Production Of Steviol Glycosides Research Articles

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

Effects of nitric oxide, spermidine, and salicylic acid signaling and their crosstalk with each other in the production of commercially important stevioside content and drought stress responses in Stevia rebaudiana bertoni

The leaves of Stevia rebaudiana Bertoni contain steviol glycosides (SGs), which provide the sweet taste of stevia. However, drought can have a negative impact on the plant's growth and development. To address this issue, signaling molecules such as sodium nitroprusside (SNP), spermidine (SPD), and salicylic acid (SA) are often applied to increase plant tolerance. However, the combined effects of these molecules have not been extensively studied. This research aimed to investigate the effects of controlled elicitation with SA, SNP, SPD, and their combinations on plant performance, SG content, and drought stress mitigation in Stevia rebaudiana under drought stress. The elicitor treatments were found to result in a significant increase in SG content, with 0.1 mM SA being the most effective treatment. Additionally, the treatments were able to reduce the stress effects on growth parameters to non-stress levels. The use of SPD, SA+SNP, and SPD+SNP on stressed plants significantly increased CAT and SOD activity, resulting in a more active antioxidant defense system that lowered MDA contents and H2O2 generation. These findings suggest that stevia cultivation with controlled elicitation could be used to improve plant growth, tolerance, and SG production under drought stress conditions.

The effect of methyl jasmonate on growth, stevioside and rebaudioside A production in Stevia rebaudiana shoot culture using mist bioreactor

Steviol glycosides (SG) from Stevia rebaudiana have been used as natural sweeteners to substitute sucrose. SG production naturally needs large biomass. Therefore, it requires large area of cultivation. In vitro cultivation can be used as an alternative to propagate Stevia, as well as for increasing the production of SG. In this experiment, we conducted Stevia propagation in bioreactor and induced SG production through methyl jasmonate (MeJa) elicitation. The objective of this study was to evaluate the effect of MeJA on plant growth and SG contents in Stevia shoot cultivated using mist bioreactor. Elicitation was carried out by MeJa spraying for 30 min every 6 h. In treatment A, we used 10 μM MeJa for 72 h and 100 μM MeJa for 24 h for treatment B. The results showed that treatment A gave higher increase in shoot growth (485%), biomass production (547%), and relative growth rate (0.13) compared to treatment B. There was no significant difference in stevioside content among treatments (A and B) and controls, meanwhile treatment B produced rebaudioside A (0.0113 mg/g) higher than treatment A (0.0062 mg/g). We concluded that treatment A was better in inducing growth and treatment B could be used to increase rebaudioside A.

Stevia Polyphenols, Their Antimicrobial and Anti-Inflammatory Properties, and Inhibitory Effect on Digestive Enzymes.

Polyphenols from stevia leaves (PPSs) are abundant byproducts from steviol glycoside production, which have been often studied as raw extracts from stevia extracts for their bioactivities. Herein, the PPSs rich in isochlorogenic acids were studied for their antimicrobial and anti-inflammatory properties, as well as their inhibitory effects on digestive enzymes. The PPSs presented stronger antibacterial activity against E. coli, S. aureus, P. aeruginosa, and B. subtilis than their antifungal activity against M. furfur and A. niger. Meanwhile, the PPSs inhibited four cancer cells by more than 60% based on their viability, in a dose-dependent manner. The PPSs presented similar IC50 values on the inhibition of digestive enzyme activities compared to epigallocatechin gallate (EGCG), but had weaker anti-inflammatory activity. Therefore, PPSs could be a potential natural alternative to antimicrobial agents. This is the first report on the bioactivity of polyphenols from stevia rebaudiana (Bertoni) leaves excluding flavonoids, and will be of benefit for understanding the role of PPSs and their application.

Influence of the Abiotic Elicitors Ag Salts of Aspartic Acid Derivatives, Self-Organized in Nanofibers with Monomeric and Dimeric Molecular Structures, on the Antioxidant Activity and Stevioside Content in Micropropagated Stevia rebaudiana Bert.

The use of nanomaterials in biotechnology for the in vitro propagation of medical plants and the accumulation of certain biologically active metabolites is becoming an efficient strategy. This study aimed to evaluate the influence of the concentration (0, 1, 10, 50, and 100 mg L-1) of two types of nanofibers on the growth characteristics, the antioxidant status, and the production of steviol glycosides in micropropagated Stevia rebaudiana Bert. plantlets. The nanofibers were synthesized by aspartic acid derivatives (L-Asp) Ag salts self-organized into nanofibers with two different molecular structures: monomeric, containing one residue of L-Asp with one hydrophilic head which bonds one Ag ion (NF1-Ag salt); and dimeric, containing two residues of L-Asp with two hydrophilic heads which bond two Ag ions (NF2-Ag salt). An increase in the shoots from the explants' number and length, biomass accumulation, and micropropagation rate was achieved in the plants treated with the NF1-Ag salt in concentrations from 1 to 50 mg L-1 after 30 days of in vitro proliferation compared to the NF2-Ag salt. In contrast, the plants grown on MS media supplemented with NF2-Ag salt exhibited an increase in the level of stevioside, rebaudioside A, and mono- (CQA) and dicaffeoylquinic (DCQA) acids as compared to the NF1-Ag salt.

Improving Steviol Glycosides Production in Stevia Rebaudian Bertoni by Plant Tissue Culture Technology

Plant tissue culture techniques have made tremendous progress in the production of various active compounds of interest, including Steviol Glycosides in Stevia. Stevia rebaudiana is a perennial herbaceous plant known for its sweet-tasting leaves that contain active substances used as natural low-calorie sweeteners called Steviol glycosides. Because of its high sweetness and low-calorie nature, it is an alternative to sugar. However, conventional cultivation of Stevia plants for the production of Steviol Glycosides faces challenges such as long growth cycles, changes in chemical composition, and environmental determinants. Therefore, plant tissue culture is an alternative for the production of large quantities of Steviol Glycosides by altering various factors such as nutrient media, growth regulators, and response. Photovoltaic and tissue culture methods have been successfully used for plant propagation as well as the production of high-quality glycosides. Through techniques such as callus culture, organogenesis, and somatic embryo culture, large quantities of cotyledons can be rapidly established from small plant parts in a relatively short time. With further advances in tissue culture techniques and an understanding of the physiology and genetics of stevia, the potential for large-scale production of glycosides using Tissue culture methods is a promising technique for the commercial use of this natural sweetener.

Cold Plasma-Induced Changes in Stevia rebaudiana Morphometric and Biochemical Parameter Correlations.

Stevia rebaudiana Bertoni is an economically important source of natural low-calorie sweeteners, steviol glycosides (SGs), with stevioside (Stev) and rebaudioside A (RebA) being the most abundant. Pre-sowing seed treatment with cold plasma (CP) was shown to stimulate SGs biosynthesis/accumulation up to several fold. This study aimed to evaluate the possibility to predict CP-induced biochemical changes in plants from morphometric parameters. Principle component analysis (PCA) was applied to two different sets of data: morphometric parameters versus SGs concentrations and ratio, and morphometric parameters versus other secondary metabolites (total phenolic content (TPC), total flavonoid content (TFC)) and antioxidant activity (AA). Seeds were treated for 2, 5 and 7 min with CP (CP2, CP5 and CP7 groups) before sowing. CP treatment stimulated SGs production. CP5 induced the highest increase of RebA, Stev and RebA+Stev concentrations (2.5-, 1.6-, and 1.8-fold, respectively). CP did not affect TPC, TFC or AA and had a duration-dependent tendency to decrease leaf dry mass and plant height. The correlation analysis of individual plant traits revealed that at least one morphometric parameter negatively correlates with Stev orRebA+Stev concentration after CP treatment.

Effects of polystyrene nanoplastics exposure on in vitro-grown Stevia rebaudiana plants

Nanoplastics (NPs) as environmental contaminants have received increased attention in recent years. Numerous studies have suggested possible negative effects of plants exposure to NPs, but more data are needed with various plants under different exposure conditions to clarify the underlying phytotoxicity mechanisms. In this study, we investigated the effect of polystyrene nanoplastics (PSNPs; 28.65 nm average diameter) exposure (10, 100 and 250 mg/L) on plant morphology and production of relevant metabolites (steviol glycosides, chlorophylls, carotenoids, and vitamins) of in vitro-grown Stevia rebaudiana plantlets. Additionally, we used dark field microscopy combined with fluorescence hyperspectral imaging for the visualization of internalized PSNPs inside plant tissues. At higher concentrations (>100 mg/L), PSNPs were shown to aggregate in roots and to be transported to leaves, having a significantly negative impact on plant growth (reduced size and biomass), while increasing the production of metabolites compared to controls, most probably because of response to stress. The production of steviol glycosides presented a biphasic dose-response suggestive of hormesis, with the highest values at 10 mg/L PSNPs (1.5–2.2-fold increase compared to controls), followed by a decline in production at higher concentrations (100 and 250 mg/L), but with values comparable to controls. These results are promising for future in vivo studies evaluating the effect of NP exposure on the production of steviol glycosides, the natural sweeteners from stevia.

Exploring Plant Tissue Culture and Steviol Glycosides Production in Stevia rebaudiana (Bert.) Bertoni: A Review

Stevia rebaudiana (Bert.) Bertoni, commonly called “sweet leaf” is a medicinally and industrially important plant known to be rich in zero-calorie natural sweetening compound(s) known as “steviol glycosides”. However, due to its poor seed germination and slow vegetative propagation, it has become rather difficult to meet the increasing global demand for Stevia-based products. Different biotechnological approaches have been developed over the past few decades to overcome these limitations and allow for mass propagation of the plant. Several protocols for in vitro organogenesis, callogenesis, and cell suspension cultures have been developed during the past few years. Apart from this, attempts have also been made to improve the production of steviol glycosides through nutrient manipulation, the use of elicitors, hairy root cultures, genetic transformation, and metabolic pathway engineering. Therefore, this review provides an up-to-date report on the applications of various biotechnological tools for mass propagation and enhanced steviol glycoside production, along with a detailed bibliometrics analysis. This review also highlights research gaps and future considerations that could be fruitful for the scientific community to delve deeper into the various unexplored aspects of the architecture and functionality of this natural sweetening plant.

Environmental life cycle assessment of production of the high intensity sweetener steviol glycosides from Stevia rebaudiana leaf grown in Europe: The SWEET project

PurposeThere is an increasing interest in the use of non-nutritive sweeteners to replace added sugar in food and beverage products for reasons of improving consumer health. Much work has been done to understand safety of sweeteners, but very little on sustainability. To address that gap, this study presents the results of a life cycle assessment (LCA) of production of rebaudioside A 60%, 95% pure (RA60) steviol glycoside mix from Stevia rebaudiana leaf grown in Europe.MethodsAn attributional cradle-to-factory-gate life cycle assessment was conducted on growing of stevia leaves and extraction of steviol glycosides in Europe. Primary data were used from a case study supply chain. Results are reported in impact categories from the ReCiPe 2016 (H) method, with focus given to global warming potential, freshwater eutrophication, water consumption, and land use. Impacts are expressed both in terms of production mass and sweetness equivalence, a common metric for understanding high intensity sweetener potency. Sweetness equivalence of RA60 is typically 200 to 300 times that of sugar. Comparison of environmental impact is made to sugar (sucrose) produced from both cane and beets. The research is part of the EU project SWEET (sweeteners and sweetness enhancers: impact on health, obesity, safety, and sustainability).Results and discussionGlobal warming potential for production of RA60 was found to be 20.25 kgCO2-eq/kgRA60 on a mass basis and 0.081 kgCO2-eq/kgSE on a sweetness equivalence basis. Field production of stevia leaves was found to be the main source of impact for most impact categories, and for all four focus categories. Extraction of the RA60 was the main source of impact for the others. Leaf processing and seedling propagation were minor contributors to life cycle impact. Removal of international transport from the supply chain reduced global warming potential by 18.8%. Compared with sugar on a sweetness equivalence basis, RA60 has approximately 5.7% to 10.2% the impact for global warming potential, 5.6% to 7.2% the impact for land use, and is lower across most other impact categories.ConclusionThis is the first LCA of steviol glycoside mix RA60 produced from leaf in Europe. The results indicate that RA60 can be used to reduce environmental impact of providing a sweet taste by replacing sugar across all impact categories. However, it is important to note that specific formulations in which RA60 is used will have a bearing on the final environmental impact of any food or beverage products. For solid foods, this requires further research.

EFFECT OF LEVEL OF PFD ON PHOTOSYNTHETIC PARAMETERS AND PRODUCTION OF STEVIOL GLYCOSIDES BY HYDROPONIC CULTURE FROM STEVIA REBAUDIANA BERTONI

Stevia rebaudiana produces steviol glycosides (GSt) 300 times sweeter higher than sugar cane. The problem is that the global demand is increasing and the agricultural production is insufficient. Is reported that Hydroponic cultivation and high irradiance increase the yield of S. rebaudiana and the production of steviol glycosides. This is due to the management and control of nutrition and the effect of light on the photosynthetic system, respectively. Therefore, the objective was to evaluate the effect on three levels of PFD (440.8, 383.9 and 273.2 μmol m-2 s) efficiency of photosystem PSII, and production of glycosides of steviol in S. rebaudiana var. Morita II at 40 and 90 days, using a NFT hydroponic culture (Nutrient Film Technique). The highest photosynthetic rate was recorded at 440.8 μmol m-2 s with 15.4 and 17.72 ppm CO2 min-1 g at 40 and 90 of culture days respectively and the higher content of steviol glycosides, with this same PFD, the lowest fluorescence emission occurred in PSII, while with low luminosity, the absorbed energy was harnessed more efficiently by the PSIIRC. The photosynthesis and production of steviol glycosides in S. rebaudiana were affected by level of PFD and culture time in a hydroponic system.

Effects of Different Elicitors on Micropropagation, Biomass and Secondary Metabolite Production of Stevia rebaudiana Bertoni-A Review.

Stevia rebaudiana Bertoni is a valuable plant whose products are increasingly used in medicine, pharmacy and the food industry. This necessitates the use of biotechnological approaches for its mass propagation. Establishing optimal conditions for in vitro cultivation is essential for obtaining high biomass and secondary metabolites production. A large number of articles considering the role of plant growth regulators and other additives in the culture medium in the growth and development of Stevia are available in the literature. However, there are no summarized data about the use of nanoparticles in Stevia tissue cultures. Therefore, this review also includes the research conducted so far on the effect of nanoparticles on Stevia micropropagation. Furthermore, the influence of different elicitors on secondary metabolite production and antioxidant activity of in vitro-cultivated Stevia plants have been discussed. By referring to the collected literature, we concluded that biotechnological approaches applied to S. rebaudiana cultivation might improve the agronomic traits of plants and steviol glycosides production.

Compost and plant growth-promoting bacteria enhanced steviol glycoside synthesis in stevia (Stevia rebaudiana Bertoni) plants by improving soil quality and regulating nitrogen uptake

Stevia ( Stevia rebaudiana Bertoni) is an important industrial crop, and its leaves contain steviol glycosides (SGs), which are valuable compounds because they are characterized by a high sweetness and low caloric content. SGs yield depends on nitrogen (N) nutrition; however, the role of N uptake in SGs synthesis needs clarification, especially in organic farming systems that depend on organic and biofertilizers. Plant growth-promoting bacteria (PGPB; i.e., Azospirillum brasilense and Bacillus circulans ) and compost were used to study the effect of N nutrition on SGs yield. The experiment consisted of five N treatments with or without PGPB inoculation. The fertilization treatments were: C = without N, T 1 = urea, T 2 = compost, T 3 = urea and compost (75% of N from urea and the rest from compost) and T 4 = urea and compost (50% of N from urea and the rest 50% from compost). Compost and PGPB increased the soil quality indicators and enhanced the synthesis of chlorophyll and nutrient uptake by stevia plants. Mixtures of compost and urea under inoculation with PGPB enhanced soil microbes and enzymatic activity. The highest significant value of nitrogen use efficiency (NUE) resulted from urea alone, while the maximum phosphorus (P) and potassium (K) uptake came from compost. Although urea led to an increase in stevia N uptake, it led to a clear decrease in SGs yield. Urea decreased the total soluble sugar and leaf C/N ratio by 29% and 31%, respectively, compared to those of C. T 4 increased the SGs concentration by 74% and 46%, respectively, compared to that of urea alone in the presence and absence of PGPB. PGPB enhanced the soil quality and promoted nutrient uptake, resulting in significant increases in SGs yield. Regulating N uptake enhanced synthesis of soluble sugars and increased the C/N ratio, which caused an increase in SGs production. The use of compost and plant growth-promoting bacteria is a suitable strategy to produce stevia plants with low rates of inorganic nitrogen without any yield loss. • Compost and plant growth-promoting bacteria (PGPB) enhances stevia growth. • Compost and PGPB reregulate N-uptake in stevia plants. • Urea decreased the total soluble sugar and leaf C/N ratio. • Regulating N-uptake increased the synthesis steviol glycosides (SGs). • The findings might be an aid for sustainable N management in stevia production.

End-point determination of the extraction processes for Stevia rebaudiana Bertoni leaves by near-infrared spectroscopy

Rebaudioside A (RA) and Stevioside (STV) are abundant steviol glycosides (SGs) contained in Stevia rebaudiana Bertoni leaves, which exhibit good stability and various pharmacological activities that have been widely developed in the food and pharmaceutical industries. However, the stevia industry still suffers from high consumption, low efficiency, and long-term dependence on the operational experience of workers. The extraction process of Stevia rebaudiana Bertoni leaves one of the fundamental units for the production of SGs, which is crucial for the homogeneous stability of the final product. The applications of near-infrared (NIR) spectroscopy combined with chemometrics in determining the end-point of the extraction process were proposed in this study. Firstly, the quantitative models were established by partial least squares regression (PLSR) and back-propagation artificial neural network (BP-ANN) to rapidly detect the changes of content RA and STV, which have good prediction results. Secondly, the qualitative analysis methods were established by moving block of standard deviation (MBSD), absolute distance of standard deviation (ADSD) and principal component analysis (PCA) to rapidly determine the extraction end-point, which MBSD method was consistent with the high-performance liquid chromatography (HPLC) method. Finally, the variation of the extraction process was revealed by Aquaphotomic to provide a microscopic perspective of water molecules for end-point determination. The HPLC method requires 30 min to determine the content, whereas NIR spectroscopy requires only 18 s to obtain a spectrum. These results indicate that the extraction end-point of Stevia rebaudiana Bertoni leaves can be determined rapidly and accurately using NIR spectroscopy, which provides a significant reference for other food, medicinal plants, and agricultural products production processes.

Critical Review on Key Approaches to Enhance Synthesis and Production of Steviol Glycosides: A Blueprint for Zero-Calorie Sweetener

Steviol glycosides (SvGls) are plant secondary metabolites belonging to the class diterpenes. SvGls naturally derived from non-calorie sugar have therapeutic properties for diabetics, carcinogens, and anti-microbials. Over the past few years, SvGls have been extensively researched because of their extraordinary sweetness without side effects. SvGls are classified into several types based on the number of sugar bases attached to the steviol. Due to the difference in glycosylation, different SvGls have different sensory properties. One of the main obstacles is the mass production of SvGls. Novel physical and chemical treatments and advanced biotechnological approaches are introduced to increase the production of SvGls. Extraction of high-purity SvGls from plants is difficult, so researchers have manipulated the metabolic pathways of engineered microorganisms for bulk production of SvGls. There are many aspects related to the biochemistry and metabolism of SvGls, but their functional relationship with the S. rebaudiana is not well understood. Thus there is a need for in-depth research in this area. This review provides the readers with an overview of the research gaps and possible promising methodologies that can be utilized in the future. To trigger more research, this review encompasses the recent developments in SvGls production and marketing.

Improvement of salt tolerance in Stevia rebaudiana by co-application of endophytic fungi and exogenous spermidine

Stevia (Stevia rebaudiana Bertoni) is an important and salt-sensitive medicinal plant used as a natural sweetener in food products. In this work, we used a pot experiment to investigate the role of microbial inoculation and exogenous polyamine spermidine (Spd) in conferring salt stress tolerance in stevia by analyzing plant growth, chlorophyll (Chl) content, Chl a fluorescence, antioxidant defense system and steviol glycosides. The inoculated plants with Piriformospora indica (Pi), Tricuderma virens (Trich) or Pi+Trich were grown in optimum conditions. At the late of vegetative stage (BBCH growth stage 47), plants sprayed with different concentrations of Spd (0, 0.75, and 1.5 mM) and then were exposed to salinity stress by irrigation with saline treatments (mix of Caspian Sea and distilled water; 0, 6, and 12 dS m−1). The results revealed that salt stress increased hydrogen peroxide (H2O2) and lipid peroxidation (Malondialdehyde: MDA), and consequently plant growth characteristics and maximum photochemical quantum yield of PSII (Fv/Fm) as well as Chl content decreased, but antioxidant enzymes activity including superoxide (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) increased in the plants. Exposure to moderate salinity increased stevioside (Stv) and rebaudioside A (Reb A) content, while these compounds were decreased in the presence of severe salinity. The endophytic symbiosis and foliar spray of Spd (especially in Pi+Trich and Spd 0.75 mM) mitigated salt stress by significant increases in antioxidant enzymes activity and a decrease in H2O2 content and lipid peroxidation. Inoculation with Pi, Trich, and especially Pi+Trich increased the Chl content, Fv/Fm, shoot dry weight, and Stv and Reb A content at all salinity levels. Consequently, combined inoculation of Pi and Trich and foliar application of Spd can be a good approach to improve tolerance, growth and steviol glycosides production of stevia in salt stress conditions.

Catalytic flexibility of rice glycosyltransferase OsUGT91C1 for the production of palatable steviol glycosides

Steviol glycosides are the intensely sweet components of extracts from Stevia rebaudiana. These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific β (1–2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa, which is efficient at catalyzing β (1–2) glucosylation. The enzyme’s ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze β (1–2) glucosylation in two distinct orientations as well as β (1–6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable β (1–2) glucosylation, eliminate β (1–6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides.

Effect of light intensity on steviol glycosides production in leaves of Stevia rebaudiana plants

Stevia rebaudiana leaf extracts contain stevioside and rebaudioside A, two steviol glycosides (SGs) used as natural sweeteners because of their non-toxic, thermally stable and non-caloric properties. Indeed, leaf extracts can be up to 300 times sweeter than sucrose. Stevioside and rebaudioside A have organoleptic differences, the first one having an undesirable bitterness and the second one a higher sweetener capacity. Selection of the S. rebaudiana varieties and the best environmental conditions that elicit higher SGs content and the appropriate composition is an important goal. In this study we quantified and compared the amount of stevioside and rebaudioside A in two of the most used S. rebaudiana cultivars, Morita II and Criolla. Our results show a strong differential ratio of stevioside and rebaudioside A accumulated in the leaf between these cultivars. The Criolla cultivar showed about 3 times more stevioside per mg of dry weight than Morita II, whereas the Morita II accumulated almost 10 times more rebaudioside A than that produced in Criolla. We observed an enhanced expression in Morita II of three genes (SrKA13H, SrUGT74G1 and SrUGT76G1) known to encode three enzymes that participate in SGs biosynthesis, likely contributing to the differences in the stevioside and rebaudioside A accumulation. Not only genetic variation can affect SGs composition, but also environmental factors and crop management. Numerous studies have shown that the light regime in which S. rebaudiana cultivars grow can affect SGs accumulation. However, the optimal light regime to increase total SGs content is currently controversial. By applying various light intensities, we detected an increase of expression of these three biosynthetic genes at higher light intensity, accompanied by higher levels of stevioside and rebaudioside A, demonstrating that light intensity influences the synthesis of SGs.

Biotechnological interventions of in vitro propagation and production ofvaluable secondary metabolites in Stevia rebaudiana.

Plant cell and tissue culture makes provision of a sustainable and nature-friendly strategy for the production of secondary metabolites, and modern progress in gene editing and genome engineering provides novel possibilities to improve both the qualitative and quantitative aspects of such phytochemicals. The ever-expanding quest for plant-based medicine to treat diabetes facilitates large-scale cultivation of Stevia rebaudiana to enhance the yield of its much-coveted low-calorie sweetener glycosides. The potential to process stevia as a "natural" product should enhance the acceptance of steviosides as a natural calorie-free sweetener especially suitable for use in diabetic and weight control drinks and foods. Besides sweetener agents, S. rebaudiana is a potent source of many antioxidant compounds and is used to cure immunodeficiencies, neurologic disorders, inflammation, diabetes mellitus, Parkinson's disease, and Alzheimer's disease. This comprehensive review presents the research outcomes of the many biotechnological interventions implicated to upscale the yield of steviol glycosides and its derivatives in in vitro cell, callus, tissue, and organ cultures with notes on the use of bioreactor and genetic engineering in relation to the production of these valuable compounds in S. rebaudiana. KEY POINTS: • Critical and updated assessment on sustainable production of steviol glycosides from Stevia rebaudiana. • In vitro propagation of S. rebaudiana and elicitation of steviol glycosides production. • Genetic fidelity and diversity assessment of S. rebaudiana using molecular markers.

In silico Characterization of UGT74G1 Protein in Stevia rebaudiana Bertoni Accession MS007

Due to its low-calorie property, Stevia rebaudiana is being promoted as an alternative sweetener for diabetic and obese patients. The steady demand in the market for high-quality stevia extracts presents a challenge for the enhanced production of steviol glycosides that are safe for human consumption. This study characterized the structure and content of the gene involved in the production of UGT74G1 protein for Stevia rebaudiana accession MS007 through in silico analysis using a transcriptome dataset of stevia MS007. Homologous search using BLASTp shows high similarity to Q6VAA6 RecName: Full=UDP-glycosyltransferase 74G1 (S. rebaudiana) as the top hit sequence. InterPro family and domain protein motif search revealed UDP-glucuronosyl/UDP-glucosyltransferase (IPR002213) and UDP-glycosyltransferase family, conserved site (IPR035595). The phylogenetic tree construction was done by selecting 14 out of 102 protein sequences from BLASTp search. The phylogenetic analysis revealed a high value of bootstrapping, which was 100, indicating the high similarity between UGT74G1 (Q6VAA6.1 and Cluster-31069.45201) in S. rebaudiana. ProtParam ExPASy, PSIPRED, and Phyre2 computed the primary, secondary, and tertiary structures for UGT74G1 protein. The UGT74G1 predicted tertiary structure scored 100.0% confidence by the single highest scoring template and coverage of 96%. The model has dimensions (Å) of X: 57.609, Y: 70.386, and Z: 58.351. Outcomes of this research will help enhance understanding UDP-glycosyltransferase 74G1 (S. rebaudiana MS007) characteristics and enhance target identification processes to improve understanding of protein-protein interaction in S. rebaudiana MS007.