricemic rats and whether this stimulation is associated with endogenous fructose production and fat accumulation. Uric acid dose-dependently stimulated aldose reductase expression in the HepG2 cells, and this stimulation was associated with endogenous fructose production and triglyceride accumulation. This stimulatory mechanism was mediated by uric acid-induced oxidative stress and stimulation of the transcription factor nuclear factor of activated T cells 5 (NFAT5). Uric acid also amplified the effects of elevated glucose levels to stimulate hepatocyte triglyceride accumulation. Hyperuricemic rats exhibited elevated hepatic aldose reductase expression, endogenous fructose accumulation, and fat buildup that was significantly reduced by co-administration of the xanthine oxidase inhibitor allopurinol. These results suggest that uric acid generated during fructose metabolism may act as a positive feedback mechanism that stimulates endogenous fructose production by stimulating aldose reductase in the polyol pathway. Our findings suggest an amplifying mechanism whereby soft drinks rich in glucose and fructose can induce NAFLD.

Deletion of four genes in Escherichia coli enables preferential consumption of xylose and secretion of glucose.

Overcoming carbon catabolite repression presents a significant challenge, largely due to the complex regulatory networks governing substrate catabolism, even in microbial cells. In this work, we have engineered an E. coli strain, which we have named X2G, that not only exhibits a reversed substrate preference for xylose over glucose, but also demonstrates an unusual ability to produce significant amounts of glucose. We obtained this non-intuitive phenotype by deleting four genes in upper central metabolism: ptsI, glk, pfkA, and zwf, which respectively encode Enzyme I of the phosphotransferase system, glucokinase, the dominant isozyme of phosphofructokinase, and glucose-6-phosphate dehydrogenase. The deletion of ptsI and glk blocks glucose uptake in E. coli, while the deletion of pfkA and zwf prevents the reassimilation of carbons through glycolysis and the oxidative pentose phosphate pathway, respectively. Our strain X2G is capable of converting 34% of the carbon it takes up as xylose into exported glucose. This corresponds to a glucose production rate of 1.4 ±â€¯0.3 mmol/gDW/h at a specific growth rate of 0.25 ±â€¯0.03 h-1, or about 1.8 ±â€¯0.1 mM of glucose accumulated for every unit increase in OD600. Despite a 22% decrease in xylose uptake rate, a 33% decrease in biomass yield, and a 52% decrease in acetate production rate relative to the wild-type, the intracellular flux profile and cofactor allocation of X2G remain largely unperturbed, as elucidated through 13C-metabolic flux analysis. Further quantification of the pool sizes of key intracellular metabolites revealed that glucose secretion by X2G is likely driven by the substantial accumulation of intracellular glucose 6-phosphate, fructose 6-phosphate, glucose and fructose at levels greater than 20x of that in wild-type E. coli. Combined, our results shed light on the flexibility of central metabolism, and the opportunities this affords for producing value-added pentose- and hexose-derived products from lignocellulosic feedstocks.

Fructose production from inulin using fungal inulinase immobilized on 3-aminopropyl-triethoxysilane functionalized multiwalled carbon nanotubes.

The main objective of the present work was to modify multiwalled carbon nanotubes (MWCNTs) using 3-aminopropyl-triethoxysilane (APTES) to generate amino-terminated surfaces for inulinase immobilization, which can be further used for fructose production. CCRD of response surface methodology was used for optimization of inulinase immobilization on MWCNTs. At optimized parameters (APTES concentration 4%; sonication time 4 h; enzyme coupling time 1.5 h and enzyme load 15 IU), maximal inulinase activity and immobilization yield was 60.7% and 74.4%, respectively. Immobilized inulinase showed same pH optima of free enzyme, while an elevation in temperature optima to 60 °C was observed after its immobilization. Immobilized inulinase also shown enhancement in pH stability and thermostability. Overall, 4.54-fold rise in half-life of inulinase was detected after immobilization at 60 °C. Km and Vmax of inulinase decreased after immobilization. Immobilized inulinase preserved 28% of its residual activity after 10 consecutive batch cycles of inulin hydrolysis for the production of fructose.

Biotechnological applications of inulin-rich feedstocks.

Inulin is a naturally occurring second largest storage polysaccharide with a wide range of applications in pharmaceutical and food industries. It is a robust polysaccharide which consists of a linear chain of ß-2, 1-linked-d-fructofuranose molecules terminated with α-d-glucose moiety at the reducing end. It is present in tubers, bulbs and tuberous roots of more than 36,000 plants belonging to both monocotyledonous and dicotyledonous families. Jerusalem artichoke, chicory, dahlia, asparagus, etc. are important inulin-rich plants. Inulin is a potent substrate and inducer for the production of inulinases. Inulin/inulin-rich feedstocks can be used for the production of fructooligosaccharides and high-fructose syrup. Additionally, inulin-rich feedstocks can also be exploited for the production of other industrially important products like acetone, butanol, bioethanol, single cell proteins, single cell oils, 2, 3-butanediol, sorbitol, mannitol, etc. Current review highlights the biotechnological potential of inulin-rich feedstocks for the production of various industrially important products.

Bioprocessing and technoeconomic feasibility analysis of simultaneous production of d-psicose and ethanol using engineered yeast strain KAM-2GD.

The objective of this study was to analyze the processing and technoeconomic feasibility of coproduction of d-psicose and ethanol in a modified dry grind ethanol process. The yeast strain was constructed by expressing d-psicose 3-epimerases (DPE) in Sachharomyces cerevisiae. The strain was capable of converting d-fructose to d-psicose at 55 °C with a conversion efficiency of 26.6%. A comprehensive process model for modified dry grind ethanol plant with 396,000 MT/yr corn processing capacity was developed using SuperPro Designer. Predicted ethanol and d-psicose yields were 390.4 L and 75.3 kg per MT of corn, with total annual production of 154.6 million L and 29,835 MT respectively. The capital investment for the plant was estimated as 150.3 million USD with total operating cost of 85.2 million USD/yr. The unit production cost and minimum selling price of d-psicose with an internal rate of return of 15% were calculated as $0.43/kg and $1.29/kg respectively.

Highly efficient production of Clostridium cellulolyticum H10 D-psicose 3-epimerase in Bacillus subtilis and use of these cells to produce D-psicose.

BACKGROUND: D-Psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to the rare sugar D-psicose, which may help prevent obesity, reduce blood sugar and blood fat, and inhibit intra-abdominal fat accumulation. RESULTS: In this study, the DPEase of Clostridium cellulolyticum H10 was expressed in the food-grade host Bacillus subtilis. Optimization of the culture medium during shake-flask experiments yielded a DPEase activity of 314 U/mL. The optimal medium included 20 g/L peptone, 15 g/L corn steep powder, 5 g/L glycerol, and 1 mM Ca2+. Controlling the carbon source concentration was important because elevated concentrations can result in catabolite metabolic suppression (CCR). To avoid CCR and increase DPEase expression, we developed a fed-batch strategy in a 3.6-L fermenter. We altered the ratio of carbon source to nitrogen source (C/N) in the feeding medium and employed a constant feeding rate (6 g/L/h). This strategy improved the DPEase activity to 2246 U/mL (7.8 g/L), which is almost 15 times higher than that observed in the original shake-flask cultures. Finally, we used the DPEase-expressing B. subtilis cells to produce D-psicose from D-fructose, and a 28% conversion yield was achieved with these cells, demonstrating their potential use in D-psicose production. CONCLUSIONS: This is the first report to enhance recombinant DPEase production in B. subtilis using efficient and convenient fermentation strategy, and the DPEase yield is three times higher than the highest yield reported to date. The recombinant B. subtilis cells were further used in the efficient synthesis of D-psicose. This study provides a basis for the industrial production of D-psicose.

[Research progress of D-psicose: function and its biosynthesis].

As the morbidity of metabolic syndrome like obesity and diabetes increases rapidly worldwide, the issue of nutrition (functional food) and health has drawn more attention. D-psicose, a rare natural ketohexose, has become a hot topic in functional food and health-care field because of its hypoglycemic and hypolipidemic function with good sweetness. This article mainly discusses the functional properties and biosynthesis research progress of D-psicose, together with the crystal structure of ketose-3-epimerase, to provide theoretical guidance for D-psicose-producing strain screening as well as improving the thermostability and catalytic efficiency of ketose-3-epimerase for industrial application.