A sustainable strategy for biosynthesis of Rebaudioside D using a novel glycosyltransferase of Solanum tuberosum.

Bioconversion of Rebaudioside D faces high-cost obstacles. Herein, a novel glycosyltransferase StUGT converting Rebaudioside A to Rebaudioside D was screened and characterized, which exhibits stronger affinity and substrate specificity for Rebaudioside A than previously reported enzymes. A whole-cell catalytic system was thus developed using the StUGT strain. The production of Rebaudioside D was enhanced significantly by enhancing cell permeability, and the maximum production of 6.12 g/L and the highest yield of 98.08% by cell catalyst was obtained by statistical-based optimization. A new cascade process utilizing this recombinant strain and E. coli expressing sucrose synthase was further established to reduce cost through replacing expensive UDPG with sucrose. A StUGT-GsSUS1 system exhibited high catalytic capability, and 5.27 g L-1 Rebaudioside D was achieved finally without UDPG addition by systematic optimization. This is the best performance reported in cell-cascaded biosynthesis, which paves a new cost-effective strategy for sustainable synthesis of scarce premium sweeteners from biomass.

Improving Thermostability and Catalytic Activity of Glycosyltransferase From Panax ginseng by Semi-Rational Design for Rebaudioside D Synthesis.

As a natural sweetener and sucrose substitute, the biosynthesis and application of steviol glycosides containing the component rebaudioside D have attracted worldwide attention. Here, a glycosyltransferase PgUGT from Panax ginseng was first reported for the biosynthesis of rebaudioside D. With the three-dimensional structures built by homology modeling and deep-learning-based modeling, PgUGT was semi-rationally designed by FireProt. After detecting 16 site-directed variants, eight of them were combined in a mutant Mut8 with both improved enzyme activity and thermostability. The enzyme activity of Mut8 was 3.2-fold higher than that of the wild type, with an increased optimum reaction temperature from 35 to 40°C. The activity of this mutant remained over 93% when incubated at 35°C for 2 h, which was 2.42 times higher than that of the wild type. Meanwhile, when the enzymes were incubated at 40°C, where the wild type was completely inactivated after 1 h, the residual activity of Mut8 retained 59.0% after 2 h. This study would provide a novel glycosyltransferase with great potential for the industrial production of rebaudioside D and other steviol glycosides.

Safety of the proposed amendment of the specifications for enzymatically produced steviol glycosides (E 960c): Rebaudioside D produced via enzymatic bioconversion of purified stevia leaf extract.

The EFSA Panel on Food Additives and Flavourings (FAF Panel) provides a scientific opinion on the safety of a proposed amendment of the specifications of enzymatically produced steviol glycosides (E 960c) with respect to the inclusion of rebaudioside D produced via enzyme-catalysed bioconversion of purified stevia leaf extract. Rebaudioside D (95% on dry basis) is produced via enzymatic bioconversion of purified stevia leaf extract using uridine diphosphate (UDP)-glucosyltransferase (UGT) and sucrose synthase enzymes produced by the genetically modified yeast K. phaffii UGT-A, that facilitates the transfer of glucose to purified stevia leaf extract via glycosidic bonds. The same enzymes from K. phaffii UGT-A may be used in the manufacturing process of the food additive, rebaudioside M produced via enzyme modification of steviol glycosides from stevia (E 960c(i)). The Panel considered that separate specifications would be needed for this food additive produced via the manufacturing process described in the current application, aligned with those already established for E 960c(i). The Panel concluded that there is no toxicological concern for Rebaudioside D produced via enzymatic bioconversion of purified stevia leaf extract using UDP-glucosyltransferase and sucrose synthase produced by a genetically modified strain of the yeast K. phaffii. However, based on the available data, the Panel could not exclude the possibility that some residual amount of DNA coding for the kanamycin resistance gene could remain in the final product. Should this gene propagate in microbiota due to the presence of recombinant DNA in the final product, this would be of concern. Therefore, the Panel concluded that the safety of Rebaudioside D produced via this enzymatic bioconversion was not sufficiently demonstrated with the available data given that the absence of recombinant DNA was not shown.

Highly efficient production of rebaudioside D enabled by structure-guided engineering of bacterial glycosyltransferase YojK.

Owing to zero-calorie, high-intensity sweetness and good taste profile, the plant-derived sweetener rebaudioside D (Reb D) has attracted great interest to replace sugars. However, low content of Reb D in stevia rebaudiana Bertoni as well as low soluble expression and enzymatic activity of plant-derived glycosyltransferase in Reb D preparation restrict its commercial usage. To address these problems, a novel glycosyltransferase YojK from Bacillus subtilis 168 with the ability to glycosylate Reb A to produce Reb D was identified. Then, structure-guided engineering was performed after solving its crystal structure. A variant YojK-I241T/G327N with 7.35-fold increase of the catalytic activity was obtained, which allowed to produce Reb D on a scale preparation with a great yield of 91.29%. Moreover, based on the results from molecular docking and molecular dynamics simulations, the improvement of enzymatic activity of YojK-I241T/G327N was ascribed to the formation of new hydrogen bonds between the enzyme and substrate or uridine diphosphate glucose. Therefore, this study provides an engineered bacterial glycosyltransferase YojK-I241T/G327N with high solubility and catalytic efficiency for potential industrial scale-production of Reb D.

Efficient synthesis of rebaudioside D2 through UGT94D1-catalyzed regio-selective glycosylation.

Steviol glycosides have been widely applied as new sweeteners in food, beverages, health care, and daily chemical industry owing to the properties of high-intensity sweetness, low calorie, and good physiological characteristics. However, most of steviol glycosides have a bitter taste. Their organoleptic properties can be effectively improved by modifying the linked glycosyl units. In this study, UGT94D1, a uridine diphosphate-dependent glycosyltransferase from Sesamum indicum, was reported to selectively glycosylate rebaudioside A (Reb A) for the synthesis of rebaudioside D2 (Reb D2). Furthermore, a cascade reaction system was constructed to synthesize Reb D2 with 94.66% yield by coupling UGT94D1 with sucrose synthase AtSuSy from Arabidopsis thaliana. Thus, our study not only introduced a practical method for the synthesis of steviol glycosides but also provided the possibility for further exploration of Reb D2.

Comparative transcriptomic of Stevia rebaudiana provides insight into rebaudioside D and rebaudioside M biosynthesis.

Rebaudioside D (Reb D) and rebaudioside M (Reb M) are commercially important low/no-calorie natural sweeteners. However, they are present in a minor proportion of all steviol glycosides (SGs) in Stevia rebaudiana Bertoni (S. rebaudiana). Strain-dependent deviation in Reb D and Reb M biosynthesis is one key breach for breeding of S. rebaudiana, which has not been studied at the transcriptional level. Herein, five different S. rebaudiana varieties with distinct SGs contents, one cultivar having high stevioside content (HST), one cultivar having high Reb A content (HRA) and three cultivars having high Reb D and Reb M content (HDM1, HDM2, HDM3), were selected for RNA-seq analysis. In total, 131,655 de novo assembled unigenes were found in the RNA-seq data. According to Reb D and Reb M content divergence of S. rebaudiana accessions, 2186 differentially expressed genes (DEGs) were selected as potential genes related to Reb D and Reb M biosynthesis. Weighted Gene Co-expression Network Analysis (WGCNA) was used to explore the genes associated with the Reb D and Reb M biosynthesis. The unigenes from the positively associated turquoise module formed a layered co-expression network. There are 7 UDP-dependent glycosyltransferases (UGT) and 76 transcription factors (TFs) distributing at different regions which represented varying coherence of Reb D and Reb M biosynthesis. Particularly, two TFs having a strong correlation with two UGTs in the network were also discovered. The present study provided a comprehensive insight into networks for regulation of Reb D and Reb M contents in S. rebaudiana.

Heterologous expression of EUGT11 from Oryza sativa in Pichia pastoris for highly efficient one-pot production of rebaudioside D from rebaudioside A.

Rebaudioside D is a promising sweetener due to its zero calorie and high sweetness. Here, a transglucosylase gene eugt11 from Oryza sativa was for the first time expressed in Pichia pastoris, and transformant XE-3 showed the highest expression levels in pH 5.5 BMMY media containing 0.75% methanol. The affinity-purified EUGT11 from XE-3 displayed the highest activity at pH 6.0-6.5 and 45 °C, compared to pH 8.5 and 35 °C for EUGT11 from Escherichia coli. One-pot synthesis with orthogonal design was employed to optimize the rebaudioside D production using XE-3, and the initial pH 7.0 of the medium appears to be a significant factor and delivers the highest conversion efficiency. A two-step temperature-control strategy was developed, and a conversion rate of 95.31% was achieved at 28/35 °C vs. 62.41% in a one-step process at 28 °C. This study provides a high-efficient whole-cell biocatalysts technology for the sweetener production.

Synthesis of rebaudioside D, using glycosyltransferase UGTSL2 and in situ UDP-glucose regeneration.

Steviol glycosides from Stevia rebaudiana leaves are used in stevia-based sweeteners for their intense sweetness and low calories. Rebaudioside D is present in leaves in minute quantities (∼0.4-0.5% w/w total dry weight), but it is ∼350 times sweeter than sucrose, and sweeter than the more abundant rebaudioside A and stevioside. In the present study, pathways for rebaudioside D synthesis and UDP-glucose recycling were developed by coupling recombinant UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Reaction parameters, including substrate ratio, sucrose concentration, temperature, crude extract concentration, and reaction time, were evaluated, and 17.4 g/l of rebaudioside D (yield = 74.6%) was obtained from 20 g/l of rebaudioside A after 20 h, using UDP or UDP-glucose in recombinant cell crude extracts. Extending the reaction time generated rebaudioside M2 from further glycosylation of rebaudioside D. Km values for UGTSL2 indicated a higher affinity for rebaudioside D than for rebaudioside A.

Beaming steviol glycoside analysis into the next dimension.

Nine state-of-the-art reversed phase (RP) columns for ultra-high performance liquid chromatography were tested for the separation of steviol glycosides. The main criteria were resolution of the critical peak pair rebaudioside A and stevioside and the retention time of rebaudioside D. Three columns yielded a resolution of 2 or more of the critical peak pair and two of them showed sufficient retention of rebaudioside D, namely 1.62 and 1.84min corresponding to retention factors of 0.98 and 1.24. The separation of nine steviol glycosides was possible in 11min with UV and MS compatible, buffer-free eluents at moderate temperature. The presented method is proposed to be adopted as a new official method.

Investigation of the solubility enhancement mechanism of rebaudioside D using a solid dispersion technique with potassium sorbate as a carrier.

Rebaudioside (Reb) D is a high intensity, natural sweetener that shows great potential for substituting sugar in sweetened beverages. However, Reb D is poorly water soluble, and thus, a solid dispersion technique was recently established to enhance its solubility. The purpose of this study was to elucidate the solubility enhancement mechanism of this solid dispersion material by employing Scanning Electron Microscopy (SEM), Raman spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR) and X-ray Diffraction (XRD). Potassium sorbate (KS) was chosen as the carrier and two different concentration ratios were investigated as solid dispersions (SD) and as physical mixtures (PM). Our data demonstrated the possible mechanism for enhancing solubility through solid dispersion through increased surface area/volume ratio and hydrogen bonding between Reb D and KS. The interaction between the two components were also related to the different concentration ratios, therefore an optimisation of the ratio is important to produce a soluble and stable complex.

Rebaudioside A and Rebaudioside D bitterness do not covary with Acesulfame K bitterness or polymorphisms in TAS2R9 and TAS2R31.

In order to reduce calories in foods and beverages, the food industry routinely uses non-nutritive sweeteners. Unfortunately, many are synthetically derived, and many consumers have a strong preference for natural sweeteners, irrespective of the safety data on synthetic non-nutritive sweeteners. Additionally, many non-nutritive sweeteners elicit aversive side tastes such as bitter and metallic in addition to sweetness. Bitterness thresholds of acesulfame-K (AceK) and saccharin are known to vary across bitter taste receptors polymorphisms in TAS2R31. RebA has shown to activate hTAS2R4 and hTAS2R14 in vitro. Here we examined bitterness and sweetness perception of natural and synthetic non-nutritive sweeteners. In a follow-up to a previous gene-association study, participants (n=122) who had been genotyped previously rated sweet, bitter and metallic sensations from rebaudioside A (RebA), rebaudioside D (RebD), aspartame, sucrose and gentiobiose in duplicate in a single session. For comparison, we also present sweet and bitter ratings of AceK collected in the original experiment for the same participants. At similar sweetness levels, aspartame elicited less bitterness than RebD, which was significantly less bitter than RebA. The bitterness of RebA and RebD showed wide variability across individuals, and bitterness ratings for these compounds were correlated. However, RebA and RebD bitterness did not covary with AceK bitterness. Likewise, single nucleotide polymorphisms (SNPs) shown previously to explain variation in the suprathreshold bitterness of AceK (rs3741845 in TAS2R9 and rs10772423 in TAS2R31) did not explain variation in RebA and RebD bitterness. Because RebA activates hT2R4 and hT2R14, a SNP in TAS2R4 previously associated with variation in bitterness perception was included here; there are no known functional SNPs for TAS2R14. In present data, a putatively functional SNP (rs2234001) in TAS2R4 did not explain variation in RebA or RebD bitterness. Collectively, these data indicate the bitterness of RebA and RebD cannot be predicted by AceK bitterness, reinforcing our view that bitterness is not a simple monolithic trait that is high or low in an individual. This also implies consumers who reject AceK may not find RebA and RebD aversive, and vice versa. Finally, RebD may be a superior natural non-nutritive sweetener to RebA, as it elicits significantly less bitterness at similar levels of sweetness.