Sweet Protein Research Articles

Effects of high hydrostatic pressure and microbial transglutaminase treatment on structure and gelation properties of sweet potato protein

The effects of high hydrostatic pressure (HHP) combined with microbial transglutaminase (MTGase) on the structure and gelation properties of sweet potato protein (SPP) were investigated. MTGase induced the formation of high molecular mass aggregates of SPP by the inter-molecular cross-linking. The average particle size of SPP increased by HHP at 250–550 MPa, being increased from 249.5 nm (0.1 MPa) to 270.2 nm (550 MPa). While it was decreased by the subsequent MTGase catalysis that showed the lowest value of 200.3 nm at 550 MPa, indicating the formation of intra-molecular linkages. The amount of sulfhydryl groups and thermal denaturation temperature were increased by HHP and MTGase, with the highest value of 3.5 μmol/g protein and 80.4 °C in MTGase catalyzed SPP with 400 MPa pretreatment. Textural properties of the gels made from HHP treated SPP or those with further MTGase catalysis were both enhanced due to the covalent linkage of disulfide bonds (-S-S-) and ε-(γ-glutaminyl) lysine isopeptide bonds. In addition, low-field NMR results showed an increase in corresponding area fractions of A2b and A21, being attributed to more bound or immobilized water fractions were trapped in the SPP gel matrix.

Use of acid whey protein concentrate as an ingredient in nonfat cup set-style yogurt

Acid whey resulting from the production of soft cheeses is a disposal problem for the dairy industry. Few uses have been found for acid whey because of its high ash content, low pH, and high organic acid content. The objective of this study was to explore the potential of recovery of whey protein from cottage cheese acid whey for use in yogurt. Cottage cheese acid whey and Cheddar cheese whey were produced from standard cottage cheese and Cheddar cheese-making procedures, respectively. The whey was separated and pasteurized by high temperature, short time pasteurization and stored at 4°C. Food-grade ammonium hydroxide was used to neutralize the acid whey to a pH of 6.4. The whey was heated to 50°C and concentrated using ultrafiltration and diafiltration with 11 polyethersulfone cartridge membrane filters (10,000-kDa cutoff) to 25% total solids and 80% protein. Skim milk was concentrated to 6% total protein. Nonfat, unflavored set-style yogurts (6.0 ± 0.1% protein, 15 ± 1.0% solids) were made from skim milk with added acid whey protein concentrate, skim milk with added sweet whey protein concentrate, or skim milk concentrate. Yogurt mixes were standardized to lactose and fat of 6.50% and 0.10%, respectively. Yogurt was fermented at 43°C to pH 4.6 and stored at 4°C. The experiment was replicated in triplicate. Titratable acidity, pH, whey separation, color, and gel strength were measured weekly in yogurts through 8 wk. Trained panel profiling was conducted on 0, 14, 28, and 56 d. Fat-free yogurts produced with added neutralized fresh liquid acid whey protein concentrate had flavor attributes similar those with added fresh liquid sweet whey protein but had lower gel strength attributes, which translated to differences in trained panel texture attributes and lower consumer liking scores for fat-free yogurt made with added acid whey protein ingredient. Difference in pH was the main contributor to texture differences, as higher pH in acid whey protein yogurts changed gel structure formation and water-holding capacity of the yogurt gel. In a second part of the study, the yogurt mix was reformulated to address texture differences. The reformulated yogurt mix at 2% milkfat and using a lower level of sweet and acid whey ingredient performed at parity with control yogurts in consumer sensory trials. Fresh liquid acid whey protein concentrates from cottage cheese manufacture can be used as a liquid protein ingredient source for manufacture of yogurt in the same factory.

Design and development of a high temperature stable sweet protein base on monellin

Abstract Monellin, one kind of most sweet proteins, could be used instead of carbohydrates in the diabetic diet and pharmaceuticals. However, the critical problem interferes with monellin usage in the food or pharmaceutical industry was its unstable at high temperatures. Here, we describe a novel method to increase the thermostability of monellin. Inspired by the high Tm value in RNA hairpin containing structure, the beta-hairpin was added between the two polypeptide chains of monellin instead of Gly-Phe dipeptide linker. Three kinds of beta-hairpin libraries were constructed and selected. Remarkably, the Tm has been increased from 54.7℃ of monellin with Gly-Phe dipeptide linker to 79.5℃, 89.1℃ and 89.4℃ of selected three mutants. And combined with E24Q/Y80R mutation, the mutated E24Q/Y80R hairpin monellin Tm was as high as 96.1℃. It was soluble even heated in boiling water for 10 min, and sweetness recovered after cooling. This new hairpin monellin may show remarkable potential for further sweeteners.

Integrative analysis reveals evolutionary patterns and potential functions of SWEET transporters in Euphorbiaceae

As a macromolecular substance, sucrose contributes to the plant growth and development. SWEET genes, a group of sugar transporters, are a recently found plant gene family and play important roles in sugar efflux, pollen nutrition, nectar secretion, phloem transport, and seed development. The SWEET genes have been identified and characterized in some plants, but the systematic study in tung tree (Vernicia fordii) was limited. Here, we identified 121 SWEETs in five Euphorbiaceae, and could be divided into four classes with 20 different motifs. Multiple sequence alignment revealed seven transmembrane helixes (TMHs) in the SWEET proteins which were created by an internal duplication of an ancestral three-TMHs unit, connected by TMH4. This study provides direct evidence for the first time for internal duplication in Euphorbiaceae. The large-scale duplication events represented the main driving force for SWEET family expansion in Euphorbiaceae. In addition, we determined the key VfSWEETs for sucrose transport from source to sink tissues in V. fordii and proposed a possible sucrose transport model, which would be helpful for understanding the mechanism of sucrose transport in V. fordii. This study provided a new insight into the evolution, expression and structural variations of SWEETs in V. fordii and four other Euphorbiaceae.

Isolation and physicochemical characterization of starches from three varieties of Ethiopian sweet potato [<i>Ipomoea batatas</i> (L.) Lam.

Starch is a polysaccharide that finds versatile applications in pharmaceutical, food and other industries. Despite the presence of different varieties of sweet potatoes in Ethiopia, the physicochemical properties of their starches have not yet been studied. The aim of this study was therefore to isolate and characterize starches from three varieties of Ethiopian sweet potatoes [Ipomoea batatas (L.) Lam.] - Awassa83, Kulfo and Tulla. The starches were isolated using 0.075% (w/v) sodium metabisulphite solution. The proximate composition of the sweet potato starches, namely, amylose, protein, lipid, and fiber content were in the range of 11.13 - 18.15, 0.05 - 0.22%, 0.16 - 0.41% and 0.37 - 0.43%, respectively. The moisture content of the starches varied from 12.41 to 13.72%. Scanning electron microscopy of the starch granules showed polygonal, round and cupoliform/bell shaped granules. The average particle size of Awassa83 starch granules was 20.9 µm while Kulfo and Tulla exhibited similar average particle size of 12.1 µm. The swelling power and solubility of the starches ranged from 14.5 g/g to 21.7 g/g and 3.05% to 9.8%, respectively. X-ray diffraction pattern of the starches exhibited C-type with maximum characteristic peaks at 15o, 17o, and 23o2θ. DSC studies provided gelatinization temperatures, i.e., onset temperature (To:61.82 - 65.27 oC), peak temperature (Tp:65.60 - 69.09 oC), and end set temperature (Te: 72.13 - 73.69 oC); The enthalpy of gelatinization ranged from 5.45 mJ/mg to 6.85 mJ/mg). Variations in pasting characteristics were observed among the starches comprising peak viscosity (787 mPa.s to 873 mPa.s), hot paste viscosity (652 mPa.s to 723 mPa.s), breakdown viscosity (135 mPa.s to 158 mPa.s), cold paste viscosity (898 mPa.s to 997 mPa.s), and setback viscosity (111 mPa.s to 127 mPa.s). The peak time ranged from 4 min to 4.53 min and pasting temp. from 71.85 oC to 74.3 oC. Awassa83 starch exhibited relatively high peak and breakdown viscosities and low setback viscosity, and it may be suitable as a thickening and gelling agent.Keywords: Ipomoea batatas, Sweet potato starch, Awassa83, Kulfo, Tulla

SWEET Uniporter Gene Family Expression Profile in the Pitcher Development in the Carnivorous Plant Nepenthes sp.

Transcriptome analysis of leaf and pitcher at different developmental stages in the carnivorous plant Nepenthes sp. identified 20 cDNAs of the SWEET gene family encoding sugar uniporters of classes I–IV. The structure of NSWEET proteins generally corresponded to the 3-1-3 scheme typical of SWEET proteins in eukaryotes. The variability in the expression of NSWEET genes in the mature leaf and in the three stages of pitcher development indicates the possible functional diversity of these genes. It has been suggested that class I transporters (NSWEET2d, 2f, and 2h) may participate in export of sugars from the leaf to the site of pitcher meristem initiation, while proteins NSWEET1, 2a, 2k (class I), 4a, 4b, 4d (II), and 12c (III) may participate in the delivery of sugars for the primary development of the pitcher. In subsequent stages, they can be replaced by NSWEET2b, 2c, 2e, 2i, 2j (class I), 4c (II), and 12b (III), delivering hexoses and sucrose to the growing pitcher. In the fully formed pitcher, proteins NSWEET12a (III), 2g (I), and 16 (IV) can export sugars from the digestive fluid to the leaf.

Identification of monellin as the first naturally derived proteinaceous allosteric agonist of metabotropic glutamate receptor 5.

Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of "selective" allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu5 ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu5 . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu5 demonstrated that monellin agonism was not mediated via the "common" allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu5 . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu5 PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu5 agonism was positively modulated by the mGlu5 pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu5 , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.

Temporal sweetness profile of the emerging sweetener MNEI in stirred yogurt

AbstractYogurt is widely consumed by people under‐controlled dietary regimes. The addition of hypocaloric sweeteners to the yogurt is typically used to make them more palatable, but this may affect the fermentation phase and/or its physical and sensory characteristics. The aim of this work was to test the possible application of an emerging sweetener, MNEI, a variant of a sweet protein, monellin. In particular, its sweetness profile was compared with that of other natural sweeteners, sucrose, xylitol, and sorbitol, in yogurt. First, determinations of iso‐sweet concentration of MNEI and of those sweeteners were performed, both for set and stirred yogurts. Then, the sweetness temporal profiles of all the sweeteners were evaluated as the mean of time–intensity data (t–I). In set yogurt, the iso‐sweet concentrations of xylitol and sorbitol, but not of MNEI, were found, whereas, in stirred yogurts, the concentrations were determined for all the sweeteners. t–I results showed that sweeteners had similar behaviors, except for the end time of sweetness perception: in particular, in stirred yogurt, MNEI resulted in a more lingering sweet aftertaste. These results suggest new applications of the sweet protein MNEI in food preparations, although the combination with other natural sweeteners could be explored.Practical applicationsYogurts are ideal products to develop potential applications of MNEI, a protein that has been demonstrated stable to acidic environments, and to our knowledge, this is the first report of the use of a sweet protein to sweeten viscous food preparations. Preliminary experimental results suggest new possible applications of sweet protein MNEI in food samples. In particular, it could be used to sweeten‐stirred yogurt.

The Effect of the Taste Receptor Protein T1R3 on the Development of Islet Tissue of the Murine Pancreas.

T1R3 protein, the main subunit of the sweet taste receptor and receptor of amino acid taste, is expressed in the epithelium of the tongue and gastrointestinal tract, in β cells of the pancreas, hypothalamus, and numerous other organs. Recently, convincing evidences on the involvement of T1R3 in the control of carbohydrate and lipid metabolism, and the control of incretin and insulin production were obtained. In the study on Tas1r3-gene knockout mouse strain and parent C57BL/6J strain as a control, the data on the effect of T1R3 on morphological characteristics of Langerhans islets in the pancreas were obtained. In Tas1r3 knockout animals, we found a reduction in the size of islets and their density in pancreatic tissue as compared to the parent strain. In addition, a decrease in the expression of active caspase-3 in the islets of gene-knockout mice was demonstrated. The data obtained indicate that the lack of functioning gene encoding sweet taste receptor protein causes a dystrophy of the islet tissue and is associated with the development of pathological changes in the pancreas specific to type 2 diabetes mellitus and obesity in humans.

Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives.

There is currently a worldwide trend to reduce sugar consumption. This trend is mostly met by the use of artificial non-nutritive sweeteners. However, these sweeteners have also been proven to have adverse health effects such as dizziness, headaches, gastrointestinal issues, and mood changes for aspartame. One of the solutions lies in the commercialization of sweet proteins, which are not associated with adverse health effects. Of these proteins, thaumatin is one of the most studied and most promising alternatives for sugars and artificial sweeteners. Since the natural production of these proteins is often too expensive, biochemical production methods are currently under investigation. With these methods, recombinant DNA technology is used for the production of sweet proteins in a host organism. The most promising host known today is the methylotrophic yeast, Pichia pastoris. This yeast has a tightly regulated methanol-induced promotor, allowing a good control over the recombinant protein production. Great efforts have been undertaken for improving the yields and purities of thaumatin productions, but a further optimization is still desired. This review focuses on (i) the motivation for using and producing sweet proteins, (ii) the properties and history of thaumatin, (iii) the production of recombinant sweet proteins, and (iv) future possibilities for process optimization based on a systems biology approach.

Production and In Vitro Gastrointestinal Digestion of Antioxidant Peptides from Enzymatic Hydrolysates of Sweet Potato Protein Affected by Pretreatment.

Effects of ultrasonication, boiling, steaming, microwaving and autoclaving pretreatments on the production of sweet potato protein hydrolysates (SPPH) by single and combined Alcalase (ALC) and Protease (PRO) were investigated, as well as antioxidant activities of SPPH subjected to in vitro gastrointestinal digestion (GID). All pretreatments significantly increased the degree of hydrolysis (DH) and antioxidant activities of SPPH by ALC, PRO and ALC + PRO in the order of autoclaving > steaming, microwaving, boiling > ultrasonication (P < 0.05). GID significantly enhanced antioxidant activities and increased MW <3kDa peptide fraction contents of all SPPH. Diverse peptides were identified as sporamin A, A precursor and sporamin B before and after GID from LC-QTOF-MS/MS analysis. Peptides with higher antioxidant amino acids of Trp, Tyr, Met, Cys, His and Phe were found after GID. There is a great potential application of SPPH as a novel food ingredient as a natural antioxidant.

Source-Sink Regulation Is Mediated by Interaction of an FT Homolog with a SWEET Protein in Potato

Potato plants form tuberous storage organs on underground modified stems called stolons. Tubers are rich in starch, proteins, and other important nutrients, making potato one of the most important staple food crops. The timing of tuber development in wild potato is regulated by day length through a mechanism that is closely related to floral transition [1, 2]. Tuberization is also known to be regulated by the availability of assimilates, in particular sucrose, the transported form of sugar, required for starch synthesis. During the onset of tuber development, the mode of sucrose unloading switches from apoplastic to symplastic [3]. Here, we show that this switch maybe mediated by the interaction between the tuberization-specific FT homolog StSP6A and the sucrose efflux transporter StSWEET11 [4]. The binding of StSP6A to StSWEET11 blocked the leakage of sucrose to the apoplast, and is therefore likely to promote symplastic sucrose transport. The direct physical interaction between StSWEET11 and StSP6A proteins represents a link between the sugar and photoperiodic pathways for the regulation of potato tuber formation. Our data suggest that a previously undiscovered function for the FT family of proteins extends their role as mobile signals to mediators of source-sink partitioning, opening the possibility for modifying source-sink interactions.

Further studies on sugar transporter (SWEET) genes in wheat (Triticum aestivum L.).

SWEET proteins represent one of the largest sugar transporter family in the plant kingdom and play crucial roles in plant development and stress responses. In the present study, a total of 108 TaSWEET genes distributed on all the 21 wheat chromosomes were identified using the latest whole genome sequence (as against 59 genes reported in an earlier report). These 108 genes included 14 of the 17 types reported in Arabidopsis and also included three novel types. Tandem duplications (22) and segmental duplications (5) played a significant role in the expansion of TaSWEET family. A number of cis-elements were also identified in the promoter regions of TaSWEET genes, indicating response of TaSWEET genes during development and also during biotic/abiotic stresses. The TaSWEET proteins carried 4-7 trans-membrane helices (TMHs) showing diversity in structure. Phylogenetic analysis using SWEET proteins of wheat and 8 other species gave four well-known clusters. Expression analysis involving both in silico and in planta indicated relatively higher expression of TaSWEET genes in water/heat sensitive and leaf rust resistant genotypes. The results provided insights into the functional role of TaSWEETs in biotic and abiotic stresses, which may further help in planning strategies to develop high yielding wheat varieties tolerant to environmental stresses.

Genome-wide characterization and expression profiling of SWEET genes in cabbage (Brassica oleracea var. capitata L.) reveal their roles in chilling and clubroot disease responses

BackgroundThe SWEET proteins are a group of sugar transporters that play a role in sugar efflux during a range of biological processes, including stress responses. However, there has been no comprehensive analysis of the SWEET family genes in Brassica oleracea (BoSWEET), and the evolutionary pattern, phylogenetic relationship, gene characteristics of BoSWEET genes and their expression patterns under biotic and abiotic stresses remain largely unexplored.ResultsA total of 30 BoSWEET genes were identified and divided into four clades in B. oleracea. Phylogenetic analysis of the BoSWEET proteins indicated that clade II formed first, followed by clade I, clade IV and clade III, successively. Clade III, the newest clade, shows signs of rapid expansion. The Ks values of the orthologous SWEET gene pairs between B. oleracea and Arabidopsis thaliana ranged from 0.30 to 0.45, which estimated that B. oleracea diverged from A. thaliana approximately 10 to 15 million years ago. Prediction of transmembrane regions showed that eight BoSWEET proteins contain one characteristic MtN3_slv domain, twenty-one contain two, and one has four. Quantitative reverse transcription-PCR (qRT-PCR) analysis revealed that five BoSWEET genes from clades III and IV exhibited reduced expression levels under chilling stress. Additionally, the expression levels of six BoSWEET genes were up-regulated in roots of a clubroot-susceptible cabbage cultivar (CS-JF1) at 7 days after inoculation with Plasmodiophora brassicae compared with uninoculated plants, indicating that these genes may play important roles in transporting sugars into sink roots associated with P. brassicae colonization in CS-JF1. Subcellular localization analysis of a subset of BoSWEET proteins indicated that they are localized in the plasma membrane.ConclusionsThis study provides important insights into the evolution of the SWEET gene family in B. oleracea and other species, and represents the first study to characterize phylogenetic relationship, gene structures and expression patterns of the BoSWEET genes. These findings provide new insights into the complex transcriptional regulation of BoSWEET genes, as well as potential candidate BoSWEET genes that promote sugar transport to enhance chilling tolerance and clubroot disease resistance in cabbage.

Improvement of thermal, microwave and ultrasonication pretreatment on the production of antioxidant peptides from sweet potato protein via in vitro gastrointestinal digestion

SummaryEffects of thermal (boiling, steaming and autoclaving), microwave and ultrasonication pretreatments on the production of sweet potato protein hydrolysates (SPPH) through in vitro gastrointestinal digestion (GID) were investigated. All pretreatments significantly increased the degree of hydrolysis (DH), antioxidant activities and molecular weight (MW) &lt;3 kDa peptide fractions contents of SPPH in the order of autoclaving &gt; microwave, steaming &gt; boiling &gt; ultrasonication (P &lt; 0.05). Correlation analysis between peptides content and antioxidant activity suggested that antioxidant activity of SPPH mainly attributed to MW &lt;3 kDa peptides. Diverse peptides ranged from 487.24 to 1477.74 Da with 7–13 amino acids were identified in the MW &lt;3 kDa peptides fraction with autoclaving pretreatment and matched sporamins A, A precursor and B sequences from LC–QTOF–MS/MS analysis. Conformational structures of nine peptides were predicted with well‐known antioxidant amino acids. There is a high potential for SPPH used as a functional supplement in food system.

Angiotensin-I Converting Enzyme Inhibitory Peptides from Sweet Sorghum Grain Protein: Optimisation of Hydrolysis Conditions and Hydrolysate Characterization

In order to utilize sweet sorghum grain protein (SSGP) for food applications, SSGP was hydrolyzed by alcalase to produce the hydrolysates with ACE-inhibitory activity. The Plackett-Burman design (PBD) was used to identify the important factors influencing the ACE-inhibitory activity of SSGP hydrolysates. The result of PBD indicated that hydrolysis temperature, pH and alcalase dosage had significant influence (P andlt; 0.05) on the ACE-inhibitory activity of SSGP hydrolysates. Response surface methodology (RSM) was applied to optimize the three significant factors. The optimum hydrolysis conditions obtained from RSM were as follows: hydrolysis temperature 56 and#176;C, pH 8.0, alcalase dosage 5200U/g. Under the optimum conditions, SSGP hydrolysates contained 24.3% 1–5 kDa (IC50=0.305 mg/ml) and 15.2% andlt;1 kDa (IC50=0.116 mg/ml) peptide fractions having potent in vitro ACE inhibitory activities. Moreover, the inhibition pattern against ACE investigated using Lineweaver–Burk plots revealed that the peptides with molecular weights above 5 kDa in the SSGP hydrolysates were non-competitive inhibitors with inhibition constants (Ki) between 1.098 and 1.171 mg/ml, while the peptides with molecular weights below 5 kDa in the hydrolysates were competitive inhibitors with Ki between 0.110 and 0.184 mg/ml. The results of this study suggest that SSGP may be considered as a source of functional ingredients for the prevention of hypertension.

Functional characterization of a reproductive tissue specific promoter fromEucalyptus camaldulensis

SWEET proteins are essential for the maintenance of nectar production, as well as seed and pollen development, in plants. A search within the Eucalyptus genome identified 52 putative genes belonging to the SWEET gene family based on sequence similarity. The expression of two of these genes, EcSWEET2 and EcSWEET5, was analyzed in vegetative and reproductive tissues of Eucalyptus camaldulensis. The expression of EcSWEET5 was specific to male reproductive tissues, and transcripts were detected only at certain stages of flower development. Tobacco Rattle Virus (TRV)-mediated suppression of EcSWEET5 resulted in a significant reduction in pollen germination percentage in Nicotiana benthamiana without adverse effect on vegetative growth. A promoter sequence 1 kb upstream of the start codon of EcSWEET5 contained many elements suggestive of pollen specificity of the promoter. This specificity was confirmed in transgenic tobacco lines harboring a GUS gene whose expression was controlled by the EcSWEET5 gene promoter. GUS expression was limited to pollen alone in transgenic tobacco as evidenced by histochemical staining. The expression of a cytotoxic gene, barnase under the control of the EcSWEET5 gene promoter, showed pollen ablation in transgenic tobacco with normal vegetative growth.

Stable expression and characterization of brazzein, thaumatin and miraculin genes related to sweet protein in transgenic lettuce

Stable expression and characterization of brazzein, thaumatin and miraculin genes related to sweet protein in transgenic lettuce

Structure basis of the improved sweetness and thermostability of a unique double-sites single-chain sweet-tasting protein monellin (MNEI) mutant

The sweet protein monellin has an intensely sweet potency but limited stability. We have identified a double-sites mutant (E2N/E23A) of the single-chain monellin (MNEI) with both improved sweetness (about 3-fold) and thermostability (10 °C). However, the structural basis of its superior properties remains elusive until now. Herein we report its crystal structure at a resolution 1.90 Å. Similar to the wild-type, E2N/E23A adopts a wedge-shaped structure consisting of a five-strand β-sheet partially “wrapped” around an α-helix. However, distinguishing parts were present in the loops region, including a remarkable conformation shift from β-strand to loop around residue R39. Molecular docking revealed the persistence of conserved protein-receptor interface and formation of new intermolecular ionic bonds in the E2N/E23A-receptor complex involving the taste-active residue R39 of the sweet protein, which could account for its significant improvement of sweetness. On the other hand, a rearrangement of intramolecular interaction network including the C-H … π bond between A23 and F89 that led to enhanced hydrophobicity in the protein core, could be correlated with its improved thermostability. Furthermore, two new sweeter mutants of MNEI were created. These findings highlight the critical roles of key sweetness determinant residue R39 and hydrophobicity at the protein core for the sweetness and thermostability of the protein, respectively, which thus provide a deeper insight for understanding the structure-function relationship of the sweet protein as well as guidance for rational design of this unique biomacromolecule.

Natural genetic variation for expression of a SWEET transporter among wild species of Solanum lycopersicum (tomato) determines the hexose composition of ripening tomato fruit.

The sugar content of Solanum lycopersicum (tomato) fruit is a primary determinant of taste and quality. Cultivated tomato fruit are characterized by near-equimolar levels of the hexoses glucose and fructose, derived from the hydrolysis of translocated sucrose. As fructose is perceived as approximately twice as sweet as glucose, increasing its concentration at the expense of glucose can improve tomato fruit taste. Introgressions of the FgrH allele from the wild species Solanum habrochaites (LA1777) into cultivated tomato increased the fructose-to-glucose ratio of the ripe fruit by reducing glucose levels and concomitantly increasing fructose levels. In order to identify the function of the Fgr gene, we combined a fine-mapping strategy with RNAseq differential expression analysis of near-isogenic tomato lines. The results indicated that a SWEET protein was strongly upregulated in the lines with a high fructose-to-glucose ratio. Overexpressing the SWEET protein in transgenic tomato plants dramatically reduced the glucose levels and increased the fructose:glucose ratio in the developing fruit, thereby proving the function of the protein. The SWEET protein was localized to the plasma membrane and expression of the SlFgr gene in a yeast line lacking native hexose transporters complemented growth with glucose, but not with fructose. These results indicate that the SlFgr gene encodes a plasma membrane-localized glucose efflux transporter of the SWEET family, the overexpression of which reduces glucose levels and may allow for increased fructose levels. This article identifies the function of the tomato Fgr gene as a SWEET transporter, the upregulation of which leads to a modified sugar accumulation pattern in the fleshy fruit. The results point to the potential of the inedible wild species to improve fruit sugar accumulation via sugar transport mechanisms.