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.

L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of Lactobacillus plantarum CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose.

We designed and constructed a green and sustainable bioprocess to efficiently coproduce D -tagatose, bioethanol, and microbial protein from whey powder. First, a one-pot biosynthesis process involving lactose hydrolysis and D -galactose redox reactions for D -tagatose production was established in vitro via a three-enzyme cascade. Second, a nicotinamide adenine dinucleotide phosphate-dependent galactitol dehydrogenase mutant, D36A/I37R, based on the nicotinamide adenine dinucleotide-dependent polyol dehydrogenase from Paracoccus denitrificans was created through rational design and screening. Moreover, an NADPH recycling module was created in the oxidoreductive pathway, and the tagatose yield increased by 3.35-fold compared with that achieved through the pathway without the cofactor cycle. The reaction process was accelerated using an enzyme assembly with a glycine-serine linker, and the tagatose production rate was 9.28-fold higher than the initial yield. Finally, Saccharomyces cerevisiae was introduced into the reaction solution, and 266.5 g of D -tagatose, 162.6 g of bioethanol, and 215.4 g of dry yeast (including 38% protein) were obtained from 1 kg of whey powder (including 810 g lactose). This study provides a promising sustainable process for functional food (D -tagatose) production. Moreover, this process fully utilized whey powder, demonstrating good atom economy.

A one-pot extraction-assisted d-galactose-to-d-tagatose isomerization strategy was proposed based on the selective extraction of d-tagatose by phenylborate anions. 4-Vinylphenylboronic acid was selected with high extraction efficiency and selectivity towards d-tagatose. The extracted sugars could be desorbed through a two-staged stripping process with the purity of d-tagatose significantly increased. In-situ extraction-assisted d-galactose-to-d-tagatose isomerization was implemented for the first time ever reported, and the effect of boron-to-sugar ratio (boron: sugar) was investigated. The conversion yield of d-tagatose at 60 °C increased from ∼ 39 % (boron: sugar = 0.5) to ∼ 56 % (boron: sugar = 1) but then decreased to ∼ 44 % (boron: sugar = 1.5). With temperature increased to 70 °C, the conversion yield of d-tagatose was further improved to ∼ 61 % (boron: sugar = 1.5), with the minimized formation of byproducts. Moreover, high purity (∼83 %) and concentrated d-tagatose solution (∼40 g/L) was obtained after sequential desorption. The proposed extraction-assisted isomerization strategy achieved improving the yield and purity of d-tagatose, proving its feasibility in industrial applications.

D-tagatose holds significant importance as a functional monosaccharide with diverse applications in food, medicine, and other fields. This study aimed to explore the oxidoreductive pathway for D-tagatose production, surpassing the contemporary isomerization-mediated biosynthesis approach in order to enhance the thermodynamic equilibrium of the reactions. Initially, a novel galactitol dehydrogenase was discovered through biochemical and bioinformatics analyses. By co-expressing the galactitol dehydrogenase and xylose reductase, the oxidoreductive pathway for D-tagatose synthesis was successfully established in Bacillus subtilis. Subsequently, pathway fine-tuning was achieved via promoter regulation and dehydrogenase-mediated cofactor regeneration, resulting in 6.75-fold higher D-tagatose compared to that produced by the strain containing the unmodified promoter. Finally, optimization of fermentation conditions and medium composition produced 39.57 g/L D-tagatose in a fed-batch experiment, with a productivity of 0.33 g/L/h and a yield of 0.55 mol/mol D-galactose. These findings highlight the potential of the constructed redox pathway as an effective approach for D-tagatose production.

Among the emerging sweeteners, d-tagatose occupies a significant niche due to its low calorific value, antidiabetic property and growth promoting effects on intestinal probiotics. Recently, the main approach for d-tagatose biosynthesis is l-arabinose isomerase-based isomerization reaction from galactose, which shows relatively low conversion rate because of unfavorable thermodynamic equilibria. Herein, oxidoreductases, d-xylose reductase and galactitol dehydrogenase, together with endogenous β-galactosidase were employed to catalyze the biosynthesis of d-tagatose from lactose with a yield of 0.282 g/g in Escherichia coli. Then, a deactivated CRISPR-associated (Cas) proteins-based DNA scaffold system was developed, which were proved to be efficient for assembling the oxidoreductases in vivo and got a 1.44-folds increase in d-tagatose titer and yield. Further, by employing d-xylose reductase with higher galactose affinity and activity, as well as overexpressing pntAB genes, the d-tagatose yield from lactose (0.484 g/g) increased to 92.0 % of the theoretical value, 1.72-times as that of original strain. Finally, whey powder, a lactose-rich food by-product, was bifunctionally utilized as an inducer and substrate. In the 5 L bioreactor, d-tagatose titer reached 32.3 g/L with little galactose detected, and the yield from lactose approached 0.402 g/g, which was the highest from waste biomass in the literature. The strategies used here might provide new insights into the biosynthesis of d-tagatose in future.

d-Tagatose is one of the several healthy sweeteners that can be a substitute for sucrose and fructose in our daily life. Whole cell-catalyzed phosphorylation and dephosphorylation previously reported by our group afford a thermodynamic-driven strategy to achieve tagatose production directly from starch with high product yields. Nonetheless, the poor structural stability of cells and difficulty in biocatalyst recycling restrict its practical application. Herein, an efficient and stable semiartificial cell factory (SACF) was developed by constructing an organosilica network (OSN) artificial shell on the cells bearing five thermophilic enzymes to produce tagatose. The OSN artificial shell, the thickness of which can be regulated by changing the tetraethyl silicate concentration, exhibited tunable permeability and superior mechanical strength. In contrast with cells, SACFs showed a relative activity of 99.5% and an extended half-life from 33.3 to 57.8 h. Over 50% of initial activity was retained after 20 reuses. The SACFs can catalyze seven consecutive reactions with tagatose yields of over 40.7% in field applications.

Rare sugar was defined as a sugar that occurs in very small quantities in nature. Among them, l-ribose and d-tagatose were of high added value and useful as pharmaceutical intermediate for anti-HBV drugs or low calorie sweetener in food industry. Bio-production of the two rare sugar from biomass waste has not been investigated. Hence, development of a feasible and efficient co-production method was of practical usage. However, lack of suitable biocatalyst has become a bottleneck. By sequence alignment and analysis, a C-terminal α-helix from l-arabinose isomerase (L-AI) family was selected as a tool for protein engineering. This α-helix was ligated to C-terminal of Lactobacillus fermentum L-AI (LFAI) and significantly enhanced its thermostability and robustness for both l-arabinose and galactose catalysis. The mutant LFAI-C4 enzyme was immobilized by alginate and antimicrobial peptide poly-l-lysine, and was used to convert pretreated corncob acid hydrolysate (PCAH) into l-ribulose and d-tagatose in the presence of boric acid. In addition, we identified and immobilized a novel thermostable mannose-6-phosphate isomerase from Bacillus subtilis (BsMPI-2) which was efficient in catalyzing retaining l-ribulose into l-ribose and showing no activity on d-tagatose. The dual immobilized enzymes (LFAI-C4 and BsMPI-2) system co-produced 191.9 g/L l-ribose and 80.1 g/L d-tagatose, respectively. Showing a total yield of 46.6% from l-arabinose to l-ribose, which was the highest among reported. The dual immobilized enzymes system preserved 82% activity after 40 batches reaction, showing excellent potentials for industrial use. This study presents a promising alternative for rare sugar production from low-value raw material and showed satisfied conversion rate, product concentration, and operation stability.

Acute respiratory distress syndrome (ARDS) is characterized by disruption of the alveolar-capillary barrier, resulting in severe alveolar edema and inflammation. D-tagatose (TAG) is a low-calorie fructose isomer with diverse biological activities whose role in ARDS has never been explored. We found that TAG protects lung tissues from injury in the oleic acid-induced rat model of ARDS. Seventeen male Sprague-Dawley rats were randomly assigned to 3 groups: Sham (n = 5), ARDS (n = 6), and TAG + ARDS (n = 6). The treatment groups were injected with oleic acid to induce ARDS, and the TAG + ARDS group was given TAG 3 days before the induction. After the treatments, the effect of TAG was evaluated by blood gas analysis and observing the gross and histological structure of the lung. The results showed that TAG significantly improved the oxygenation function, reduced the respiratory acidosis and the inflammatory response. TAG also improved the vascular permeability in ARDS rats and promoted the differentiation of alveolar type II cells, maintaining the stability of the alveolar structure. This protective effect of TAG on the lung may be achieved by activating the PTEN/PI3K/AKT pathway. Thus, TAG protects against oleic acid-induced ARDS in rats, suggesting a new clinical strategy for treating the condition.

Impurity sugars produced in upstream process of functional sugars are significantly impacting the product quality. In this work, the effect of congeners (d-maltose (d-MAL), d-fructose (d-FRU), d-glucose (d-GLU)) on primary and secondary nucleation of d-tagatose (d-TAG) crystals was investigated. The impurity sugars showed an inhibition on primary nucleation of d-TAG crystals, while a promotion on the secondary nucleation of d-TAG. Interestingly, the impact of impurity sugars on d-TAG crystal growth was similar to that on primary nucleation. The diffusion ability, hydrogen bonding forming ability, interaction energy of d-TAG crystal surfaces and impurity sugars were evaluated by molecular dynamics (MD) simulations to reveal the nucleation and growth behavior. Based on the above findings, we designed the d-TAG crystallization experiments, and obtained d-TAG crystals with uniform particle size distribution and regular morphology. This study helps to understand the influence of impurity sugars on crystallization, guiding the industrial manufacturing of functional sugars.