Production Of Thaumatin Research Articles

Life cycle assessment of the sweetness enhancer thaumatin (E957) produced from Thaumatococcus daniellii fruit foraged from West Africa: The SWEET project

Replacing added sugar with non-nutritive sweeteners and sweetness enhancers is of increasing interest due to the negative health effects of excess sugar consumption. Much has been done to understand health and safety of such sweetening additives, but little on their sustainability. This study, part of the Horizon 2020 SWEET project, presents results from the first life cycle assessment of the sweetness enhancer thaumatin, produced from Thaumatococcus daniellii fruit, from forests in West Africa and extracted in the United Kingdom. Thaumatin is used in formulations to increase perceived sweetness of added sugar, allowing some to be removed. Environmental impact is reported for multiple impact categories from the ReCiPe 2016 (H) method, focusing on global warming potential, land use, water consumption, and freshwater eutrophication. Impacts are expressed in terms of product mass and sweetness equivalence. Global warming potential for production of thaumatin is found to be 719.2 kgCO2-eq/kg. When thaumatin replaces 20% of added sugar, environmental impact for a given sweetness is found to reduce by an average of 19.4% across all impact categories. International transport is a major contributor to global warming potential, as is aril removal from the fruit to freshwater eutrophication and water use, and fruit foraging to land use. However, land use is identified as a key area of future research to improve uncertainty in the data. Results show that thaumatin can be used to reduce the environmental impact of providing sweet taste in food and beverage products.

Overview of Thaumatococcus Daniellii Plant, History, Uses, Benefits, and Characterization

Thaumatococcus daniellii (Benn.) Benth, known as “katempe” or “katemfe”. It grows in humid tropical forests and the coastal areas of West Africa, especially in Nigeria, Ghana, Central African Republic, Uganda, and Cote d'Ivoire. T. daniellii contains chemical compounds that have several uses in many fields and contain thaumatin protein, which plays an essential role in the food industry, used as a natural sweetener, and pharmaceutical industry. T. daniellii can play a significant role in economic growth in many countries in which it grows. This study summarises some crucial aspects of T. daniellii. As the study highlight, some of the chemical components are contained in the plant. In addition to the other medicinal benefits and applications used from T. daniellii. the study presented the importance of the plant in the production of thaumatin and highlighted the two types of this protein and the difference between them in the arrangement of amino acids.

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris

Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/gDCW per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris.

Sweetest Protein -Thaumatin

Thaumatin, commercially available under the trade name Talin, is known as a sweetest compound (100,000 times sweeter than sucrose on molar basis) which can be used not only as a sweetener but also as a flavor enhancer. This sweetener is a protein found in plant Thaumatococcus daniellii. Biotechnology has also seen as alternative method of production of thaumatin. The plant source of this protein is limited and very expensive. Although the sweetener is the sweetest one, its application is not so common because of its liquorice taste and delayed sweetness. This article reviews brie y on the property, production, application and safety aspects of Thaumatin which is known as a sweetest compound till date. DOI: http://dx.doi.org/10.3126/jfstn.v7i0.11210 J. Food Sci. Technol. Nepal, Vol. 7 (112-118), 2012

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.

Plant bioreactors – the taste of sweet success

Thaumatins are intensely sweet proteins (3000 times sweeter than the same weight of sucrose) that are found in the arils of the tropical perennial plant Thaumatococcus daniellii Benth and are produced commercially by aqueous extraction from the fruits. The proteins are widely used as sweeteners and flavor enhancers in the food industry, and the European Food Safety Association (EFSA) has recently confirmed that their use as feed additive (1 to 5 mg/kg complete feed) is safe for all animal species. Given the large market for sweeteners and flavor enhancers, thaumatins could become increasingly important in the food and feed additives sector. In this issue of Biotechnology Journal, a study examines the production of thaumatin in tobacco hairy root cultures.

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.

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 α-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.

Silencing of the aspergillopepsin B (pepB) gene of Aspergillus awamori by antisense RNA expression or protease removal by gene disruption results in a large increase in thaumatin production.

Aspergillopepsin B was identified in culture broths of Aspergillus awamori by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopepsin B. The pepB gene encoding aspergillopepsin B of A. awamori was cloned and characterized. It is located in chromosome IV of A. awamori, as shown by pulsed-field gel electrophoresis, and encodes a protein of 282 amino acids with high similarity to the aspergillopepsin B of Aspergillus niger var. macrosporus. The pepB gene is expressed at high rates as a monocistronic 1.0-kb transcript in media with casein at acidic pH values. An antisense cassette constructed by inserting the pepB gene in the antisense orientation downstream from the gpdA promoter resulted in a good level of antisense mRNA, as shown by reverse transcription-PCR. Partial silencing of the pepB gene by the antisense mRNA resulted in a 31% increase in thaumatin yield. However, significant residual degradation of thaumatin still occurred. To completely remove aspergillopepsin B, the pepB gene was deleted by double crossover. Two of the selected transformants lacked the endogenous pepB gene and did not form aspergillopepsin B. Thaumatin yields increased by between 45% in transformant APB 7/25 and 125% in transformant 7/36 with respect to the parental strain. Reduction of proteolytic degradation by gene silencing with antisense mRNA or total removal of the aspergillopepsin B by directed gene deletion was a very useful method for improving thaumatin production in A. awamori.

A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspegillus awamori.

Thaumatin, a 22-kDa protein containing eight disulfide bonds, is secreted by the filamentous fungus Aspergillus awamori at levels which are dependent upon the extent of overexpression of protein disulfide isomerase (PDIA). Additional copies of the PDIA-encoding gene pdiA were introduced into a strain of A. awamori that expresses a cassette encoding thaumatin. Transformants with different levels of pdiA mRNA and measured PDIA levels were chosen for examination of the impact that PDIA levels had on thaumatin secretion. The secretion of two native proteins, alpha-amylase and acid phosphatase, was also examined in relation to varying levels of PDIA. Over a range of PDIA levels of 1-8, relative to the native level in strains with just one copy of the pdiA gene, the fraction of alpha-amylase and acid phosphatase in the total secreted protein was unaffected. In contrast, a peak level of thaumatin, about 5-fold higher than in the strain with one copy of pdiA, was found in strains with a relative PDIA level of between two and four. Improved thaumatin production was confirmed in 5-1 fermenters using a strain of A. awamori with six pdiA gene copies, containing 3.2-fold higher levels of PDIA than wild-type strains.

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.

Issues and advances in the use of transgenic organisms for the production of thaumatin, the intensely sweet protein from Thaumatococcus danielli.

The thaumatins are a class of intensely sweet proteins isolated from the fruit of the tropical plant Thaumatococcus danielli. Thaumatin is approved for use in many countries and has application as both a flavor enhancer and a high-intensity sweetener. The supply of naturally occurring thaumatin is limited, which has prompted extensive research into its synthesis via transgenic organisms. The gene encoding thaumatin has been introduced into various microorganisms under transcriptional control of heterologous promoters. Yields to date have been low, but the factors governing more efficient microbial production have been identified. Continued research should allow microbial yields to be improved to commercially viable levels. The unique properties of thaumatin as a food additive could well be exploited by the food industry. Alternatively, the thaumatin gene could be engineered directly into selected fruit and vegetable crops to improve their flavor and sweetness.

Expression and Secretion of Thaumatin fromAspergillus oryzae

The plant protein thaumatin has been expressed and secreted in Aspergillus oryzae. The thaumatin II cDNA was expresed via a Saccharomyces cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter, and its secretion is apparently directed into the medium by the native plant signal sequence. The S. cerevisiae GPD promoter is properly regulated in A. oryzae; the addition of glucose stimulates the production of thaumatin. A 22-kDa immunoreactive protein indistinguishable from mature thaumatin is present in the culture medium. A slightly larger, 25-kDa protein is observed in cell extracts and may represent the unprocessed form of the protein. Maximum accumulation of the extracellular immunoreactive protein is observed after two days and no further production is observed.

Expression and secretion of thaumatin from Aspergillus oryzae.

The plant protein thaumatin has been expressed and secreted in Aspergillus oryzae. The thaumatin II cDNA was expresed via a Saccharomyces cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter, and its secretion is apparently directed into the medium by the native plant signal sequence. The S. cerevisiae GPD promoter is properly regulated in A. oryzae; the addition of glucose stimulates the production of thaumatin. A 22-kDa immunoreactive protein indistinguishable from mature thaumatin is present in the culture medium. A slightly larger, 25-kDa protein is observed in cell extracts and may represent the unprocessed form of the protein. Maximum accumulation of the extracellular immunoreactive protein is observed after two days and no further production is observed.

Expression of foreign proteins in microorganisms.

The various alternative strategies for the expression of heterologous proteins in microorganisms are reviewed. To illustrate how these general considerations can be addressed in particular cases, the expression of chimeric human-mouse antibodies in Escherichia coli and the production of thaumatin, an intensely sweet plant protein, in yeast, are described.