BACKGROUND: The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer-Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L-1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.
RESULTS: Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer-Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW-1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW- 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.
CONCLUSIONS: Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the β-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the β-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

BACKGROUND: Dendrobium officinale Kimura et Migo, a renowned traditional Chinese orchid herb esteemed for its significant horticultural and medicinal value, thrives in adverse habitats and contends with various abiotic or biotic stresses. Acid invertases (AINV) are widely considered enzymes involved in regulating sucrose metabolism and have been revealed to participate in plant responses to environmental stress. Although members of AINV gene family have been identified and characterized in multiple plant genomes, detailed information regarding this gene family and its expression patterns remains unknown in D. officinale, despite their significance in polysaccharide biosynthesis.
RESULTS: This study systematically analyzed the D. officinale genome and identified four DoAINV genes, which were classified into two subfamilies based on subcellular prediction and phylogenetic analysis. Comparison of gene structures and conserved motifs in DoAINV genes indicated a high-level conservation during their evolution history. The conserved amino acids and domains of DoAINV proteins were identified as pivotal for their functional roles. Additionally, cis-elements associated with responses to abiotic and biotic stress were found to be the most prevalent motif in all DoAINV genes, indicating their responsiveness to stress. Furthermore, bioinformatics analysis of transcriptome data, validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct organ-specific expression patterns of DoAINV genes across various tissues and in response to abiotic stress. Examination of soluble sugar content and interaction networks provided insights into stress release and sucrose metabolism.
CONCLUSIONS: DoAINV genes are implicated in various activities including growth and development, stress response, and polysaccharide biosynthesis. These findings provide valuable insights into the AINV gene amily of D. officinale and will aid in further elucidating the functions of DoAINV genes.

This article examined the effect of geographical (different climate conditions) and floral origins on some quality parameters of honey including the activity of diastase enzyme. Moreover, some non-quality parameters were investigated such as the pH, fructose, glucose, ratio of fructose/glucose and invertase. The honey samples were collected from Asir (cold climate) and Jazan (hot climate) regions at the southwestern part of Saudi Arabia. The geographical origin significantly affected the mean value moisture of the Acacia honey (p-value = 0.02), conductivity of the polyfloral honey (p-value = 0.03), sucrose of the Acacia honey (p-value = 0.02), diastase activity of the Acacia (p-value = 0.001), Ziziphus (p-value = 0.046) and polyfloral honey (p-value ≤ 0.001), fructose of the Acacia honey (p-value = 0.01), glucose of the Ziziphus honey (p-value = 0.03), fructose/ glucose ratio of the Ziziphus honey (p-value = 0.035), and invertase activity of the polyfloral honey (p-value ≤ 0.001). Regarding the effect of the floral origin of the honey from Asir region, the sucrose percentage of the Acacia honey was significantly more than that of the polyfloral honey (p- value = 0.003), the diastase activity of the Acacia honey was significantly more than its activity in the Ziziphus honey (p- value = 0.044), glucose percentage of the Ziziphus honey was significantly more the glucose percentage of the Acacia honey (p-value = 0.009) and the fructose/ glucose ratio of the Ziziphus honey was significantly more than that of the Acacia and polyforal honeys (p-value = 0.011 and p-value = 0.045, respectively). Concerning the significant effects of the floral origin on the quality parameters of the honey samples from Jazan region, the moisture of the Ziziphus honey was significantly increased when compared to the moisture of the Acacia honey (p-value = 0.038), the acidity of the polfloral honey was significantly more than the acidity of the Acacia honey (p-value = 0.049), the sum of fructose and glucose of the polyfloral honey was significantly increased compared to that of the Acacia honey (p-value = 0.015), the pH of the Ziziphus hiney was significantly more than the pH of the polyfloral honey (0.011) and the fructose of the polfloral honey was significantly more than that of the Acacia honey (p-value = 0.031). The effect of the geographical origin of the honey samples on their quality parameters depends on their floral origin and the effect of their floral origin differs according to their geographical origin. This article suggests considering collectively the geographical and floral origins effect when developing honey standards. However, the Codex standards for honey started considering this issue when it changed the standard concentration of HMF in honey from not more than 80-40 mg/Kg for honeys from cold climate and 80 mg/Kg for honeys from hot climates.

BACKGROUND: Aspergillus niger ATCC 20611 is an industrially important fructooligosaccharides (FOS) producer since it produces the β-fructofuranosidase with superior transglycosylation activity, which is responsible for the conversion of sucrose to FOS accompanied by the by-product (glucose) generation. This study aims to consume glucose to enhance the content of FOS by heterologously expressing glucose oxidase and peroxidase in engineered A. niger.
RESULTS: Glucose oxidase was successfully expressed and co-localized with β-fructofuranosidase in mycelia. These mycelia were applied to synthesis of FOS, which possessed an increased purity of 60.63% from 52.07%. Furthermore, peroxidase was expressed in A. niger and reached 7.70 U/g, which could remove the potential inhibitor of glucose oxidase to facilitate the FOS synthesis. Finally, the glucose oxidase-expressing strain and the peroxidase-expressing strain were jointly used to synthesize FOS, which content achieved 71.00%.
CONCLUSIONS: This strategy allows for obtaining high-content FOS by the multiple enzymes expressed in the industrial fungus, avoiding additional purification processes used in the production of oligosaccharides. This study not only facilitated the high-purity FOS synthesis, but also demonstrated the potential of A. niger ATCC 20611 as an enzyme-producing cell factory.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to minimize sprouting and losses due to disease. However, cold temperatures strongly induce the expression of the potato vacuolar invertase gene (VInv) and cause reducing sugar accumulation. This process, referred to as \"cold-induced sweetening,\" is a major postharvest problem for the potato industry. We discovered that the cold-induced expression of VInv is controlled by a 200 bp enhancer, VInvIn2En, located in its second intron. We identified several DNA motifs in VInvIn2En that bind transcription factors involved in the plant cold stress response. Mutation of these DNA motifs abolished VInvIn2En function as a transcriptional enhancer. We developed VInvIn2En deletion lines in both diploid and tetraploid potato using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. VInv transcription in cold-stored tubers was significantly reduced in the deletion lines. Interestingly, the VInvIn2En sequence is highly conserved among distantly related Solanum species, including tomato (Solanum lycopersicum) and other non-tuber-bearing species. We conclude that the VInv gene and the VInvIn2En enhancer have adopted distinct roles in the cold stress response in tubers of tuber-bearing Solanum species.

Arrow bamboo (Fargesia nitida) is a pioneer plant in secondary forest succession in the Sichuan Province mountains. To comprehensively investigate the microbial communities and their functional variations in different rhizocompartments (root endosphere, rhizosphere, and root zone) of arrow bamboo (Fargesia nitida), a high-throughput metagenomic study was conducted in the present study. The results showed that the abundances of the dominant bacterial phyla Proteobacteria and Actinobacteria in the bamboo root endosphere were significantly lower than those in the rhizosphere and root zones. In contrast, the dominant fungal phyla, Ascomycota and Basidiomycota, showed the opposite tendency. Lower microbial diversity, different taxonomic composition and functional profiles, and a greater abundance of genes involved in nitrogen fixation (nifB), cellulose degradation (beta-glucosidase), and cellobiose transport (cellulose 1, 4-beta-cellobiosidase) were found in the bamboo root endosphere than in the other rhizocompartments. Greater soil total carbon, total nitrogen, NH4+-N, microbial biomass carbon, and greater activities of invertase and urease were found in the bamboo root zone than in the adjacent soil (spruce root zone). In contrast, the soil microbial community and functional profiles were similar. At the phylum level, invertase was significantly related to 31 microbial taxa, and the effect of NH4+-N on the microbial community composition was greater than that of NO3--N. The soil physicochemical properties and enzyme activities were significantly correlated with microbial function. These results indicate that the root endosphere microbiomes of arrow bamboo were strongly selected by the host plant, which caused changes in the soil nutrient properties in the subalpine coniferous forest.

Light plays a dominant role in the biosynthesis and accumulation of photosynthetic products. However, the metabolism and translocation of photosynthetic products in plants under different light spectra remain elusive. In this study, tomato (Solanum lycopersicum L.) seedlings were treated with different light spectra delivered by light-emitting diodes (LEDs) with the same photosynthetic photon flux density at 300 μmol m-2 s-1, including monochromatic red (660 nm, R), blue (450 nm, B), sun-like white (W, 380-780 nm), or a combination of R and B lights (R:B = 1:1, RB). Compared with W, the biomass distribution ratio for leaves under R, B, and RB decreased by 5.01-9.53%, while the ratio for stems and roots increased by 3.71-6.92% and 0.14-2.81%, respectively. The photosynthetic carbon distribution expressed as 13C enrichment was higher in stems and roots under RB and R, while B led to more 13C transported from leaves and enriched in stems when compared with W. Meanwhile, RB led to significant increases in the activities of phosphate synthase (SPS), sucrose synthase (SS), vacuolar acid invertase (VI), and neutral invertase (NI). The R was more efficient in increasing the activity of SPS and SS, while B was more effective in promoting the activity of VI and NI. The transcript levels of SPS, SS3, NI6, and VI were upregulated under R, B, and RB. However, the transcript patterns of SPS, SS3, NI6, and VI were not consistent with the changes in their encoded enzymes, especially the transcript patterns of SPS and SS3. Our study suggests that the red- and blue-light-induced long-distance and short-distance transport of photosynthetic products in plants, respectively, might result from different regulation of sucrose-metabolizing enzymes from transcriptional and post-transcriptional levels.

To evaluated the effects melatonin (MT) on the sugar and acid metabolism of early-ripening peach fruits, the concentration of 150 μmol/L MT was sprayed on the leaves of peach trees. MT increased the contents of total soluble sugar and sucrose in peach fruits during the whole ripening period, and increased the contents of glucose and sorbitol at the mature stage. During the whole ripening period, MT also increased the activities of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase and the relative expression levels of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase genes, while decreased the activity of sorbitol oxidase and the relative expression level of sorbitol dehydrogenase to some extent. Moreover, MT decreased the contents of total organic acid, malic acid, and citric acid at mature stage. At mature stage, MT decreased the activities of citrate synthetase and phosphoenolpyruvate carboxylase and the relative expression levels of citrate synthetase and phosphoenolpyruvate carboxylase genes, while increased the relative expression levels of Nicotinamide adenine dinucleotide phosphate (NADP+)-malic enzyme, malate dehydrogenase, and aconitase genes. Therefore, MT promotes the sugar accumulation and organic acid degradation in early-ripening peach fruits.