Genetic engineering of thaumatin

Thaumatin Expression in Prokaryotes

Due to the strict growth conditions of Thaumatococcus daniellii and its inability to bear fruit outside its native region, extracting thaumatin from the plant cannot meet market demands, resulting in high market prices[1]. As a result, researchers have turned to genetic engineering to produce thaumatin. Thaumatin genes have been used to transform various microorganisms, fungi, and higher plants, achieving some progress[1].

In 1982, Edens et al. cloned the preprothaumatin cDNA gene into Escherichia coli (E. coli) and expressed it under the control of lactose and tryptophan promoters[1]. However, the expression product had a low yield (500 molecules/cell) and a larger molecular weight than expected, with no sweetness[1]. It is speculated that E. coli could not correctly process the precursor thaumatin, retaining the extra peptides at the N-terminus and C-terminus[1]. In 2000, Daniell et al. used the reduced/oxidized glutathione system to refold thaumatin recombinant protein in E. coli, obtaining a sweet-tasting mature protein with a concentration of 40 mg/L[1]. Generally, proteins expressed in E. coli through genetic engineering often exist as inactive, insoluble inclusion bodies, making Daniell’s experiment particularly significant[1].

Thaumatin Expression in Bacillus subtilis

In 1988, Illingworth fused the α-amylase gene and thaumatin II cDNA gene in Bacillus subtilis for expression[1]. The result was not only a low yield but also an inactive expression product[1]. Despite these challenges, researchers have continued to explore biotechnological methods to extract and concentrate thaumatin in various organisms[2].

Thaumatin Production in Transgenic Plants

Transgenic plants have been explored as a potential source of thaumatin production, with some success in barley (Hordeum vulgare)[2]. Thaumatin expressed in transgenic barley plants has shown high yields and sufficient sweetness and stability[2]. This approach offers a promising alternative to traditional extraction methods and can help meet the increasing demand for natural sweeteners in the food and pharmaceutical industries[1][3].

Genetic engineering of thaumatin-Xi'an Lyphar Biotech Co., Ltd

Genetic engineering of thaumatin

Thaumatin Expression in Fungi

Thaumatin expression in fungi mainly focuses on yeast and filamentous fungi. After decades of research, thaumatin production and protein activity in fungi, especially filamentous fungi, have significantly improved[1]. Edens et al. transformed the preprothaumatin cDNA into Saccharomyces cerevisiae under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter[1] and Kluyveromyces lactis[1]. Although the expression level was low (3,000 molecules/cell), yeast could accurately cleave the N-terminal signal peptide of preprothaumatin[1].

Synthetic Thaumatin Gene in Yeast

In 1988, Lee synthesized the thaumatin II gene according to yeast-preferred codons and transformed it into yeast[1]. The expression was controlled by the 3-phosphoglycerate kinase promoter and terminator, resulting in a high yield (20% of insoluble protein) but insoluble thaumatin[1]. It is speculated that the reducing environment inside the cell might have prevented the formation of disulfide bonds[1].

Thaumatin Expression in Filamentous Fungi

Subsequently, Illingworth expressed thaumatin in Streptomyces lividans[1], and Hahm expressed thaumatin in Aspergillus oryzae[1]. Unfortunately, the products had low yields and no sweetness[1]. However, due to the gradual optimization of the filamentous fungal expression system for producing recombinant thaumatin, the yield of thaumatin in filamentous fungi has gradually increased[1]. In 1997, Faus obtained thaumatin with a yield of 2 mg/L in Penicillium roquefortii[1]. In 1998, Faus et al. constructed a thaumatin II gene expression vector and transferred it into Aspergillus niger var. awamori, obtaining sweet-tasting thaumatin with a concentration of 5-7 mg/L[1]. In 1999, Moralejo et al. constructed different thaumatin expression cassettes in Aspergillus niger, achieving a maximum yield of 15 mg/L[1].

Thaumatin Expression in Plants

Thaumatin genes have been used to transform various plants to improve their taste, in addition to being transformed into microorganisms and fungi[1]. In 1990, Witty first cloned thaumatin II cDNA into the CaMV 35S promoter shuttle vector pWIT2[1]. Using the hairy root transformation technique and Ri plasmid-mediated transformation, thaumatin II cDNA was introduced into Solanum tuberosum cv. Iwa, resulting in regenerated plants with a sweet taste[1]. However, the expression level was low, at only 3×10-8 mol/L[1].

Significance of Thaumatin Transformation in Plants

Witty’s work has far-reaching implications. Firstly, this was the first time the sweet characteristics of T. daniellii were expressed in a high-yielding temperate crop, making it possible to extract thaumatin in large quantities from transgenic plants[1]. Secondly, the successful transformation of thaumatin into potatoes suggests that other crops, such as strawberries, watermelons, kiwifruit, cucumbers, and apples, are also suitable for exogenous thaumatin expression, providing the possibility of improving the taste of various crops[1].

Thaumatin Gene in Other Plants

Since then, the thaumatin gene has been successfully introduced into plants such as cucumbers[2], apples, carrots, peaches, and strawberries[1]. In China, researchers have also introduced exogenous thaumatin genes into domestic potatoes[1] and tobacco[1], achieving some success. Additionally, attempts are being made to introduce the thaumatin gene into kale[1] and corn[1].

what are the benefits of using transgenic plants to produce thaumatin

There are several benefits of using transgenic plants to produce thaumatin. Firstly, it provides an alternative source of thaumatin, as the natural sources are limited

1. This can help meet the increasing demand for this low-calorie sugar substitute in the food and pharmaceutical industries

1. Secondly, transgenic plants can potentially produce thaumatin in large quantities, making it more cost-effective and sustainable compared to extracting it from its natural sources

2. Thirdly, transgenic plants can be engineered to express thaumatin with desired properties, such as improved sweetness and stability

2. Lastly, using transgenic plants to produce thaumatin can also help improve the taste of various crops, such as tomatoes, potatoes, cucumbers, and apples

3, which can enhance their market value and consumer appeal.

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