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This article presents the results of a comprehensive toxicity assessment of brazzein and monellin, yeast-produced recombinant sweet-tasting proteins. Excessive sugar consumption is one of the leading dietary and nutritional problems in the world, resulting in health complications such as obesity, high blood pressure, and cardiovascular disease. Although artificial small-molecule sweeteners widely replace sugar in food, their safety and long-term health effects remain debatable. Many sweet-tasting proteins, including thaumatin, miraculin, pentadin, curculin, mabinlin, brazzein, and monellin have been found in tropical plants. These proteins, such as brazzein and monellin, are thousands-fold sweeter than sucrose. Multiple reports have presented preparations of recombinant sweet-tasting proteins. A thorough and comprehensive assessment of their toxicity and safety is necessary to introduce and apply sweet-tasting proteins in the food industry. We experimentally assessed acute, subchronic, and chronic toxicity effects, as well as allergenic and mutagenic properties of recombinant brazzein and monellin. Our study was performed on three mammalian species (mice, rats, and guinea pigs). Assessment of animals' physiological, biochemical, hematological, morphological, and behavioral indices allows us to assert that monellin and brazzein are safe and nontoxic for the mammalian organism, which opens vast opportunities for their application in the food industry as sugar alternatives. [ABSTRACT FROM AUTHOR]

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 pha#ii (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 n and favourable growth of the host organism. To achieve this, the most recommended media formulations for K. pham 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. [ABSTRACT FROM AUTHOR]

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 identfication and quantification of recombinant thaumatin n 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. [ABSTRACT FROM AUTHOR]

This article presents the results of a comprehensive toxicity assessment of brazzein and monellin, yeast-produced recombinant sweet-tasting proteins. Excessive sugar consumption is one of the leading dietary and nutritional problems in the world, resulting in health complications such as obesity, high blood pressure, and cardiovascular disease. Although artificial small-molecule sweeteners widely replace sugar in food, their safety and long-term health effects remain debatable. Many sweet-tasting proteins, including thaumatin, miraculin, pentadin, curculin, mabinlin, brazzein, and monellin have been found in tropical plants. These proteins, such as brazzein and monellin, are thousands-fold sweeter than sucrose. Multiple reports have presented preparations of recombinant sweet-tasting proteins. A thorough and comprehensive assessment of their toxicity and safety is necessary to introduce and apply sweet-tasting proteins in the food industry. We experimentally assessed acute, subchronic, and chronic toxicity effects, as well as allergenic and mutagenic properties of recombinant brazzein and monellin. Our study was performed on three mammalian species (mice, rats, and guinea pigs). Assessment of animals’ physiological, biochemical, hematological, morphological, and behavioral indices allows us to assert that monellin and brazzein are safe and nontoxic for the mammalian organism, which opens vast opportunities for their application in the food industry as sugar alternatives.

Honey truffle sweetener (HTS), a 121 amino acid protein is identified as a high-intensity sweetener found naturally occurring in the Hungarian Sweet Truffle Mattirolomyces terfezioides , an edible mushroom used in regional diets. The protein is intensely sweet, but the truffle is difficult to cultivate; therefore, the protein was systematically characterized, and the gene coding for the protein was expressed in a commonly used host yeast Komagataella phaffii . The heterologously expressed protein maintained the structural characteristics and sweet taste of the truffle. Preliminary safety evaluations for use as a food ingredient were performed on the protein including digestibility and in silico approaches for predicting the allergenicity and toxicity of the protein. HTS is predicted to be nonallergenic, nontoxic, and readily digestible. This protein is readily produced by precision fermentation of the host yeast, making it a potential replacement for both added sugars and small molecule high-intensity sweeteners in food.

Saccharomyces cerevisiae is an attractive host for the expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilizes type IIS restriction enzymes to facilitate an efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of 16 well-characterized SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion promoting signals are validated by using five different proteins of interest. Comparison of intracellular and secreted protein levels reveals the optimal secretion promoting sequence for each individual protein. Large, protein of interest-specific variations in secretion efficiency are observed. SP sequences are also used with the five surface display anchors, and the combination of SP and anchor protein proves critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs and TFPs and anchor proteins for optimal secretion and/or surface display of a given protein of interest in S. cerevisiae .

The excessive consumption of sugar-containing foods contributes to the development of a number of diseases, including obesity, diabetes mellitus, etc. As a substitute for sugar, people with diabetes mellitus and obesity most often use sweeteners. Sweet proteins, in particular brazzein, are an alternative to synthetic sweeteners that have natural origin, are broken down in the intestines along with food proteins, and do not affect blood sugar and insulin levels. The purpose of the review was to analyze the available data on the sweet protein brazzein, its physical and chemical properties, existing biotechnological methods of production, and prospects for application in the food industry in order to further develop an optimized heterologous expression system. Material and methods . Google Scholar, Scopus, Web of Science, PubMed, RSCI and eLibrary.ru databases were used for collecting and analyzing literature. Search depth - 30 years. Results . Numerous studies of the physical and chemical properties of brazzein have demonstrated its high potential for use in the food industry. In particular, a short amino acid sequence, thermal stability, the ability to maintain its structure and sweet properties in a wide pH range, hypoallergenicity, lack of genotoxicity, and an extremely high level of sweetness compared to sucrose allow us to conclude that its use is promising. Mutant variants of brazzein have been generated, the sweetest of which (with three amino acid substitutions H31R/E36D/E41A) exceeds sucrose sweetness by 22 500 times. To date, various systems for the expression of recombinant brazzein have already been developed, in which bacteria (Escherichia coli, Lactococcus lactis, Bacillus licheniformis), yeast (Komagataella phaffii, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum, Daucus carota) and animals (Mus musculus) have been used. Conclusion . Due to its high sweetness, organoleptic properties and long history of human consumption, brazzein can be considered as a promising natural sweetener. Despite the short peptide sequence, the production of the recombinant protein faced a number of problems, including low protein yield (for example, it could only be detected in mouse milk by Western blot hybridization) and loss of sweetness. Thus, further optimization of the process is necessary for widespread brazzein use in the food industry, which includes the selection of an adequate producer and the use of extracellular expression systems to reduce the final cost of the product., Competing Interests: The authors declare no conflict of interest., (Copyright© GEOTAR-Media Publishing Group.)

The analysis of the morbidity in the population of the Russian Federation showed the presence of a significant number of people with chronic non-infection pathologies, including obesity, diabetes mellitus, whose diet should include food with reduced energy value. For this purpose, food containing sweeteners are widely used. At the same time, the range of sweeteners permitted for the usage in food industry does not always allow achieving the desired technological effect. In this regard, it is of interest to search for new types of sweeteners with the necessary organoleptic and technological properties. Such sweeteners include a number of proteins with a sweet taste. The purpose of the study was to analyze the available data on the possibility of using protein-based sweeteners in the food industry and their human health safety. Material and methods . The article presents an analysis and generalization of published data from Scopus, Web of Science, PubMed, RSCI, Cyberleninka, Google Scholar, as well as provisions of domestic and international regulatory and legislative documents. Results . An analysis of the biological and technological properties of sweeteners based on sweet-tasting proteins (thaumatin, brazzein, curculin, manbilin, miraculin, monellin, pentadin) and their production methods has been carried out. Evidence is presented of the possibility of safe use in food, not only of thaumatin (E957), but also of such proteins as brazzein and monellin. Other sweet-tasting proteins are also of interest for their use as sweeteners and substances modifying food taste. It has been shown that at present the biological properties and mechanism of action of various types of sweet proteins on the human body have not been studied sufficiently. In addition, the use of only plant raw materials for the production of these proteins will not allow their wide application in food industry, which is a limitation for their use as food additives - sweeteners. There are reports on the possibility of including sweet proteins in the treatment protocol for various chronic non-infection diseases, including oncological diseases. Conclusion . The conducted analysis of the properties of sweet taste proteins showed the prospects of their use in food industry as sweeteners, substances modifying food taste and bioactive substances. Due to the fact that an increase in the production of sweet proteins is possible only in the case of using biotechnological methods, producer strains and sweeteners obtained by microbial synthesis can be applied in food industry only after assessing the risks to human health and establishing regulations for their safe use., Competing Interests: Authors declare no conflict of interests., (Copyright© GEOTAR-Media Publishing Group.)

[Display omitted] • Protein sweeteners are healthier than synthetic ones. • Synthetic dipeptide sweeteners alter gut micro biota. • Sweeteners prevent dental caries. • Sweeteners decrease calories intake. • Aspartame metabolites lead to several drawbacks. Artificial sweeteners have become increasingly popular worldwide owing to their lower calorie content in addition to the claims of health benefits such as weight control, blood glucose level regulation in diabetics, and protection against dental caries. Nevertheless, there is still controversy regarding their safety, especially when administered over the long term, taking into account that most of the safety studies are based on animal models and only a few human studies. This review focuses on low-calorie protein/peptide sweeteners. These include artificial sweeteners, i.e. aspartame, advantame, neotame, and alitame which are synthetic, versus those of natural origin such as thaumatin, monellin, brazzein, pentadin, mabinlin, curculin, and egg white lysozyme. We conducted a systematic literature survey to ensure the accuracy of the data regarding the chemical properties, synthesis, and industrial applications. The health benefits and safety of these sweeteners in humans are presented for the first time in context to their metabolic profiles and gut interaction. [ABSTRACT FROM AUTHOR]

Optogenetics has great potential for biotechnology and metabolic engineering due to the cost-effective control of cellular activities. The usage of optogenetics techniques for the biosynthesis of bioactive molecules ensures reduced costs and enhanced regulatory possibilities. This requires development of efficient methods for light-delivery during a production process in a fermenter. Here, we benchmarked the fermenter production of a low-caloric sweetener in Saccharomyces cerevisiae with optogenetic tools against the production in small scale cell culture flasks. An expression system based on the light-controlled interaction between Cry2 and Cib1 was used for sweet-protein production. Optimization of the fermenter process was achieved by increasing the light-flux during the production phase to circumvent shading by yeast cells at high densities. Maximal amounts of the sweet-protein were produced in a pre-stationary growth phase, whereas at later stages, a decay in protein abundance was observable. Our investigation showcases the upscaling of an optogenetic production process from small flasks to a bioreactor. Optogenetic-controlled production in a fermenter is highly cost-effective due to the cheap inducer and therefore a viable alternative to chemicals for a process that requires an induction step., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Owing to various undesirable health effects of sugar overconsumption, joint efforts are being made by industrial sectors and regulatory authorities to reduce sugar consumption practices, worldwide. Artificial sweeteners are considered potential substitutes in several products, e.g., sugar alcohols (polyols), high-fructose corn syrup, powdered drink mixes, and other beverages. Nevertheless, their long-standing health effects continue to be debatable. Consequently, growing interest has been shifted in producing non-caloric sweetenersfrom renewable resources to meet consumers' dietary requirements. Except for the lysozyme protein, various sweet proteins including thaumatin, mabinlin, brazzein, monellin, miraculin, pentadin, and curculin have been identified in tropical plants. Given the high cost and challenging extortion of natural resources, producing these sweet proteins using engineered microbial hosts, such as Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis represents an appealing choice. Engineering techniques can be applied for large-scale biosynthesis of proteins, which can be used in biopharmaceutical, food, diagnostic, and medicine industries. Nevertheless, extensive work needs to be undertaken to address technical challenges in microbial production of sweet-tasting proteins in bulk. This review spotlights historical aspects, physicochemical properties (taste, safety, stability, solubility, and cost), and recombinant biosynthesis of sweet proteins. Moreover, future opportunities for process improvement based on metabolic engineering strategies are also discussed.