Sucrose 1-fructosyltransferase Research Articles

Fructooligosaccharides: Production by recombinant fructosyltransferase from Festuca arundinacea in a continuous reactor and kinetic modeling profile

In the quest for maximum conversion of sucrose into 1-kestose using fructosyltransferases in a continuous reactor, consideration of the reversible reactions at competing concentrations of sucrose and 1-kestose is crucial. In this study, a kinetic model based on a combined approach of rapid equilibrium and steady-state kinetics was developed to predict the profile of FOS production using recombinant sucrose: sucrose 1-fructosyltransferase from Festuca arundinacea produced in Pichia pastoris. The purified enzyme selectively converted sucrose into 1-kestose with a final yield of 0.66 g/g sucrose consumed. The reaction rate laws adequately described the kinetic profile of the substrate and the products and accurately predicted the composition of the reaction mixture. The model was fitted to experimental data obtained at three enzyme dosages and three initial sucrose concentrations. The model could predict the effect of substrate concentration on initial rate kinetics, half-saturation constant, and transferase to hydrolase ratio. For the known values of initial sucrose and enzyme concentration, the optimum reaction time and the required dilution rate were predicted for the development of a continuous process to produce 1-kestose-enriched short-chain FOS. A volumetric productivity of ∼110 g/L/h of 1-kestose with 40 % carbohydrate composition in the permeate flux was demonstrated.

Modified fructan accumulation through overexpression of wheat fructan biosynthesis pathway fusion genes Ta1SST:Ta6SFT

BackgroundFructans are water-soluble carbohydrates that accumulate in wheat and are thought to contribute to a pool of stored carbon reserves used in grain filling and tolerance to abiotic stress.ResultsIn this study, transgenic wheat plants were engineered to overexpress a fusion of two fructan biosynthesis pathway genes, wheat sucrose: sucrose 1-fructosyltransferase (Ta1SST) and wheat sucrose: fructan 6-fructosyltransferase (Ta6SFT), regulated by a wheat ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (TaRbcS) gene promoter. We have shown that T4 generation transgene-homozygous single-copy events accumulated more fructan polymers in leaf, stem and grain when compared in the same tissues from transgene null lines. Under water-deficit (WD) conditions, transgenic wheat plants showed an increased accumulation of fructan polymers with a high degree of polymerisation (DP) when compared to non-transgenic plants. In wheat grain of a transgenic event, increased deposition of particular fructan polymers such as, DP4 was observed.ConclusionsThis study demonstrated that the tissue-regulated expression of a gene fusion between Ta1SST and Ta6SFT resulted in modified fructan accumulation in transgenic wheat plants and was influenced by water-deficit stress conditions.

Rational design and immobilization of a recombinant sucrose: Sucrose 1-fructosyltransferase on Sepabeads® and ReliZyme™ supports for short-chain fructooligosaccharides production

The recombinant sucrose:sucrose 1-fructosyltransferase from Schedonorus arundinaceus (Sa1-SSTrec) produces short-chainfructooligosaccharides (scFOS). In this work, Sa1-SSTrec was covalently or hydrophobic immobilized on Sepabeads® and ReliZyme™ supports. The possible clusters and the parameters related to the immobilization process were predicted by rational design of immobilized derivatives (RDID). Immobilization was performed at pH 7.0 and the three immobilized derivatives with higher experimental enzymatic activity (one of each epoxy-activated, amino-activated and hydrophobic supports: Sepabeads® EC-HFA/S, ReliZyme™ EA403/M and Sepabeads® EC-BU/M, respectively) were selected. The mismatch between some predictions and experimental results were explained by probable limitations of the RDID strategy. The thermodynamic parameters determined at 40, 50 and 60ºC, under not reactive conditions (i.e. in absence of sucrose), showed high thermal stability of Sa1-SSTrec after immobilization on the Sepabeads®EC-HFA/S. Batch experiments with this support, at 45ºC reached similar total scFOS (1-kestotriose and 1,1-kestotetraose) than the soluble enzyme, but scFOS productivity was higher for the immobilized biocatalyst. Operational stability of the immobilized biocatalyst after 4 cycles dropped to about 60% and 25% of its initial value at 30 and 45ºC, respectively, probably by denaturation at these temperatures. The obtained results suggest that the immobilized enzyme could be effectively exploited for industrial production of 1-kestotriose.

Integrated Transcriptomics and Metabolomics Analysis of the Fructan Metabolism Response to Low-Temperature Stress in Garlic.

As the main reserve carbohydrate in garlic, fructan contributes to garlic's yield and quality formation. Numerous studies have shown that plant fructan metabolism induces a stress response to adverse environments. However, the transcriptional regulation mechanism of garlic fructan in low-temperature environments is still unknown. In this study, the fructan metabolism of garlic seedlings under low-temperature stress was revealed by transcriptome and metabolome approaches. With the extension of stress time, the number of differentially expressed genes and metabolites increased. Using weighted gene co-expression network analysis (WGCNA), three key enzyme genes related to fructan metabolism were screened (a total of 12 transcripts): sucrose: sucrose 1-fructosyltransferase (1-SST) gene; fructan: fructan 6G fructosyltransferase (6G-FFT) gene; and fructan 1-exohydrolase (1-FEH) gene. Finally, two hub genes were obtained, namely Cluster-4573.161559 (6G-FFT) and Cluster-4573.153574 (1-FEH). The correlation network and metabolic heat map analysis between fructan genes and carbohydrate metabolites indicate that the expression of key enzyme genes in fructan metabolism plays a positive promoting role in the fructan response to low temperatures in garlic. The number of genes associated with the key enzyme of fructan metabolism in trehalose 6-phosphate was the highest, and the accumulation of trehalose 6-phosphate content may mainly depend on the key enzyme genes of fructan metabolism rather than the enzyme genes in its own synthesis pathway. This study not only obtained the key genes of fructan metabolism in garlic seedlings responding to low temperatures but also preliminarily analyzed its regulatory mechanism, providing an important theoretical basis for further elucidating the cold resistance mechanism of garlic fructan metabolism.

Ectopic Expression of the Allium cepa 1-SST Gene in Cotton Improves Drought Tolerance and Yield Under Drought Stress in the Field.

In some plants, sucrose: sucrose 1-fructosyltransferase (1-SST) is the first irreversible key enzyme in fructan biosynthesis. Studies have shown that fructan accumulation enhances abiotic stress tolerance of plants. To investigate the role of 1-SST in drought stress responses, a total of 37 cotton plants expressing a 1-SST gene from Allium cepa were developed by Agrobacterium-mediated transformation. Under drought stress in the field, compared with wild-type, ectopic expression of Ac1-SST in cotton resulted in significantly higher soluble sugars (especially 1-kestose), proline and relative water contents, as well as decreased malondialdehyde content, which contributed to maintaining intracellular osmoregulation and reducing membrane damage. In addition, ectopic expression of Ac1-SST in cotton significantly improved the photosynthesis rate, performance of PSII (including Pn, Fv/Fm, WUE, ΦPSII, and PItotal) and plant growth under drought stress. Furthermore, compared with the wild-type, under the droughted field, the yield loss per square meter of transgenic cotton was reduced by an average of 20.9% over two consecutive years. Our results indicate that the Ac1-SST gene can be used to improve drought tolerance and yield of cotton varieties, and might also be a promising drought-resistant gene for improving other crop varieties.

Regioselective synthesis of 6’’-O-lauroyl-1-kestose and 6’’’-O-lauroylnystose by sequential enzymatic reactions of transfructosylation and acylation

Fructan fatty acid esters are amphiphilic compounds with potential applications in the food industry due to their surface-active properties on the air/water interface. In this work, the regioselective synthesis of fatty acid esters of short-chain inulin-type fructooligosaccharides (FOS) was carried out in a two-step process comprising sequential enzymatic reactions of transfructosylation and acylation. In the first step, Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) converted sucrose (600 g/L) into the trisaccharide 1-kestose and the tetrasaccharide nystose in a ratio 9:1 with their sum accounting for 53% (w/w) of total carbohydrates. In the second step, the FOS mixture was transesterified with vinyl laurate in 2-methyl-2-butanol by immobilized Candida antarctica lipase B (CALB) (EC.3.1.1.3). NMR analysis of the synthesized monolaurate FOS esters revealed a regioselective acylation of the 6-OH of the terminal fructosyl moiety of both 1-kestose and nystose. The hydrophilic-lipophilic balance (HLB) values of 6’’-O-lauroyl-1-kestose and 6’’’-O-lauroylnystose were 14.7 and 15.7, respectively; which suggests their use as oil in water (O/W) emulsifiers. The main compound 6’’-O-lauroyl-1-kestose with critical micelle concentration (CMC) of 0.6 mM and surface tension of 45.8 mN/m proved to be a more efficient surfactant than 6’’’-O-lauroylnystose (CMC 5.38 mM) and surface tension 36.26 mN/m). We report a regioselective method for the synthesis of lauryl-FOS using the abundant and renewable resource sucrose as the starting substrate.

Fructooligosaccharides production by immobilized Pichia pastoris cells expressing Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase.

Fructooligosaccharides (FOSs)—fructose-based oligosaccharides—are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

Enzymatic synthesis of phlorizin fructosides

Phlorizin is a low soluble dihydrochalcone with relevant pharmacological properties. In this study, enzymatic fructosylation was approached to enhance the water solubility of phlorizin, and consequently its bioavailability. Three enzymes were assayed for phlorizin fructosylation in aqueous reactions using sucrose as fructosyl donor. Levansucrase (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (Gd_LsdA) was 6.5–fold more efficient than invertase (EC 3.2.1.26) from Rhodotorula mucilaginosa (Rh_Inv), while sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) from Schedonorus arundinaceus (Sa_1-SST) failed to modify the non-sugar acceptor. Gd_LsdA synthesized series of phlorizin mono- di- and tri-fructosides with maximal conversion efficiency of 73 %. The three most abundant products were identified by ESI-MS and NMR analysis as β-D-fructofuranosyl-(2→6)-phlorizin (P1a), phlorizin-4'-O-β-D-fructofuranosyl-(2→6)-D-fructofuranoside (P2c) and phlorizin-4-O-monofructofuranoside (P1b), respectively. Purified P1a was 16 times (30.57 g L−1 at 25 °C) more soluble in water than natural phlorizin (1.93 g L−1 at 25 °C) and exhibited 44.56 % free radical scavenging activity. Gd_LsdA is an attractive candidate enzyme for the scaled synthesis of phlorizin fructosides in the absence of co-solvent.

Genome-wide association study reveals the genetic complexity of fructan accumulation patterns in barley grain.

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the 'high' set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley.

Genome-wide characterization of early response genes to abscisic acid coordinating multiple pathways in Aegilops tauschii

The diploid wild goat grass Aegilops tauschii (Ae. tauschii, 2n = 14; DD), as the D-sub genome of common wheat, provides rich germplasm resources for many aspects of wheat breeding. Abscisic acid (ABA) is an essential phytohormone that plays a pivotal role in plant adaptation to abiotic stresses. However, the gene regulation network of Ae. tauschii in response to ABA stress remains unclear. Here, we conducted a time-course strand-specific RNA-sequencing study to globally profile the transcriptome that responded to ABA treatment in Ae. tauschii. We identified 4818 differentially expressed transcription units/genes with time-point-specific induction/repression patterns. Using functional annotation, one-to-one ortholog and comparative transcriptome profiling analyses, we identified 319 ABA-responsive Ae. tauschii orthologs that were also induced/repressed under ABA treatment in hexaploid wheat. On the quantitative trait loci (QTL) used in wheat marker-assisted breeding, we found that the ABA-responsive expression patterns of eight Ae. tauschii orthologs were associated with drought stress tolerance, flowering process and/or grain quality. Of them, the ABA-responsive gene encoding sucrose:sucrose 1-fructosyltransferase in fructan and glucose metabolism pathways showed the most significant association with wheat drought tolerance. The characterization of ABA early-responsive genes in this study provides valuable information for exploring the molecular functions of the regulatory genes and will assist in wheat breeding.

Decreased expression of fructosyltransferase genes in asparagus roots may contribute to efficient fructan degradation during asparagus spear harvesting

Asparagus (Asparagus officinalis L.) accumulates inulin and inulin neoseries-type fructans in root, which are synthesized by three fructosyltransferases—sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan:fructan 6G-fructosyltransferase (6G-FFT, EC 2.4.1.243). Fructans in roots are considered as energy sources for emerging of spears, and it has been demonstrated that a gradual decrease in root fructan content occurs during the spear harvesting season (budding and shooting up period). However, the roles of certain three fructosyltransferases during the harvest season have not yet been elucidated. Here, we investigated the variation in enzymatic activities and gene expression levels of three fructosyltransferases and examined sugar contents in roots before and during the spear harvest period. Two cDNAs, aoft2 and aoft3, were isolated from the cDNA library of roots. The respective recombinant proteins (rAoFT2 and rAoFT3), produced by Pichia pastoris, were characterized: rAoFT2 showed 1-FFT activity (producing nystose from 1-kestose), whereas rAoFT3 showed 1-SST activity (producing 1-kestose from sucrose). These reaction profiles of recombinant proteins were similar to those of native enzymes purified previously. These results indicate that aoft2 and aoft3 encoding 1-FFT and 1-SST are involved in fructan synthesis in roots. A gradual downregulation of fructosyltransferase genes and activity of respective enzymes was observed in roots during the harvest period, which also coincided with the decrease in fructooligosaccharides and increase in fructose due to fructan exohydrolase activity. These findings suggest that downregulation of fructosyltransferases genes during harvest time may contribute to efficient degradation of fructan required for the emergence of spears.

Whole-Transcriptome Analysis Unveils the Synchronized Activities of Genes for Fructans in Developing Tubers of the Jerusalem Artichoke.

Helianthus tuberosus L., known as the Jerusalem artichoke, is a hexaploid plant species, adapted to low-nutrient soils, that accumulates high levels of inulin in its tubers. Inulin is a fructose-based polysaccharide used either as dietary fiber or for the production of bioethanol. Key enzymes involved in inulin biosynthesis are well known. However, the gene networks underpinning tuber development and inulin accumulation in H. tuberous remain elusive. To fill this gap, we selected 6,365 expressed sequence tags (ESTs) from an H. tuberosus library to set up a microarray platform and record their expression across three tuber developmental stages, when rhizomes start enlarging (T0), at maximum tuber elongation rate (T3), and at tuber physiological maturity (Tm), in “VR” and “K8-HS142”clones. The former was selected as an early tuberizing and the latter as a late-tuberizing clone. We quantified inulin and starch levels, and qRT-PCR confirmed the expression of critical genes accounting for inulin biosynthesis. The microarray analysis revealed that the differences in morphological and physiological traits between tubers of the two clones are genetically determined since T0 and that is relatively low the number of differentially expressed ESTs across the stages shared between the clones (93). The expression of ESTs for sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT), the two critical genes for fructans polymerization, resulted to be temporarily synchronized and mirror the progress of inulin accumulation and stretching. The expression of ESTs for starch biosynthesis was insignificant throughout the developmental stages of the clones in line with the negligible level of starch into their mature tubers, where inulin was the dominant polysaccharide. Overall, our study disclosed candidate genes underpinning the development and storage of carbohydrates in the tubers of two H. tuberosus clones. A model according to which the steady-state levels of 1-SST and 1-FFT transcripts are developmentally controlled and might represent a limiting factor for inulin accumulation has been provided. Our finding may have significant repercussions for breeding clones with improved levels of inulin for food and chemical industry.

Primer Design for Isolation of Sucrose:Sucrose 1-Fructosyltransferase (1-SST) Gene from Gembili (Dioscorea esculenta)

Recent studies have reported the presence of inulin, a prebiotic polysaccharide, in gembili (Dioscorea esculenta). Sucrose:sucrose 1-fructosyltransferase (1-SST) is an enzyme that catalyzes the first step of inulin biosynthesis. The identification of this enzyme would be the foundation to improve the yield of inulin in gembili; to modify 1-SST gene for this purpose, its sequence must first be determined. This study aimed to design primers to isolate 1-SST gene from D. esculenta. The primers were designed by using the whole-genome sequence of Dioscorea rotundata due to the lack of genomic information on D. esculenta. Sequences from chromosome 6 and 11 were used as the template of primer design in which four pairs of primers were selected. Amplification products with expected size were gel-purified, then the targets were sequenced and analyzed in-silico. As a result, one of the primer pairs had successfully isolated vacuolar invertase gene, which is closely related to 1-SST gene. On the other hand, the other primer pairs showed either negative or false-positive result. Using the current strategy, 1-SST gene sequence from D. esculenta has not been successfully isolated, thus other approaches in primer design should be considered in further studies.
 Keywords: 1-SST, Dioscorea esculenta, Gembili, Inulin, Primer design

Expression of Jerusalem artichoke (Helianthus tuberosus L.) fructosyltransferases, and high fructan accumulation in potato tubers

Fructans are polymers of fructose that are present as storage carbohydrates in various plants. Jerusalem artichoke (Helianthus tuberosus L.) contains a high amount of inulin. Two enzymes are involved in inulin biosynthesis. The sucrose:sucrose 1-fructosyltransferase (1-SST) enzyme mainly catalyzes the synthesis of 1-kestose from sucrose. In the next step, fructan:fructan 1-fructosyltransferase (1-FFT) catalyzes the synthesis of inulin from 1-kestose. In this study, the Ht1-SST and Ht1-FFT genes were isolated from Jerusalem artichoke and expressed in potato (Solanum tuberosum L.), either separately or together, via Agrobacterium-mediated transformation. Transgenic potato tubers overexpressing Ht1-SST accumulated 1-kestose to a high level (up to 3.36 mg/g), while tubers overexpressing both Ht1-SST and Ht1-FFT accumulated up to 3.14 mg/g short-chain inulin-type fructans, with the degree of polymerization (DP) ranging from 3 to 5, excluding high DP inulins. Transgenic potato plants accumulated fructo-oligosaccharides to a high level, following the fructan biosynthetic pathway of Jerusalem artichoke, and therefore present a high potential for the mass production of inulin through established potato breeding and cultivation methods.

Label-free quantitative proteomic analysis determines changes in amino acid and carbohydrate metabolism in three cultivars of Jerusalem artichoke tubers

Jerusalem artichoke (JA) tubers are an important bio-economy developing crop because of its invaluable bioproducts in both food and biofuel aspects. However, the molecular mechanism of its tuberization, and the differences among different cultivars have been little studied to date. Therefore, here we selected PJA, DJA, and HJA cultivars of JA tubers, showing variations in their tuber epidermal pigmentation, underground tuberization, and inulin content. A comparative proteome analysis led to the identification of 402 proteins in the tubers of which 114 were significantly modulated among different cultivars. Gene Ontology (GO) analysis showed proteins related to the biosynthesis of amino acids and carbohydrate metabolism were differentially modulated in the tubers of three cultivars. Results from the inulin content measurement and proteome analysis suggest that Sucrose:sucrose 1-fructosyltransferase (1-SST) prioritizes inulin biosynthesis rather than rate-limiting enzyme fructan:fructan 1-fructosyltransferases (1-FFT). Furthermore, we confirmed the relationship between transcript-protein expression levels was in discord within inulin biosynthesis enzymes 1-SST and 1-FFT with the terms in previous RT-qPCR results using the same tubers. Our data represent the first report of comparative tuber proteome profiling of different JA and provide the metabolic and molecular basis for understanding carbohydrate metabolism in the tuber tissue.

Review and in silico analysis of fermentation, bioenergy, fiber, and biopolymer genes of biotechnological interest in Agave L. for genetic improvement and biocatalysis.

Several of the over 200 known species of Agave L. are currently used for production of distilled beverages and biopolymers. The plants live in a wide range of stressful environments as a result of their resistance to abiotic stress (drought, salinity, and extreme temperature) and pathogens, which gives the genus potential for germplasm conservation and biotechnological applications that may minimize economic losses as a result of the global climate change. However, the limited knowledge in the genus of genome structure and organization hampers development of potential improved biotechnological applications by means of genetic manipulation and biocatalysis. We reviewed Agave and plant sequences in the GenBank NCBI database for identifying genes with biotechnological potential for fermentation, bioenergy, fiber improvement, and in vivo plant biopolymer production. Three-dimensional modeling of enzyme structures in plant accessions revealed structural differences in sucrose 1-fructosyltransferase, fructan 1-fructosyltransferase, fructan exohydrolase (1-FEH), cellulose synthase (CES), and glucanases (EGases) with possible effects in fructan, sugar, and biopolymer production. Although the coding genes of FEH and enzymes involved in biopolymer production (CES, sucrose synthase, and EGases) remain unidentified in Agave L., our results could aid isolation of such genes in Agave. By comparing nucleotide and amino acid sequences in accessions of Agave and other plants, knowledge may be gained about transcriptional regulation and enzymatic activity factors. Future study is needed of biotechnological application of Agave genes for crop breeding aided by genetic engineering and biocatalysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1314-1334, 2018.

Accumulation of water-soluble carbohydrates and gene expression in wheat stems correlates with drought resistance

In order to understand the effects of sugar metabolism on drought resistance in wheat, two wheat cultivars with different levels of drought resistance were used in this study. We investigated the accumulation pattern of water-soluble carbohydrates (WSC) and expression profiles of twelve fructan metabolism-related genes in peduncle (PED), penultimate (PEN), and lower internode (LOW) stem tissues under drought stress. LH7, a higher drought-resistance cultivar, contained a higher stem dry weight and higher content of WSC in PED, PEN, and LOW tissues, while XN979, a lower drought-resistance cultivar, contained lower values. The tissues from LOW internodes had the highest WSC content, while PED had the lowest. The mRNA levels of genes encoding fructan synthesis-related enzymes, sucrose: sucrose 1-fructosyltransferase (1-SST), sucrose: fructan 6-fructosyltransferase (6-SFT), and fructan: fructan 1- fructosyltransferase (1-FFT) showed higher expression levels at early time points following stress, whilst the genes encoding degradation-related enzymes, fructan exohydrolases (1-FEH), and invertase (INV), showed higher expression at a later time point. Compared with XN979, LH7 showed higher expression levels of genes encoding fructan synthesis-related enzymes at all growth stages, whilst the expression of 1-FEH-W3, 6-FEH, and INV3 were higher at a later stage; these expression levels would benefit fructan accumulation and remobilization at early and later stages, respectively. Drought stress induced most of fructan metabolism related genes expression level decreasing in LH7 PED, but enhancing in LH7 LOW part at early time points following stress. The results confirm that there are complex, coordinated expression patterns of fructan synthesis- and degradation-related genes in stems under drought stress. In summary, 1–SST-A2, 6-SFT, 1-FFT-A, 1-FEH-W3, 6-FEH, and INV3 play important roles in fructan accumulation. In addition, higher expression of genes related to fructan synthesis and degradation occurs during early and later stages of drought stress, respectively, enhancing the drought resistance of wheat cultivar LH7.

Characterization of Fructan Metabolism During Jerusalem Artichoke (Helianthus tuberosus L.) Germination.

The inulin-type fructans in Jerusalem artichoke (Helianthus tuberosus L.) tubers exhibit different degrees of polymerization and are critical for germination. We aimed to characterize the sugar metabolism dynamics in the tubers without bud eyes or shoots (T) and BE/S of indoor- and field-grown Jerusalem artichokes during germination. Ht1-FEH II and Ht1-FEH III (1-fructan exohydrolases II and III, inulin-degrading enzymes) expression increased 5 days after planting indoors, whereas Ht1-FEH II expression increased 72 days after planting in the field in T and BE/S. Ht1-SST (sucrose:sucrose 1-fructosyl transferase, inulin synthesis initiator), and Ht1-FFT (fructan:fructan 1-fructosyl transferase, inulin elongator) expression generally decreased in indoor-grown T. The enzyme activities of 1-FEH and 1-FFT were unchanged during germination in both indoor- and field-grown T and BE/S, whereas 1-SST activity decreased in indoor-grown T, while 1-FEH and 1-FFT activities increased as a function of germination time in BE/S of both indoor- and field-grown tubers. The total soluble sugar content gradually decreased in T after germination indoors or in the field, while at the end of germination, the sucrose and fructan contents decreased, and fructose content increased in the field. The enzyme activities of soluble vacuolar (VI) or neutral invertase (NI) did not change significantly, except at the late germination stage. Sucrose synthase (SS) and sucrose-phosphate synthase (SPS) activities were not significantly changed in T and BE/S in indoor-grown artichokes, while SS activity gradually increased, and SPS activity gradually decreased in field-grown artichokes, alongside sucrose degradation. Compared to T, BE/S generally had higher enzyme activities of 1-FEH and 1-FFT, promoting inulin hydrolysis. This work shows that the process of tuber germination is similar indoors and in the field, and germination studies can therefore be conducted in either environment.

Fructan dynamics and antioxidant capacity of 4-day-old seedlings of wheat (Triticum aestivum) cultivars during drought stress and recovery.

One of the inevitable consequences of drought stress is enhanced production of reactive oxygen species (ROS). Fructan might function as effective candidate for capturing ROS in a wide range of stresses. Herein, 4-day-old seedlings of drought-tolerant and -sensitive wheat cultivars were exposed to drought stress for 7 days by water cessation, followed by further 7 days re-watering. The content, metabolism, related enzymes activity, degree of polymerisation (DP) and antioxidant capacity of fructan were compared in the two cultivars. High resolution HPAEC-PAD analysis of fructan showed an increase in the activities of fructan: fructan 1-fructosyltransferase (1-FFT) in the tolerant cultivar and sucrose: sucrose 1-fructosyltransferase (1-SST) and 1-FFT in the sensitive cultivar under drought condition. The activity of fructan exohydrolase (FEH) did not show any significant change in tolerant cultivar, but decreased in a sensitive one. In comparison with the sensitive cultivar, the tolerant one accumulated fructan (0.9% of dry matter) with higher degree of polymerisation (10.67±1.1), accompanied by increased OH radical scavenging activity, during drought condition. In regard to the fact that OH radical is the most prevalent ROS in damaging membrane lipids, the results suggest that fructans play a crucial role in the tolerance of wheat seedlings against drought stress.

Fructooligosaccharides production by Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase constitutively expressed to high levels in Pichia pastoris

The non-saccharolytic yeast Pichia pastoris was engineered to express constitutively the mature region of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Tall fescue (Schedonorus arundinaceus). The increase of the transgene dosage from one to nine copies enhanced 7.9-fold the recombinant enzyme (Sa1-SSTrec) yield without causing cell toxicity. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (38%) and extracellular release (62%) of Sa1-SSTrec to an overall activity of 102.1 U/ml when biomass reached (106 g/l, dry weight) in fed-batch fermentation using cane sugar for cell growth. The volumetric productivity of the nine-copy clone PGFT6x-308 at the end of fermentation (72 h) was 1422.2 U/l/h. Sa1-SSTrec purified from the culture supernatant was a monomeric glycoprotein optimally active at pH 5.0–6.0 and 45–50 °C. The removal of N-linked oligosaccharides by Endo Hf treatment decreased the enzyme stability but had no effect on the substrate and product specificities. Sa1-SSTrec converted sucrose (600 g/l) into 1-kestose (GF2) and nystose (GF3) in a ratio 9:1 with their sum representing 55–60% (w/w) of the total carbohydrates in the reaction mixture. Variations in the sucrose (100–800 g/l) or enzyme (1.5–15 units per gram of substrate) concentrations kept unaltered the product profile. Sa1-SSTrec is an attractive candidate enzyme for the industrial production of short-chain fructooligosaccharides, most particularly 1-kestose.