Sweet-tasting Protein Thaumatin Research Articles

Gastric digestion of the sweet-tasting plant protein thaumatin releases bitter peptides that reduce H. pylori induced pro-inflammatory IL-17A release via the TAS2R16 bitter taste receptor

About half of the world's population is infected with the bacterium Helicobacter pylori. For colonization, the bacterium neutralizes the low gastric pH and recruits immune cells to the stomach. The immune cells secrete cytokines, i.e., the pro-inflammatory IL-17A, which directly or indirectly damage surface epithelial cells. Since (I) dietary proteins are known to be digested into bitter tasting peptides in the gastric lumen, and (II) bitter tasting compounds have been demonstrated to reduce the release of pro-inflammatory cytokines through functional involvement of bitter taste receptors (TAS2Rs), we hypothesized that the sweet-tasting plant protein thaumatin would be cleaved into anti-inflammatory bitter peptides during gastric digestion. Using immortalized human parietal cells (HGT-1 cells), we demonstrated a bitter taste receptor TAS2R16-dependent reduction of a H. pylori-evoked IL-17A release by up to 89.7 ± 21.9% (p ≤ 0.01). Functional involvement of TAS2R16 was demonstrated by the study of specific antagonists and siRNA knock-down experiments.

Effect of the consumption of brazzein and monellin, two recombinant sweet-tasting proteins, on rat gut microbiota.

Sweet-tasting proteins (SPs) are proteins of plant origin initially isolated from tropical fruits. They are thousands of times sweeter than sucrose and most artificial sweeteners. SPs are a class of proteins capable of causing a sweet taste sensation in humans when interacting with the T1R2/T1R3 receptor. SP thaumatin has already been introduced in the food industry in some countries. Other SPs, such as monellin and brazzein, are promising products. An important stage in researching SPs, in addition to confirming the absence of toxicity, mutagenicity, oncogenicity, and allergenic effects, is studying their influence on gut microbiota. In this paper we describe changes in the composition of rat gut microbiota after six months of consuming one of two recombinant SPs-brazzein or monellin. A full length 16S gene sequencing method was used for DNA library barcoding. The MaAsLin2 analysis results showed noticeable fluctuations in the relative abundances of Anaerocella delicata in brazzein-fed rat microbiota, and of Anaerutruncus rubiinfantis in monellin-fed rat microbiota, which, however, did not exceed the standard deviation. The sucrose-fed group was associated with an increase in the relative abundance of Faecalibaculum rodentium, which may contribute to obesity. Overall, prolonged consumption of the sweet proteins brazzein and monellin did not significantly change rat microbiota and did not result in the appearance of opportunistic microbiota. This provides additional evidence for the safety of these potential sweeteners.

Effect of Transgenesis on mRNA and miRNA Profiles in Cucumber Fruits Expressing Thaumatin II.

Transgenic plants are commonly used in breeding programs because of the various features that can be introduced. However, unintended effects caused by genetic transformation are still a topic of concern. This makes research on the nutritional safety of transgenic crop plants extremely interesting. Cucumber (Cucumis sativus L.) is a crop that is grown worldwide. The aim of this study was to identify and characterize differentially expressed genes and regulatory miRNAs in transgenic cucumber fruits that contain the thaumatin II gene, which encodes the sweet-tasting protein thaumatin II, by NGS sequencing. We compared the fruit transcriptomes and miRNomes of three transgenic cucumber lines with wild-type cucumber. In total, we found 47 differentially expressed genes between control and all three transgenic lines. We performed the bioinformatic functional analysis and gene ontology classification. We also identified 12 differentially regulated miRNAs, from which three can influence the two targets (assigned as DEGs) in one of the studied transgenic lines (line 224). We found that the transformation of cucumber with thaumatin II and expression of the transgene had minimal impact on gene expression and epigenetic regulation by miRNA, in the cucumber fruits.

A thaumatin-like protein of Ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic Arabidopsis.

Plant often responds to fungal pathogens by expressing a group of proteins known as pathogenesis-related proteins (PRs). The expression of PR is mediated through pathogen-induced signal-transduction pathways that are fine-tuned by phytohormones such as methyl jasmonate (MeJA). Here, we report functional characterization of an Ocimum basilicum PR5 family member (ObTLP1) that was identified from a MeJA-responsive expression sequence tag collection. ObTLP1 encodes a 226 amino acid polypeptide that showed sequence and structural similarities with a sweet-tasting protein thaumatin of Thaumatococcus danielli and also with a stress-responsive protein osmotin of Nicotiana tabacum. The expression of ObTLP1 in O. basilicum was found to be organ-preferential under unstressed condition, and responsive to biotic and abiotic stresses, and multiple phytohormone elicitations. Bacterially-expressed recombinant ObTLP1 inhibited mycelial growth of the phytopathogenic fungi, Scleretonia sclerotiorum and Botrytis cinerea; thereby, suggesting its antifungal activity. Ectopic expression of ObTLP1 in Arabidopsis led to enhanced tolerance to S. sclerotiorum and B. cinerea infections, and also to dehydration and salt stress. Moreover, induced expression of the defense marker genes suggested up-regulation of the defense-response pathways in ObTLP1-expressing Arabidopsis upon fungal challenge. Thus, ObTLP1 might be useful for providing tolerance to the fungal pathogens and abiotic stresses in crops.

Genetically Modified Crops Expressing 35S‐Thaumatin II Transgene: Sensory Properties and Food Safety Aspects

Abstract: Obesity and diabetes have led consumers to increasingly seek out products containing low‐calorie sweeteners. In this context, the intense sweeteners, especially plant proteins that interact with the taste receptors, are playing a growing role. Among the sweet‐tasting proteins thaumatin seems the most promising. Production of thaumatin by transgenic plants might be an interesting alternative to the extraction of this product from the natural source. Another possible application of the unusual properties of thaumatin is to produce transgenic plants containing a protein capable of inducing a sweet taste phenotype. The taste of 5 crop species, cucumber, pear, potato, strawberry, and tomato, was improved by expressing the thaumatin II gene under control of the 35S promoter. Only in the case of transgenic cucumber were sensory assessments carried out for several cultivation cycles. Also, processed fruits of transgenic cucumber, as well as fresh‐cut transgenic cucumber salads, were the subject of sensory analysis. Transgenic cucumber expression of the thaumatin II gene resulted not only in sweeter taste, in comparison with the control, but also in higher aroma acceptability. Therefore, the authors of this article have summarized sensory properties of transgenic crops expressing the thaumatin II gene, placing special emphasis on the sensory characteristics of transgenic cucumber. Consumers often perceive disparagingly food deriving from genetically modified (GM) plants, partly because of some concerns about unintended effects. To address such concerns, this article also summarizes the food safety evaluation of transgenic crops expressing the thaumatin II gene. Future research directions are suggested.

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50nM. Although the sweetness remains when thaumatin is heated at 80°C for 4h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0Å. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a Cα atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154–164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the β-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.

Production and secretion of recombinant thaumatin in tobacco hairy root cultures

Production of recombinant proteins in plant cell or organ cultures and their secretion into the plant cell culture medium simplify the purification procedure and increase protein yield. In this study, the sweet-tasting protein thaumatin I was expressed and successfully secreted from tobacco hairy root cultures. The presence of an ER signal peptide appears to be crucial for the secretion of thaumatin: without an ER signal peptide, no thaumatin was detectable in the spent medium, whereas inclusion of the ER signal peptide calreticulin fused to the N terminus of thaumatin led to the secretion of thaumatin into the spent medium of hairy root cultures at concentrations of up to 0.21 mg/L. Extracellular thaumatin levels reached a maximum after 30 days (stationary phase) and the subsequent decline was linked to the rapid increase of proteases in the medium. Significant amounts of thaumatin were trapped in the apoplastic space of the root cells. The addition of polyvinylpyrrolidone and sodium chloride into the culture medium led to an increase of extracellular thaumatin amounts up to 1.4 and 2.63 mg/L, respectively. Thaumatin production compares well with yields from other transgenic plants, so that tobacco hairy roots can be considered an alternative production platform of thaumatin.

Crystal structure of the sweet-tasting protein thaumatin II at 1.27 Å

Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 Å. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the Cα atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.

High-resolution structure of the recombinant sweet-tasting protein thaumatin I.

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å. The model was refined with anisotropic B parameters and riding H atoms. A comparison of the diffraction data and refinement statistics for recombinant thaumatin I with those for plant thaumatin I revealed no significant differences in the diffraction data. The R values for recombinant thaumatin I and plant thaumatin I (F(o) > 4σ) were 9.11% and 9.91%, respectively, indicating the final model to be of good quality. Notably, the electron-density maps around Asn46 and Ser63, which differ between thaumatin variants, were significantly improved. Furthermore, a number of H atoms became visible in an OMIT map and could be assigned. The high-quality structure of recombinant thaumatin with H atoms should provide details about sweetness determinants in thaumatin and provide valuable insights into the mechanism of its interaction with taste receptors.

High-yield Secretion of the Recombinant Sweet-Tasting Protein Thaumatin I

Thaumatin, an intensely sweet-tasting protein used as a sweetener, was secreted by the methylotrophic yeast Pichia pastoris. Approximately 100 mg L-1 of recombinant thaumatin I was obtained using an expression vector which possesses three copies of the thaumatin gene containing the 22-amino acid pre-sequence. Expression yield was about three-fold higher than when the α-factor secretion signal from Saccharomyces cerevisiae was used. The circular dichroism and tryptophan fluorescence spectra for recombinant thaumatin I were almost the same as those for plant thaumatin. Large amounts of homogeneous recombinant thaumatin allowed for preparation of high-quality crystals in the presence of cryoprotective glycerol used in high-resolution x-ray structural analysis to help further understand the perception of the sweet taste of thaumatin.

Interactions of the Sweet-Tasting Proteins Thaumatin and Lysozyme with the Human Sweet-Taste Receptor

This study investigated the sweetness of the sweet-tasting protein thaumatin and lysozyme by both an in vitro cell-based assay and an in vivo sensory analysis to elucidate the differences between in vitro and in vivo response profiles. Hek293 cells were constructed that stably expressed the human T1R2+T1R3 sweet-taste receptor, and their responses to thaumatin and lysozyme were analyzed by monitoring the levels of intracellular cAMP. The results indicated that thaumatin and lysozyme as well as aspartame induced a decrease in the intracellular cAMP accumulation of the T1R2+T1R3-transfected cells and that EC(50) values of thaumatin and lysozyme determined by cell-based assay are well-consistent with the results of the sweetness threshold value determined by sensory analysis in the presence of 140 mM NaCl. The results of both in vitro and in vivo experiments confirmed that the sweetness inhibitor lactisole significantly suppressed the sweetness of thaumatin and lysozyme.

Copper co-crystallization and divalent metal salts cross-influence effect: A new optimization tool improving crystal morphology and diffraction quality

The effect of several metal cations (Cu 2+, Cd 2+, Co 2+, Ba 2+) was tested in attempts to improve crystallization and verify a newly discovered cross-crystallization alias cross-influence procedure (CIP) with two selected proteins; di-heme cytochrome c 4 from anaerobic purple sulphur bacterium Thiocapsa roseopersicina and sweet-tasting protein thaumatin from the African berry Thaumatococcus daniellii. Divalent cupric cations (Cu 2+) promoted the most dramatic improvement in crystal morphology, internal packing and diffraction quality. This investigation qualitatively established the influence of cupric cations on the crystal growth with use of CIP. The CIP procedure is based on using a set of additives included in additional droplets, effecting the same reservoir through slightly varying pH and vapour pressure of water over the neighbouring drop containing protein. It was found that influence of Cu 2+ ions produced evidently different outer morphology and internal packing of thaumatin crystals (hexagonal prism). Usually their shape is presented as a tetragonal bipyramids. In the case of cytochrome, the good diffractable crystals were obtained only by using CIP with metal-ion salts. Newly grown crystals (hexagonal prisms) of thaumatin and cytochrome displayed as the same tetragonal system with different unit cell parameters and diffracted up to 1.7 Å. From the analysis of copper influence and CIP effect, we propose that the copper addition with CIP provides useful technique to modify crystal morphology and improve diffraction quality.

Purification of an osmotin-like protein from the seeds of Benincasa hispida and cloning of the gene encoding this protein

A pathogenesis-related (PR) protein was purified from the seeds of Benincasa hispida, which is a medicinal plant and a member of the Cucurbitaceae family. Purification was achieved by using a procedure consisting of an acid treatment step followed by two chromatography steps. The protein is a basic protein with molecular mass of ∼28 kDa. The sequences of the N-terminal 30 amino acids and four peptides generated from protease digestion were determined. These sequences indicated that the protein is an osmotin-like protein (OLP). Osmotin and OLPs are members of the thaumatin-like, PR-5 family of the PR proteins. A genomic clone of the gene encoding the protein was isolated and sequenced. The predicted protein has a signal peptide of 18 amino acids, and the mature protein has a molecular mass of 24.8 kDa with an isoelectric point of 7.67. The protein has 17 cysteine residues, of which 16 appear in the same positions as those appear in the sweet-tasting protein thaumatin and several other thaumatin-like proteins. Southern hybridization analysis indicated that the gene encoding the protein is a single copy gene. A computer-generated, three-dimensional model of the protein is presented.

Overexpression and lack of degradation of thaumatin in an aspergillopepsin A-defective mutant of Aspergillus awamori containing an insertion in the pepA gene.

A gene encoding the sweet-tasting protein thaumatin (tha) with optimized codon usage was expressed in Aspergillus awamori. Mutants of A. awamori with reduced proteolytic activity were isolated. One of these mutants, named lpr66, contained an insertion of about 200 bp in the pepA gene, resulting in an inactive aspergillopepsin A. In vitro thaumatin degradation tests confirmed that culture broths of mutant lpr66 showed only a small thaumatin-degrading activity. A. awamori lpr66 has been used as host strain for thaumatin expression cassettes containing the tha gene under the control of either the cahB (cephalosporin acetylhydrolase) promoter of Acremonium chrysogenum or the gdhA (glutamate dehydrogenase) promoter of Aspergillus awamori. Residual proteolytic activities were repressed by using a mixture of glucose and sucrose as carbon sources and L-asparagine as nitrogen source. Degradation of thaumatin by acidic proteases was prevented by maintaining the pH value at 6.2 in the fermentor. Expression of cassettes containing the gdhA promoter was optimal in ammonium sulfate as nitrogen source, whereas transformants expressing the tha gene from the cahB promoter yielded higher thaumatin levels using L-asparagine as nitrogen source. Under optimal fermentation conditions, yields of 105 mg thaumatin/l were obtained, thus making this fermentation a process of industrial interest.

Refolding the sweet-tasting protein thaumatin II from insoluble inclusion bodies synthesised in Escherichia coli

A synthetic gene encoding the amino acid sequence of the strongly sweet-tasting protein thaumatin II has been expressed in Escherichia coli. The recombinant protein has been renatured from inclusion bodies using a reduced/oxidized glutathione system to yield a purified protein preparation that is indistinguishable from native thaumatin with respect to its biochemical, spectroscopic and organoleptic properties.

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori.

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5-7 mg/l. This recombinant thaumatin is sweet.

Expression of a synthetic gene encoding the sweet-tasting protein thaumatin in the filamentous fungus Penicillium roquefortii

A synthetic gene encoding the amino acid sequence of the sweet-tasting protein thaumatin II has been expressed in Penicillium roquefortii. Using several different expression cassettes thaumatin was expressed either intracellularly or extracellularly, at concentrations of 1–2 mg thaumatin/l.

Expression of a Synthetic Gene Encoding the Sweet-Tasting Protein Thaumatin inEscherichia coli

A synthetic gene encoding the aminoacid sequence of the sweet-tasting protein thaumatin II has been assembled and expressed inEscherichia coli.Immunoblotting analysis shows that the expressed recombinant thaumatin has the same molecular weight as the protein from its natural source, the plantThaumatoccocus daniellii Benth.

Possible active site of the sweet-tasting protein thaumatin.

Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

Structures of three crystal forms of the sweet protein thaumatin.

Three crystal forms of the sweet-tasting protein thaumatin from the African berry Thaumatococcus daniellii have been grown. These include two naturally occurring isoforms, A and B, that differ by a single amino acid, and a recombinant form of isoform B expressed in yeast. The crystals are of space groups C2 with a = 117.7, b = 44.9, c = 38.0 A, and beta = 94.0 degrees, P2(1)2(1)2(1) with a = 44.3, b = 63.7 and c = 72.7 A, and a tetragonal form P4(1)2(1)2 with a = b = 58.6 and c = 151.8 A. The structures of all three crystals have been solved by molecular replacement and subsequently refined to R factors of 0.184 for the monoclinic at 2.6 A, 0.165 for the orthorhombic at 1.75 A, and 0.181 for the tetragonal, also at 1.75 A resolution. No solvent was included in the monoclinic crystal while 123 and 105 water molecules were included in the higher resolution orthorhombic and tetragonal structures, respectively. A bound tartrate molecule was also clearly visible in the tetragonal structure. The r.m.s. deviations between molecular structures in the three crystals range from 0.6 to 0.7 A for Calpha atoms, and 1.1 to 1.3 A for all atoms. This is comparable to the r.m.s. deviation between the three structures and the starting model. Nevertheless, several peptide loops show particularly large variations from the initial model.