Recombinant Thaumatin Research Articles

Optimal culture medium selection and supplementation for recombinant thaumatin II production by Komagataella phaffii

Sweet protein thaumatin, a promising alternative to table sugar, can overcome several drawbacks imposed by other sugar alternatives in the market today. Recombinant technology involving Komagataella phaffii (previously known as Pichia pastoris) has the potential to facilitate a uniform and large-scale production of this protein. To ensure maximal yield from the bioprocess, this study performed a screening to identify the media composition that exhibits the highest secretion yield of recombinant thaumatin II and favourable growth of the host organism. To achieve this, the most recommended media formulations for K. phaffii namely, BSM, FM22, MGY, BMGY, MBSM and d’Anjou media were investigated. It was identified from this study that the highest yields of recombinant thaumatin II were achieved from BMGY, FM22 and BSM media, irrespective of not exhibiting the highest cell growth. Despite exhibiting a lower cell density than FM22, the BMGY medium resulted in 62.79 mg/L of recombinant thaumatin II, which is 1–2 times higher than the former. This observation facilitated an understanding that the optimal medium for cell growth is not necessarily optimal for recombinant protein production. Moreover, a comparison between baffled and non-baffled flasks demonstrated that the former leads to a higher secretion yield, thereby indicating the importance of sufficient oxygen transfer into the medium. Furthermore, casamino acids and vitamins were supplemented to the media, where the latter contributed to an increased yield of thaumatin. As such, the highest protein yield of 68.60 mg/L was achieved for BMGY supplemented with vitamins.

Determination of recombinant thaumatin II secreted by Pichia pastoris using reversed-phase high-performance liquid chromatography

The manufacturing of recombinant proteins is increasing phenomenally, and yeasts serve as one of the most popular platforms for the large-scale production of these industrially relevant products. Protein analysis is a traditional step for the control and optimisation of such bioprocesses and often necessitates the combination of laborious sample treatments and expensive instruments. A hassle-free and cost-effective approach is always preferred for routine analyses and instigates a need for replacing these laborious tasks. In this study, a fast, simple, and reliable RP-HPLC method for the determination of the upcoming sweetener in the market, the sweet protein thaumatin was established. There is currently no reference method available for the quantification of this protein in fermentation samples. Due to an increasing industrial relevance foreseen for this sweetener, Pichia pastoris was used as a host for its bioproduction. The developed analytical method does not require a pre-treatment step of the biological sample and enables an easy quantification using a conventionally available HPLC system. The method facilitates the identification and quantification of recombinant thaumatin II and was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines(ICH, 2021). The developed technique enables the monitoring of thaumatin levels in fermentations, thereby facilitating bioprocess control and optimisation.

Effects of Temperature and pH on Recombinant Thaumatin II Production by Pichia pastoris

The sweet protein thaumatin is emerging as a promising sugar replacer in the market today, especially in the food and beverage sector. Rising demand for its production necessitates the large-scale extraction of this protein from its natural plant source, which can be limited in terms of raw material availability and production costs. Using a recombinant production technique via a yeast platform, specifically, Pichia pastoris, is more promising to achieve the product economically while maintaining batch-to-batch consistency. However, the bioproduction of recombinant proteins requires the identification of optimal process variables, constituting the maximal yield of the product of interest. These variables have a direct effect on the growth of the host organism and the secretion levels of the recombinant protein. In this study, two important environmental factors, pH, and temperature were assessed by cultivating P. pastoris in shake flasks to understand their influence on growth and the production levels of thaumatin II protein. The results from the pH study indicate that P. pastoris attained a higher viable cell density and secretion of protein at pH 6.0 compared to 5.0 when grown at 30 °C. Furthermore, within the three levels of temperatures investigated when grown at pH 6.0, the protein levels were the highest at 30 °C compared to 20 and 25 °C, whereas 25 °C exhibited the highest viable cell density. Interestingly, the trend observed from the qualitative effects of temperature and pH occurred in all the media that was investigated. These results broaden our understanding of how pH and temperature adjustment during P. pastoris cultivation aid in enhancing the production yields of thaumatin II prior to optimising the fed batch bioreactor operation.

An improved process for the production of highly purified recombinant thaumatin tagged-variants

The sweetest tasting molecule known is the protein thaumatin, first isolated from the katemfe fruit, Thaumatococcus daniellii. Thaumatin is used in the food and beverage industry as a low-calorie sugar substitute. Thaumatin interacts with taste receptors in the oral cavity eliciting a persistent sweet taste and a bitter, liquorice flavor. Recombinant thaumatin was expressed in Pichia pastoris and through a co-expression strategy with a molecular chaperone, yields of one engineered thaumatin variant increased by greater than two-fold. A detailed purification strategy for thaumatin is reported resulting in a homogenous sample recovered at a yield of 42%. The recombinant thaumatins were extensively characterised using size exclusion chromatography for homogeneity, reversed-phase HPLC for purity (99%), peptide digest LC–MS/MS for sequence determination, and circular dichroism and tryptophan fluorescence spectroscopies for conformational characterisation. These new thaumatin variants are amenable for bioconjugation, providing chemical biology tools for thaumatin:taste receptor interaction studies.

Purification and characterization of recombinant supersweet protein thaumatin II from tomato fruit

Thaumatin, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), is a promising zero-calorie sweetener for use in the food and pharmaceutical industries. Due to limited natural sources of thaumatin, its production using transgenic plants is an advantageous alternative. We report a simple protocol for purification of recombinant thaumatin II from transgenic tomato. Thaumatin was extracted from ripe tomato fruit in a low-salt buffer and purified on an SP-Sephacryl column. Recombinant thaumatin yield averaged 50 mg/kg fresh fruit. MALDI-MS analysis showed correct processing of thaumatin in tomato plants. The recombinant thaumatin was indistinguishable from the native protein in a taste test. The purified tomato-derived thaumatin had an intrinsic sweetness with a threshold value in taste tests of around 50 nM. These results demonstrate the potential of an expression system based on transgenic tomato plants for production of recombinant thaumatin for the food and pharmaceutical industries.

Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

Atomic structure of recombinant thaumatin II reveals flexible conformations in two residues critical for sweetness and three consecutive glycine residues

Thaumatin, an intensely sweet-tasting protein used as a sweetener, elicits a sweet taste at 50 nM. Although two major variants designated thaumatin I and thaumatin II exist in plants, there have been few dedicated thaumatin II structural studies and, to date, data beyond atomic resolution had not been obtained. To identify the detailed structural properties explaining why thaumatin elicits a sweet taste, the structure of recombinant thaumatin II was determined at the resolution of 0.99 Å. Atomic resolution structural analysis with riding hydrogen atoms illustrated the differences in the direction of the side-chains more precisely and the electron density maps of the C-terminal regions were markedly improved. Though it had been suggested that the three consecutive glycine residues (G142-G143-G144) have highly flexible conformations, G143, the central glycine residue was successfully modelled in two conformations for the first time. Furthermore, the side chain r.m.s.d. values for two residues (R67 and R82) critical for sweetness exhibited substantially higher values, suggesting that these residues are highly disordered. These results demonstrated that the flexible conformations in two critical residues favoring their interaction with sweet taste receptors are prominent features of the intensely sweet taste of thaumatin.

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.

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.

Expression pattern of the pre-prothaumatin II gene under the control of the CaMV 35S promoter in transgenic cucumber (Cucumis sativus L.) flower buds and fruits

Thaumatin II is an extremely sweet-tasting protein produced by fruits of the West African shrub Thaumatococcus daniellii Benth, so it can be used in biotechnology to improve the tastes of various plant products. This study is concerned with the spatial and temporal aspects of expression of the 35S-pre-prothaumatin II chimeric gene in flower buds and fruits of transgenic cucumber (Cucumis sativus L.) line 225. The activity of the 35S promoter in organs of line 225 was compared with its activity in 2 other transgenic lines. The accumulation of recombinant thaumatin varied spatially in flower bud tissues of transgenic lines. We found that these differences in the spatial accumulation of transgenic protein concerned the ovary of female buds and the perianth of male buds. In contrast to flower parts, recombinant thaumatin was found in nearly all parts of the young fruit from the transgenic plants. The pre-prothaumatin II gene expression was detected at a very early developmental stage in male buds, and its pattern was rather conserved as the buds aged. The expression of the transgene was also detected in vascular tissues of examined organs but was undetectable in pollen grains, in agreement with the generally held view that the CaMV 35S promoter is virtually silent in pollen. Immunocytochemical analyses of sections of control organs revealed endogenous homolog(s) of thaumatin when using polyclonal antisera, but not when using monoclonal antibodies for recombinant thaumatin detection in transgenic cucumber.

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.

Thaumatin‐like proteins – a new family of pollen and fruit allergens

Thaumatin‐like proteins – a new family of pollen and fruit allergens

Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage.

Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (tha) encoding the sweet protein thaumatin II were used to overexpress this protein in Aspergillus awamori lpr66, a PepA protease-deficient strain. The best expression results were obtained with the gdhA promoter of A. awamori or with the gpdA promoter of Aspergillus nidulans. There was good correlation of tha gene dosage, transcript levels, and thaumatin secretion. The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h. The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components. In all cases, cleavage of the fused protein occurred at the KEX recognition sequence. This work provides new expression systems in A. awamori that result in very high levels of thaumatin production.

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 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.

Secretion of the sweet-tasting plant protein thaumatin byStreptomyces lividans

To produce and direct the export inStreptomyces lividans of the sweet plant protein thaumatin, thaumatin II cDNA was fused in the correct reading frame to the β-galactosidase leader peptide, under the control of the β-galactosidase promoter and ribosome binding site. The export of the recombinant thaumatin may allow the correct formation of the thaumatin disulfide bonds. The recombinant thaumatin was purified from the medium on an S-Sepharose column and detected with western blots by sheep α-thaumatin antibodies. The recombinant thaumatin was the same size as authentic thaumatin and changed position on an acrylamide gel in response to reduction by 2-mercaptoethanol in the same manner.