Recent developments in the characterization and biotechnological production of sweet-tasting proteins

 The state of the art regarding the six known sweet-tasting proteins (thaumatin, monellin, mabinlin, pentadin, brazzein and curculin) and the taste-modifying protein miraculin is reviewed. Their biochemical properties, molecular genetics and biotechnological production are assessed. All of these proteins have been isolated from plants that grow in tropical rainforests. They share no sequence homology or structural similarities. Nonetheless, one of them, thaumatin, shares extensive homology with certain non-sweet proteins found in other plants. The potential industrial applications of the sweet-tasting proteins are also discussed, placing special emphasis on the barriers that a recombinant product of these characteristics will have to overcome before it reaches the market. Received: 14 June 1999 / Received revision: 30 August 1999 / Accepted: 3 September 1999     

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.     

The complete amino-acid sequence of the sweet protein thaumatin I.

The primary structure of the sweet-tasting protein thaumatin has been elucidated. The protein consists of a single polypeptide chain of 207 residues. The sequence of the N-terminal part of the chain was determined by sequenator analysis. As the protein contains only one methionine residue, it was possible to deduce the N-terminal sequence of the C-terminal cyanogen bromide fragment by automatic sequencing of the cyanogen-bromide-cleaved, succinylated protein. To arrive at the sequence of the whole protein tryptic and Staphylococcus protease peptides, together with chymotryptic peptides and a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole) fragment were also sequenced. Comparing the amino acid sequence of thaumatin with that of the other sweet-tasting protein, monellin, we have located five sets of identical tripeptides. Since immunological cross-reactivity of thaumatin antibodies with monellin has recently been described, one or more of these tripeptides might be part of a common antibody recombination site and possibly be involved in the interaction with the sweet-taste receptor.     

Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.     

Microbial synthesis of the sweet-tasting plant protein thaumatin

Thaumatin, the intensely sweet-tasting protein from the African plant, Thaumatococcus daniellii, is a potential low-caloric substitute for sugar and an attractive compound for studying the structural aspects of sweetness perception. Since thaumatin has other properties, such as flavour enhancement, it could also be used in products other than low-calorie foods. Recombinant DNA techniques could be used to synthesize this protein in quantities sufficient for such applications.     

Complete amino Acid sequence of soybean leaf p21 : similarity to the thaumatin-like polypeptides

A polypeptide structurally related to the thaumatin family of proteins has been purified from soybean (Glycine max) leaves and the complete amino acid sequence has been determined. The mature protein, which we have termed P21, has a calculated molecular weight of 21,461 and an isoelectric point of 4.6. The soybean protein shows 64% amino acid identity with thaumatin, a sweet-tasting protein found in the West African shrub Thaumatococcus danielli, and as much as 71% identity with thaumatin-like polypeptides present in tobacco and maize.     

The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin

Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50 nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2–T1R3. Chimeric human– mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2– mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners.     

Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris

Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.     

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.     

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

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 ?. 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.     

Cloning of the thaumatin I cDNA and characterization of recombinant thaumatin I secreted by Pichia pastoris

Thaumatin is a sweet-tasting protein comprising a mixture of some variants. The major variants are thaumatins I and II. Although the amino acid sequence of thaumatin I was known and the nucleotide sequence of cDNA of thaumatin II was elucidated, the nucleotide sequence of thaumatin I has been controversial. We have cloned two thaumatin cDNAs from the fruit of Thaumatococcus daniellii Benth. One is the same nucleotide sequence as that of thaumatin II already reported, and the other is a novel nucleotide sequence. The amino acid sequence deduced from the novel cDNA was the same amino acid sequence as that of thaumatin I, the only exception being the residue at position 113 (Asp instead of Asn), indicating that the novel thaumatin cDNA is that for thaumatin I. This thaumatin I cDNA was transformed into Pichia pastoris X-33, and the recombinant thaumatin I expressed was purified and characterized. The threshold value of sweetness of the recombinant thaumatin I was the same as that of the plant thaumatin I, although several unexpected amino acid residues were attached to the N-terminal of the recombinant thaumatin I. These indicate that the N-terminal portion of thaumatin is not critical for the elicitation of sweetness.     

Assignment of the disulphide bonds in the sweet-tasting protein thaumatin I

The disulphide linkages of the 16 half-cystine residues in the sweet-tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high-performance liquid chromatography and peptide mapping by paper high-voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9-204, 56-66, 71-77, 121-193, 126-177, 134-149, 145-158 and 159-164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys-145 and Cys-158.     

Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris

Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris. The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro alpha-mating factor secretion signal. Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule. The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule. Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II. These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin.     

Photoaffinity labeling of thaumatin-binding protein in monkey circumvallate papillae

Thaumatin I is an intensely sweet-tasting protein. It was photo-crosslinked with taste papillae of crab-eating monkey by using a conjugated photo-affinity reagent [3H]azidobenzoylthaumatin I. Serial sections of SDS-polyacrylamide gel electrophoresis of the 0.1 M sodium phosphate buffer-soluble fraction from taste papillae had a large peak of radioactivity at the Mr region of approx. 70,000; fractions from non-taste papillae did not. Excess unlabeled thaumatin I reduced the photo-crosslinking at the 70 kDa region; acetylated thaumatin I (which is not sweet) did not. The results show that taste papillae of the monkey contain a protein of Mr approx. 50,000, which binds to thaumatin I (Mr 22,209) but not to completely acetylated thaumatin I. The possibility that the thaumatin-binding protein is a sweet receptor protein is discussed.     

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. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.     

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.     

Introduction of a negative charge at Arg82 in thaumatin abolished responses to human T1R2-T1R3 sweet receptors

Thaumatin, an intensely sweet-tasting protein, elicits a sweet-taste sensation at a level as low as 50 nM. Although previous sensory analyses have suggested that Lys67 and Arg82 are important to the sweetness of thaumatin, the exact effects of each residue on sweet receptors are still unknown. In the present study, various mutants of thaumatin altered at Arg82 as well as Lys67 were prepared and their sweetness levels were quantitatively evaluated by cell-based assays using HEK293 cells expressing human sweet receptors. Mutations at Arg82 had a more deteriorative effect on sweetness than mutations at Lys67. Particularly, a charge inversion at Arg82 (R82E) resulted in an abolishment of the response to sweet receptors even at a concentration as high as 1 mM. These results indicate that Arg82 plays a central role in determining the sweetness of thaumatin. A strict spatial charge location at residue 82 appears to be required for interaction with sweet receptors.     

Structural investigation of the sweet-tasting proteins thaumatin and monellin by immunological studies

Rabbit antibodies were produced against thaumatin I, a sweet-tastingprotein from plant origin using the technique of double diffusionin agar. Cross-reactions were observed between these antibodiesand thaumatin I, monellin and chemically modified thaumatins.No cross-reaction was observed between the antibodies of thaumatinI and the not sweet-tasting iodinated monellin. This lack ofcross-reaction may be due to the fact that iodination splitsmonellin into its A and B chain, resulting in a disturbanceof the tertiary structure of the molecule. The appearance ofprecipitation lines from thaumatin I as well as from monellinin reaction with the antibodies of thaumatin in the immunodiffusionassay indicates that thaumatin and monellin are immunologicallyclosely related. An identical conformational antigenic determinantin both molecules is probably responsible for this relationship.It is tentatively concluded that the identical conformationaldeterminant coincides with the active site responsible for thesweet-taste sensation.