D-allose, a typical rare sugar: properties, applications, and biosynthetic advances and challenges.

D-allose, a C-3 epimer of D-glucose and an aldose-ketose isomer of D-allulose, exhibits 80% of sucrose's sweetness while being remarkably low in calories and nontoxic, making it an appealing sucrose substitute. Its diverse physiological functions, particularly potent anticancer and antitumor effects, render it a promising candidate for clinical treatment, garnering sustained attention. However, its limited availability in natural sources and the challenges associated with chemical synthesis necessitate exploring biosynthetic strategies to enhance production. This overview encapsulates recent advancements in D-allose's physicochemical properties, physiological functions, applications, and biosynthesis. It also briefly discusses the crucial role of understanding aldoketose isomerase structure and optimizing its performance in D-allose synthesis. Furthermore, it delves into the challenges and future perspectives in D-allose bioproduction. Early efforts focused on identifying and characterizing enzymes responsible for D-allose production, followed by detailed crystal structure analysis to improve performance through molecular modification. Strategies such as enzyme immobilization and implementing multi-enzyme cascade reactions, utilizing more cost-effective feedstocks, were explored. Despite progress, challenges remain, including the lack of efficient high-throughput screening methods for enzyme modification, the need for food-grade expression systems, the establishment of ordered substrate channels in multi-enzyme cascade reactions, and the development of downstream separation and purification processes.

Exploiting the biocatalytic potential of co-expressed l-fucose isomerase and d-tagatose 3-epimerase for the biosynthesis of 6-deoxy-l-sorbose.

6-Deoxy-l-sorbose (6-DLS) is an imperative rare sugar employed in food, agriculture, pharmaceutical and cosmetic industeries. However, it is a synthetic and very expensive rare sugars, previously synthesized by chemo-enzymatic methods through a long chain of chemical processes. Recently, enzymatic synthesis of rare sugars has attracted a lot of attention due to its advantages over synthetic methods. In this work, a promising approach for the synthesis of 6-DLS from an inexpensive sugar l-fucose was identified. The genes for l-fucose isomerase from Paenibacillus rhizosphaerae (Pr-LFI) and genes for d-tagatose-3-epimerase from Caballeronia fortuita (Cf-DTE) have been used for cloning and co-expression in Escherichia coli, developed a recombinant plasmid harboring pANY1-Pr-LFI/Cf-DTE vector. The recombinant co-expression system exhibited an optimum activity at 50 °C of temperature and pH 6.5 in the presence of Co2+ metal ion which inflated the catalytic activity by 6.8 folds as compared to control group with no metal ions. The recombinant co-expressed system was stable up to more than 50 % relative activity after 12 h and revealed a melting temperature (Tm) of 63.38 °C exhibiting half-life of 13.17 h at 50 °C. The co-expression system exhibited, 4.93, 11.41 and 16.21 g/L of 6-DLS production from initial l-fucose concentration of 30, 70 and 100 g/L, which equates to conversion yield of 16.44 %, 16.30 % and 16.21 % respectively. Generally, this study offers a promising strategy for the biological production of 6-DLS from an inexpensive substrate l-fucose in slightly acidic conditions with the aid of co-expression system harboring Pr-LFI and CF-DTE genes.

Characterization of l-fucose isomerase from Paenibacillus rhizosphaerae to produce l-fuculose from hydrolyzed fucoidan and commercial fucose.

AIMS: l-Fuculose is a valuable rare sugar that is used to treat a variety of ailments, including HIV, cancer, Hepatitis B, human lysosomal disease (fucosidosis), and cardio-protective medications. The enzymatic approach for the production of l-fuculose using l-fucose as a substrate would be an advantageous method with a wide range of industrial applications. The objective of this study is the characterization of recombinant l-fucose isomerase from Paenibacillus rhizosphaerae (Pa-LFI) for the production of l-fuculose from an inexpensive and natural source (fucoidan) as well as its comparison with commercial l-fucose (Sigma-Aldrich). METHODS AND RESULTS: Fucoidan, a fucose-containing polysaccharide (FPs), was isolated from Undaria pinnatifida, subsequently hydrolyzed, and characterized before the enzymatic production of l-fuculose. The results elaborate that FPs contain 35.9% of fucose along with other kinds of monosaccharides. The purified Pa-LFI exhibited a single band at 65 kDa and showed it as a hexamer with a native molecular mass of 396 kDa. The highest activity of 104.5 U mg-1 of Pa-LFI was perceived at a temperature of 50°C and pH 6.5 in the presence of 1 mM of Mn2+. The Pa-LFI revealed a melting temperature (Tm) of 75°C and a half-life of 12.6 h at 50°C. It exhibited that Pa-LFI with aldose substrate (l-fucose), has a stronger isomerizing activity, disclosing Km,kcat, and kcat/Km 86.2 mM, 32 831 min-1, and 335 min-1 mM-1, respectively. After reaching equilibrium, Pa-LFI efficiently catalyzed the reaction to convert l-fucose into l-fuculose and the conversion ratios of l-fuculose from 100 g L-1 of FPs and commercial fucose were around 6% (5.6 g L-1) and 30% (30.2 g L-1), respectively. CONCLUSIONS: According to the findings of the current study, the Pa-LFI will be useful in the manufacturing of l-fuculose using an effective and easy approach that produces no by-products.

Fucoidan-based nanomaterial and its multifunctional role for pharmaceutical and biomedical applications.

Fucoidans are promising sulfated polysaccharides isolated from marine sources that have piqued the interest of scientists in recent years due to their widespread use as a bioactive substance. Bioactive coatings and films, unsurprisingly, have seized these substances to create novel, culinary, therapeutic, and diagnostic bioactive nanomaterials. The applications of fucoidan and its composite nanomaterials have a wide variety of food as well as pharmacological properties, including anti-oxidative, anti-inflammatory, anti-cancer, anti-thrombic, anti-coagulant, immunoregulatory, and anti-viral properties. Blends of fucoidan with other biopolymers such as chitosan, alginate, curdlan, starch, etc., have shown promising coating and film-forming capabilities. A blending of biopolymers is a recommended approach to improve their anticipated properties. This review focuses on the fundamental knowledge and current development of fucoidan, fucoidan-based composite material for bioactive coatings and films, and their biological properties. In this article, fucoidan-based edible bioactive coatings and films expressed excellent mechanical strength that can prolong the shelf-life of food products and maintain their biodegradability. Additionally, these coatings and films showed numerous applications in the biomedical field and contribute to the economy. We hope this review can deliver the theoretical basis for the development of fucoidan-based bioactive material and films.

In vitroassessment of green polyhydroxybutyrate/chitosan blend loaded with kaempferol nanocrystals as a potential dressing for infected wounds.

Despite the major medical advancements in recent decades, treating infected wounds successfully remains a challenge. In this research, a functional blend of Polyhydroxybutyrate (PHB) and Chitosan (Cs) was developed for wound infection mitigation with tailored biological and physicochemical properties. Water insoluble kaempferol (KPF) was pre-formulated to water soluble KPF nanocrystals (KPF-NCs) with fine particle size of 145 ± 11 nm, and high colloidal stability (-31 ± 0.4 mV) to improve its drug transdermal delivery. PHB-Cs-KPF-NCs (1:2 ratio) film owned the best physical properties in terms of high breathability, thermal stability and mechanical strength (33 ± 1 MPa). Besides, XRD and FTIR findings indicated the interaction between Cs, PHB and KPF, reducing the film crystallinity. The scanning electron microscopy of the film displayed a highly interconnected porous morphology. KPF-NCs were integrated in PHB-Cs matrix with a marked encapsulation efficiency of 96.6%. The enhanced drug-loading film showed a sustain release pattern of KPF-NCs over 48 h. Interestingly, the developed blend possessed an impressive blood clotting capacity within 20 min. Furthermore, we presented a new naturally-sourced mixture of Cs+KPF-NCs with powerful antibacterial effects against MDRStaphylococcus aureusandAcentibacter baumanniiat very low concentrations. The membrane evidenced a remarkable antibacterial naturein vitrowith almost 100% cell viability reduction against the study strains after 48 h. By virtue of these advantages, this green blend is highly proposed for optimal wound care.

Compact optically pumped cesium beam atomic clock with a 5-day frequency stability of 7×10-15.

Compared to other commercial atomic clocks in the time keeping field, the greatest advantage of cesium beam atomic clocks is their superior long-term stability. Compared to magnetic state-selection clocks, optically pumped cesium beam atomic clocks have more interacting atoms, which results in better stability potential. To achieve good long-term stability, we propose methods including stabilization of laser power and reconstruction of circuits. They play a key role in the long-term stability of cesium beam atomic clocks. After 75 days of continuous running and measurement, we released the 5-day stability results (7×10-15 Allan deviation) of our optically pumped cesium beam atomic clock. To the best of our knowledge, this is the best 5-day stability result ever reported for compact optically pumped cesium beam atomic clocks.

Application of nonthermal processing technologies in extracting and modifying polysaccharides: A critical review.

Polysaccharides are natural polymer compounds widely distributed in plants, animals, and microorganisms, most of which have a broad spectrum of biological activities to promote human health. They could also be used as texture modifiers in food industry due to their excellent rheological and mechanical properties. Many researchers have shown that nonthermal processing technologies have numerous advantages, such as high extraction efficiency, short extraction time, and environmental friendliness, in the extraction of polysaccharides compared with the traditional extraction methods. Moreover, nonthermal technologies could effectively change the physicochemical properties and structural characteristics of polysaccharides to improve their biological activities or processing properties. Therefore, a comprehensive summary about the extraction and modification of polysaccharides by nonthermal technologies, including ultrasound, high hydrostatic pressure, pulsed electric fields, and cold plasma, was provided in this review. In particular, the underlying mechanisms, processing operations, and current application status of these technologies were discussed. In addition, the applications of combining nonthermal techniques with other technological methods in polysaccharide extraction and modification were briefly introduced.

The Draft Genome Sequence of Clostridium butyricum QXYZ514, a Potent Bacterium for Converting Glycerol into Fuels and Bioproducts in the Waste-Based Biorefinery.

An anaerobic bacterium Clostridium buturicum QXYZ514 was isolated from a pond soil sample located neighboring to a biodiesel factory. This bacterium possesses excellent metabolic features for converting biodiesel-derived glycerol into various bioproducts, including 1,3-propanediol, butyrate, lactate, and acetate, and fuels, like ethanol and butanol. To further improve the yield of the target products and minimize the production of the by-products, the whole genome sequence of this multipurpose strain might provide necessary genetic information, and hence, the complete genome of QXYZ514 strain was sequenced using the PacBio RS II sequencing method. According to the complete genome sequence, the genome of QXYZ514 consisted of two circular chromosomes with a total of 4,636,461 bp, where GC content was found to be 28.76%. Major predicted features of the genome included a total of 4220 coding sequences (CDS), 87 tRNAs genes, and 36 rRNAs genes, which were annotated with the help of different databases for a better understanding of this strain. Six possible CRISPR components were also predicted in the genome. The exploration of the complete genome sequence of the QXYZ514 strain would have the potential to enrich the diversity of this species, and to recognize some significant hydrolytic enzymes, which could provide the references for overcoming the bottlenecks in the biorefinery usage of this bacterium in the valorization of biodiesel-derived glycerol.

Influence of nutrient supplementation and starvation conditions on the biomass and lipid productivities of Micractinium reisseri grown in wastewater for biodiesel production.

Generation of biodiesel from microalgae grown in wastewater can offer a cost-effective approach, whilst wastewaters usually do not contain the optimum concentrations of the essential nutrients and carbon sources that result in lowering the productivities of biomass and lipid. This study aimed to overcome this limitation by manipulating wastewater with various concentrations of nutrients (NO3-, PO43-, Cl- and SO42-) and three carbon sources either individually or in combination to cultivate Micractinium reisseri for biodiesel production. Initially, various dilutions of wastewater were tested and a concentration up to 75% of wastewater showed the highest biomass productivity (0.076 g L-1 d-1) and lipid productivity (0.014 g L-1 d-1). The optimum manipulating conditions for maximum lipid production and the highest productivity required 50% decrease in phosphorous from the concentration of the control medium and supplementation with 1.0 g L-1 of glucose. Under this condition, biomass and lipid productivities increased by 1.7 and 4-folds, respectively, compared to those observed in the control. Furthermore, phosphorous starvation condition in the presence of glucose significantly improved fatty acid profile in the biomass and biodiesel quality related parameters.

Enhancing 2-Ketogluconate Production of Pseudomonas plecoglossicida JUIM01 by Maintaining the Carbon Catabolite Repression of 2-Ketogluconate Metabolism.

2-Ketogluconate (2KGA) is an organic acid that is important for pharmaceutical, cosmetic, and environmental applications. Pseudomonas plecoglossicida JUIM01 strain is an important industrial 2KGA producer in China. In this paper, we found that P. plecoglossicida JUIM01 could convert glucose to 2KGA extracellularly, and the formed 2KGA was subsequently consumed after glucose was exhausted during the fermentation process. Experiments of glucose and 2KGA supplementation during fermentation process revealed that, only when glucose was exhausted, the strain started to consume the product 2KGA. Then, the mechanism of this phenomenon was investigated at transcription and protein levels, and the results indicated that P. plecoglossicida JUIM01 possesses carbon catabolite repression of 2KGA metabolism by glucose. Next, increasing the supply of glucose could attenuate 2KGA consumption and enhance the 2KGA yield from glucose. Finally, fed-batch fermentation of P. plecoglossicida JUIM01 resulted in 205.67 g/L of 2KGA with a productivity of 6.86 g/L/h and yield of 0.953 g/g glucose. These results can provide references for the industrial fermentation production of 2KGA and other fermentation products.

Genetically Engineered Strains: Application and Advances for 1,3-Propanediol Production from Glycerol.

1,3-Propanediol (1,3-PD) is one of the most important chemicals widely used as monomers for synthesis of some commercially valuable products, including cosmetics, foods, lubricants and medicines. Although 1,3-PD can be synthesized both chemically and biosynthetically, the latter offers more merits over chemical approach as it is economically viable, environmentally friendly and easy to carry out. The biosynthesis of 1,3-PD can be done by transforming glycerol or other similar substrates using some bacteria, such as Clostridium butyricum and Klebsiella pneumoniae. However, these natural microorganisms pose some bottlenecks like low productivity and metabolite inhibition. To overcome these problems, recent research efforts have been focused more on the development of new strains by modifying the genome through different techniques, such as mutagenesis and genetic engineering. Genetically engineered strains obtained by various strategies cannot only gain higher yield than wild types, but also overcome some of the barriers in production by the latter. This review paper presents an overview on the recent advances in the technological approaches to develop genetically engineered microorganisms for efficient biosynthesis of 1,3-PD.

Laser frequency stabilization using a dispersive line shape induced by Doppler Effect.

We report a simple and robust Doppler-free spectroscopic technique to stabilize a laser frequency to the atomic transition. By employing Doppler Effect on the atomic beam, we obtained a very stable dispersive signal with a high signal-to-noise ratio and no Doppler-background, which served as an error signal to electronically stabilize a laser frequency without modulation. For validating the performance of this technique, we locked a DFB laser to the (133)Cs D2 line and observed an efficient suppression of the frequency noise and a long-term reduction of the frequency drifts in a laboratory environment.

Enhanced D-arabitol production by Zygosaccharomyces rouxii JM-C46: isolation of strains and process of repeated-batch fermentation.

A new strain producing high yield of D-arabitol was isolated from hyperosmotic environments and the ITS rDNA sequencing analysis revealed it as Zygosaccharomyces rouxii. In addition, using a pH control and repeated-batch fermentation strategy in a 5-L reactor, the maximum yield and the highest volumetric productivity of D-arabitol were 93.48 ± 2.79 g/L and 1.143 g/L h, respectively. Volumetric productivity was successfully improved from 0.86 to 1.143 g/L h, which was increased by 32.9 % after 72 h of fermentation. Z. rouxii JM-C46 has potential to be used for D-arabitol and xylitol production from glucose via D-arabitol route.

Fermentative Production of Ergothioneine by Exploring Novel Biosynthetic Pathway and Remodulating Precursor Synthesis Pathways.

Ergothioneine (EGT) is a naturally occurring derivative of histidine with diverse applications in the medicine, cosmetic, and food industries. Nevertheless, its sustainable biosynthesis faces hurdles due to the limited biosynthetic pathways, complex metabolic network of precursors, and high cost associated with fermentation. Herein, efforts were made to address these limitations first by reconstructing a novel EGT biosynthetic pathway from Methylobacterium aquaticum in Escherichia coli and optimizing it through plasmid copy number. Subsequently, the supply of precursor amino acids was promoted by engineering the global regulator, recruiting mutant resistant to feedback inhibition, and blocking competitive pathways. These metabolic modifications resulted in a significant improvement in EGT production, increasing from 35 to 130 mg/L, representing a remarkable increase of 271.4%. Furthermore, an economical medium was developed by replacing yeast extract with corn steep liquor, a byproduct of wet milling of corn. Finally, the production of EGT reached 595 mg/L with a productivity of 8.2 mg/L/h by exploiting fed-batch fermentation in a 10 L bioreactor. This study paves the way for exploring and modulating a de novo biosynthetic pathway for efficient and low-cost fermentative production of EGT.

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools.

The CRISPR/Cas9 systems have been developed as tools for genetic engineering and metabolic engineering in various organisms. In this review, various aspects of CRISPR/Cas9 in Saccharomyces cerevisiae, from basic principles to practical applications, have been summarized. First, a comprehensive review has been conducted on the history of CRISPR/Cas9, successful cases of gene disruptions, and efficiencies of multiple DNA fragment insertions. Such advanced systems have accelerated the development of microbial engineering by reducing time and labor, and have enhanced the understanding of molecular genetics. Furthermore, the research progress of the CRISPR/Cas9-based systems in the production of high-value-added chemicals and the improvement of stress tolerance in S. cerevisiae have been summarized, which should have an important reference value for genetic and synthetic biology studies based on S. cerevisiae.

Metabolic Remodulation of Chassis and Corn Stover Bioprocessing to Unlock 3-Hydroxypropionic Acid Biosynthesis from Agrowaste-Derived Substrates.

Embracing the principles of sustainable development, the valorization of agrowastes into value-added chemicals has nowadays received significant attention worldwide. Herein, Escherichia coli was metabolically rewired to convert cellulosic hydrolysate of corn stover into a key platform chemical, namely, 3-hydroxypropionic acid (3-HP). First, the heterologous pathways were introduced into E. coli by coexpressing glycerol-3-P dehydrogenase and glycerol-3-P phosphatase in both single and fusion (gpdp12) forms, making the strain capable of synthesizing glycerol from glucose. Subsequently, a glycerol dehydratase (DhaB123-gdrAB) and an aldehyde dehydrogenase (GabD4) were overexpressed to convert glycerol into 3-HP. A fine-tuning between glycerol synthesis and its conversion into 3-HP was successfully established by 5'-untranslated region engineering of gpdp12 and dhaB123-gdrAB. The strain was further metabolically modulated to successfully prevent glycerol flux outside the cell and into the central metabolism. The finally remodulated chassis produced 32.91 g/L 3-HP from the cellulosic hydrolysate of stover during fed-batch fermentation.

Efficient Fermentative Production of ß-Alanine from Glucose through Multidimensional Engineering of Escherichia coli.

ß-Alanine, a valuable ß-type amino acid, is experiencing increased demand due to its multifaceted applications in food flavoring, nutritional supplements, pharmaceuticals, and the chemical industry. Nevertheless, the sustainable biosynthesis of ß-alanine currently faces challenges due to the scarcity of robust strains, attributed to the complexities of modulating multiple genes and the inherent physiological constraints. Here, systems metabolic engineering was implemented in Escherichia coli to overcome these limitations. First, an efficient l-aspartate-α-decarboxylase (ADC) was recruited for ß-alanine biosynthesis. To conserve phosphoenolpyruvate flux, we subsequently modified the endogenous glucose assimilation system by inactivating the phosphotransferase system (PTS) and introducing an alternative non-PTS system, which increased ß-alanine production to 1.70 g/L. The supply of key precursors, oxaloacetate and l-aspartate, was synergistically improved through comprehensive modulation, including strengthening main flux and blocking bypass metabolism, which significantly increased the ß-alanine titer to 3.43 g/L. Next, the expression of ADC was optimized by promoter and untranslated region (UTR) engineering. Further transport engineering, which involved disrupting ß-alanine importer CycA and heterologously expressing ß-alanine exporter NCgI0580, improved ß-alanine production to 8.48 g/L. Additionally, corn steep liquor was used to develop a cost-effective medium. The final strain produced 74.03 g/L ß-alanine with a yield of 0.57 mol/mol glucose during fed-batch fermentation.

Random mutagenesis and transcriptomics-guided rational engineering in Zygosaccharomyces rouxii for elevating D-arabitol biosynthesis.

D-arabitol, a versatile compound with applications in food, pharmaceutical, and biochemical industries, faces challenges in biomanufacturing due to poor chassis performance and unclear synthesis mechanisms. This study aimed to enhance the performance of Zygosaccharomyces rouxii to improve D-arabitol production. Firstly, a mutant strain Z. rouxii M075 obtained via atmospheric and room temperature plasma-mediated mutagenesis yielded 42.0 g/L of D-arabitol at 96 h, with about 50 % increase. Transcriptome-guided metabolic engineering of pathway key enzymes co-expression produced strain ZR-M3, reaching 48.9 g/L D-arabitol after 96 h fermentation. Finally, under optimized conditions, fed-batch fermentation of ZR-M3 in a 5 L bioreactor yielded an impressive D-arabitol titer of 152.8 g/L at 192 h, with a productivity of 0.8 g/L/h. This study highlights promising advancements in enhancing D-arabitol production, offering potential for more efficient biomanufacturing processes and wider industrial applications.

Characterization of a novel multifunctional ß-glucosidase/xylanase/feruloyl esterase and its effects on improving the quality of Longjing tea.

Herein, a novel multifunctional enzyme ß-glucosidase/xylanase/feruloyl esterase (GXF) was constructed by fusion of ß-glucosidase and bifunctional xylanase/feruloyl esterase. The activities of ß-glucosidase, xylanase, feruloyl esterase and acetyl xylan esterase displayed by GXF were 67.18 %, 49.54 %, 38.92 % and 23.54 %, respectively, higher than that of the corresponding single functional enzymes. Moreover, the GXF performed better in enhancing aroma and quality of Longjing tea than the single functional enzymes and their mixtures. After treatment with GXF, the grassy and floral odors of tea infusion were significantly improved. Moreover, GXF treatment could improve concentrations of flavonoid aglycones of myricetin, kaempferol and quercetin by 68.1-, 81.42- and 77.39-fold, respectively. In addition, GXF could accelerate the release of reducing sugars, ferulic acid and xylo-oligosaccharides by 9.48-, 8.25- and 4.11-fold, respectively. This multifunctional enzyme may have potential applications in other fields such as food production and biomass degradation.

Sustainable bio-manufacturing of D-arabitol through combinatorial engineering of Zygosaccharomyces rouxii, bioprocess optimization and downstream separation.

Biosynthesis of D-arabitol, a high value-added platform chemical, from renewable carbon sources provides a sustainable and eco-friendly alternative to the chemical industry. Here, a robust brewing yeast, Zygosaccharomyces rouxii, capable of naturally producing D-arabitol was rewired through genome sequencing-based metabolic engineering. The recombinant Z. rouxii obtained by reinforcing the native D-xylulose pathway, improving reductive power of the rate-limiting step, and inhibiting the shunt pathway, produced 73.61% higher D-arabitol than the parent strain. Subsequently, optimization of the fermentation medium composition for the engineered strain provided 137.36 g/L D-arabitol, with a productivity of 0.64 g/L/h in a fed-batch experiment. Finally, the downstream separation of D-arabitol from the complex fermentation broth using an ethanol precipitation method provided a purity of 96.53%. This study highlights the importance of D-xylulose pathway modification in D-arabitol biosynthesis, and pave a complete and efficient way for the sustainable manufacturing of this value-added compound from biosynthesis to preparation.