Designed microbial platforms for tunable production of rare sugars

L-Rhamnose isomerase (L-RI, EC 5.3.1.14), catalyzing the isomerization between L-rhamnose and L-rhamnulose, plays an important role in microbial L-rhamnose metabolism and thus occurs in a wide range of microorganisms. It attracts more and more attention because of its broad substrate specificity and its great potential in enzymatic production of various rare sugars. In this article, the enzymatic properties of various reported L-RIs were compared in detail, and their applications in the production of L-rhamnulose and various rare sugars including D-allose, D-gulose, L-lyxose, L-mannose, L-talose, and L-galactose were also reviewed.

Macroalgae-derived rare sugars: Applications and catalytic synthesis

Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-ribose-5-phosphate isomerase and D-mannose-6-phosphate isomerase to produce D-allose and L-ribose, respectively.

Characterization of a d-tagatose 3-epimerase from Caballeronia fortuita and its application in rare sugar production

d-Allulose is an attractive rare sugar that can be used as a low-calorie sweetener with significant health benefits. To meet the increasing market demands, it is necessary to develop an efficient and extensive microbial fermentation platform for the synthesis of d-allulose. Here, we applied a comprehensive systematic engineering strategy in Bacillus subtilis WB600 by introducing d-allulose 3-epimerase (DAEase), combined with the deactivation of fruA, levDEFG, and gmuE, to balance the metabolic network for the efficient production of d-allulose. This resulting strain initially produced 3.24 g/L of d-allulose with a yield of 0.93 g of d-allulose/g d-fructose. We further screened and obtained a suitable dual promoter combination and performed fine-tuning of its spacer region. After 64 h of fed-batch fermentation, the optimized engineered B. subtilis produced d-allulose at titers of 74.2 g/L with a yield of 0.93 g/g and a conversion rate of 27.6%. This d-allulose production strain is a promising platform for the industrial production of rare sugar.

Izumoring: A Novel and Complete Strategy for Bioproduction of Rare Sugars

Izumoring: A novel and complete strategy for bioproduction of rare sugars

A review on l-ribose isomerases for the biocatalytic production of l-ribose and l-ribulose

Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to give different functional groups, the silica nanoparticle derivatized with epoxy groups showed the highest immobilization efficiency (92%). The thermostability of ReXDH was improved more than tenfold by immobilization on epoxy-silica nanoparticles; the t(1/2) of the ReXDH was enhanced from 120 min to 1,410 min at 40 °C and from 30 min to 450 min at 50 °C. The K(m) of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into L-xylulose. A∼71% conversion and a productivity of 10.7 g h(-1)l(-1) were achieved when the immobilized ReXDH was employed to catalyze the biotransformation of xylitol to L-xylulose, a sugar that has been used in medicine and in the diagnosis of hepatitis. These results suggest that immobilization of ReXDH onto epoxy-silica nanoparticles has potential industrial application in rare sugar production.

Except Itea virginica, Itea ilicifilia, and Itea yunnanensis Franch, there had been few reports of other itea plants related to rare sugars. Likewise, little had been known about the antioxidants in itea plants. In this study, the rare sugars, phenolic profiles, as well as their antioxidant activities in the itea leaves, which were from Itea virginica, Itea oblonga Hand.-Mazz., and Itea yunnanensis Franch, were determined and compared. The first discovery of D-psicose and allitol in Itea oblonga Hand.-Mazz. further demonstrated the great potential application of itea plants in the production of rare sugars. Moreover, the leaves of Itea virginica and Itea oblonga Hand.-Mazz. showed good antioxidant activities in the assays of free radical scavenging activities and reducing power. The high performance liquid chromatography-tandem mass spectrometry experiment revealed the presence of several flavonoid glycosides as major antioxidant components in these leaves. These results indicated that the itea plants, especially Itea virginica, could be used for potential antioxidants in addition to functional sweeteners.

D-Allose, a rare sugar, has gained significant attention not only as a low-calorie sweetener but also for its anticancer, antitumor, anti-inflammatory, antioxidant, and other pharmaceutical properties. Despite its potential, achieving high-level biosynthesis of D-allose remains challenging due to inefficient biocatalysts, low conversion rates, and the high cost of substrates. Here, we explored the food-grade coexpression of Blautia produca D-allulose 3-epimerase (Bp-DAE) and Bacillus subtilis L-rhamnose isomerase (BsL-RI) within a single cell using B. subtilis WB800N as the host. Using this system, D-allose was synthesized via a simple, cost-effective, one-pot enzymatic process, employing whole cells as catalysts and D-fructose as the substrate. The system exhibited optimal activity at 65 °C, pH 8.5, with 1 mM Mn2+ and 20 g/L of whole-cell dry weight. Initial production reached 12.5 g/L D-allose with a 12.5% yield from 100 g/L D-fructose. Optimization of dual promoter combinations enhanced production, achieving 15.0, 29.1, and 43.2 g/L D-allose from 100, 200, and 300 g/L D-fructose, with yields of 15.00, 14.55, and 14.40%, respectively. This D-allose production biocatalyst offers a scalable and economically viable platform for the industrial production of rare sugar.

Monosaccharides and their derivatives which hardly exist in nature are so-called "rare sugars". Rare sugars have significance not only in food industries but also pharmaceutical industries. We discovered a novel L-ribose isomerase from Cellulomonas parahominis (CpL-RbI, 249 amino acids), which catalyzes the reversible isomerization between L-ribose and L-ribulose, L-allose and L-psicose, and D-talose and D-tagatose. Since CpL-RbI has a broad substrate specificity, it is useful for the production of various rare sugars. To elucidate the molecular basis of unique enzymatic properties of CpL-RbI, we determined its crystal structure. The N-terminal His-tagged CpL-RbI overexpressed in Escherichia coli was purified using a nickel affinity column. Crystals of CpL-RbI were obtained from a reservoir solution of 0.1 M sodium acetate trihydrate (pH 4.6) with 3.9 M ammonium acetate, by a hanging-drop vapor-diffusion method at 293 K (Space group C2221, a = 76.8, b = 88.6, c = 152.3 Å). X-ray diffraction data were collected up to 2.10 Å resolution using a Rigaku R-AXIS VII on a RA-Micro7HF rotating anode generator (40 kV, 30 mA) at 100 K. The structure was solved by a molecular replacement method with a structure of Acinetobacter sp L-ribose isomerase (4NS7) as a search model, and refined to R-factor of 0.227. CpL-RbI had a cupin-type beta-barrel structure, and the catalytic site was found between two large beta-sheets with a bound metal ion (Fig. 1). There were two protein molecules in an asymmetric unit, forming a homo-dimer with a non-crystallographic 2-fold symmetry (Fig.1). Furthermore, the PISA server showed that two dimers in crystal were associated to form a stable tetramer. Complex structures with substrates, L-ribose, L-allose and L-psicose, were also successfully determined. We will discuss a broad substrate specificity and catalytic reaction mechanism of CpL-RbI based on its three-dimensional structure.

The electrochemical oxidation of biomass for the production of value-added chemicals represents a promising approach in the field of sustainable chemistry. In this study, we investigated the electrochemical conversion of d-glucose, a biomass-derived compound, using boron-doped diamond (BDD) electrodes under constant applied current (10 mA) or potentials (1.5-3.0 V vs Ag/AgCl). The reaction products were analyzed using high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) measurements, employing both p-aminobenzoic acid ethyl ester (ABEE) and l-tryptophan amide labeling methods to enable characterization. The results demonstrated that the BDD electrodes achieved 95.9% d-glucose degradation and successfully generated various rare sugars, including d-arabinose (0.126 mmol/L), d-erythrose (0.0544 mmol/L), d-glyceraldehyde, and l-glyceraldehyde (combined 0.148 mmol/L). Under identical conditions, Pt electrodes as a control showed only 10.2% d-glucose degradation with significantly lower rare sugar yields. The applied potential significantly influenced the product distribution, with optimal rare sugar production observed at 2.5 V vs Ag/AgCl, reflecting a balance between glucose oxidation and product degradation. Mechanistic studies suggest that the formation of rare sugars involves a series of oxidation and decarboxylation reactions, facilitated by electrochemically generated active species. The superior performance of the BDD electrodes is attributed to their wide potential window, efficient generation of oxidizing species, and unique surface characteristics. This research provides new insights into the electrochemical transformation of biomass-derived compounds and demonstrates the potential for sustainable production of high-value rare sugars, opening avenues for applications in food science, pharmaceuticals, and green chemistry.

Ribose-5-phosphate isomerase (Rpi, EC 5.3.1.6) is widespread in microorganisms, animals, and plants. It has a pivotal role in the pentose phosphate pathway and responsible for catalyzing the isomerization between D-ribulose 5-phosphate and D-ribose 5-phosphate. In recent years, Rpi has received considerable attention as a multipurpose biocatalyst for production of rare sugars, including D-allose, L-rhamnulose, L-lyxose, and L-tagatose. Besides, it has been thought of as a potential drug target in the treatment of trypanosomatid-caused diseases such as Chagas' disease, leishmaniasis, and human African trypanosomiasis. Despite increased research activities, up to now, no systematic review of Rpi has been published. To fill this gap, this paper provides detailed information about the enzymatic properties of various Rpis. Furthermore, structural features, catalytic mechanism, and molecular modifications of Rpis are summarized based on extensive crystal structure research. Additionally, the applications of Rpi in rare sugar production and the role of Rpi in trypanocidal drug design are reviewed. Key points • Fundamental properties of various ribose-5-phosphate isomerases (Rpis). • Differences in crystal structure and catalytic mechanism between RpiA and RpiB. • Application of Rpi as a rare sugar producer and a potential drug target.

Carbohydrates are much more than just a source of energy as they also mediate a variety of recognition processes that are central to human health. As such, saccharides can be applied in the food and pharmaceutical industries to stimulate our immune system (e.g., prebiotics), to control diabetes (e.g., low-calorie sweeteners), or as building blocks for anticancer and antiviral drugs (e.g., L: -nucleosides). Unfortunately, only a small number of all possible monosaccharides are found in nature in sufficient amounts to allow their commercial exploitation. Consequently, so-called rare sugars have to be produced by (bio)chemical processes starting from cheap and widely available substrates. Three enzyme classes that can be used for rare sugar production are keto-aldol isomerases, epimerases, and oxidoreductases. In this review, the recent developments in rare sugar production with these biocatalysts are discussed.