Enzymatic Synthesis of Novel Terpenoid Glycoside Derivatives Decorated with N-Acetylglucosamine Catalyzed by UGT74AC1.

The addition of the O-linked N-acetylglucosamine (O-GlcNAc) is a significant modification for active molecules, such as proteins, carbohydrates, and natural products. However, the synthesis of terpenoid glycoside derivatives decorated with GlcNAc remains a challenging task due to the absence of glycosyltransferases, key enzymes for catalyzing the transfer of GlcNAc to terpenoids. In this study, we demonstrated that the enzyme mutant UGT74AC1T79Y/L48M/R28H/L109I/S15A/M76L/H47R efficiently transferred GlcNAc from uridine diphosphate (UDP)-GlcNAc to a variety of terpenoids. This powerful enzyme was employed to synthesize GlcNAc-decorated derivatives of terpenoids, including mogrol, steviol, andrographolide, protopanaxadiol, glycyrrhetinic acid, ursolic acid, and betulinic acid for the first time. To unravel the mechanism of UDP-GlcNAc recognition, we determined the X-ray crystal structure of the inactivated mutant UGT74AC1His18A/Asp111A in complex with UDP-GlcNAc at a resolution of 1.66 Å. Through molecular dynamic simulation and activity analysis, we revealed the molecular mechanism and catalytically important amino acids directly involved in the recognition of UDP-GlcNAc. Overall, this study not only provided a potent biocatalyst capable of glycodiversifying natural products but also elucidated the structural basis for UDP-GlcNAc recognition by glycosyltransferases.

[Construction and culture condition optimization of a Saccharomyces cerevisiae strain for production of galactitol].

Galactitol, a rare sugar alcohol, has promising potential in the food industry and pharmaceutical field. The available industrial production methods rely on harsh hydrogenation processes, which incur high costs and environmental concerns. It is urgent to develop environmentally friendly and efficient biosynthesis technologies. In this study, a xylose reductase named AnXR derived from Aspergillus niger CBS 513.88 was identified and characterized for the enzymatic properties. AnXR exhibited the highest activity at 25 ℃ and pH 8.0, and it belonged to the NADPH-dependent aldose reductase family. To engineer a strain for galactitol production, we deleted the galactokinase (GAL1) gene in Saccharomyes cerevisiae by using the recombinant gene technology, which significantly reduced the metabolic utilization of D-galactose by host cells. Subsequently, we introduced the gene encoding AnXR into this modified strain, creating an engineered strain capable of catalyzing the conversion of D-galactose into galactitol. Furthermore, we optimized the whole-cell catalysis conditions for the engineered strain, which achieved a maximum galactitol yield of 12.10 g/L. Finally, we tested the reduction ability of the strain for other monosaccharides and discovered that it could produce functional sugar alcohols such as xylitol and arabinitol. The engineered strain demonstrates efficient biotransformation capabilities for galactitol and other functional sugar alcohols, representing a significant advancement in environmentally sustainable production practices.

Extracellular polymeric substances and microbial community shift during the start-up of a single-stage partial nitritation/anammox process.

A sequencing batch reactor (SBR) was operated to investigate variations of extracellular polymeric substances (EPS) and microbial community during the start-up of the single-stage partial nitritation/anammox (SPN/A) process at intermittent aeration mode. The SPN/A system was successfully started on day 34, and the nitrogen removal efficiency and total nitrogen loading rate were 82.29% and 0.31 kg N/(m3 ·day), respectively. Furthermore, the relationship between the protein secondary structures and microbial aggregation was strongly related. The α-helix/ (ß-sheet + random coil) ratios increased obviously from 0.20 ± 0.03 to 0.23 ± 0.01, with the sludge aggregation mean size increased from 56 to 107 µm during the start-up of SPN/A. During the start-up of SPN/A, Candidatus Kuenenia was the primary anammox bacteria, whereas Nitrospira was the main functional bacteria of nitrite-oxidizing bacteria. Correlation between the microbial community and EPS components was performed. The EPS and microbial community played important roles in keeping stable nitrogen removal and the formation of sludge granules. PRACTITIONER POINTS: Intermittent aeration strategy promoted SPN/A system start-up. EPS composition and protein secondary structure were related with the sludge disintegration and aggregation. Microbial community shift existed and promoted the stability of sludge and reactor performance during SPN/A start-up.

Ginsenoside Rb1, a principal effective ingredient of Panax notoginseng, produces pain antihypersensitivity by spinal microglial dynorphin A expression.

Panax notoginseng (Chinese ginseng, Sanqi), one of the major ginseng species, has been traditionally used to alleviate different types of chronic pain. The raw P. notoginseng powder is commonly available in China as a non-prescription drug to treat various aliments including arthritic pain. However, strong scientific evidence is needed to illustrate its pain antihypersensitive effects, effective ingredients and mechanism of action. The oral P. notoginseng powder dose-dependently alleviated formalin-induced tonic hyperalgesia, and its total ginsenosides remarkably inhibited neuropathic pain hypersensitivity. Ginsenoside Rb1, the most abundant ginsenoside of P. notoginseng, dose-dependently produced neuropathic pain antihypersensitivity. Conversely, ginsenosides Rg1, Re and notoginseng R1, the other major saponins from P. notoginseng, failed to inhibit formalin-induced tonic pain or mechanical allodynia in neuropathic pain. Ginsenoside Rb1 metabolites ginsenosides Rg3, Compound-K and protopanaxadiol also had similar antineuropathic pain efficacy to ginsenoside Rb1. Additionally, intrathecal ginsenoside Rb1 specifically stimulated dynorphin A expression which was colocalized with microglia but not neurons or astrocytes in the spinal dorsal horn and primary cultured cells. Pretreatment with microglial metabolic inhibitor minocycline, dynorphin A antiserum and specific κ-opioid receptor antagonist GNTI completely blocked Rb1-induced mechanical antiallodynia in neuropathic pain. Furthermore, the specific glucocorticoid receptor (GR) antagonist Dex-21-mesylate (but not GPR30 estrogen receptor antagonist G15) also entirely attenuated ginsenoside Rb1-related antineuropathic pain effects. All these results, for the first time, show that P. notoginseng alleviates neuropathic pain and ginsenoside Rb1 is its principal effective ingredient. Furthermore, ginsenoside Rb1 inhibits neuropathic pain by stimulation of spinal microglial dynorphin A expression following GR activation.

Engineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars.

Naturally, haloacid dehalogenase superfamily phosphatases have been evolved with broad substrate promiscuity; however, strong specificity to a particular substrate is required for developing thermodynamically driven routes for manufacturing sugars. How to alter the intrinsic substrate promiscuity of phosphatases and fit the "one enzyme-one substrate" model remains a challenge. Herein, we report the structure-guided engineering of a phosphatase, and successfully provide variants with tailor-made preference for three widespread phosphorylated sugars, namely, glucose 6-phosphate, fructose 6-phosphate, and mannose 6-phosphate, while simultaneously enhancement in catalytic efficiency. A 12000-fold switch from unfavorite substrate to dedicated one is generated. Molecular dynamics simulations reveal the origin of improved activity and substrate specificity. Furthermore, we develop four coordinated multienzyme systems and accomplish the conversion of inexpensive sucrose and starch to fructose and mannose in excellent yield of 94-96%. This innovative sugar-biosynthesis strategy overcomes the reaction equilibrium of isomerization and provides the promise of high-yield manufacturing of other monosaccharides and polyols.

Supercritical Fluid CO2 Extraction and Microcapsule Preparation of Lycium barbarum Residue Oil Rich in Zeaxanthin Dipalmitate.

The scope of this investigation aimed at obtaining and stabilizing bioactive products derived from Lycium barbarum seeds and peels, which were the byproducts in the processing of fruit juice. Zeaxanthin dipalmitate is a major carotenoid, comprising approximately 80% of the total carotenoid content in the seeds and peels. The method of obtainment was supercritical fluid CO2 extraction, studying different parameters that affect the oil yield and content of zeaxanthin dipalmitate. The optimized protocol to enact successful supercritical fluid CO2 extraction included optimum extraction pressure of 250 bar, temperature at 60 °C over a time span of 2.0 h, and a CO2 flow of 30 g/min, together with the use of a cosolvent (2% ethanol). The yields of oil and zeaxanthin dipalmitate under these optimal conditions were 17 g/100 g and 0.08 g/100 g, respectively. The unsaturated fatty acids were primarily linoleic acid (C18:2), oleic acid (C18:1), and γ-linolenic acid (C18:3), with their contents being as high as 91.85 ± 0.27% of the total fatty acids. The extract was a red-colored oil that was consequently microencapsulated through spray-drying with octenylsuccinate starch, gum arabic, and maltodextrin (13.5:7.5:3, w/w) as wall materials to circumvent lipid disintegration during storage and add to fruit juice in a dissolved form. The mass ratio of core material and wall material was 4:1. These materials exhibited the highest microencapsulation efficiency (92.83 ± 0.13%), with a moisture content of 1.98 ± 0.05% and solubility of 66.22 ± 0.24%. The peroxide content level within the microencapsulated zeaxanthin dipalmitate-rich oil remained at one part per eight in comparison to the unencapsulated oil, following fast-tracked oxidation at 60 °C for 6 weeks. This indicated the potential oxidation stability properties of microcapsule powders. Consequently, this microencapsulated powder has good prospects for development, and can be utilized for a vast spectrum of consumer health and beauty products.

Novel catalytic property of fructose-6-phosphate aldolase in directly conversion of two 1-hydroxyalkanones to diketones.

Asymmetric CC bond formation catalyzed by aldolases requires the supplementation of nucleophiles and receptors in the reaction medium. However, aldol condensation using a single ketone as substrate has never been reported yet. In this work, we discovered that d-fructose-6-phosphate aldolase (FSA) could convert two 1-hydroxyalkanones, such as hydroxyacetone (HA) and 1-hydroxy-2-butanone, into two type of diketones. The initial product synthesis rate increased 3-fold and the yield reached to 56 %, when pure oxygen was directly inputted into the reaction medium. The results confirmed that oxygen participated in this reaction and hydrogen peroxide was generated. Metal ions Co2+ and Cu2+ remarkably increased the conversion yield compared with the control. For this reaction mechanism, we conjectured that HA may be oxidized to methylglyoxal by enzyme FSA in the presence of oxygen in the medium, and then FSA catalyzes the aldol addition between HA and its oxidative product MG to form diketone products. The obtained diketones could serve as important precursors for preparing furans and pyrroles.

VaAPRT3 Gene is Associated With Sex Determination in Vitis amurensis.

In the past decade, progress has been made in sex determination mechanism in Vitis. However, genes responsible for sexual differentiation and its mechanism in V. amurensis remain unknown. Here, we identify a sex determination candidate gene coding adenine phosphoribosyl transferase 3 (VaAPRT3) in V. amurensis. Cloning and sequencing of the VaAPRT3 gene allowed us to develop a molecular marker able to discriminate female individuals from males or hermaphrodites based on a 22-bp InDel. Gene expression and endogenous cytokinin content analysis revealed that the VaAPRT3 gene is involved in sex determination or, to be precise, in female organ differentiation, through regulating cytokinin metabolism in V. amurensis. This study enlarged the understanding of sex determination mechanism in the genus Vitis, and the sex marker could be used as a helpful tool for sexual identification in breeding programs as well as in investigation and collection of V. amurensis germplasms.

High-Yield Biosynthesis of Glucosylglycerol through Coupling Phosphorolysis and Transglycosylation Reactions.

Glucosylglycerol is a powerful osmolyte that has attracted attention as a useful moisturizing ingredient in the cosmetic industry. This study demonstrates two artificially designed synthetic routes for manufacturing glucosylglycerol by combining phosphorolysis and transglycosylation reactions. The overall Gibbs energy change of the synthetic routes was negative, indicating that they are thermodynamically favorable. In vitro biosystems were constructed through combining the phosphorolysis ability of sucrose/maltose phosphorylase and the transglycosylation capacity of glucosylglycerol phosphorylases from different organisms. A near-stoichiometric conversion of sucrose and glycerol with a high product yield of 98% was achieved under optimal reaction conditions. The large-scale glucosylglycerol production of this biosystem was investigated under a high concentration of substrates (2 mol/L sucrose and 2.4 mol/L glycerol), and the titer reached 1.78 mol/L (452 g/L) with a productivity of 24.3 g/L/h. To the best of our knowledge, this value presented the highest glucosylglycerol production level until now, which indicated a great industrial application potential for glucosylglycerol manufacturing.

Evaluation of Contrast-enhanced Transcranial Color-coded Duplex Sonography (CE-TCCD) Applied in Stroke Patients with Intracranial Collateral Circulation.

Background and Introduction: Collateral circulation is very crucial for the prognosis of stroke patients. Transcranial color-coded duplexsonography (TCCD) is used widely to evaluate the intracranial arterial blood flow. However, approximately 20% - 30% of the patients with cerebral infarction cannot be detected via TCCD due to the interruption of thickened temporal bones. We assessed the diagnostic efficacy of contrast-enhanced transcranial color-coded duplexsonography (CE-TCCD) in stroke patients with limited bone windows. METHODS: CE-TCCD was applied to 70 patients (51 males and 19 females) who presented with ischemic symptoms, to detect the openness of the anterior communicating artery (ACoA) and posterior communicating artery (PCoA) of the Willis ring before Computed Tomography angiography (CTA) or Magnetic Resonance Angiography (MRA) examination. The results from CETCCD is used to compare with CTA/MRA result to verify the diagnostic efficacy. RESULTS: Forty-one communicating artery openings were detected by CE-TCCD, among which 37 were PCoA and 4 were ACoA. Among the 70 patients, 23 of 70 patients indicated severe stenosis within intracranial and/or extracranial arteries. Eighteen out of the 23 patients showed collateral circulation, accounting for 78.3% (18/23). Moderate stenosis were 23 cases in total, in which 7 cases showed collateral circulation, accounting for 30.4% (7/23). Slight stenosis were 24 cases in total, none of which showed collateral circulation. CONCLUSION: In the stroke patients with limited bone windows, CE-TCCD can evaluate intracranial collateral circulation.

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies.

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed "starch-mannose-fermentation" biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.

Production of neoagarobiose from agar through a dual-enzyme and two-stage hydrolysis strategy.

The oligosaccharides from agar hydrolysis have special biological activities, and exhibit application prospects in cosmetic, food and pharmaceutical industry. In this study, two novel ß-agarases (AgaA and AgaB) were screened and characterized. It was found that the AgaA was an endo-type agarase which could efficiently hydrolyzed agar or agarose to form neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6), while the AgaB was an exo-type and bifunctional enzyme that showed activities towards both agarose and porphyran. Based on the properties of the two enzymes, we developed modular strategy for enzymatic production of neoagarobiose through a two-stage hydrolysis reaction. The cheap substrate agar was first liquefied by AgaA at high temperature to form neoagaroligosaccharides, which together with the sulfated polysaccharides were homogenized by AgaB to form neoagarobiose as the final product. High concentration of agar (10 g/L) was almost completely converted into neoagarobiose with high purity.

The Characterization and Modification of a Novel Bifunctional and Robust Alginate Lyase Derived from Marinimicrobium sp. H1.

Alginase lyase is an important enzyme for the preparation of alginate oligosaccharides (AOS), that possess special biological activities and is widely used in various fields, such as medicine, food, and chemical industry. In this study, a novel bifunctional alginate lyase (AlgH) belonging to the PL7 family was screened and characterized. The AlgH exhibited the highest activity at 45 °C and pH 10.0, and was an alkaline enzyme that was stable at pH 6.0-10.0. The enzyme showed no significant dependence on metal ions, and exhibited unchanged activity at high concentration of NaCl. To determine the function of non-catalytic domains in the multi-domain enzyme, the recombinant AlgH-I containing only the catalysis domain and AlgH-II containing the catalysis domain and the carbohydrate binding module (CBM) domain were constructed and characterized. The results showed that the activity and thermostability of the reconstructed enzymes were significantly improved by deletion of the F5/8 type C domain. On the other hand, the substrate specificity and the mode of action of the reconstructed enzymes showed no change. Alginate could be completely degraded by the full-length and modified enzymes, and the main end-products were alginate disaccharide, trisaccharide, and tetrasaccharide. Due to the thermo and pH-stability, salt-tolerance, and bifunctionality, the modified alginate lyase was a robust enzyme which could be applied in industrial production of AOS.

Circular RNA hsa_circ_0007142 Is Upregulated and Targets miR-103a-2-5p in Colorectal Cancer.

Circular RNAs (circRNAs) are a large class of endogenous noncoding RNAs that regulate gene expression and mainly function as microRNA sponges. This study aimed to explore the aberrant expression of circRNAs in colorectal cancer (CRC). Using a circRNA microarray, we identified 892 differentially expressed circRNAs between six pairs of CRC and adjacent paracancerous tissues. Among them, hsa_circ_0007142 was significantly upregulated. Further analysis in 50 CRC clinical samples revealed that hsa_circ_0007142 upregulation was associated with poor differentiation and lymphatic metastasis of CRC. Bioinformatic analysis and luciferase reporter assay showed that hsa_circ_0007142 targeted miR-103a-2-5p in CRC cells. Moreover, the silencing of hsa_circ_0007142 by siRNAs decreased the proliferation, migration, and invasion of HT-29 and HCT-116 cells. Taken together, these findings suggest that hsa_circ_0007142 is upregulated in CRC and targets miR-103a-2-5p to promote CRC.

Multi-enzyme systems and recombinant cells for synthesis of valuable saccharides: Advances and perspectives.

Saccharides have recently attracted considerable attention because of their biological functions and potential applications in the pharmaceutical, cosmetic and food industries. Over the decades, a large amount of enzymes involved in saccharide synthesis have been discovered and characterised with the aid of available genome sequences. The advancement of metabolic engineering and synthetic biology strategies facilitated the artificial pathway design and construction for production of multiple sugars in vitro and in vivo based on those characterized enzymes. This review presented a panoramic view of enzymes related to saccharide synthesis and gave the detailed information. Furthermore, we provide an extensive overview of the recent advances in the construction of cell-free reaction systems and engineering of microbial cells for the production of natural or unnatural saccharides. In addition, the future trends in the synthesis of sugars with high structural diversity through the combination of multiple pathways are presented and evaluated.

The development of low-calorie sugar and functional jujube food using biological transformation and fermentation coupling technology.

Jujube juice has been used as ingredient in a range of foods and dietary supplements. In this study, an enzyme transformation and fermentation coupling technology was applied to increase the nutritional value of concentrated/extracted Jinsi jujube juice. Two enzymes, D-glucose isomerase (GI) and D-allulose 3-epimerase (DAE), were employed to convert the glucose and fructose to a low-calorie sweeter D-allulose with a concentration of 110 g/L in jujube juice. Furthermore, the mixed cultures of Pediococcus pentosaceus PC-5 and Lactobacillus plantarum M were employed to increase the content of nutrition components related to bioactivities and flavor volatiles in jujube juice. Accordingly, this fermentation accumulated 100 mg/L gamma-aminobutyric acid (GABA), which has neurotransmission, hypotension, diuretic, and tranquilizer effects, and increased the content of branched-chain amino acids (BCAAs) and many free amino acids (Asp, Glu, Gly, and Ala) at different level. The fermentation not only maintained the concentration of native functional components such as cyclic adenosine monophosphate (cAMP) and minerals, but also increased the content of iron (Fe2+) and zinc (Zn2+), which have blood and eyesight tonic function. The value-added jujube juice might serve as a low-calorie and probiotic functional beverage and show high application potential in food industry.

Development of food-grade expression system for d-allulose 3-epimerase preparation with tandem isoenzyme genes in Corynebacterium glutamicum and its application in conversion of cane molasses to D-allulose.

D-Allulose 3-epimerase (DAE) has been applied to produce D-allulose, a low-calorie and functional sweetener. In this study, a new DAE from Paenibacillus senegalensis was characterized in Escherichia coli. Furthermore, we presented a tandem isoenzyme gene expression strategy to express multiple DAEs in one cell and construct food-grade expression systems based on Corynebacterium glutamicum. Seventeen expression cassettes based on three DAE genes from different organisms were constructed. Among all recombinant strains, DAE16 harboring three DAE genes in an expression vector exhibited the highest enzyme activity with 22.7 U/mg. Whole-cell transformation of DAE16 produced 225 g/L D-allulose with a volumetric productivity of 353 g·g -1 ·hr -1 . The catalytic efficiency of strain C-DAE9 integrating total 11 DAE genes in chromosome was 16.4-fold higher than strains carrying one DAE. Fed-batch culture of C-DAE9 gave enzyme activity of 44,700 U/L. We also expressed a thermostable invertase in C. glutamicum and obtained enzyme activity of 29 U/mg. Immobilized cells expressing DAE or invertase exhibited 80% of retained activity after 30 cycles of catalytic reactions. Those immobilized cells were coupled to produce 61.2 g/L D-allulose from cane molasses in a two-step reaction process. This study provided an efficient approach for enzyme preparation and allowed access to produce D-allulose from other abundant and low-cost feedstock enriched with sucrose.

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System.

Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained.IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

Prebiotic, Immunomodulating, and Antifatigue Effects of Konjac Oligosaccharide.

Irregular and long time work schedules not only makes people feel fatigue, but also brings great risks of diseases, due to gastrointestinal disorder and immune dysfunction. Therefore, it has positive significance to help challenged people stay energetic and healthy with food supplement. Konjac oligosaccharide has shown various physiological benefits and been recommended in the fortification of functional foods. However, there have been few reports on its application aimed to simultaneously relieve physical fatigue and keep body healthy. In this paper, the potential prebiotic, immunoregulatory, and antifatigue activities of konjac oligosaccharide were evaluated in vitro and in vivo. The results showed that konjac oligosaccharide could promote probiotics growth and short chain fatty acids production in mice cecum. At the concentration of 50 to 200 µg/mL, konjac oligosaccharide could activate murine macrophage RAW 264.7 to secret NO and cytokines of IL-10 and IL-6. Moreover, this oligosaccharide could alleviate physical fatigue by prolonging exhaustive time, improving the level of superoxide dismutases and glutathione peroxidase, increasing the content of blood glucose, and decreasing the content of blood urea nitrogen. The results suggested that konjac oligosaccharide had prebiotic, immunoregulatory, and antifatigue effects, providing its application potential in functional food aimed at people with irregular and long time work.

Biosynthesis of dendroketose from different carbon sources using in vitro and in vivo metabolic engineering strategies.

BACKGROUND: Asymmetric aldol-type C-C bond formation with ketones used as electrophilic receptor remains a challenging reaction for aldolases as biocatalysts. To date, only one kind of dihydroxyacetone phosphate (DHAP)-dependent aldolases has been discovered and applied to synthesize branched-chain sugars directly using DHAP and dihydroxyacetone (DHA) as substrate. However, the unstable and high-cost properties of DHAP limit large-scale application. Therefore, biosynthesis of branched-chain sugar from low-cost and abundant carbon sources is essential. RESULTS: The detailed catalytic property of l-rhamnulose-1-phosphate aldolase (RhaD) and l-fuculose-1-phosphate aldolase (FucA) from Escherichia coli in catalyzing the aldol reactions with DHA as electrophilic receptors was characterized. Furthermore, we calculated the Bürgi-Dunitz trajectory using molecular dynamics simulations, thereby revealing the original sources of the catalytic efficiency of RhaD and FucA. A multi-enzyme reaction system composed of formolase, DHA kinase, RhaD, fructose-1-phosphatase, and polyphosphate kinase was constructed to in vitro produce dendroketose, a branched-chain sugar, from one-carbon formaldehyde. The conversion rate reached 86% through employing a one-pot, two-stage reaction process. Moreover, we constructed two artificial pathways in Corynebacterium glutamicum to obtain this product in vivo starting from glucose or glycerol. Fermentation with glycerol as feedstock produced 6.4 g/L dendroketose with a yield of 0.45 mol/mol glycerol, representing 90% of the maximum theoretical value. Additionally, the dendroketose production reached 36.3 g/L with a yield of 0.46 mol/mol glucose when glucose served as the sole carbon resource. CONCLUSIONS: The detailed enzyme kinetics data of the two DHAP-dependent aldolases with DHA as electrophilic receptors were presented in this study. In addition, insights into this catalytic property were given via in silico simulations. Moreover, the cost-effective synthesis of dendroketose starting from one-, three-, and six-carbon resources was achieved through in vivo and in vitro metabolic engineering strategies. This rare branched-chain ketohexose may serve as precursor to prepare 4-hydroxymethylfurfural and branched-chain alkanes using chemical method.